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1
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Rashid F, Zaongo SD, Iqbal H, Harypursat V, Song F, Chen Y. Interactions between HIV proteins and host restriction factors: implications for potential therapeutic intervention in HIV infection. Front Immunol 2024; 15:1390650. [PMID: 39221250 PMCID: PMC11361988 DOI: 10.3389/fimmu.2024.1390650] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2024] [Accepted: 07/22/2024] [Indexed: 09/04/2024] Open
Abstract
Different host proteins target different HIV proteins and antagonize their functions, depending on the stage of the HIV life cycle and the stage of infection. Concurrently, HIV proteins also target and antagonize various different host proteins to facilitate HIV replication within host cells. The preceding quite specific area of knowledge in HIV pathogenesis, however, remains insufficiently understood. We therefore propose, in this review article, to examine and discuss the HIV proteins that counteract those host restriction proteins which results directly in increased infectivity of HIV. We elaborate on HIV proteins that antagonize host cellular proteins to promote HIV replication, and thus HIV infection. We examine the functions and mechanisms via which Nef, Vif, Vpu, Env, Vpr, and Vpx counteract host proteins such as Ser5, PSGL-1, IFITMS, A3G, tetherin, GBP5, SAMHD1, STING, HUSH, REAF, and TET2 to increase HIV infectivity. Nef antagonizes three host proteins, viz., Ser5, PSGL1, and IFITIMs, while Vpx also antagonizes three host restriction factors, viz., SAMHD1, STING, and HUSH complex; therefore, these proteins may be potential candidates for therapeutic intervention in HIV infection. Tetherin is targeted by Vpu and Env, PSGL1 is targeted by Nef and Vpu, while Ser5 is targeted by Nef and Env proteins. Finally, conclusive remarks and future perspectives are also presented.
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Affiliation(s)
- Farooq Rashid
- Department of Infectious Diseases, Chongqing Public Health Medical Center, Chongqing, China
| | - Silvere D. Zaongo
- Department of Infectious Diseases, Chongqing Public Health Medical Center, Chongqing, China
| | - Hifza Iqbal
- School of science, University of Management and Technology, Lahore, Pakistan
| | - Vijay Harypursat
- Department of Infectious Diseases, Chongqing Public Health Medical Center, Chongqing, China
| | - Fangzhou Song
- Basic Medicine College, Chongqing Medical University, Chongqing, China
| | - Yaokai Chen
- Department of Infectious Diseases, Chongqing Public Health Medical Center, Chongqing, China
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2
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Judith D, Versapuech M, Bejjani F, Palaric M, Verlhac P, Kuster A, Lepont L, Gallois-Montbrun S, Janvier K, Berlioz-Torrent C. ATG5 selectively engages virus-tethered BST2/tetherin in an LC3C-associated pathway. Proc Natl Acad Sci U S A 2023; 120:e2217451120. [PMID: 37155854 PMCID: PMC10193943 DOI: 10.1073/pnas.2217451120] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2022] [Accepted: 03/15/2023] [Indexed: 05/10/2023] Open
Abstract
Bone marrow stromal antigen 2 (BST2)/tetherin is a restriction factor that reduces HIV-1 dissemination by tethering virus at the cell surface. BST2 also acts as a sensor of HIV-1 budding, establishing a cellular antiviral state. The HIV-1 Vpu protein antagonizes BST2 antiviral functions via multiple mechanisms, including the subversion of an LC3C-associated pathway, a key cell intrinsic antimicrobial mechanism. Here, we describe the first step of this viral-induced LC3C-associated process. This process is initiated at the plasma membrane through the recognition and internalization of virus-tethered BST2 by ATG5, an autophagy protein. ATG5 and BST2 assemble as a complex, independently of the viral protein Vpu and ahead of the recruitment of the ATG protein LC3C. The conjugation of ATG5 with ATG12 is dispensable for this interaction. ATG5 recognizes cysteine-linked homodimerized BST2 and specifically engages phosphorylated BST2 tethering viruses at the plasma membrane, in an LC3C-associated pathway. We also found that this LC3C-associated pathway is used by Vpu to attenuate the inflammatory responses mediated by virion retention. Overall, we highlight that by targeting BST2 tethering viruses, ATG5 acts as a signaling scaffold to trigger an LC3C-associated pathway induced by HIV-1 infection.
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Affiliation(s)
- Delphine Judith
- Université Paris Cité, Institut Cochin, INSERM, CNRS, F-75014Paris, France
| | - Margaux Versapuech
- Université Paris Cité, Institut Cochin, INSERM, CNRS, F-75014Paris, France
| | - Fabienne Bejjani
- Université Paris Cité, Institut Cochin, INSERM, CNRS, F-75014Paris, France
| | - Marjory Palaric
- Université Paris Cité, Institut Cochin, INSERM, CNRS, F-75014Paris, France
| | - Pauline Verlhac
- Université Paris Cité, Institut Cochin, INSERM, CNRS, F-75014Paris, France
| | - Aurelia Kuster
- Université Paris Cité, Institut Cochin, INSERM, CNRS, F-75014Paris, France
| | - Leslie Lepont
- Université Paris Cité, Institut Cochin, INSERM, CNRS, F-75014Paris, France
| | | | - Katy Janvier
- Université Paris Cité, Institut Cochin, INSERM, CNRS, F-75014Paris, France
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3
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Upadhyay C, Rao PG, Feyznezhad R. Dual Role of HIV-1 Envelope Signal Peptide in Immune Evasion. Viruses 2022; 14:v14040808. [PMID: 35458538 PMCID: PMC9030904 DOI: 10.3390/v14040808] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2022] [Revised: 04/08/2022] [Accepted: 04/11/2022] [Indexed: 12/10/2022] Open
Abstract
HIV-1 Env signal peptide (SP) is an important contributor to Env functions. Env is generated from Vpu/Env encoded bicistronic mRNA such that the 5′ end of Env-N-terminus, that encodes for Env-SP overlaps with 3′ end of Vpu. Env SP displays high sequence diversity, which translates into high variability in Vpu sequence. This study aimed to understand the effect of sequence polymorphism in the Vpu-Env overlapping region (VEOR) on the functions of two vital viral proteins: Vpu and Env. We used infectious molecular clone pNL4.3-CMU06 and swapped its SP (or VEOR) with that from other HIV-1 isolates. Swapping VEOR did not affect virus production in the absence of tetherin however, presence of tetherin significantly altered the release of virus progeny. VEOR also altered Vpu’s ability to downregulate CD4 and tetherin. We next tested the effect of these swaps on Env functions. Analyzing the binding of monoclonal antibodies to membrane embedded Env revealed changes in the antigenic landscape of swapped Envs. These swaps affected the oligosaccharide composition of Env-N-glycans as shown by changes in DC-SIGN-mediated virus transmission. Our study suggests that genetic diversity in VEOR plays an important role in the differential pathogenesis and also assist in immune evasion by altering Env epitope exposure.
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4
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Stoneham CA, Langer S, De Jesus PD, Wozniak JM, Lapek J, Deerinck T, Thor A, Pache L, Chanda SK, Gonzalez DJ, Ellisman M, Guatelli J. A combined EM and proteomic analysis places HIV-1 Vpu at the crossroads of retromer and ESCRT complexes: PTPN23 is a Vpu-cofactor. PLoS Pathog 2021; 17:e1009409. [PMID: 34843601 PMCID: PMC8659692 DOI: 10.1371/journal.ppat.1009409] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2021] [Revised: 12/09/2021] [Accepted: 10/08/2021] [Indexed: 11/21/2022] Open
Abstract
The HIV-1 accessory protein Vpu modulates membrane protein trafficking and degradation to provide evasion of immune surveillance. Targets of Vpu include CD4, HLAs, and BST-2. Several cellular pathways co-opted by Vpu have been identified, but the picture of Vpu's itinerary and activities within membrane systems remains incomplete. Here, we used fusion proteins of Vpu and the enzyme ascorbate peroxidase (APEX2) to compare the ultrastructural locations and the proximal proteomes of wild type Vpu and Vpu-mutants. The proximity-omes of the proteins correlated with their ultrastructural locations and placed wild type Vpu near both retromer and ESCRT-0 complexes. Hierarchical clustering of protein abundances across the mutants was essential to interpreting the data and identified Vpu degradation-targets including CD4, HLA-C, and SEC12 as well as Vpu-cofactors including HGS, STAM, clathrin, and PTPN23, an ALIX-like protein. The Vpu-directed degradation of BST-2 was supported by STAM and PTPN23 and to a much lesser extent by the retromer subunits Vps35 and SNX3. PTPN23 also supported the Vpu-directed decrease in CD4 at the cell surface. These data suggest that Vpu directs targets from sorting endosomes to degradation at multi-vesicular bodies via ESCRT-0 and PTPN23.
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Affiliation(s)
- Charlotte A. Stoneham
- Department of Medicine, University of California, San Diego School of Medicine and Veterans Affairs San Diego Healthcare System, La Jolla, California, United States of America
| | - Simon Langer
- Infectious and Inflammatory Disease Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California, United States of America
| | - Paul D. De Jesus
- Infectious and Inflammatory Disease Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California, United States of America
| | - Jacob M. Wozniak
- Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California, United States of America
| | - John Lapek
- Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California, United States of America
| | - Thomas Deerinck
- National Center for Microscopy and Imaging Research, Center for Research on Biological Systems, University of California, San Diego, School of Medicine, La Jolla, California, United States of America
| | - Andrea Thor
- National Center for Microscopy and Imaging Research, Center for Research on Biological Systems, University of California, San Diego, School of Medicine, La Jolla, California, United States of America
| | - Lars Pache
- Infectious and Inflammatory Disease Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California, United States of America
| | - Sumit K. Chanda
- Infectious and Inflammatory Disease Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California, United States of America
| | - David J. Gonzalez
- Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California, United States of America
| | - Mark Ellisman
- National Center for Microscopy and Imaging Research, Center for Research on Biological Systems, University of California, San Diego, School of Medicine, La Jolla, California, United States of America
- Department of Neurosciences, University of California, San Diego School of Medicine, La Jolla, California, United States of America
| | - John Guatelli
- Department of Medicine, University of California, San Diego School of Medicine and Veterans Affairs San Diego Healthcare System, La Jolla, California, United States of America
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5
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Li B, Dong X, Zhang W, Chen T, Yu B, Zhao W, Yang Y, Wang X, Hu Q, Wang X. High-Throughput NanoBiT-Based Screening for Inhibitors of HIV-1 Vpu and Host BST-2 Protein Interaction. Int J Mol Sci 2021; 22:ijms22179308. [PMID: 34502213 PMCID: PMC8431494 DOI: 10.3390/ijms22179308] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2021] [Revised: 08/20/2021] [Accepted: 08/25/2021] [Indexed: 12/18/2022] Open
Abstract
Bone marrow stromal cell antigen 2 (BST-2), also known as CD317 or tetherin, has been identified as a host restriction factor that suppresses the release of enveloped viruses from host cells by physically tethering viral particles to the cell surface; however, this host defense can be subverted by multiple viruses. For example, human immunodeficiency virus (HIV)-1 encodes a specific accessory protein, viral protein U (Vpu), to counteract BST-2 by binding to it and directing its lysosomal degradation. Thus, blocking the interaction between Vpu and BST-2 will provide a promising strategy for anti-HIV therapy. Here, we report a NanoLuc Binary Technology (NanoBiT)-based high-throughput screening assay to detect inhibitors that disrupt the Vpu-BST-2 interaction. Out of more than 1000 compounds screened, four inhibitors were identified with strong activity at nontoxic concentrations. In subsequent cell-based BST-2 degradation assays, inhibitor Y-39983 HCl restored the cell-surface and total cellular level of BST-2 in the presence of Vpu. Furthermore, the Vpu-mediated enhancement of pesudotyped viral particle production was inhibited by Y-39983 HCl. Our findings indicate that our newly developed assay can be used for the discovery of potential antiviral molecules with novel mechanisms of action.
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Affiliation(s)
- Boye Li
- The Faculty of Environment and Life, Beijing University of Technology, Beijing 100124, China; (B.L.); (X.D.); (W.Z.); (T.C.); (B.Y.); (W.Z.); (Y.Y.); (X.W.)
- Center of Excellence for Environmental Safety and Biological Effects, Beijing Key Laboratory for Green Catalysis and Separation, Department of Chemistry and Biology, Beijing University of Technology, Beijing 100124, China
- Beijing International Science and Technology Cooperation Base of Antivirus Drug, Beijing University of Technology, Beijing 100124, China
| | - Xiaoxiao Dong
- The Faculty of Environment and Life, Beijing University of Technology, Beijing 100124, China; (B.L.); (X.D.); (W.Z.); (T.C.); (B.Y.); (W.Z.); (Y.Y.); (X.W.)
- Beijing International Science and Technology Cooperation Base of Antivirus Drug, Beijing University of Technology, Beijing 100124, China
| | - Wenmei Zhang
- The Faculty of Environment and Life, Beijing University of Technology, Beijing 100124, China; (B.L.); (X.D.); (W.Z.); (T.C.); (B.Y.); (W.Z.); (Y.Y.); (X.W.)
- Center of Excellence for Environmental Safety and Biological Effects, Beijing Key Laboratory for Green Catalysis and Separation, Department of Chemistry and Biology, Beijing University of Technology, Beijing 100124, China
- Beijing International Science and Technology Cooperation Base of Antivirus Drug, Beijing University of Technology, Beijing 100124, China
| | - Tian Chen
- The Faculty of Environment and Life, Beijing University of Technology, Beijing 100124, China; (B.L.); (X.D.); (W.Z.); (T.C.); (B.Y.); (W.Z.); (Y.Y.); (X.W.)
- Beijing International Science and Technology Cooperation Base of Antivirus Drug, Beijing University of Technology, Beijing 100124, China
| | - Boyang Yu
- The Faculty of Environment and Life, Beijing University of Technology, Beijing 100124, China; (B.L.); (X.D.); (W.Z.); (T.C.); (B.Y.); (W.Z.); (Y.Y.); (X.W.)
- Beijing International Science and Technology Cooperation Base of Antivirus Drug, Beijing University of Technology, Beijing 100124, China
| | - Wenyue Zhao
- The Faculty of Environment and Life, Beijing University of Technology, Beijing 100124, China; (B.L.); (X.D.); (W.Z.); (T.C.); (B.Y.); (W.Z.); (Y.Y.); (X.W.)
