• HSV-1
• ICP0
• beta-barrel
• crystal structure
• dimerization
• novel fold

• P20 GM113117 NIGMS NIH HHS
• P30 GM110761 NIGMS NIH HHS
• T32 GM132061 NIGMS NIH HHS

• E3 ubiquitin ligase
• HSV-1
• ICP0
• herpes simplex virus
• innate immunity
• interferon-beta
• interferons
• viral gene expression

• Amino Acids
• Antiviral Agents/pharmacology
• Herpesvirus 1, Human/genetics
• Humans
• Immediate-Early Proteins/genetics
• Interferon Type I/immunology
• Latent Infection/virology
• Ubiquitin-Protein Ligases/genetics
• Viral Proteins/genetics

• P20 GM103418 NIGMS NIH HHS
• R01 AI048633 NIAID NIH HHS
• R01 AI072357 NIAID NIH HHS
• T32 AI007110 NIAID NIH HHS
• HHS | NIH | National Institute of General Medical Sciences (NIGMS)
• HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)
• University of Kansas (KU)

• ICP0
• herpes simplex virus
• interactome
• proteomics
• ubiquitinome

Promyelocytic leukemia nuclear bodies (PML NBs) are multi-protein assemblies representing distinct sub-nuclear structures. As phase-separated molecular condensates, PML NBs exhibit liquid droplet-like consistency. A key organizer of the assembly and dynamics of PML NBs is the ubiquitin-like SUMO modification system. SUMO is covalently attached to PML and other core components of PML NBs thereby exhibiting a glue-like function by providing multivalent interactions with proteins containing SUMO interacting motifs (SIMs). PML NBs serve as the catalytic center for nuclear SUMOylation and SUMO-SIM interactions are essential for protein assembly within these structures. Importantly, however, formation of SUMO chains on PML and other PML NB-associated proteins triggers ubiquitylation and proteasomal degradation which coincide with disruption of these nuclear condensates. To date, a plethora of nuclear activities such as transcriptional and post-transcriptional regulation of gene expression, apoptosis, senescence, cell cycle control, DNA damage response, and DNA replication have been associated with PML NBs. Not surprisingly, therefore, SUMO-dependent PML NB integrity has been implicated in regulating many physiological processes including tumor suppression, metabolism, drug-resistance, development, cellular stemness, and anti-pathogen immune response. The interplay between PML NBs and viral infection is multifaceted. As a part of the cellular antiviral defense strategy, PML NB components are crucial restriction factors for many viruses and a mutual positive correlation has been found to exist between PML NBs and the interferon response. Viruses, in turn, have developed counterstrategies for disarming PML NB associated immune defense measures. On the other end of the spectrum, certain viruses are known to usurp specific PML NB components for successful replication and disruption of these sub-nuclear foci has recently been linked to the stimulation rather than curtailment of antiviral gene repertoire. Importantly, the ability of invading virions to manipulate the host SUMO modification machinery is essential for this interplay between PML NB integrity and viruses. Moreover, compelling evidence is emerging in favor of bacterial pathogens to negotiate with the SUMO system thereby modulating PML NB-directed intrinsic and innate immunity. In the current context, we will present an updated account of the dynamic intricacies between cellular PML NBs as the nuclear SUMO modification hotspots and immune regulatory mechanisms in response to viral and bacterial pathogens.

• bacteria
• intrinsic and innate immune response
• promyelocytic leukemia nuclear bodies
• small ubiquitin-related modifier
• viruses

Nuclear domains 10 (ND10), a.k.a. promyelocytic leukemia nuclear bodies (PML-NBs), are membraneless subnuclear domains that are highly dynamic in their protein composition in response to cellular cues. They are known to be involved in many key cellular processes including DNA damage response, transcription regulation, apoptosis, oncogenesis, and antiviral defenses. The diversity and dynamics of ND10 residents enable them to play seemingly opposite roles under different physiological conditions. Although the molecular mechanisms are not completely clear, the pro- and anti-cancer effects of ND10 have been well established in tumorigenesis. However, in herpesvirus research, until the recently emerged evidence of pro-viral contributions, ND10 nuclear bodies have been generally recognized as part of the intrinsic antiviral defenses that converge to the incoming viral DNA to inhibit the viral gene expression. In this review, we evaluate the newly discovered pro-infection influences of ND10 in various human herpesviruses and analyze their molecular foundation along with the traditional antiviral functions of ND10. We hope to shed light on the explicit role of ND10 in both the lytic and latent cycles of herpesvirus infection, which is imperative to the delineation of herpes pathogenesis and the development of prophylactic/therapeutic treatments for herpetic diseases.