- Beijing International Science and Technology Cooperation Base of Antivirus Drug, Beijing University of Technology, Beijing 100124, China
| | - Yishu Yang
- The Faculty of Environment and Life, Beijing University of Technology, Beijing 100124, China; (B.L.); (X.D.); (W.Z.); (T.C.); (B.Y.); (W.Z.); (Y.Y.); (X.W.)
- Beijing International Science and Technology Cooperation Base of Antivirus Drug, Beijing University of Technology, Beijing 100124, China
| | - Xiaoli Wang
- The Faculty of Environment and Life, Beijing University of Technology, Beijing 100124, China; (B.L.); (X.D.); (W.Z.); (T.C.); (B.Y.); (W.Z.); (Y.Y.); (X.W.)
- Beijing International Science and Technology Cooperation Base of Antivirus Drug, Beijing University of Technology, Beijing 100124, China
| | - Qin Hu
- The Faculty of Environment and Life, Beijing University of Technology, Beijing 100124, China; (B.L.); (X.D.); (W.Z.); (T.C.); (B.Y.); (W.Z.); (Y.Y.); (X.W.)
- Beijing International Science and Technology Cooperation Base of Antivirus Drug, Beijing University of Technology, Beijing 100124, China
- Correspondence: (Q.H.); (X.W.)
| | - Xiayan Wang
- The Faculty of Environment and Life, Beijing University of Technology, Beijing 100124, China; (B.L.); (X.D.); (W.Z.); (T.C.); (B.Y.); (W.Z.); (Y.Y.); (X.W.)
- Center of Excellence for Environmental Safety and Biological Effects, Beijing Key Laboratory for Green Catalysis and Separation, Department of Chemistry and Biology, Beijing University of Technology, Beijing 100124, China
- Correspondence: (Q.H.); (X.W.)
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6
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Khan N, Geiger JD. Role of Viral Protein U (Vpu) in HIV-1 Infection and Pathogenesis. Viruses 2021; 13:1466. [PMID: 34452331 PMCID: PMC8402909 DOI: 10.3390/v13081466] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2021] [Revised: 07/16/2021] [Accepted: 07/24/2021] [Indexed: 12/11/2022] Open
Abstract
Human immunodeficiency virus (HIV)-1 and HIV-2 originated from cross-species transmission of simian immunodeficiency viruses (SIVs). Most of these transfers resulted in limited spread of these viruses to humans. However, one transmission event involving SIVcpz from chimpanzees gave rise to group M HIV-1, with M being the principal strain of HIV-1 responsible for the AIDS pandemic. Vpu is an HIV-1 accessory protein generated from Env/Vpu encoded bicistronic mRNA and localized in cytosolic and membrane regions of cells capable of being infected by HIV-1 and that regulate HIV-1 infection and transmission by downregulating BST-2, CD4 proteins levels, and immune evasion. This review will focus of critical aspects of Vpu including its zoonosis, the adaptive hurdles to cross-species transmission, and future perspectives and broad implications of Vpu in HIV-1 infection and dissemination.
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Affiliation(s)
| | - Jonathan D. Geiger
- Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, 504 Hamline Street, Room 110, Grand Forks, ND 58203, USA;
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7
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Planas D, Fert A, Zhang Y, Goulet JP, Richard J, Finzi A, Ruiz MJ, Marchand LR, Chatterjee D, Chen H, Wiche Salinas TR, Gosselin A, Cohen EA, Routy JP, Chomont N, Ancuta P. Pharmacological Inhibition of PPARy Boosts HIV Reactivation and Th17 Effector Functions, While Preventing Progeny Virion Release and de novo Infection. Pathog Immun 2020; 5:177-239. [PMID: 33089034 PMCID: PMC7556414 DOI: 10.20411/pai.v5i1.348] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2020] [Accepted: 04/04/2020] [Indexed: 01/02/2023] Open
Abstract
The frequency and functions of Th17-polarized
CCR6+RORyt+CD4+ T cells are rapidly
compromised upon HIV infection and are not restored with long-term viral
suppressive antiretroviral therapy (ART). In line with this, Th17 cells
represent selective HIV-1 infection targets mainly at mucosal sites, with
long-lived Th17 subsets carrying replication-competent HIV-DNA during ART.
Therefore, novel Th17-specific therapeutic interventions are needed as a
supplement of ART to reach the goal of HIV remission/cure. Th17 cells express
high levels of peroxisome proliferator-activated receptor gamma
(PPARy), which acts as a transcriptional repressor of the HIV provirus and the
rorc gene, which encodes for the Th17-specific master
regulator RORyt. Thus, we hypothesized that the pharmacological inhibition of
PPARy will facilitate HIV reservoir reactivation while enhancing Th17 effector
functions. Consistent with this prediction, the PPARy antagonist T0070907
significantly increased HIV transcription (cell-associated HIV-RNA) and
RORyt-mediated Th17 effector functions (IL-17A). Unexpectedly, the PPARy
antagonism limited HIV outgrowth from cells of ART-treated people living with
HIV (PLWH), as well as HIV replication in vitro.
Mechanistically, PPARy inhibition in CCR6+CD4+ T cells
induced the upregulation of transcripts linked to Th17-polarisation (RORyt,
STAT3, BCL6 IL-17A/F, IL-21) and HIV transcription (NCOA1-3, CDK9, HTATIP2).
Interestingly, several transcripts involved in HIV-restriction were upregulated
(Caveolin-1, TRIM22, TRIM5α, BST2, miR-29), whereas HIV permissiveness
transcripts were downregulated (CCR5, furin), consistent with the decrease in
HIV outgrowth/replication. Finally, PPARy inhibition increased intracellular
HIV-p24 expression and prevented BST-2 downregulation on infected T cells,
suggesting that progeny virion release is restricted by BST-2-dependent
mechanisms. These results provide a strong rationale for considering PPARy
antagonism as a novel strategy for HIV-reservoir purging and restoring
Th17-mediated mucosal immunity in ART-treated PLWH.
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Affiliation(s)
- Delphine Planas
- Département de microbiologie, infectiologie et immunologie; Faculté de médecine; Université de Montréal; Montréal, Québec, Canada.,Centre de recherche du CHUM; Montréal, Québec, Canada
| | - Augustine Fert
- Département de microbiologie, infectiologie et immunologie; Faculté de médecine; Université de Montréal; Montréal, Québec, Canada.,Centre de recherche du CHUM; Montréal, Québec, Canada
| | - Yuwei Zhang
- Département de microbiologie, infectiologie et immunologie; Faculté de médecine; Université de Montréal; Montréal, Québec, Canada.,Centre de recherche du CHUM; Montréal, Québec, Canada
| | | | - Jonathan Richard
- Département de microbiologie, infectiologie et immunologie; Faculté de médecine; Université de Montréal; Montréal, Québec, Canada.,Centre de recherche du CHUM; Montréal, Québec, Canada
| | - Andrés Finzi
- Département de microbiologie, infectiologie et immunologie; Faculté de médecine; Université de Montréal; Montréal, Québec, Canada.,Centre de recherche du CHUM; Montréal, Québec, Canada
| | - Maria Julia Ruiz
- Département de microbiologie, infectiologie et immunologie; Faculté de médecine; Université de Montréal; Montréal, Québec, Canada.,Centre de recherche du CHUM; Montréal, Québec, Canada
| | | | - Debashree Chatterjee
- Département de microbiologie, infectiologie et immunologie; Faculté de médecine; Université de Montréal; Montréal, Québec, Canada.,Centre de recherche du CHUM; Montréal, Québec, Canada
| | - Huicheng Chen
- Département de microbiologie, infectiologie et immunologie; Faculté de médecine; Université de Montréal; Montréal, Québec, Canada.,Centre de recherche du CHUM; Montréal, Québec, Canada
| | - Tomas Raul Wiche Salinas
- Département de microbiologie, infectiologie et immunologie; Faculté de médecine; Université de Montréal; Montréal, Québec, Canada.,Centre de recherche du CHUM; Montréal, Québec, Canada
| | - Annie Gosselin
- Département de microbiologie, infectiologie et immunologie; Faculté de médecine; Université de Montréal; Montréal, Québec, Canada.,Centre de recherche du CHUM; Montréal, Québec, Canada
| | - Eric A Cohen
- Institut de recherches cliniques de Montréal; Montréal, Québec, Canada
| | - Jean-Pierre Routy
- Chronic Viral Illness Service; Division of Hematology; McGill University Health Centre-Glen site; Montreal, Québec, Canada
| | - Nicolas Chomont
- Département de microbiologie, infectiologie et immunologie; Faculté de médecine; Université de Montréal; Montréal, Québec, Canada.,Centre de recherche du CHUM; Montréal, Québec, Canada
| | - Petronela Ancuta
- Département de microbiologie, infectiologie et immunologie; Faculté de médecine; Université de Montréal; Montréal, Québec, Canada.,Centre de recherche du CHUM; Montréal, Québec, Canada
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8
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Zadeh VR, Urata S, Sakaguchi M, Yasuda J. Human BST-2/tetherin inhibits Junin virus release from host cells and its inhibition is partially counteracted by viral nucleoprotein. J Gen Virol 2020; 101:573-586. [PMID: 32375950 DOI: 10.1099/jgv.0.001414] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023] Open
Abstract
Bone marrow stromal cell antigen-2 (BST-2), also known as tetherin, is an interferon-inducible membrane-associated protein. It effectively targets enveloped viruses at the release step of progeny viruses from host cells, thereby restricting the further spread of viral infection. Junin virus (JUNV) is a member of Arenaviridae, which causes Argentine haemorrhagic fever that is associated with a high rate of mortality. In this study, we examined the effect of human BST-2 on the replication and propagation of JUNV. The production of JUNV Z-mediated virus-like particles (VLPs) was significantly inhibited by over-expression of BST-2. Electron microscopy analysis revealed that BST-2 functions by forming a physical link that directly retains VLPs on the cell surface. Infection using JUNV showed that infectious JUNV production was moderately inhibited by endogenous or exogenous BST-2. We also observed that JUNV infection triggers an intense interferon response, causing an upregulation of BST-2, in infected cells. However, the expression of cell surface BST-2 was reduced upon infection. Furthermore, the expression of JUNV nucleoprotein (NP) partially recovered VLP production from BST-2 restriction, suggesting that the NP functions as an antagonist against antiviral effect of BST-2. We further showed that JUNV NP also rescued the production of Ebola virus VP40-mediated VLP from BST-2 restriction as a broad spectrum BST-2 antagonist. To our knowledge, this is the first report showing that an arenavirus protein counteracts the antiviral function of BST-2.
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Affiliation(s)
- Vahid Rajabali Zadeh
- Program for Nurturing Global Leaders in Tropical and Emerging Communicable Diseases, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.,Department of Emerging Infectious Diseases, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, Japan
| | - Shuzo Urata
- National Research Center for the Control and Prevention of Infectious Diseases (CCPID), Nagasaki University, Nagasaki, Japan.,Department of Emerging Infectious Diseases, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, Japan
| | - Miako Sakaguchi
- Central Laboratory, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, Japan
| | - Jiro Yasuda
- National Research Center for the Control and Prevention of Infectious Diseases (CCPID), Nagasaki University, Nagasaki, Japan.,Program for Nurturing Global Leaders in Tropical and Emerging Communicable Diseases, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.,Department of Emerging Infectious Diseases, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, Japan
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9
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Prévost J, Pickering S, Mumby MJ, Medjahed H, Gendron-Lepage G, Delgado GG, Dirk BS, Dikeakos JD, Stürzel CM, Sauter D, Kirchhoff F, Bibollet-Ruche F, Hahn BH, Dubé M, Kaufmann DE, Neil SJD, Finzi A, Richard J. Upregulation of BST-2 by Type I Interferons Reduces the Capacity of Vpu To Protect HIV-1-Infected Cells from NK Cell Responses. mBio 2019; 10:e01113-19. [PMID: 31213558 PMCID: PMC6581860 DOI: 10.1128/mbio.01113-19] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2019] [Accepted: 05/09/2019] [Indexed: 01/03/2023] Open
Abstract
The HIV-1 accessory protein Vpu enhances viral release by counteracting the restriction factor BST-2. Furthermore, Vpu promotes NK cell evasion by downmodulating cell surface NTB-A and PVR, known ligands of the NK cell receptors NTB-A and DNAM-1, respectively. While it has been established that Vpu's transmembrane domain (TMD) is required for the interaction and intracellular sequestration of BST-2, NTB-A, and PVR, it remains unclear how Vpu manages to target these proteins simultaneously. In this study, we show that upon upregulation, BST-2 is preferentially downregulated by Vpu over its other TMD substrates. We found that type I interferon (IFN)-mediated BST-2 upregulation greatly impairs the ability of Vpu to downregulate NTB-A and PVR. Our results suggest that occupation of Vpu by BST-2 affects its ability to downregulate other TMD substrates. Accordingly, knockdown of BST-2 increases Vpu's potency to downmodulate NTB-A and PVR in the presence of type I IFN treatment. Moreover, we show that expression of human BST-2, but not that of the macaque orthologue, decreases Vpu's capacity to downregulate NTB-A. Importantly, we show that type I IFNs efficiently sensitize HIV-1-infected cells to NTB-A- and DNAM-1-mediated direct and antibody-dependent NK cell responses. Altogether, our results reveal that type I IFNs decrease Vpu's polyfunctionality, thus reducing its capacity to protect HIV-1-infected cells from NK cell responses.IMPORTANCE The restriction factor BST-2 and the NK cell ligands NTB-A and PVR are among a growing list of membrane proteins found to be downregulated by HIV-1 Vpu. BST-2 antagonism enhances viral release, while NTB-A and PVR downmodulation contributes to NK cell evasion. However, it remains unclear how Vpu can target multiple cellular factors simultaneously. Here we provide evidence that under physiological conditions, BST-2 is preferentially targeted by Vpu over NTB-A and PVR. Specifically, we show that type I IFNs decrease Vpu's polyfunctionality by upregulating BST-2, thus reducing its capacity to protect HIV-1-infected cells from NK cell responses. This indicates that there is a hierarchy of Vpu substrates upon IFN treatment, revealing that for the virus, targeting BST-2 as part of its resistance to IFN takes precedence over evading NK cell responses. This reveals a potential weakness in HIV-1's immunoevasion mechanisms that may be exploited therapeutically to harness NK cell responses against HIV-1.
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Affiliation(s)
- Jérémie Prévost
- Centre de Recherche du CHUM, Montreal, Quebec, Canada
- Département de Microbiologie, Infectiologie et Immunologie, Université de Montréal, Montreal, Québec, Canada
| | - Suzanne Pickering
- Department of Infectious Disease, King's College London School of Life Sciences and Medicine, Guy's Hospital, London, United Kingdom
| | - Mitchell J Mumby
- Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada
| | | | | | | | - Brennan S Dirk
- Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada
| | - Jimmy D Dikeakos
- Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada
| | - Christina M Stürzel
- Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany
| | - Daniel Sauter
- Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany
| | - Frank Kirchhoff
- Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany
| | - Frederic Bibollet-Ruche
- Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
- Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - Beatrice H Hahn
- Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
- Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - Mathieu Dubé
- Centre de Recherche du CHUM, Montreal, Quebec, Canada
| | - Daniel E Kaufmann
- Centre de Recherche du CHUM, Montreal, Quebec, Canada
- Department of Medicine, Université de Montréal, Montreal, Quebec, Canada
- Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, California, USA
| | - Stuart J D Neil
- Department of Infectious Disease, King's College London School of Life Sciences and Medicine, Guy's Hospital, London, United Kingdom
| | - Andrés Finzi
- Centre de Recherche du CHUM, Montreal, Quebec, Canada
- Département de Microbiologie, Infectiologie et Immunologie, Université de Montréal, Montreal, Québec, Canada
- Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada
| | - Jonathan Richard
- Centre de Recherche du CHUM, Montreal, Quebec, Canada
- Département de Microbiologie, Infectiologie et Immunologie, Université de Montréal, Montreal, Québec, Canada
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10
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Sharma S, Jafari M, Bangar A, William K, Guatelli J, Lewinski MK. The C-Terminal End of HIV-1 Vpu Has a Clade-Specific Determinant That Antagonizes BST-2 and Facilitates Virion Release. J Virol 2019; 93:e02315-18. [PMID: 30867310 PMCID: PMC6532089 DOI: 10.1128/jvi.02315-18] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2018] [Accepted: 03/06/2019] [Indexed: 12/12/2022] Open
Abstract
The cellular protein bone marrow stromal antigen-2 (BST-2)/tetherin acts against a variety of enveloped viruses by restricting their release from the plasma membrane. The HIV-1 accessory protein Vpu counteracts BST-2 by downregulating it from the cell surface and displacing it from virion assembly sites. Previous comparisons of Vpus from transmitted/founder viruses and between viruses isolated during acute and chronic infection led to the identification of a tryptophan at position 76 in Vpu (W76) as a key determinant for the displacement of BST-2 from virion assembly sites. Although present in Vpus from clades B, D, and G, W76 is absent from Vpus from clades A, C, and H. Mutagenesis of the C-terminal region of Vpu from two clade C viruses led to the identification of a conserved LL sequence that is functionally analogous to W76 of clade B. Alanine substitution of these leucines partially impaired virion release. This impairment was even greater when the mutations were combined with mutations of the Vpu β-TrCP binding site, resulting in Vpu proteins that induced high surface levels of BST-2 and reduced the efficiency of virion release to less than that of virus lacking vpu Microscopy confirmed that these C-terminal leucines in clade C Vpu, like W76 in clade B, contribute to virion release by supporting the displacement of BST-2 from virion assembly sites. These results suggest that although encoded differently, the ability of Vpu to displace BST-2 from sites of virion assembly on the plasma membrane is evolutionarily conserved among clade B and C HIV-1 isolates.IMPORTANCE Although targeted by a variety of restriction mechanisms, HIV-1 establishes chronic infection in most cases, in part due to the counteraction of these host defenses by viral accessory proteins. Using conserved motifs, the accessory proteins exploit the cellular machinery to degrade or mistraffic host restriction factors, thereby counteracting them. The Vpu protein counteracts the virion-tethering factor BST-2 in part by displacing it from virion assembly sites along the plasma membrane, but a previously identified determinant of that activity is clade specific at the level of protein sequence and not found in the clade C viruses that dominate the pandemic. Here, we show that clade C Vpu provides this activity via a leucine-containing sequence rather than the tryptophan-containing sequence found in clade B Vpu. This difference seems likely to reflect the different evolutionary paths taken by clade B and clade C HIV-1 in human populations.
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Affiliation(s)
- Shilpi Sharma
- Department of Medicine, University of California San Diego, La Jolla, California, USA
| | - Moein Jafari
- Department of Medicine, University of California San Diego, La Jolla, California, USA
| | - Amandip Bangar
- Department of Medicine, University of California San Diego, La Jolla, California, USA
| | - Karen William
- Department of Medicine, University of California San Diego, La Jolla, California, USA
| | - John Guatelli
- Department of Medicine, University of California San Diego, La Jolla, California, USA
- VA San Diego Healthcare System, San Diego, California, USA
| | - Mary K Lewinski
- Department of Medicine, University of California San Diego, La Jolla, California, USA
- VA San Diego Healthcare System, San Diego, California, USA
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11
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βTrCP is Required for HIV-1 Vpu Modulation of CD4, GaLV Env, and BST-2/Tetherin. Viruses 2018; 10:v10100573. [PMID: 30347660 PMCID: PMC6212966 DOI: 10.3390/v10100573] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2018] [Revised: 10/10/2018] [Accepted: 10/16/2018] [Indexed: 12/27/2022] Open
Abstract
The Human immunodeficiency virus-1 (HIV-1) accessory protein Vpu modulates numerous proteins, including the host proteins CD4 and BST-2/tetherin. Vpu interacts with the Skp, Cullin, F-Box (SCF) ubiquitin ligase through interactions with the F-Box protein βTrCP (1 and/or 2). This interaction is dependent on phosphorylation of S52,56 in Vpu. Mutation of S52,56, or inhibition of the SCF, abolishes most Vpu activity against CD4 and partly reduces activity against BST-2/tetherin. Recently, Vpu has also been reported to interact with the clathrin adapter proteins AP-1 and AP-2, and these interactions were also found to be required for BST-2/tetherin antagonism in an S52,56 -dependent manner. In assays where HIV-1 is pseudotyped with gibbon ape leukemia virus (GaLV Env), Vpu has also been found to prevent GaLV Env from being incorporated into viral particles, but the mechanism for this antagonism is not fully understood. To clarify the role of the βTrCPs in Vpu function we used CRISPR/Cas9 to generate a clonal cell line lacking both βTrCP-1 and -2. Vpu activity against CD4 and GaLV Env was abolished in this cell line, and activity against BST-2/tetherin reduced significantly. Mutation of the S52,56 residues no longer affected Vpu activity against BST-2/tetherin in this cell line. These data suggest that the primary role of the S52,56 residues in antagonism of CD4, GaLV Env, and BST-2/tetherin is to recruit the SCF/βTrCP ubiquitin ligase.
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12
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Abstract
Viroporins are short polypeptides encoded by viruses. These small membrane proteins assemble into oligomers that can permeabilize cellular lipid bilayers, disrupting the physiology of the host to the advantage of the virus. Consequently, efforts during the last few decades have been focused towards the discovery of viroporin channel inhibitors, but in general these have not been successful to produce licensed drugs. Viroporins are also involved in viral pathogenesis by engaging in critical interactions with viral proteins, or disrupting normal host cellular pathways through coordinated interactions with host proteins. These protein-protein interactions (PPIs) may become alternative attractive drug targets for the development of antivirals. In this sense, while thus far most antiviral molecules have targeted viral proteins, focus is moving towards targeting host proteins that are essential for virus replication. In principle, this largely would overcome the problem of resistance, with the possibility of using repositioned existing drugs. The precise role of these PPIs, their strain- and host- specificities, and the structural determination of the complexes involved, are areas that will keep the fields of virology and structural biology occupied for years to come. In the present review, we provide an update of the efforts in the characterization of the main PPIs for most viroporins, as well as the role of viroporins in these PPIs interactions.
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Affiliation(s)
| | - David Bhella
- MRC-University of Glasgow Centre for Virus Research, Glasgow, UK
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13
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Jain P, Boso G, Langer S, Soonthornvacharin S, De Jesus PD, Nguyen Q, Olivieri KC, Portillo AJ, Yoh SM, Pache L, Chanda SK. Large-Scale Arrayed Analysis of Protein Degradation Reveals Cellular Targets for HIV-1 Vpu. Cell Rep 2018; 22:2493-2503. [PMID: 29490283 PMCID: PMC5916846 DOI: 10.1016/j.celrep.2018.01.091] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2016] [Revised: 11/03/2017] [Accepted: 01/30/2018] [Indexed: 11/28/2022] Open
Abstract
Accessory proteins of lentiviruses, such as HIV-1, target cellular restriction factors to enhance viral replication. Systematic analyses of proteins that are targeted for degradation by HIV-1 accessory proteins may provide a better understanding of viral immune evasion strategies. Here, we describe a high-throughput platform developed to study cellular protein stability in a highly parallelized matrix format. We used this approach to identify cellular targets of the HIV-1 accessory protein Vpu through arrayed coexpression with 433 interferon-stimulated genes, followed by differential fluorescent labeling and automated image analysis. Among the previously unreported Vpu targets identified by this approach, we find that the E2 ligase mediating ISG15 conjugation, UBE2L6, and the transmembrane protein PLP2 are targeted by Vpu during HIV-1 infection to facilitate late-stage replication. This study provides a framework for the systematic and high-throughput evaluation of protein stability and establishes a more comprehensive portrait of cellular Vpu targets.
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Affiliation(s)
- Prashant Jain
- Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Guney Boso
- Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Simon Langer
- Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Stephen Soonthornvacharin
- Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Paul D De Jesus
- Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Quy Nguyen
- Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Kevin C Olivieri
- Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Alex J Portillo
- Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Sunnie M Yoh
- Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Lars Pache
- Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
| | - Sumit K Chanda
- Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
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14
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Waheed AA, Gitzen A, Swiderski M, Freed EO. High-Mannose But Not Complex-Type Glycosylation of Tetherin Is Required for Restriction of HIV-1 Release. Viruses 2018; 10:v10010026. [PMID: 29303997 PMCID: PMC5795439 DOI: 10.3390/v10010026] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2017] [Revised: 12/28/2017] [Accepted: 12/31/2017] [Indexed: 11/16/2022] Open
Abstract
Tetherin is an interferon-inducible antiviral protein that inhibits the release of a broad spectrum of enveloped viruses by retaining virions at the surface of infected cells. While the role of specific tetherin domains in antiviral activity is clearly established, the role of glycosylation in tetherin function is not clear. In this study, we carried out a detailed investigation of this question by using tetherin variants in which one or both sites of N-linked glycosylation were mutated (N65A, N92A, and N65,92A), and chemical inhibitors that prevent glycosylation at specific stages of oligosaccharide were added or modified. The single N-linked glycosylation mutants, N65A and N92A, efficiently inhibited the release of Vpu-defective human immunodeficiency virus type 1 (HIV-1). In contrast, the non-glycosylated double mutant, N65,92A, lost its ability to block HIV-1 release. The inability of the N65,92A mutant to inhibit HIV-1 release is associated with a lack of cell-surface expression. A role for glycosylation in cell-surface tetherin expression is supported by tunicamycin treatment, which inhibits the first step of N-linked glycosylation and impairs both cell-surface expression and antiviral activity. Inhibition of complex-type glycosylation with kifunensine, an inhibitor of the oligosaccharide processing enzyme mannosidase 1, had no effect on either the cell-surface expression or antiviral activity of tetherin. These results demonstrate that high-mannose modification of a single asparagine residue is necessary and sufficient, while complex-type glycosylation is dispensable, for cell-surface tetherin expression and antiviral activity.
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Affiliation(s)
- Abdul A Waheed
- Virus-Cell Interaction Section, HIV Dynamics and Replication Program, National Cancer Institute, Bldg. 535, Room 108B, 1050 Boyles St., Frederick, MD 21702-1201, USA.
| | - Ariana Gitzen
- Virus-Cell Interaction Section, HIV Dynamics and Replication Program, National Cancer Institute, Bldg. 535, Room 108B, 1050 Boyles St., Frederick, MD 21702-1201, USA.
| | - Maya Swiderski
- Virus-Cell Interaction Section, HIV Dynamics and Replication Program, National Cancer Institute, Bldg. 535, Room 108B, 1050 Boyles St., Frederick, MD 21702-1201, USA.
| | - Eric O Freed
- Virus-Cell Interaction Section, HIV Dynamics and Replication Program, National Cancer Institute, Bldg. 535, Room 108B, 1050 Boyles St., Frederick, MD 21702-1201, USA.
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15
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Foster TL, Pickering S, Neil SJD. Inhibiting the Ins and Outs of HIV Replication: Cell-Intrinsic Antiretroviral Restrictions at the Plasma Membrane. Front Immunol 2018; 8:1853. [PMID: 29354117 PMCID: PMC5758531 DOI: 10.3389/fimmu.2017.01853] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2017] [Accepted: 12/07/2017] [Indexed: 01/01/2023] Open
Abstract
Like all viruses, human immunodeficiency viruses (HIVs) and their primate lentivirus relatives must enter cells in order to replicate and, once produced, new virions need to exit to spread to new targets. These processes require the virus to cross the plasma membrane of the cell twice: once via fusion mediated by the envelope glycoprotein to deliver the viral core into the cytosol; and secondly by ESCRT-mediated scission of budding virions during release. This physical barrier thus presents a perfect location for host antiviral restrictions that target enveloped viruses in general. In this review we will examine the current understanding of innate host antiviral defences that inhibit these essential replicative steps of primate lentiviruses associated with the plasma membrane, the mechanism by which these viruses have adapted to evade such defences, and the role that this virus/host battleground plays in the transmission and pathogenesis of HIV/AIDS.
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Affiliation(s)
- Toshana L Foster
- Department of Infectious Disease, School of Immunology and Microbial Sciences, King's College London, London, United Kingdom
| | - Suzanne Pickering
- Department of Infectious Disease, School of Immunology and Microbial Sciences, King's College London, London, United Kingdom
| | - Stuart J D Neil
- Department of Infectious Disease, School of Immunology and Microbial Sciences, King's College London, London, United Kingdom
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16
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Brinkmann C, Hoffmann M, Lübke A, Nehlmeier I, Krämer-Kühl A, Winkler M, Pöhlmann S. The glycoprotein of vesicular stomatitis virus promotes release of virus-like particles from tetherin-positive cells. PLoS One 2017; 12:e0189073. [PMID: 29216247 PMCID: PMC5720808 DOI: 10.1371/journal.pone.0189073] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2017] [Accepted: 11/17/2017] [Indexed: 11/26/2022] Open
Abstract
Vesicular stomatitis virus (VSV) release from infected cells is inhibited by the interferon (IFN)-inducible antiviral host cell factor tetherin (BST-2, CD317). However, several viruses encode tetherin antagonists and it is at present unknown whether residual VSV spread in tetherin-positive cells is also promoted by a virus-encoded tetherin antagonist. Here, we show that the viral glycoprotein (VSV-G) antagonizes tetherin in transfected cells, although with reduced efficiency as compared to the HIV-1 Vpu protein. Tetherin antagonism did not involve alteration of tetherin expression and was partially dependent on a GXXXG motif in the transmembrane domain of VSV-G. However, mutation of the GXXXG motif did not modulate tetherin sensitivity of infectious VSV. These results identify VSV-G as a tetherin antagonist in transfected cells but fail to provide evidence for a contribution of tetherin antagonism to viral spread.