• ND10
• PML nuclear bodies
• antiviral defense
• herpesvirus
• virus-host interaction

• HSV-1 based therapies
• HSV-1 egress
• HSV-1 envelopment
• HSV-1 non-essential proteins
• gene silencing
• innate immunity

• Animals
• Endonucleases/genetics
• Endonucleases/metabolism
• Gene Expression Regulation, Viral
• Gene Silencing
• Herpes Simplex/immunology
• Herpes Simplex/metabolism
• Herpes Simplex/virology
• Herpesvirus 1, Human/physiology
• Host-Pathogen Interactions/genetics
• Host-Pathogen Interactions/immunology
• Humans
• Nucleic Acids/metabolism
• Phosphotransferases/genetics
• Phosphotransferases/metabolism
• Trans-Activators
• Viral Proteins/genetics
• Viral Proteins/metabolism
• Viral Regulatory and Accessory Proteins/genetics
• Viral Regulatory and Accessory Proteins/metabolism
• Virulence
• Virus Replication

• R21 AI144883 NIAID NIH HHS
• R21AI144883 Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases
• P20GM113117 NIGMS NIH HHS

Herpes simplex virus (HSV) infections are among the most common viral infections and usually last for a lifetime. The virus can potentially be controlled with vaccines since humans are the only known host. However, despite the development and trial of many vaccines, this has not yet been possible. This is normally attributed to the high latency potential of the virus. Numerous immune cells, particularly the natural killer cells and interferon gamma and pathways that are used by the body to fight HSV infections have been identified. On the other hand, the virus has developed different mechanisms, including using different microRNAs to inhibit apoptosis and autophagy to avoid clearance and aid latency induction. Both traditional and new methods of vaccine development, including the use of live attenuated vaccines, replication incompetent vaccines, subunit vaccines and recombinant DNA vaccines are now being employed to develop an effective vaccine against the virus. We conclude that this review has contributed to a better understanding of the interplay between the immune system and the virus, which is necessary for the development of an effective vaccine against HSV.

• herpes simplex virus
• immune response
• immune system
• vaccine

•

ICP0 is a viral E3 ubiquitin ligase that promotes HSV-1 infection.

Herpes simplex virus 1 (HSV-1) hijacks ubiquitination machinery to modify the cellular proteome to create an environment permissive for virus replication. HSV-1 encodes its own RING-finger E3 ubiquitin (Ub) ligase, Infected Cell Protein 0 (ICP0), that directly interfaces with component proteins of the Ub pathway to inactivate host immune defences and cellular processes that restrict the progression of HSV-1 infection. Consequently, ICP0 plays a critical role in the infectious cycle of HSV-1 that is required to promote the efficient onset of lytic infection and productive reactivation of viral genomes from latency. This review will describe the current knowledge regarding the biochemical properties and known substrates of ICP0 during HSV-1 infection. We will highlight the gaps in the characterization of ICP0 function and propose future areas of research required to understand fully the biological properties of this important HSV-1 regulatory protein.

• Chromatin
• HSV-1
• ICP0
• Immunity
• PML-NBs
• Ubiquitin

• ISA virus
• Maqui
• NADPH oxidase complex
• NP
• ROS
• SUMOylation

Reoviruses are potential anticancer agents due to their ability to induce cell death in tumor cells. Grass carp reovirus (GCRV) is one of the best characterized models on reovirus pathogenesis in vitro. However, there is little known about how SUMOylation affects reovirus pathogenesis. The SUMO conjugating enzyme 9 (Ubc9) determines the targets of SUMOylation. Here, the protein interactions between reovirus outer fiber proteins, specifically GCRV-104 VP55, and Ubc9 were probed using a yeast two-hybrid system. The N-terminal coiled-coil domain of VP55, containing a single lysine residue, was responsible for the interaction between VP55 and Ubc9 in yeast. In solid phase binding assays, a single amino acid mutation (K87R) prevented Ubc9 from binding to VP55. Overexpression of Ubc9 enhanced GCRV-104 infection efficiency, and knockdown of Ubc9 in CIK cells inhibited viral replication, which suggested that Ubc9 was a proviral factor. Furthermore, Ubc9 was shown to bind outer fiber proteins from type II GCRV, avian reovirus and mammalian reovirus in yeast. To our knowledge, this is the first study to show that Ubc9 binds to reovirus outer-fiber proteins and likely contributes to efficient orthoreovirus replication. These results suggest that SUMOylation modifications could be targeted to improve the therapeutic efficacy of oncolytic reovirus.