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Affiliation(s)
| | - Markus Hoffmann
- Infection Biology Unit, German Primate Center, Kellnerweg 4, Göttingen, Germany
| | - Anastasia Lübke
- Infection Biology Unit, German Primate Center, Kellnerweg 4, Göttingen, Germany
| | - Inga Nehlmeier
- Infection Biology Unit, German Primate Center, Kellnerweg 4, Göttingen, Germany
| | - Annika Krämer-Kühl
- Infection Biology Unit, German Primate Center, Kellnerweg 4, Göttingen, Germany
| | - Michael Winkler
- Infection Biology Unit, German Primate Center, Kellnerweg 4, Göttingen, Germany
| | - Stefan Pöhlmann
- Infection Biology Unit, German Primate Center, Kellnerweg 4, Göttingen, Germany
- * E-mail:
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17
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Paliwal D, Joshi P, Panda SK. Hepatitis E Virus (HEV) egress: Role of BST2 (Tetherin) and interferon induced long non- coding RNA (lncRNA) BISPR. PLoS One 2017; 12:e0187334. [PMID: 29091957 PMCID: PMC5665557 DOI: 10.1371/journal.pone.0187334] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2017] [Accepted: 10/18/2017] [Indexed: 12/18/2022] Open
Abstract
Background The biology of Hepatitis E Virus (HEV), a common cause of epidemic and sporadic hepatitis, is still being explored. HEV exits liver through bile, a process which is essential for its natural transmission by feco-oral route. Though the process of this polarised HEV egress is not known in detail, HEV pORF3 and hepatocyte actin cytoskeleton have been shown to play a role. Methods Our transcriptome analysis in Hepatitis E virus (HEV) replicon transfected Huh7 cells at 24 and 72 hrs indicated that at 24hrs, both LncBISPR and BST2, expressed by a bidirectional promoter were highly upregulated whereas at 72 hrs, BST2 expression was comparatively reduced accompanied by normal levels of BISPR. These findings were confirmed by qPCR analysis. Co-localisation of BST2 and HEV pORF2 was confirmed in HEV transfected Huh7 by confocal microscopy. To investigate the role of BISPR/BST2 in HEV life cycle, particularly virus egress, we generated Huh7 cells with ~8kb deletion in BISPR gene using Crispr-Cas9 system. The deletion was confirmed by PCR screening, Sanger sequencing and Real time PCR. Virus egress in ΔBISPR Huh7 and Huh7 cells was compared by measuring HEV positive strand RNA copy numbers in cell lysates and culture supernatants at 24 and 72 hrs post HEV replicon transfection and further validated by western blot for HEV pORF2 capsid protein. Results ΔBISPR Huh7 cells showed ~8 fold increase in virus egress at 24 hrs compared to Huh7 cells. No significant difference in virus egress was observed at 72hrs. Immunohistochemistry in histologically normal liver and HEV associated acute liver failure revealed BST2 overexpression in HEV infected hepatocytes and a dominant canalicular BST2 distribution in normal liver in addition to the cytoplasmic localisation reported in literature. Conclusions These findings lead us to believe that BISPR and BST2 may regulate egress of HEV virions into bile in vivo. This system may also be used to scale up virus production in vitro.
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Affiliation(s)
- Daizy Paliwal
- Department of Pathology, All India Institute of Medical Sciences, New Delhi, India
| | - Prashant Joshi
- Department of Pathology, All India Institute of Medical Sciences, New Delhi, India
| | - Subrat Kumar Panda
- Department of Pathology, All India Institute of Medical Sciences, New Delhi, India
- * E-mail:
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18
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Cooperation of the Ebola Virus Proteins VP40 and GP 1,2 with BST2 To Activate NF-κB Independently of Virus-Like Particle Trapping. J Virol 2017; 91:JVI.01308-17. [PMID: 28878074 DOI: 10.1128/jvi.01308-17] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2017] [Accepted: 08/28/2017] [Indexed: 11/20/2022] Open
Abstract
BST2 is a host protein with dual functions in response to viral infections: it traps newly assembled enveloped virions at the plasma membrane in infected cells, and it induces NF-κB activity, especially in the context of retroviral assembly. In this study, we examined whether Ebola virus proteins affect BST2-mediated induction of NF-κB. We found that the Ebola virus matrix protein, VP40, and envelope glycoprotein, GP, each cooperate with BST2 to induce NF-κB activity, with maximal activity when all three proteins are expressed. Unlike human immunodeficiency virus type 1 Vpu protein, which antagonizes both virion entrapment and the activation of NF-κB by BST2, Ebola virus GP does not inhibit NF-κB signaling even while it antagonizes the entrapment of virus-like particles. GP from Reston ebolavirus, a nonpathogenic species in humans, showed a phenotype similar to that of GP from Zaire ebolavirus, a highly pathogenic species, in terms of both the activation of NF-κB and the antagonism of virion entrapment. Although Ebola virus VP40 and GP both activate NF-κB independently of BST2, VP40 is the more potent activator. Activation of NF-κB by the Ebola virus proteins either alone or together with BST2 requires the canonical NF-κB signaling pathway. Mechanistically, the maximal NF-κB activation by GP, VP40, and BST2 together requires the ectodomain cysteines needed for BST2 dimerization, the putative BST2 tetramerization residue L70, and Y6 of a potential hemi-ITAM motif in BST2's cytoplasmic domain. BST2 with a glycosylphosphatidylinositol (GPI) anchor signal deletion, which is not expressed at the plasma membrane and is unable to entrap virions, activated NF-κB in concert with the Ebola virus proteins at least as effectively as wild-type BST2. Signaling by the GPI anchor mutant also depended on Y6 of BST2. Overall, our data show that activation of NF-κB by BST2 is independent of virion entrapment in the case of Ebola virus. Nonetheless, BST2 may induce or amplify proinflammatory signaling during Ebola virus infection, potentially contributing to the dysregulated cytokine response that is a hallmark of Ebola virus disease.IMPORTANCE Understanding how the host responds to viral infections informs the development of therapeutics and vaccines. We asked how proinflammatory signaling by the host protein BST2/tetherin, which is mediated by the transcription factor NF-κB, responds to Ebola virus proteins. Although the Ebola virus envelope glycoprotein (GP1,2) antagonizes the trapping of newly formed virions at the plasma membrane by BST2, we found that it does not inhibit BST2's ability to induce NF-κB activity. This distinguishes GP1,2 from the HIV-1 protein Vpu, the prototype BST2 antagonist, which inhibits both virion entrapment and the induction of NF-κB activity. Ebola virus GP1,2, the Ebola virus matrix protein VP40, and BST2 are at least additive with respect to the induction of NF-κB activity. The effects of these proteins converge on an intracellular signaling pathway that depends on a protein modification termed neddylation. Better mechanistic understanding of these phenomena could provide targets for therapies that modulate the inflammatory response during Ebola virus disease.
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19
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Stoneham CA, Singh R, Jia X, Xiong Y, Guatelli J. Endocytic activity of HIV-1 Vpu: Phosphoserine-dependent interactions with clathrin adaptors. Traffic 2017; 18:545-561. [PMID: 28504462 DOI: 10.1111/tra.12495] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2016] [Revised: 05/09/2017] [Accepted: 05/10/2017] [Indexed: 10/19/2022]
Abstract
HIV-1 Vpu modulates cellular transmembrane proteins to optimize viral replication and provide immune-evasion, triggering ubiquitin-mediated degradation of some targets but also modulating endosomal trafficking to deplete them from the plasma membrane. Interactions between Vpu and the heterotetrameric clathrin adaptor protein (AP) complexes AP-1 and AP-2 have been described, yet the molecular basis and functional roles of such interactions are incompletely defined. To investigate the trafficking signals encoded by Vpu, we fused the cytoplasmic domain (CD) of Vpu to the extracellular and transmembrane domains of the CD8 α-chain. CD8-VpuCD was rapidly endocytosed in a clathrin- and AP-2-dependent manner. Multiple determinants within the Vpu CD contributed to endocytic activity, including phosphoserines of the β-TrCP binding site and a leucine-based ExxxLV motif. Using recombinant proteins, we confirmed ExxxLV-dependent binding of the Vpu CD to the α/σ2 subunit hemicomplex of AP-2 and showed that this is enhanced by serine-phosphorylation. Remarkably, the Vpu CD also bound directly to the medium (μ) subunits of AP-2 and AP-1; this interaction was dependent on serine-phosphorylation of Vpu and on basic residues in the μ subunits. We propose that the flexibility with which Vpu binds AP complexes broadens the range of cellular targets that it can misdirect to the virus' advantage.
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Affiliation(s)
- Charlotte A Stoneham
- Department of Medicine, University of California at San Diego, La Jolla, California.,VA San Diego Healthcare System, San Diego, California
| | - Rajendra Singh
- Department of Medicine, University of California at San Diego, La Jolla, California.,VA San Diego Healthcare System, San Diego, California
| | - Xiaofei Jia
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut
| | - Yong Xiong
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut
| | - John Guatelli
- Department of Medicine, University of California at San Diego, La Jolla, California.,VA San Diego Healthcare System, San Diego, California
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20
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Roy N, Pacini G, Berlioz-Torrent C, Janvier K. Characterization of E3 ligases involved in lysosomal sorting of the HIV-1 restriction factor BST2. J Cell Sci 2017; 130:1596-1611. [PMID: 28320822 PMCID: PMC5450231 DOI: 10.1242/jcs.195412] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2016] [Accepted: 02/17/2017] [Indexed: 12/17/2022] Open
Abstract
The cellular protein BST2 (also known as tetherin) acts as a major intrinsic antiviral protein that prevents the release of enveloped viruses by trapping nascent viral particles at the surface of infected cells. Viruses have evolved specific strategies to displace BST2 from viral budding sites in order to promote virus egress. In HIV-1, the accessory protein Vpu counters BST2 antiviral activity and promotes sorting of BST2 for lysosomal degradation. Vpu increases polyubiquitylation of BST2, a post-translation modification required for Vpu-induced BST2 downregulation, through recruitment of the E3 ligase complex SCF adaptors β-TrCP1 and β-TrCP2 (two isoforms encoded by BTRC and FBXW11, respectively). Herein, we further investigate the role of the ubiquitylation machinery in the lysosomal sorting of BST2. Using a small siRNA screen, we highlighted two additional regulators of BST2 constitutive ubiquitylation and sorting to the lysosomes: the E3 ubiquitin ligases NEDD4 and MARCH8. Interestingly, Vpu does not hijack the cellular machinery that is constitutively involved in BST2 ubiquitylation to sort BST2 for degradation in the lysosomes but instead promotes the recognition of BST2 by β-TrCP proteins. Altogether, our results provide further understanding of the mechanisms underlying BST2 turnover in cells. Highlighted Article: We identify two E3 ubiquitin ligases, NEDD4 and MARCH8, as regulators of BST2 (tetherin) – a protein that restricts viral release; we thus provide further understanding of the mechanisms underlying BST2 turnover in cells.
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Affiliation(s)
- Nicolas Roy
- Inserm, U1016, Institut Cochin, Paris, France.,CNRS, UMR8104, Paris, France.,Université Paris Descartes, Sorbonne Paris Cité, Paris, France
| | - Grégory Pacini
- Inserm, U1016, Institut Cochin, Paris, France.,CNRS, UMR8104, Paris, France.,Université Paris Descartes, Sorbonne Paris Cité, Paris, France
| | - Clarisse Berlioz-Torrent
- Inserm, U1016, Institut Cochin, Paris, France.,CNRS, UMR8104, Paris, France.,Université Paris Descartes, Sorbonne Paris Cité, Paris, France
| | - Katy Janvier
- Inserm, U1016, Institut Cochin, Paris, France .,CNRS, UMR8104, Paris, France.,Université Paris Descartes, Sorbonne Paris Cité, Paris, France
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21
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Lukhele S, Cohen ÉA. Conserved residues within the HIV-1 Vpu transmembrane-proximal hinge region modulate BST2 binding and antagonism. Retrovirology 2017; 14:18. [PMID: 28288652 PMCID: PMC5348903 DOI: 10.1186/s12977-017-0345-6] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2017] [Accepted: 03/05/2017] [Indexed: 11/10/2022] Open
Abstract
Background BST2 inhibits HIV-1 release by tethering nascent virions to the surface of infected cells. HIV-1 Vpu overcomes this restriction by removing BST2 from viral budding sites via BST2 intracellular trapping and sequestration, surface downregulation and/or displacement mechanisms. Vpu is composed of a short luminal tail, a transmembrane domain (TMD) and a cytoplasmic hinge region that is followed by two helices. BST2 counteraction relies on the ability of Vpu to physically bind BST2 through TMD interactions and recruit the clathrin-dependent trafficking machinery via a canonical acidic di-leucine signalling motif within the helix-2 of Vpu. The highly conserved Vpu transmembrane-proximal hinge region encompasses residues that resemble an acidic leucine-based trafficking motif, whose functional roles are currently ill-defined. In this study, we investigated the contribution of these residues towards Vpu-mediated BST2 antagonism. Results We show that while these conserved residues have no intrinsic activity on the cellular distribution of Vpu in the absence of BST2, they regulate the ability of Vpu to bind to BST2 and, consequently, govern both BST2-dependent trafficking properties of the protein as well as its co-localization with BST2. Moreover, these residues, particularly a glutamic acid residue positioned immediately following the TMD, are a determinant not only for efficient targeting of BST2, but also binding and degradation of CD4, another host membrane protein targeted by Vpu. Mechanistically, our data are consistent with a role of these residues in the maintenance of the Vpu TMD conformational configuration such that interactions with membrane-associated host targets are favoured. Conclusions Altogether, this work demonstrates an important regulatory role of the transmembrane-proximal Vpu hinge region residues towards enabling the protein to efficiently engage its target host proteins. Thus, this highly conserved, cytosolic Vpu hinge region may represent an attractive target for the development of anti-Vpu inhibitors. Electronic supplementary material The online version of this article (doi:10.1186/s12977-017-0345-6) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Sabelo Lukhele
- Laboratory of Human Retrovirology, Institut de Recherches Cliniques de Montréal (IRCM), 110, Pine Avenue West, Montreal, QC, H2W 1R7, Canada.,Division of Experimental Medicine, McGill University, Montreal, QC, H3A 1A3, Canada
| | - Éric A Cohen
- Laboratory of Human Retrovirology, Institut de Recherches Cliniques de Montréal (IRCM), 110, Pine Avenue West, Montreal, QC, H2W 1R7, Canada. .,Division of Experimental Medicine, McGill University, Montreal, QC, H3A 1A3, Canada. .,Department of Microbiology, Infectiology and Immunology, Université de Montréal, Montreal, QC, H3T 1J4, Canada.
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22
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Differential Control of BST2 Restriction and Plasmacytoid Dendritic Cell Antiviral Response by Antagonists Encoded by HIV-1 Group M and O Strains. J Virol 2016; 90:10236-10246. [PMID: 27581991 DOI: 10.1128/jvi.01131-16] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2016] [Accepted: 08/29/2016] [Indexed: 12/15/2022] Open
Abstract
BST2/tetherin is a type I interferon (IFN-I)-stimulated host factor that restricts the release of HIV-1 by entrapping budding virions at the cell surface. This membrane-associated protein can also engage and activate the plasmacytoid dendritic cell (pDC)-specific immunoglobulin-like transcript 7 (ILT7) inhibitory receptor to downregulate the IFN-I response by pDCs. Pandemic HIV-1 group M uses Vpu (M-Vpu) to counteract the two BST2 isoforms (long and short) that are expressed in human cells. M-Vpu efficiently downregulates surface long BST2, while it displaces short BST2 molecules away from viral assembly sites. We recently found that this attribute is used by M-Vpu to activate the BST2/ILT7-dependent negative-feedback pathway and to suppress pDC IFN-I responses during sensing of infected cells. However, whether this property is conserved in endemic HIV-1 group O, which has evolved Nef (O-Nef) to counteract specifically the long BST2 isoform, remains unknown. In the present study, we validated that O-Nefs have the capacity to downregulate surface BST2 and enhance HIV-1 particle release although less efficiently than M-Vpu. In contrast to M-Vpu, O-Nef did not efficiently enhance viral spread in T cell culture or displace short BST2 from viral assembly sites to prevent its occlusion by tethered HIV-1 particles. Consequently, O-Nef impairs the ability of BST2 to activate negative ILT7 signaling to suppress the IFN-I response by pDC-containing peripheral blood mononuclear cells (PBMCs) during sensing of infected cells. These distinctive features of BST2 counteraction by O-Nefs may in part explain the limited spread of HIV-1 group O in the human population. IMPORTANCE The geographical distributions and prevalences of different HIV-1 groups show large variations. Understanding drivers of distinctive viral spread may aid in the development of therapeutic strategies for controlling the spread of HIV-1 pandemic strains. The differential spread of HIV-1 groups appears to be linked to their capacities to antagonize the long and short isoforms of the BST2 restriction factor. We found that the endemic HIV-1 group O-encoded BST2 antagonist Nef is unable to counteract the restriction mediated by short BST2, a condition that impairs its ability to activate ILT7 and suppress pDC antiviral responses. This is in contrast to the pandemic HIV-1 group M-specified BST2 countermeasure Vpu, which displays a diverse array of mechanisms to counteract short and long BST2 isoforms, an attribute that allows the effective control of pDC antiviral responses. These findings may help explain the limited spread of HIV-1 group O as well as the continued predominance of HIV-1 group M throughout the world.
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HIV-1 Vpu Antagonizes CD317/Tetherin by Adaptor Protein-1-Mediated Exclusion from Virus Assembly Sites. J Virol 2016; 90:6709-6723. [PMID: 27170757 DOI: 10.1128/jvi.00504-16] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2016] [Accepted: 05/06/2016] [Indexed: 01/22/2023] Open
Abstract
UNLABELLED The host cell restriction factor CD317/tetherin traps virions at the surface of producer cells to prevent their release. The HIV-1 accessory protein Vpu antagonizes this restriction. Vpu reduces the cell surface density of the restriction factor and targets it for degradation; however, these activities are dispensable for enhancing particle release. Instead, Vpu has been suggested to antagonize CD317/tetherin by preventing recycling of internalized CD317/tetherin to the cell surface, blocking anterograde transport of newly synthesized CD317/tetherin, and/or displacing the restriction factor from virus assembly sites at the plasma membrane. At the molecular level, antagonism relies on the physical interaction of Vpu with CD317/tetherin. Recent findings suggested that phosphorylation of a diserine motif enables Vpu to bind to adaptor protein 1 (AP-1) trafficking complexes via two independent interaction motifs and to couple CD317/tetherin to the endocytic machinery. Here, we used a panel of Vpu proteins with specific mutations in individual interaction motifs to define which interactions are required for antagonism of CD317/tetherin. Impairing recycling or anterograde transport of CD317/tetherin to the plasma membrane was insufficient for antagonism. In contrast, excluding CD317/tetherin from HIV-1 assembly sites depended on Vpu motifs for interaction with AP-1 and CD317/tetherin and correlated with antagonism of the particle release restriction. Consistently, interference with AP-1 function or its expression blocked these Vpu activities. Our results define displacement from HIV-1 assembly sites as active principle of CD317/tetherin antagonism by Vpu and support a role of tripartite complexes between Vpu, AP-1, and CD317/tetherin in this process. IMPORTANCE CD317/tetherin poses an intrinsic barrier to human immunodeficiency virus type 1 (HIV-1) replication in human cells by trapping virus particles at the surface of producer cells and thereby preventing their release. The viral protein Vpu antagonizes this restriction, and molecular interactions with the restriction factor and adaptor protein complex 1 (AP-1) were suggested to mediate this activity. Vpu modulates intracellular trafficking of CD317/tetherin and excludes the restriction factor from HIV-1 assembly sites at the plasma membrane, but the relative contribution of these effects to antagonism remain elusive. Using a panel of Vpu mutants, as well as interference with AP-1 function and expression, we show here that Vpu antagonizes CD317/tetherin by blocking its recruitment to viral assembly sites in an AP-1-dependent manner. These results refine our understanding of the molecular mechanisms of CD317/tetherin antagonism and suggest complexes of Vpu with the restriction factor and AP-1 as targets for potential therapeutic intervention.
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24
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Rapiteanu R, Davis LJ, Williamson JC, Timms RT, Paul Luzio J, Lehner PJ. A Genetic Screen Identifies a Critical Role for the WDR81-WDR91 Complex in the Trafficking and Degradation of Tetherin. Traffic 2016; 17:940-58. [PMID: 27126989 PMCID: PMC5025723 DOI: 10.1111/tra.12409] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2016] [Revised: 04/26/2016] [Accepted: 04/26/2016] [Indexed: 12/24/2022]
Abstract
Tetherin (BST2/CD317) is a viral restriction factor that anchors enveloped viruses to host cells and limits viral spread. The HIV‐1 Vpu accessory protein counteracts tetherin by decreasing its cell surface expression and targeting it for ubiquitin‐dependent endolysosomal degradation. Although the Vpu‐mediated downregulation of tetherin has been extensively studied, the molecular details are not completely elucidated. We therefore used a forward genetic screen in human haploid KBM7 cells to identify novel genes required for tetherin trafficking. Our screen identified WDR81 as a novel gene required for tetherin trafficking and degradation in both the presence and absence of Vpu. WDR81 is a BEACH‐domain containing protein that is also required for the degradation of EGF‐stimulated epidermal growth factor receptor (EGFR) and functions in a complex with the WDR91 protein. In the absence of WDR81 the endolysosomal compartment appears swollen, with enlarged early and late endosomes and reduced delivery of endocytosed dextran to cathepsin‐active lysosomes. Our data suggest a role for the WDR81‐WDR91 complex in the fusion of endolysosomal compartments and the absence of WDR81 leads to impaired receptor trafficking and degradation.
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Affiliation(s)
- Radu Rapiteanu
- Cambridge Institute for Medical Research, University of Cambridge School of Clinical Medicine, Wellcome Trust/MRC Building Biomedical Campus, Cambridge, CB2 0XY, UK
| | - Luther J Davis
- Departments of Medicine and Clinical Biochemistry, University of Cambridge School of Clinical Medicine, Wellcome Trust/MRC Building Biomedical Campus, Cambridge, CB2 0XY, UK
| | - James C Williamson
- Cambridge Institute for Medical Research, University of Cambridge School of Clinical Medicine, Wellcome Trust/MRC Building Biomedical Campus, Cambridge, CB2 0XY, UK
| | - Richard T Timms
- Cambridge Institute for Medical Research, University of Cambridge School of Clinical Medicine, Wellcome Trust/MRC Building Biomedical Campus, Cambridge, CB2 0XY, UK
| | - J Paul Luzio
- Departments of Medicine and Clinical Biochemistry, University of Cambridge School of Clinical Medicine, Wellcome Trust/MRC Building Biomedical Campus, Cambridge, CB2 0XY, UK
| | - Paul J Lehner
- Cambridge Institute for Medical Research, University of Cambridge School of Clinical Medicine, Wellcome Trust/MRC Building Biomedical Campus, Cambridge, CB2 0XY, UK
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25
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Remodeling of the Host Cell Plasma Membrane by HIV-1 Nef and Vpu: A Strategy to Ensure Viral Fitness and Persistence. Viruses 2016; 8:67. [PMID: 26950141 PMCID: PMC4810257 DOI: 10.3390/v8030067] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2016] [Revised: 02/09/2016] [Accepted: 02/16/2016] [Indexed: 02/07/2023] Open
Abstract
The plasma membrane protects the cell from its surroundings and regulates cellular communication, homing, and metabolism. Not surprisingly, the composition of this membrane is highly controlled through the vesicular trafficking of proteins to and from the cell surface. As intracellular pathogens, most viruses exploit the host plasma membrane to promote viral replication while avoiding immune detection. This is particularly true for the enveloped human immunodeficiency virus (HIV), which assembles and obtains its lipid shell directly at the plasma membrane. HIV-1 encodes two proteins, negative factor (Nef) and viral protein U (Vpu), which function primarily by altering the quantity and localization of cell surface molecules to increase virus fitness despite host antiviral immune responses. These proteins are expressed at different stages in the HIV-1 life cycle and employ a variety of mechanisms to target both unique and redundant surface proteins, including the viral receptor CD4, host restriction factors, immunoreceptors, homing molecules, tetraspanins and membrane transporters. In this review, we discuss recent progress in the study of the Nef and Vpu targeting of host membrane proteins with an emphasis on how remodeling of the cell membrane allows HIV-1 to avoid host antiviral immune responses leading to the establishment of systemic and persistent infection.
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26
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Dotson D, Woodruff EA, Villalta F, Dong X. Filamin A Is Involved in HIV-1 Vpu-mediated Evasion of Host Restriction by Modulating Tetherin Expression. J Biol Chem 2016; 291:4236-46. [PMID: 26742839 DOI: 10.1074/jbc.m115.708123] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2015] [Indexed: 11/06/2022] Open
Abstract
Tetherin, also known as bone marrow stromal antigen 2 (BST-2), inhibits the release of a wide range of enveloped viruses, including human immunodeficiency virus, type 1 (HIV-1) by directly tethering nascent virions to the surface of infected cells. The HIV-1 accessary protein Vpu counteracts tetherin restriction via sequestration, down-regulation, and/or displacement mechanisms to remove tetherin from sites of virus budding. However, the exact mechanism of Vpu-mediated antagonism of tetherin restriction remains to be fully understood. Here we report a novel role for the actin cross-linking regulator filamin A (FLNa) in Vpu anti-tetherin activities. We demonstrate that FLNa associates with tetherin and that FLNa modulates tetherin turnover. FLNa deficiency was found to enhance cell surface and steady-state levels of tetherin expression. In contrast, we observed that overexpression of FLNa reduced tetherin expression levels both on the plasma membrane and in intracellular compartments. Although FLNb shows high amino acid sequence similarity with FLNa, we reveal that only FLNa, but not FLNb, plays an essential role in tetherin turnover. We further showed that FLNa deficiency inhibited Vpu-mediated enhancement of virus release through interfering with the activity of Vpu to down-regulate cellular tetherin. Taken together, our studies suggest that Vpu hijacks the FLNa function in the modulation of tetherin to neutralize the antiviral factor tetherin. These findings may provide novel strategies for the treatment of HIV-1 infection.
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Affiliation(s)
- Dominique Dotson
- From the Department of Microbiology and Immunology and Center for AIDS Health Disparities Research, School of Medicine, Meharry Medical College, Nashville, Tennessee 37208
| | - Elvin A Woodruff
- From the Department of Microbiology and Immunology and Center for AIDS Health Disparities Research, School of Medicine, Meharry Medical College, Nashville, Tennessee 37208
| | - Fernando Villalta
- From the Department of Microbiology and Immunology and Center for AIDS Health Disparities Research, School of Medicine, Meharry Medical College, Nashville, Tennessee 37208
| | - Xinhong Dong
- From the Department of Microbiology and Immunology and Center for AIDS Health Disparities Research, School of Medicine, Meharry Medical College, Nashville, Tennessee 37208
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HIV-1 Adapts To Replicate in Cells Expressing Common Marmoset APOBEC3G and BST2. J Virol 2015; 90:725-40. [PMID: 26512082 DOI: 10.1128/jvi.02431-15] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2015] [Accepted: 10/16/2015] [Indexed: 12/11/2022] Open
Abstract
UNLABELLED Previous studies have shown that a major block to HIV-1 replication in common marmosets operates at the level of viral entry and that this block can be overcome by adaptation of the virus in tissue-cultured cells. However, our current studies indicate that HIV-1 encounters additional postentry blocks in common marmoset peripheral blood mononuclear cells. Here, we show that the common marmoset APOBEC3G (A3G) and BST2 proteins block HIV-1 in cell cultures. Using a directed-evolution method that takes advantage of the natural ability of HIV-1 to mutate during replication, we have been able to overcome these blocks in tissue-cultured cells. In the adapted viruses, specific changes were observed in gag, vif, env, and nef. The contribution of these changes to virus replication in the presence of the A3G and BST2 restriction factors was studied. We found that certain amino acid changes in Vif and Env that arise during adaptation to marmoset A3G and BST2 allow the virus to replicate in the presence of these restriction factors. The changes in Vif reduce expression levels and encapsidation of marmoset APOBEC3G, while the changes in Env increase viral fitness and discretely favor cell-to-cell transmission of the virus, allowing viral escape from these restriction factors. IMPORTANCE HIV-1 can infect only humans and chimpanzees. The main reason for this narrow tropism is the presence in many species of dominant-acting factors, known as restriction factors, that block viral replication in a species-specific way. We have been exploring the blocks to HIV-1 in common marmosets, with the ultimate goal of developing a new animal model of HIV-1 infection in these monkeys. In this study, we observed that common marmoset APOBEC3G and BST2, two known restriction factors, are able to block HIV-1 in cell cultures. We have adapted HIV-1 to replicate in the presence of these restriction factors and have characterized the mechanisms of escape. These studies can help in the development of a novel animal model for in vivo infection of marmosets with HIV-1-like viruses.