• SUMOylation
• Ubc9
• outer-fiber protein
• reovirus

• Cell Line
• Cell Line, Transformed
• Cells, Cultured
• Click Chemistry
• Gene Deletion
• Gene Expression Regulation, Viral/drug effects
• Herpes Simplex/drug therapy
• Herpes Simplex/metabolism
• Herpes Simplex/pathology
• Herpes Simplex/virology
• Herpesvirus 1, Human/growth & development
• Herpesvirus 1, Human/physiology
• Host-Pathogen Interactions/drug effects
• Humans
• Immunity, Innate/drug effects
• Inclusion Bodies, Viral/drug effects
• Inclusion Bodies, Viral/metabolism
• Inclusion Bodies, Viral/pathology
• Inclusion Bodies, Viral/virology
• Kinetics
• Lysogeny/drug effects
• Mutation
• Nuclear Proteins/antagonists & inhibitors
• Nuclear Proteins/genetics
• Nuclear Proteins/metabolism
• Phosphoproteins/antagonists & inhibitors
• Phosphoproteins/genetics
• Phosphoproteins/metabolism
• Promyelocytic Leukemia Protein/antagonists & inhibitors
• Promyelocytic Leukemia Protein/genetics
• Promyelocytic Leukemia Protein/metabolism
• RNA Interference
• Reverse Transcriptase Inhibitors/pharmacology
• Ubiquitin-Protein Ligases/genetics
• Ubiquitin-Protein Ligases/metabolism
• Viral Proteins/genetics
• Viral Proteins/metabolism
• Virus Internalization/drug effects
• Virus Replication/drug effects

• MC_UP_A550_1030 Medical Research Council
• MC_UU_12012/5 Medical Research Council
• MC_UU_12014/5 Medical Research Council
• Saudi Arabian Cultural Bureau

Viruses have evolved elaborate means to regulate diverse cellular pathways in order to create a cellular environment that facilitates viral survival and reproduction. This includes enhancing viral macromolecular synthesis and assembly, as well as preventing antiviral responses, including intrinsic, innate, and adaptive immunity. There are numerous mechanisms by which viruses mediate their effects on the host cell, and this includes targeting various cellular post-translational modification systems, including sumoylation. The wide-ranging impact of sumoylation on cellular processes such as transcriptional regulation, apoptosis, stress response, and cell cycle control makes it an attractive target for viral dysregulation. To date, proteins from both RNA and DNA virus families have been shown to be modified by SUMO conjugation, and this modification appears critical for viral protein function. More interestingly, members of the several viral families have been shown to modulate sumoylation, including papillomaviruses, adenoviruses, herpesviruses, orthomyxoviruses, filoviruses, and picornaviruses. This chapter will focus on mechanisms by which sumoylation both impacts human viruses and is used by viruses to promote viral infection and disease.

• DNA viruses
• RNA viruses
• SUMO
• Ubc9

Herpes simplex virus 1 (HSV-1) is a ubiquitous human pathogen that establishes latent infection in ganglia neurons. Its unique life cycle requires a balanced “conquer and compromise” strategy to deal with the host anti-viral defenses. One of HSV-1 α (immediate early) gene products, infected cell protein 0 (ICP0), is a multifunctional protein that interacts with and modulates a wide range of cellular defensive pathways. These pathways may locate in different cell compartments, which then migrate or exchange factors upon stimulation, for the purpose of a concerted and effective defense. ICP0 is able to simultaneously attack multiple host pathways by either degrading key restrictive factors or modifying repressive complexes. This is a viral protein that contains an E3 ubiquitin ligase, translocates among different cell compartments and interacts with major defensive complexes. The multiple functional domains of ICP0 can work independently and at the same time coordinate with each other. Dissecting the functional domains of ICP0 and delineating the coordination of these domains will help us understand HSV-1 pathogenicity as well as host defense mechanisms. This article focuses on describing individual ICP0 domains, their biochemical properties and their implication in HSV-1 infection. By putting individual domain functions back into the picture of host anti-viral defense network, this review seeks to elaborate the complex interactions between HSV-1 and its host.