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A multi-scale mathematical modeling framework to investigate anti-viral therapeutic opportunities in targeting HIV-1 accessory proteins. J Theor Biol 2015; 386:89-104. [PMID: 26385832 DOI: 10.1016/j.jtbi.2015.08.032] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2015] [Revised: 08/13/2015] [Accepted: 08/31/2015] [Indexed: 02/03/2023]
Abstract
Human immunodeficiency virus-1 (HIV-1) employs accessory proteins to evade innate immune responses by neutralizing the anti-viral activity of host restriction factors. Apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G, A3G) and bone marrow stromal cell antigen 2 (BST2) are host resistance factors that potentially inhibit HIV-1 infection. BST2 reduces viral production by tethering budding HIV-1 particles to virus producing cells, while A3G inhibits the reverse transcription (RT) process and induces viral genome hypermutation through cytidine deamination, generating fewer replication competent progeny virus. Two HIV-1 proteins counter these cellular restriction factors: Vpu, which reduces surface BST2, and Vif, which degrades cellular A3G. The contest between these host and viral proteins influences whether HIV-1 infection is established and progresses towards AIDS. In this work, we present an age-structured multi-scale viral dynamics model of in vivo HIV-1 infection. We integrated the intracellular dynamics of anti-viral activity of the host factors and their neutralization by HIV-1 accessory proteins into the virus/cell population dynamics model. We calculate the basic reproductive ratio (Ro) as a function of host-viral protein interaction coefficients, and numerically simulated the multi-scale model to understand HIV-1 dynamics following host factor-induced perturbations. We found that reducing the influence of Vpu triggers a drop in Ro, revealing the impact of BST2 on viral infection control. Reducing Vif׳s effect reveals the restrictive efficacy of A3G in blocking RT and in inducing lethal hypermutations, however, neither of these factors alone is sufficient to fully restrict HIV-1 infection. Interestingly, our model further predicts that BST2 and A3G function synergistically, and delineates their relative contribution in limiting HIV-1 infection and disease progression. We provide a robust modeling framework for devising novel combination therapies that target HIV-1 accessory proteins and boost antiviral activity of host factors.
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Serine Phosphorylation of HIV-1 Vpu and Its Binding to Tetherin Regulates Interaction with Clathrin Adaptors. PLoS Pathog 2015; 11:e1005141. [PMID: 26317613 PMCID: PMC4552633 DOI: 10.1371/journal.ppat.1005141] [Citation(s) in RCA: 51] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2015] [Accepted: 08/11/2015] [Indexed: 02/07/2023] Open
Abstract
HIV-1 Vpu prevents incorporation of tetherin (BST2/ CD317) into budding virions and targets it for ESCRT-dependent endosomal degradation via a clathrin-dependent process. This requires a variant acidic dileucine-sorting motif (ExxxLV) in Vpu. Structural studies demonstrate that recombinant Vpu/tetherin fusions can form a ternary complex with the clathrin adaptor AP-1. However, open questions still exist about Vpu’s mechanism of action. Particularly, whether endosomal degradation and the recruitment of the E3 ubiquitin ligase SCFβTRCP1/2 to a conserved phosphorylated binding site, DSGNES, are required for antagonism. Re-evaluation of the phenotype of Vpu phosphorylation mutants and naturally occurring allelic variants reveals that the requirement for the Vpu phosphoserine motif in tetherin antagonism is dissociable from SCFβTRCP1/2 and ESCRT-dependent tetherin degradation. Vpu phospho-mutants phenocopy ExxxLV mutants, and can be rescued by direct clathrin interaction in the absence of SCFβTRCP1/2 recruitment. Moreover, we demonstrate physical interaction between Vpu and AP-1 or AP-2 in cells. This requires Vpu/tetherin transmembrane domain interactions as well as the ExxxLV motif. Importantly, it also requires the Vpu phosphoserine motif and adjacent acidic residues. Taken together these data explain the discordance between the role of SCFβTRCP1/2 and Vpu phosphorylation in tetherin antagonism, and indicate that phosphorylation of Vpu in Vpu/tetherin complexes regulates promiscuous recruitment of adaptors, implicating clathrin-dependent sorting as an essential first step in tetherin antagonism. Counteraction of tetherin, a host antiviral protein that blocks viral release from infected cells, is an essential attribute of HIV-1 and its related viruses. The HIV-1 accessory protein Vpu binds to tetherin, preventing its incorporation into viral particles, and targets it for ubiquitin-dependent degradation. This involves mis-trafficking of tetherin by a Vpu-dependent mechanism through the engagement of clathrin adaptor proteins. Although structural evidence exists for Vpu and tetherin interacting with clathrin adaptor 1 (AP-1), evidence that it is required for Vpu-mediated tetherin counteraction is still lacking. Tetherin degradation by Vpu also requires an E3 ubiquitin ligase, SCFβTRCP1/2 that binds to phosphorylated serine residues in the Vpu cytoplasmic tail. Again, discrepancies exist about the importance of this interaction in tetherin’s counteraction. Here we show that Vpu phosphorylation, in combination with its physical interaction with tetherin, regulates interaction with both AP-1 and the other major cellular clathrin adaptor, AP-2. These interactions can be decoupled from SCFβTRCP1/2 recruitment, thus indicating clathrin-dependent mis-trafficking as a critical step in tetherin antagonism by Vpu. Additionally, the ability to interact both with AP-1 and AP-2 in a tetherin-dependent manner indicates a redundancy in host cofactors used by Vpu that explains disparate previous observations of its mechanism of action.
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30
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Ramirez PW, DePaula-Silva AB, Szaniawski M, Barker E, Bosque A, Planelles V. HIV-1 Vpu utilizes both cullin-RING ligase (CRL) dependent and independent mechanisms to downmodulate host proteins. Retrovirology 2015. [PMID: 26215564 PMCID: PMC4517359 DOI: 10.1186/s12977-015-0192-2] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
Background Hijacking of the cullin-RING E3 ubiquitin ligase (CRL) machinery is a common mechanism employed by diverse groups of viruses for the efficient counteraction and degradation of host proteins. In particular, HIV-1 Vpu usurps the SCFβ-TrCP E3 ubiquitin ligase complex to mark CD4 for degradation by the 26S proteasome. Vpu also interacts with and downmodulates a number of other host proteins, including the restriction factor BST-2. However, whether Vpu primarily relies on a cullin-dependent or -independent mechanism to antagonize its cellular targets has not been fully elucidated. Results We utilized a sulphamate AMP analog, MLN4924, to effectively block the activation of CRLs within infected primary CD4+ T cells. MLN4924 treatment, in a dose dependent manner, efficiently relieved surface downmodulation and degradation of CD4 by NL4-3 Vpu. MLN4924 inhibition was highly specific, as this inhibitor had no effect on Nef’s ability to downregulate CD4, which is accomplished by a CRL-independent mechanism. In contrast, NL4-3 Vpu’s capacity to downregulate BST-2, NTB-A and CCR7 was not inhibited by the drug. Vpu’s from both a transmitted founder (T/F) and chronic carrier (CC) virus preserved the ability to downregulate BST-2 in the presence of MLN4924. Finally, depletion of cellular pools of cullin 1 attenuated Vpu’s ability to decrease CD4 but not BST-2 surface levels. Conclusions We conclude that Vpu employs both CRL-dependent and CRL-independent modes of action against host proteins. Notably, we also establish that Vpu-mediated reduction of BST-2 from the cell surface is independent of β-TrCP and the CRL- machinery and this function is conserved by Vpu’s from primary isolates. Therefore, potential therapies aimed at antagonizing the activities of Vpu may need to address these distinct mechanisms of action in order to achieve a maximal effect.
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Affiliation(s)
- Peter W Ramirez
- Division of Microbiology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, 84112, USA.
| | - Ana Beatriz DePaula-Silva
- Division of Microbiology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, 84112, USA.
| | - Matt Szaniawski
- Division of Microbiology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, 84112, USA.
| | - Edward Barker
- Department of Immunology and Microbiology, Rush University Medical Center, Chicago, IL, 60612, USA.
| | - Alberto Bosque
- Division of Microbiology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, 84112, USA.
| | - Vicente Planelles
- Division of Microbiology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, 84112, USA.
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Bego MG, Côté É, Aschman N, Mercier J, Weissenhorn W, Cohen ÉA. Vpu Exploits the Cross-Talk between BST2 and the ILT7 Receptor to Suppress Anti-HIV-1 Responses by Plasmacytoid Dendritic Cells. PLoS Pathog 2015; 11:e1005024. [PMID: 26172439 PMCID: PMC4501562 DOI: 10.1371/journal.ppat.1005024] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2015] [Accepted: 06/16/2015] [Indexed: 11/25/2022] Open
Abstract
Plasmacytoid dendritic cells (pDCs) constitute a major source of type-I interferon (IFN-I) production during acute HIV infection. Their activation results primarily from TLR7-mediated sensing of HIV-infected cells. However, the interactions between HIV-infected T cells and pDCs that modulate this sensing process remain poorly understood. BST2/Tetherin is a restriction factor that inhibits HIV release by cross-linking virions onto infected cell surface. BST2 was also shown to engage the ILT7 pDC-specific inhibitory receptor and repress TLR7/9-mediated IFN-I production by activated pDCs. Here, we show that Vpu, the HIV-1 antagonist of BST2, suppresses TLR7-mediated IFN-I production by pDC through a mechanism that relies on the interaction of BST2 on HIV-producing cells with ILT7. Even though Vpu downregulates surface BST2 as a mean to counteract the restriction on HIV-1 release, we also find that the viral protein re-locates remaining BST2 molecules outside viral assembly sites where they are free to bind and activate ILT7 upon cell-to-cell contact. This study shows that through a targeted regulation of surface BST2, Vpu promotes HIV-1 release and limits pDC antiviral responses upon sensing of infected cells. This mechanism of innate immune evasion is likely to be important for an efficient early viral dissemination during acute infection. Plasmacytoid dendritic cells (pDCs) produce large quantities of type I interferon (IFN-I) upon stimulation by many viruses, including HIV. Their activation is very effective following cell contacts with HIV-1-infected CD4+ T cells. We investigated whether HIV-1 could regulate the antiviral responses of pDCs triggered upon sensing of infected cells. We show that HIV-1 suppresses the levels of IFN-I produced by pDCs through a process that requires expression of the Vpu accessory protein in virus-producing cells. A well-described role of Vpu is to promote efficient HIV-1 production by counteracting BST2, a host factor that entraps nascent viral particle at the cell surface. Apart from its antiviral activity, BST2 was reported to inhibit IFN-I production by pDCs through binding and activation of the ILT7 pDC-specific inhibitory receptor. Our results reveal that through a highly sophisticated targeted regulation of BST2 levels at the surface of infected cells, Vpu promotes HIV-1 release and limits IFN-I production by pDCs via the negative signaling exerted by the BST2-ILT7 pair. Overall, this study sheds light on a novel Vpu-BST2 interaction that allows HIV-1 to escape pDC antiviral responses. This modulation of pDC antiviral response by HIV Vpu may facilitate the initial viral expansion during acute infection.
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Affiliation(s)
- Mariana G. Bego
- Institut de Recherches Cliniques de Montréal (IRCM), Montreal, Quebec, Canada
| | - Édouard Côté
- Institut de Recherches Cliniques de Montréal (IRCM), Montreal, Quebec, Canada
| | - Nick Aschman
- Université Grenoble Alpes, Unit of Virus Host Cell Interactions (UVHCI), CNRS, UVHCI, Grenoble, France
| | - Johanne Mercier
- Institut de Recherches Cliniques de Montréal (IRCM), Montreal, Quebec, Canada
| | - Winfried Weissenhorn
- Université Grenoble Alpes, Unit of Virus Host Cell Interactions (UVHCI), CNRS, UVHCI, Grenoble, France
| | - Éric A. Cohen
- Institut de Recherches Cliniques de Montréal (IRCM), Montreal, Quebec, Canada
- Department of Microbiology, Infectiology and Immunology, Université de Montréal, Montreal, Quebec, Canada
- * E-mail:
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HIV-1 Nef and Vpu are functionally redundant broad-spectrum modulators of cell surface receptors, including tetraspanins. J Virol 2014; 88:14241-57. [PMID: 25275127 DOI: 10.1128/jvi.02333-14] [Citation(s) in RCA: 67] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
UNLABELLED HIV-1 Nef and Vpu are thought to optimize virus replication in the infected host, at least in part via their ability to interfere with vesicular host cell trafficking. Despite the use of distinct molecular mechanisms, Nef and Vpu share specificity for some molecules such as CD4 and major histocompatibility complex class I (MHC-I), while disruption of intracellular transport of the host cell restriction factor CD317/tetherin represents a specialized activity of Vpu not exerted by HIV-1 Nef. To establish a profile of host cell receptors whose intracellular transport is affected by Nef, Vpu, or both, we comprehensively analyzed the effect of these accessory viral proteins on cell surface receptor levels on A3.01 T lymphocytes. Thirty-six out of 105 detectable receptors were significantly downregulated by HIV-1 Nef, revealing a previously unappreciated scope with which HIV-1 Nef remodels the cell surface of infected cells. Remarkably, the effects of HIV-1 Vpu on host cell receptor exposure largely matched those of HIV-1 Nef in breadth and specificity (32 of 105, all also targeted by Nef), even though the magnitude was generally less pronounced. Of particular note, cell surface exposure of all members of the tetraspanin (TSPAN) protein family analyzed was reduced by both Nef and Vpu, and the viral proteins triggered the enrichment of TSPANs in a perinuclear area of the cell. While Vpu displayed significant colocalization and physical association with TSPANs, interactions of Nef with TSPANs were less robust. TSPANs thus emerge as a major target of deregulation in host cell vesicular transport by HIV-1 Nef and Vpu. The conservation of this activity in two independent accessory proteins suggests its importance for the spread of HIV-1 in the infected host. IMPORTANCE In this paper, we define that HIV-1 Nef and Vpu display a surprising functional overlap and affect the cell surface exposure of a previously unexpected breadth of cellular receptors. Our analyses furthermore identify the tetraspanin protein family as a previously unrecognized target of Nef and Vpu activity. These findings have implications for the interpretation of effects detected for these accessory gene products on individual host cell receptors and illustrate the coevolution of Nef and Vpu function.
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Hasan Z, Kamori D, Ueno T. Role of host immune responses in sequence variability of HIV-1 Vpu. World J Immunol 2014; 4:107-115. [DOI: 10.5411/wji.v4.i2.107] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/07/2014] [Revised: 04/19/2014] [Accepted: 06/16/2014] [Indexed: 02/05/2023] Open
Abstract
Viral protein U (Vpu) is an accessory protein associated with two main functions important in human immunodeficiency virus type 1 (HIV-1) replication and dissemination; these are down-regulation of CD4 receptor through mediating its proteasomal degradation and enhancement of virion release by antagonizing tetherin/BST2. It is also well established that Vpu is one of the most highly variable proteins in the HIV-1 proteome. However it is still unclear what drives Vpu sequence variability, whether Vpu acquires polymorphisms as a means of immune escape, functional advantage, or otherwise. It is assumed that the host-pathogen interaction is a cause of polymorphic phenotype of Vpu and that the resulting functional heterogeneity of Vpu may have critical significance in vivo. In order to comprehensively understand Vpu variability, it is important to integrate at the population level the genetic association approaches to identify specific amino acid residues and the immune escape kinetics which may impose Vpu functional constraints in vivo. This review will focus on HIV-1 accessory protein Vpu in the context of its sequence variability at population level and also bring forward evidence on the role of the host immune responses in driving Vpu sequence variability; we will also highlight the recent findings that illustrate Vpu functional implication in HIV-1 pathogenesis.
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Roy N, Pacini G, Berlioz-Torrent C, Janvier K. Mechanisms underlying HIV-1 Vpu-mediated viral egress. Front Microbiol 2014; 5:177. [PMID: 24822052 PMCID: PMC4013480 DOI: 10.3389/fmicb.2014.00177] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2014] [Accepted: 03/31/2014] [Indexed: 11/13/2022] Open
Abstract
Viruses such as lentiviruses that are responsible for long lasting infections have to evade several levels of cellular immune mechanisms to persist and efficiently disseminate in the host. Over the past decades, much evidence has emerged regarding the major role of accessory proteins of primate lentiviruses, human immunodeficiency virus and simian immunodeficiency virus, in viral evasion from the host immune defense. This short review will provide an overview of the mechanism whereby the accessory protein Vpu contributes to this escape. Vpu is a multifunctional protein that was shown to contribute to viral egress by down-regulating several mediators of the immune system such as CD4, CD1d, NTB-A and the restriction factor BST2. The mechanisms underlying its activity are not fully characterized but rely on its ability to interfere with the host machinery regulating protein turnover and vesicular trafficking. This review will focus on our current understanding of the mechanisms whereby Vpu down-regulates CD4 and BST2 expression levels to favor viral egress.
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Affiliation(s)
- Nicolas Roy
- INSERM U1016, Institut Cochin Paris, France ; CNRS UMR8104 Paris, France ; Université Paris Descartes Paris, France
| | - Grégory Pacini
- INSERM U1016, Institut Cochin Paris, France ; CNRS UMR8104 Paris, France ; Université Paris Descartes Paris, France
| | - Clarisse Berlioz-Torrent
- INSERM U1016, Institut Cochin Paris, France ; CNRS UMR8104 Paris, France ; Université Paris Descartes Paris, France
| | - Katy Janvier
- INSERM U1016, Institut Cochin Paris, France ; CNRS UMR8104 Paris, France ; Université Paris Descartes Paris, France
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Jia X, Weber E, Tokarev A, Lewinski M, Rizk M, Suarez M, Guatelli J, Xiong Y. Structural basis of HIV-1 Vpu-mediated BST2 antagonism via hijacking of the clathrin adaptor protein complex 1. eLife 2014; 3:e02362. [PMID: 24843023 PMCID: PMC4018625 DOI: 10.7554/elife.02362] [Citation(s) in RCA: 82] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
BST2/tetherin, an antiviral restriction factor, inhibits the release of enveloped viruses from the cell surface. Human immunodeficiency virus-1 (HIV-1) antagonizes BST2 through viral protein u (Vpu), which downregulates BST2 from the cell surface. We report the crystal structure of a protein complex containing Vpu and BST2 cytoplasmic domains and the core of the clathrin adaptor protein complex 1 (AP1). This, together with our biochemical and functional validations, reveals how Vpu hijacks the AP1-dependent membrane trafficking pathways to mistraffick BST2. Vpu mimics a canonical acidic dileucine-sorting motif to bind AP1 in the cytosol, while simultaneously interacting with BST2 in the membrane. These interactions enable Vpu to build on an intrinsic interaction between BST2 and AP1, presumably causing the observed retention of BST2 in juxtanuclear endosomes and stimulating its degradation in lysosomes. The ability of Vpu to hijack AP-dependent trafficking pathways suggests a potential common theme for Vpu-mediated downregulation of host proteins. DOI:http://dx.doi.org/10.7554/eLife.02362.001 HIV is a retrovirus that attacks the immune system, making the body increasingly susceptible to opportunistic infections and disease and eventually leading to AIDS. While antiretroviral drugs have allowed people with AIDS to live longer, there is no cure or vaccine for HIV. Two types of HIV exist, with HIV-1 being much more common and pathogenic than HIV-2. Like other ‘complex’ retroviruses, the HIV-1 genome contains genes that encode various proteins that allow the virus to disrupt the immune response of the host it is attacking. Viral protein u is a protein encoded by HIV-1 (but not HIV-2) that counteracts an antiviral protein called BST2 in the host. BST2, which is part of the host's innate immune response, prevents newly formed viruses from leaving the surface of infected cells. By counteracting BST2, viral protein u allows the virus to spread in the host more efficiently. Like many proteins, newly produced BST2 is packaged inside structures called vesicles in a part of the cell called the trans-Golgi network, and then sent to its destination. Complexes formed by various proteins make sure that the vesicles take their cargo to their correct destinations within the cell. Two adaptor protein complexes—known as AP1 and AP2—are thought to be involved the transport of BST2. However, it is not known how viral protein u stops BST2 from reaching the cell surface, or how it decreases the amount of BST2 in the cell as a whole. Jia et al. show how viral protein u and BST2 jointly interact with AP1. This interaction leads to the mistrafficking and degradation of BST2 and the counteraction of its antiviral activity. DOI:http://dx.doi.org/10.7554/eLife.02362.002
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Affiliation(s)
- Xiaofei Jia
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, United States
| | - Erin Weber
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, United States
| | - Andrey Tokarev
- Department of Medicine, University of California San Diego, La Jolla, United States The VA San Diego Healthcare System, San Diego, United States
| | - Mary Lewinski
- Department of Medicine, University of California San Diego, La Jolla, United States The VA San Diego Healthcare System, San Diego, United States
| | - Maryan Rizk
- Department of Medicine, University of California San Diego, La Jolla, United States The VA San Diego Healthcare System, San Diego, United States
| | - Marissa Suarez
- Department of Medicine, University of California San Diego, La Jolla, United States The VA San Diego Healthcare System, San Diego, United States
| | - John Guatelli
- Department of Medicine, University of California San Diego, La Jolla, United States The VA San Diego Healthcare System, San Diego, United States
| | - Yong Xiong
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, United States
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Abstract
Many viruses encode short transmembrane proteins that play vital roles in virus replication or virulence. Because many of these proteins are less than 50 amino acids long and not homologous to cellular proteins, their open reading frames were often overlooked during the initial annotation of viral genomes. Some of these proteins oligomerize in membranes and form ion channels. Other miniproteins bind to cellular transmembrane proteins and modulate their activity, whereas still others have an unknown mechanism of action. Based on the underlying principles of transmembrane miniprotein structure, it is possible to build artificial small transmembrane proteins that modulate a variety of biological processes. These findings suggest that short transmembrane proteins provide a versatile mechanism to regulate a wide range of cellular activities, and we speculate that cells also express many similar proteins that have not yet been discovered.
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Affiliation(s)
- Daniel DiMaio
- Department of Genetics, Yale School of Medicine, New Haven, Connecticut 06520;
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Differential sensitivities of tetherin isoforms to counteraction by primate lentiviruses. J Virol 2014; 88:5845-58. [PMID: 24623426 DOI: 10.1128/jvi.03818-13] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
UNLABELLED The mammalian antiviral membrane protein tetherin (BST2/CD317) can be expressed as two isoforms derived from differential translational initiation. The shorter isoform of the human protein (S-tetherin) lacks the first 12 amino acids of the longer (L-tetherin) cytoplasmic tail, which includes a tyrosine motif that acts as both an endocytic recycling signal and a determinant of virus-induced NF-κB activation. S-tetherin is also reported to be less sensitive to the prototypic viral antagonist human immunodeficiency virus type 1 (HIV-1) Vpu. Here we analyzed the relative sensitivities of L- and S-tetherins to primate lentiviral countermeasures. We show that the reduced sensitivity of S-tetherin to HIV-1 Vpu is a feature of all group M proteins, including those of transmitted founder viruses, primarily because it cannot be targeted for endosomal degradation owing to the truncation of its cytoplasmic tail. In contrast, both isoforms of the human and rhesus macaque tetherins display the same sensitivity to nondegradative lentiviral countermeasures of HIV-2 and SIVmac, respectively. Surprisingly, however, the Vpu proteins encoded by simian immunodeficiency viruses (SIVs) of African guenons, as well as that from recently isolated highly pathogenic HIV-1 group N, do not discriminate between tetherin isoforms. Together, these data suggest that the group M HIV-1 Vpu primarily adapted to target L-tetherin upon zoonotic transmission from chimpanzees, and further, we speculate that functions specifically associated with this isoform, such as proinflammatory signaling, play key roles in human tetherin's antiviral function in vivo. IMPORTANCE The ability of HIV-1 and related viruses to counteract a host antiviral protein, tetherin, is strictly maintained. The adaptation of the HIV-1 Vpu protein to counteract human tetherin is thought to have been one of the key events in the establishment of the HIV/AIDS pandemic. Recent evidence shows that tetherin is expressed as two isoforms and that Vpu preferentially targets the longer form. Here we show that unlike other virus-encoded countermeasures, such as those from primate viruses related to HIV-1, the enhanced ability to counteract the long tetherin isoform is conserved among HIV-1 strains that make up the majority of the human pandemic. This correlates with the ability of Vpu to induce long tetherin degradation. We speculate that functions associated with the human version of this isoform, such as an inflammatory signaling capacity, selected for Vpu's enhanced targeting of long tetherin during its adaptation to humans.
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Strebel K. HIV accessory proteins versus host restriction factors. Curr Opin Virol 2013; 3:692-9. [PMID: 24246762 PMCID: PMC3855913 DOI: 10.1016/j.coviro.2013.08.004] [Citation(s) in RCA: 94] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2013] [Revised: 08/05/2013] [Accepted: 08/06/2013] [Indexed: 02/05/2023]
Abstract
Primate immunodeficiency viruses, including HIV-1, are characterized by the presence of accessory genes such as vif, vpr, vpx, vpu, and nef. Current knowledge indicates that none of the primate lentiviral accessory proteins has enzymatic activity. Instead, these proteins interact with cellular ligands to either act as adapter molecules to redirect the normal function of host factors for virus-specific purposes or to inhibit a normal host function by mediating degradation or causing intracellular mislocalization/sequestration of the factors involved. This review aims at providing an update of our current understanding of how Vif, Vpu, and Vpx control the cellular restriction factors APOBEC3G, BST-2, and SAMHD1, respectively.
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Affiliation(s)
- Klaus Strebel
- Laboratory of Molecular Microbiology, Viral Biochemistry Section, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892-0460, United States.
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Efficient BST2 antagonism by Vpu is critical for early HIV-1 dissemination in humanized mice. Retrovirology 2013; 10:128. [PMID: 24195843 PMCID: PMC4226203 DOI: 10.1186/1742-4690-10-128] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2013] [Accepted: 10/27/2013] [Indexed: 11/30/2022] Open
Abstract
Background Vpu is a multifunctional accessory protein that enhances the release of HIV-1 by counteracting the entrapment of nascent virions on infected cell surface mediated by BST2/Tetherin. Vpu-mediated BST2 antagonism involves physical association with BST2 and subsequent mislocalization of the restriction factor to intracellular compartments followed by SCF(β-TrCP) E3 ligase-dependent lysosomal degradation. Apart from BST2 antagonism, Vpu also induces down regulation of several immune molecules, including CD4 and SLAMF6/NTB-A, to evade host immune responses and promote viral dissemination. However, it should be noted that the multiple functions of Vpu have been studied in cell-based assays, and thus it remains unclear how Vpu influences the dynamic of HIV-1 infection in in vivo conditions. Results Using a humanized mouse model of acute infection as well as CCR5-tropic HIV-1 that lack Vpu or encode WT Vpu or Vpu with mutations in the β-TrCP binding domain, we provide evidence that Vpu-mediated BST2 antagonism plays a crucial role in establishing early plasma viremia and viral dissemination. Interestingly, we also find that efficient HIV-1 release and dissemination are directly related to functional strength of Vpu in antagonizing BST2. Thus, reduced antagonism of BST2 due to β-TrCP binding domain mutations results in decreased plasma viremia and frequency of infected T cells, highlighting the importance of Vpu-mediated β-TrCP-dependent BST-2 degradation for optimal initial viral propagation. Conclusions Overall, our findings suggest that BST2 antagonism by Vpu is critical for efficient early viral expansion and dissemination during acute infection and as such is likely to confer HIV-1 increased transmission fitness.