• Subcellular translocation
• Herpes simplex virus 1
• Infected cell protein 0
• E3 ubiquitin ligase
• Protein modification
• ND10 nuclear bodies
• Chromatin repression

• R01 AI118992 NIAID NIH HHS

Covalent linkage to members of the small ubiquitin-like (SUMO) family of proteins is an important mechanism by which the functions of many cellular proteins are regulated. Sumoylation has roles in the control of protein stability, activity and localization, and is involved in the regulation of transcription, gene expression, chromatin structure, nuclear transport and RNA metabolism. Sumoylation is also linked, both positively and negatively, with the replication of many different viruses both in terms of modification of viral proteins and modulation of sumoylated cellular proteins that influence the efficiency of infection. One prominent example of the latter is the widespread reduction in the levels of cellular sumoylated species induced by herpes simplex virus type 1 (HSV-1) ubiquitin ligase ICP0. This activity correlates with relief from intrinsic immunity antiviral defence mechanisms. Previous work has shown that ICP0 is selective in substrate choice, with some sumoylated proteins such the promyelocytic leukemia protein PML being extremely sensitive, while RanGAP is completely resistant. Here we present a comprehensive proteomic analysis of changes in the cellular SUMO2 proteome during HSV-1 infection. Amongst the 877 potentially sumoylated species detected, we identified 124 whose abundance was decreased by a factor of 3 or more by the virus, several of which were validated by western blot and expression analysis. We found many previously undescribed substrates of ICP0 whose degradation occurs by a range of mechanisms, influenced or not by sumoylation and/or the SUMO2 interaction motif within ICP0. Many of these proteins are known or are predicted to be involved in the regulation of transcription, chromatin assembly or modification. These results present novel insights into mechanisms and host cell proteins that might influence the efficiency of HSV-1 infection.

Proteins are subject to many types of modification that regulate their functions and which are applied after their initial synthesis in the cell. One such modification is known as sumoylation, the covalent linkage of a small ubiquitin-like protein to a wide variety of substrate proteins. Sumoylation is involved in the regulation of many cellular pathways, including transcription, DNA repair, chromatin modification and defence to viral infections. Several viruses have connections with sumoylation, either through modification of their own proteins or in changing the sumoylation status of cellular proteins in ways that may be beneficial for infection. Herpes simplex virus type 1 (HSV-1) causes a widespread reduction in uncharacterized sumoylated cellular protein species, an effect that is caused by one of its key regulatory proteins (ICP0), which also induces the degradation of a number of repressive cellular proteins and thereby stimulates efficient infection. This study describes a comprehensive analysis of cellular proteins whose sumoylation status is altered by HSV-1 infection. Of 877 putative cellular sumoylation substrates, we found 124 whose sumoylation status reduces at least three-fold during infection. We validated the behavior of several such proteins and identified amongst them several novel targets of ICP0 activity with predicted repressive properties.

•

Application of near-infrared imaging to quantify ubiquitin biochemistry in vitro.

The ubiquitin–proteasome system is an essential cellular process that plays a fundamental role in the regulation of protein stability. This pathway is tightly controlled by a sequential cascade of enzymatic steps that culminates in the formation of a poly-ubiquitin chain onto the substrate protein targeted for 26S proteasome degradation. Through a process of co-evolution viruses have evolved mechanisms to utilize or suppress this pathway in order to enhance their replication and spread. One of the first proteins to be expressed during herpes simplex virus 1 (HSV-1) infection is ICP0, a viral RING-finger E3 ubiquitin ligase that targets a variety of cellular proteins for ubiquitination and proteasome-dependent degradation. This activity is required in order for ICP0 to efficiently stimulate the onset of HSV-1 lytic infection and viral reactivation from latency. While it is clear that the RING-finger domain of ICP0 plays an important role in the biology of HSV-1, methods for accurately quantifying its biochemical activity are currently lacking. Here we describe a protocol that enables the quantitative measurement of the ubiquitin ligase activity of ICP0 using near-infrared (IR) western blot imaging. The use of such imaging technology provides an accurate means to examine the biochemical and kinetic parameters of RING-finger ubiquitin ligases in solution, and may provide significant application for inhibitor studies.

• HSV-1
• ICP0
• Infrared
• LI-COR
• RING
• Ubiquitin

• Antigens, Nuclear/metabolism
• Autoantigens/metabolism
• Blotting, Western
• Cell Nucleus Structures/metabolism
• DNA Primers/genetics
• Gene Expression Regulation, Viral/genetics
• Gene Expression Regulation, Viral/physiology
• Herpes Simplex/genetics
• Herpes Simplex/metabolism
• Humans
• Immediate-Early Proteins/genetics
• Immediate-Early Proteins/metabolism
• Immunoprecipitation
• Microscopy, Confocal
• Nuclear Proteins/metabolism
• Polymerase Chain Reaction
• Promyelocytic Leukemia Protein
• Transcription Factors/metabolism
• Tumor Suppressor Proteins/metabolism
• Ubiquitin-Protein Ligases/genetics
• Ubiquitin-Protein Ligases/metabolism