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Rollason R, Dunstan K, Billcliff PG, Bishop P, Gleeson P, Wise H, Digard P, Banting G. Expression of HIV-1 Vpu leads to loss of the viral restriction factor CD317/Tetherin from lipid rafts and its enhanced lysosomal degradation. PLoS One 2013; 8:e75680. [PMID: 24086611 PMCID: PMC3782430 DOI: 10.1371/journal.pone.0075680] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2012] [Accepted: 08/20/2013] [Indexed: 01/01/2023] Open
Abstract
CD317/tetherin (aka BST2 or HM1.24 antigen) is an interferon inducible membrane protein present in regions of the lipid bilayer enriched in sphingolipids and cholesterol (often termed lipid rafts). It has been implicated in an eclectic mix of cellular processes including, most notably, the retention of fully formed viral particles at the surface of cells infected with HIV and other enveloped viruses. Expression of the HIV viral accessory protein Vpu has been shown to lead to intracellular sequestration and degradation of tetherin, thereby counteracting the inhibition of viral release. There is evidence that tetherin interacts directly with Vpu, but it remains unclear where in the cell this interaction occurs or if Vpu expression affects the lipid raft localisation of tetherin. We have addressed these points using biochemical and cell imaging approaches focused on endogenous rather than ectopically over-expressed tetherin. We find i) no evidence for an interaction between Vpu and endogenous tetherin at the cell surface, ii) the vast majority of endogenous tetherin that is at the cell surface in control cells is in lipid rafts, iii) internalised tetherin is present in non-raft fractions, iv) expression of Vpu in cells expressing endogenous tetherin leads to the loss of tetherin from lipid rafts, v) internalised tetherin enters early endosomes, and late endosomes, in both control cells and cells expressing Vpu, but the proportion of tetherin molecules destined for degradation rather than recycling is increased in cells expressing Vpu vi) lysosomes are the primary site for degradation of endogenous tetherin in cells expressing Vpu. Our studies underlie the importance of studying endogenous tetherin and let us propose a model in which Vpu intercepts newly internalised tetherin and diverts it for lysosomal destruction rather than recycling to the cell surface.
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Affiliation(s)
- Ruth Rollason
- School of Biochemistry, University of Bristol, Bristol, United Kingdom
| | - Katie Dunstan
- School of Biochemistry, University of Bristol, Bristol, United Kingdom
| | | | - Paul Bishop
- School of Biochemistry, University of Bristol, Bristol, United Kingdom
| | - Paul Gleeson
- Bio21 Institute, University of Melbourne, Melbourne, Australia
| | - Helen Wise
- The Roslin Institute, University of Edinburgh, Edinburgh, United Kingdom
| | - Paul Digard
- The Roslin Institute, University of Edinburgh, Edinburgh, United Kingdom
| | - George Banting
- School of Biochemistry, University of Bristol, Bristol, United Kingdom
- * E-mail:
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Strebel K. HIV-1 Vpu - an ion channel in search of a job. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2013; 1838:1074-81. [PMID: 23831603 DOI: 10.1016/j.bbamem.2013.06.029] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/09/2013] [Revised: 06/18/2013] [Accepted: 06/22/2013] [Indexed: 12/22/2022]
Abstract
Vpu is a small membrane protein encoded by HIV-1 and some SIV isolates. The protein is best known for its ability to degrade CD4 and to enhance the release of progeny virions from infected cells. However, Vpu also promotes host-cell apoptosis by deregulating the NFκB signaling pathway and it assembles into cation-conducting membrane pores. This review summarizes our current understanding of these various functions of Vpu with particular emphasis on recent progress in the Vpu field. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking.
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Affiliation(s)
- Klaus Strebel
- Viral Biochemistry Section, Laboratory of Molecular Microbiology, NIAID, NIH Bldg. 4, Room 310, 4 Center Drive MSC 0460, Bethesda, MD 20892-0460, USA.
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Abstract
Tetherin (BST2/CD317) has emerged as a key host cell defense molecule, inhibiting the release and spread of diverse enveloped virions from infected cells. In this chapter, I review the molecular and cellular basis for tetherin's antiviral activities and the function of virally encoded countermeasures that disrupt its function. I further describe recent advances in our understanding of tetherin's associated role in viral pattern recognition and the evidence for its role in limiting viral pathogenesis in vivo.
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Affiliation(s)
- Stuart J D Neil
- Department of Infectious Disease, King's College London School of Medicine, London, UK.
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Zheng YH, Jeang KT, Tokunaga K. Host restriction factors in retroviral infection: promises in virus-host interaction. Retrovirology 2012; 9:112. [PMID: 23254112 PMCID: PMC3549941 DOI: 10.1186/1742-4690-9-112] [Citation(s) in RCA: 75] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2012] [Accepted: 12/09/2012] [Indexed: 01/19/2023] Open
Abstract
Retroviruses have an intricate life cycle. There is much to be learned from studying retrovirus-host interactions. Among retroviruses, the primate lentiviruses have one of the more complex genome structures with three categories of viral genes: structural, regulatory, and accessory genes. Over time, we have gained increasing understanding of the lentivirus life cycle from studying host factors that support virus replication. Similarly, studies on host restriction factors that inhibit viral replication have also made significant contributions to our knowledge. Here, we review recent progress on the rapidly growing field of restriction factors, focusing on the antiretroviral activities of APOBEC3G, TRIM5, tetherin, SAMHD1, MOV10, and cellular microRNAs (miRNAs), and the counter-activities of Vif, Vpu, Vpr, Vpx, and Nef.
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Affiliation(s)
- Yong-Hui Zheng
- Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA
| | | | - Kenzo Tokunaga
- Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan
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Lucas TM, Janaka SK, Stephens EB, Johnson MC. Vpu downmodulates two distinct targets, tetherin and gibbon ape leukemia virus envelope, through shared features in the Vpu cytoplasmic tail. PLoS One 2012; 7:e51741. [PMID: 23284757 PMCID: PMC3526647 DOI: 10.1371/journal.pone.0051741] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2012] [Accepted: 11/09/2012] [Indexed: 11/21/2022] Open
Abstract
During human immunodeficiency virus-1 (HIV-1) assembly, the host proteins CD4 (the HIV-1 receptor) and tetherin (an interferon stimulated anti-viral protein) both reduce viral fitness. The HIV-1 accessory gene Vpu counteracts both of these proteins, but it is thought to do so through two distinct mechanisms. Modulation of CD4 likely occurs through proteasomal degradation from the endoplasmic reticulum. The exact mechanism of tetherin modulation is less clear, with possible roles for degradation and alteration of protein transport to the plasma membrane. Most investigations of Vpu function have used different assays for CD4 and tetherin. In addition, many of these investigations used exogenously expressed Vpu, which could result in variable expression levels. Thus, few studies have investigated these two Vpu functions in parallel assays, making direct comparisons difficult. Here, we present results from a rapid assay used to simultaneously investigate Vpu-targeting of both tetherin and a viral glycoprotein, gibbon ape leukemia virus envelope (GaLV Env). We previously reported that Vpu modulates GaLV Env and prevents its incorporation into HIV-1 particles through a recognition motif similar to that found in CD4. Using this assay, we performed a comprehensive mutagenic scan of Vpu in its native proviral context to identify features required for both types of activity. We observed considerable overlap in the Vpu sequences required to modulate tetherin and GaLV Env. We found that features in the cytoplasmic tail of Vpu, specifically within the cytoplasmic tail hinge region, were required for modulation of both tetherin and GaLV Env. Interestingly, these same regions features have been determined to be critical for CD4 downmodulation. We also observed a role for the transmembrane domain in the restriction of tetherin, as previously reported, but not of GaLV Env. We propose that Vpu may target both proteins in a mechanistically similar manner, albeit in different cellular locations.
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Affiliation(s)
- Tiffany M. Lucas
- Department of Molecular Microbiology and Immunology, Christopher S. Bond Life Science Center, University of Missouri-School of Medicine, Columbia, Missouri, United States of America
| | - Sanath K. Janaka
- Department of Molecular Microbiology and Immunology, Christopher S. Bond Life Science Center, University of Missouri-School of Medicine, Columbia, Missouri, United States of America
| | - Edward B. Stephens
- Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, United States of America
| | - Marc C. Johnson
- Department of Molecular Microbiology and Immunology, Christopher S. Bond Life Science Center, University of Missouri-School of Medicine, Columbia, Missouri, United States of America
- * E-mail:
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45
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Abstract
Cellular proteins called "restriction factors" can serve as powerful blockades to HIV replication, but the virus possesses elaborate strategies to circumvent these barriers. First, we discuss general hallmarks of a restriction factor. Second, we review how the viral Vif protein protects the viral genome from lethal levels of cDNA deamination by promoting APOBEC3 protein degradation; how the viral Vpu, Env, and Nef proteins facilitate internalization and degradation of the virus-tethering protein BST-2/tetherin; and how the viral Vpx protein prevents the premature termination of reverse transcription by degrading the dNTPase SAMHD1. These HIV restriction and counter-restriction mechanisms suggest strategies for new therapeutic interventions.
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Affiliation(s)
- Reuben S Harris
- Department of Biochemistry, Molecular Biology and Biophysics, Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota 55455, USA.
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Galão R, Le Tortorec A, Pickering S, Kueck T, Neil S. Innate sensing of HIV-1 assembly by Tetherin induces NFκB-dependent proinflammatory responses. Cell Host Microbe 2012; 12:633-44. [PMID: 23159053 PMCID: PMC3556742 DOI: 10.1016/j.chom.2012.10.007] [Citation(s) in RCA: 231] [Impact Index Per Article: 17.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2012] [Revised: 08/04/2012] [Accepted: 10/01/2012] [Indexed: 11/29/2022]
Abstract
Antiviral proteins that recognize pathogen-specific or aberrantly located molecular motifs are perfectly positioned to act as pattern-recognition receptors and signal to the immune system. Here we investigated whether the interferon-induced viral restriction factor tetherin (CD317/BST2), which is known to inhibit HIV-1 particle release by physically tethering virions to the cell surface, has such a signaling role. We find that upon restriction of Vpu-defective HIV-1, tetherin acts as a virus sensor to induce NFκB-dependent proinflammatory gene expression. Signaling requires both tetherin's extracellular domain involved in virion retention and determinants in the cytoplasmic tail, including an endocytic motif, although signaling is independent of virion endocytosis. Furthermore, recruitment of the TNF-receptor-associated factor TRAF6 and activation of the mitogen-activated protein kinase TAK1 are critical for signaling. Human tetherin's ability to mediate efficient signaling may have arisen as a result of a five amino acid deletion that occurred in hominids after their divergence from chimpanzees.
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Affiliation(s)
- Rui Pedro Galão
- Department of Infectious Disease, King’s College London School of Medicine, Guy’s Hospital, London SE1 9RT, UK
| | - Anna Le Tortorec
- Department of Infectious Disease, King’s College London School of Medicine, Guy’s Hospital, London SE1 9RT, UK
| | - Suzanne Pickering
- Department of Infectious Disease, King’s College London School of Medicine, Guy’s Hospital, London SE1 9RT, UK
| | - Tonya Kueck
- Department of Infectious Disease, King’s College London School of Medicine, Guy’s Hospital, London SE1 9RT, UK
| | - Stuart J.D. Neil
- Department of Infectious Disease, King’s College London School of Medicine, Guy’s Hospital, London SE1 9RT, UK
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Abstract
Viroporins are small virally encoded hydrophobic proteins that oligomerize in the membrane of host cells, leading to the formation of hydrophilic pores. This activity modifies several cellular functions, including membrane permeability, Ca2+ homeostasis, membrane remodelling and glycoprotein trafficking. A classification scheme for viroporins is proposed on the basis of their structure and membrane topology. Thus, class I and class II viroporins are defined according to the number of transmembrane domains in the protein (one and two, respectively), and subclasses are defined according to their orientation in the membrane. The main function of viroporins during viral replication is to participate in virion morphogenesis and release from host cells. In addition, some viroporins are involved in viral entry and genome replication. The structure and activity of several viroporins, such as picornavirus protein 2B (P2B), influenza A virus matrix protein 2 (M2), hepatitis C virus p7 and HIV-1 viral protein U (Vpu), have been analysed in detail. New members of this expanding family of viral proteins have been described, from both RNA and DNA viruses. In addition to having a common general structure, all of these new viroporins have the ability to increase membrane permeability. Viroporins represent ideal targets to block viral replication and the spread of infection. Although a number of selective inhibitors of viroporin ion channels have been analysed in detail, optimized screening systems promise to provide new and more potent antiviral compounds in the near future. Viroporins belong to a growing family of virally encoded proteins that form aqueous channels in the membranes of host cells. Here, Carrasco and colleagues review the structure and diverse biological functions of these proteins during the viral life cycle, as well as their potential as antiviral therapeutic targets. Viroporins are small, hydrophobic proteins that are encoded by a wide range of clinically relevant animal viruses. When these proteins oligomerize in host cell membranes, they form hydrophilic pores that disrupt a number of physiological properties of the cell. Viroporins are crucial for viral pathogenicity owing to their involvement in several diverse steps of the viral life cycle. Thus, these viral proteins, which include influenza A virus matrix protein 2 (M2), HIV-1 viral protein U (Vpu) and hepatitis C virus p7, represent ideal targets for therapeutic intervention, and several compounds that block their pore-forming activity have been identified. Here, we review recent studies in the field that have advanced our knowledge of the structure and function of this expanding family of viral proteins.
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Gustin JK, Douglas JL, Bai Y, Moses AV. Ubiquitination of BST-2 protein by HIV-1 Vpu protein does not require lysine, serine, or threonine residues within the BST-2 cytoplasmic domain. J Biol Chem 2012; 287:14837-50. [PMID: 22383521 DOI: 10.1074/jbc.m112.349928] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The cellular protein BST-2/CD317/Tetherin has been shown to inhibit the release of HIV-1 and other enveloped viruses from infected cells. The HIV-1 accessory protein Vpu binds to both BST-2 and βTrCP, a substrate-recognition subunit for the SCF (Skip1-Cullin1-F-box protein) E3 ubiquitin ligase complex. This interaction leads to both the degradation of BST-2 and the enhancement of viral egress. Recently BST-2 was shown to be ubiquitinated in this process. Here we have confirmed the Vpu- and βTrCP-dependent multi/polyubiquitination of BST-2. Ubiquitinated BST-2 accumulated in cells treated with a lysosomal inhibitor but not a proteasomal inhibitor. Additionally, we observed that a BST-2 mutant deleted for its cytosolically exposed lysine residues is also ubiquitinated. Subsequent experiments suggested that Vpu promotes BST-2 ubiquitination upon amino acid residues bearing hydroxyl- but not thiol-bearing side chains. However, a BST-2 mutant bearing substitutions for its cytoplasmically exposed Ser, Thr, and Lys residues was still down-regulated, ubiquitinated, and degraded in a Vpu-dependent manner. Our results suggest that Vpu may target either the BST-2 cytoplasmic Tyr residues or the NH(2) terminus itself for ubiquitination.
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Affiliation(s)
- Jean K Gustin
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, Oregon 97006, USA.
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