Influenza Viruses have always been a major health concern due to their highly contagious nature. The PB1-F2 viroporin encoded by the influenza A virus is known to be a pro-apoptotic protein involved in cell death induction of the host immune cells. The structural arrangement and the mode of action of PB1-F2 viroporin have not been fully understood yet. Nonetheless, there is limited information on the oligomeric state of PB1-F2 and its possible role in the pore formation which could act as a channel for ion transport. The probable oligomeric structural existences of the viroporin and their channel-like behavior need to be explored in light of experimental reports cited in the literature. In our study, we report on the structural and dynamical properties of the trimeric and tetrameric state of PB1-F2, investigated by molecular dynamics simulations with improved sampling of conformational states as the initial focus of the study is to establish a rationale for their existence in a lipid environment. The simulation study provides detailed information on the mitochondrial membrane permeation pathway which causes the leakage of mitochondrial contents like cytochrome C and induces apoptosis. By focusing on low-order oligomers, trimer, and tetramer, we have identified key pore-forming characteristics that serve as a foundation for understanding the pro-apoptotic activity of PB1-F2. The structural and dynamical properties of these states were evaluated in the light of experimental reports, which reveal the tetrameric form to be the preferable state in the lipid environment, demonstrating superior structural stability, effective channel symmetry, and ion permeation compared to the higher-order oligomers besides trimer including pentameric and hexameric assemblies. The simulation results also explore the typical ion transportation criteria based on finding a less energetic barrier for ions/water molecules crossing the membrane.

We investigated several small viral proteins that reside and function in cellular membranes. These proteins belong to the viroporin family because they assemble into ion-conducting oligomers. However, despite forming similar oligomeric structures with analogous functions, these proteins have diverse amino acid sequences. In particular, the amino acid compositions of the proposed channel-forming transmembrane (TM) helices are vastly different-some contain residues (e.g., His, Trp, Asp, Ser) that could facilitate cation transport. Still, other viroporins' TM helices encompass exclusively hydrophobic residues; therefore, it is difficult to explain their channels' activity, unless other mechanisms (e.g., involving a negative lipid headgroups and/or membrane destabilization) take place. For this study, we selected the M2, Vpu, E, p13II, p7, and 2B proteins from the influenza A, HIV-1, human T-cell leukemia, hepatitis C, and picorna viruses, respectively. We provide a brief overview of the current knowledge about these proteins' structures as well as remaining questions about more comprehensive understanding of their structures, conformational dynamics, and function. Finally, we outline strategies to utilize a multi-prong structural and computational approach to overcome current deficiencies in the knowledge about these proteins.

SARS-CoV-2 infection affects the respiratory system but also many tissues and organs that may be adversely compromised. Accordingly, recent evidence has assessed virus ability to infect different cell phenotypes, translate viral proteins and promote virus replication. Among them, Envelope (E) proteins sustain virus replication, promote inflammatory processes and remodelling of host cells. However, despite advances on structure and sequence, E-protein specific location and effects in human host cells are still controversial and poorly investigated. Using lentiviral vectors, we established HEK293 and hiPS cell lines stably expressing E-protein. Immunocytochemistry showed E-protein mainly locates within the endoplasmic reticulum, the ERGIC and the Golgi compartments, while only HEK293 cells display some protein staining in cell periphery suggesting a possible insertion into the plasmalemma. Electrophysiological recordings in HEK293 cells revealed E-protein self-assembles in the plasma membrane to mediate a cation efflux pore that is sensitive to amantadine blockade. Calcium fluorescence imaging in HEK293 and hiPS cells demonstrated E-protein expression induces a marked depletion of thapsigargin-sensitive intracellular calcium stores. The altered calcium homeostasis associates to reduced cell metabolic activity, mitochondrial potential, proliferation rate and promotes ER stress. Finally, trilineage differentiation of hiPS cells indicated E-protein expression preserves cell pluripotency while selectively impairs mesodermal differentiation. These results unveil a critical role of stable E-viroporin expression that through alteration of ER Ca²⁺ homeostasis, metabolic activity and induction of ER stress affects important cellular functions, including the differentiative process from pluripotent to mesodermal progenitors, a critical cell population in self-repair and homeostasis of most human tissue and organs.

A major hindrance to the identification of new drug targets and the large-scale testing of new or existing compound libraries against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is that research on the virus is restricted to biosafety level 3 (BSL-3) laboratories. In such cases, BSL-2 surrogate systems or chimeric and attenuated versions of the virus are developed for safer, faster, and cheaper examination of the stages of the virus life cycle and specific drug targets. In this study, we describe a BSL-2 chimeric viral system utilizing a Sindbis virus background as a tool to study one such target, the SARS-CoV-2 Envelope (E) protein channel activity. This protein is fully conserved between SARS-CoV and SARS-CoV-2 variants of concern (VOCs), except for a threonine to isoleucine mutation in the Omicron variant, making the E ion channel domain an attractive antiviral target for combination therapy. Using a BSL-2-chimeric system, we have been able to show similar inhibition profiles using channel inhibitors as previously reported for E-channel inhibition in authentic SARS-CoV-2. This system has the potential to allow faster initial screening of E-channel inhibitors and can be useful in developing broad-spectrum antivirals against viral channel proteins.IMPORTANCEDespite its importance in viral infections, no antivirals exist against the ion channel activity of the SARS-CoV-2 Envelope (E) protein. The E protein is highly conserved among SARS-CoV-2 variants, making it an attractive target for antiviral therapies. Research on SARS-CoV-2 is restricted to BSL-3 laboratories, creating a bottleneck for screening potential antiviral compounds. This study presents a BSL-2 chimeric system using a Sindbis virus background to study the ion channel activity of the E protein. This novel BSL-2 system bypasses this limitation, offering a safer and faster approach for the initial screening of ion channel inhibitors. By replicating the channel inhibition profiles of authentic SARS-CoV-2 in a more accessible system, this research paves the way for the development of broad-spectrum antivirals against viral channel proteins, potentially expediting the discovery of life-saving treatments for COVID-19 and other viral diseases.

SARS-CoV-2 hijacks multiple organelles for virion assembly, of which the mechanisms have not been fully understood. Here, we identified a SARS-CoV-2-driven membrane structure named the 3a dense body (3DB). 3DBs are unusual electron-dense and dynamic structures driven by the accessory protein ORF3a via remodeling a specific subset of the trans-Golgi network (TGN) and early endosomal membrane. 3DB formation is conserved in related bat and pangolin coronaviruses but was lost during the evolution to SARS-CoV. During SARS-CoV-2 infection, 3DB recruits the viral structural proteins spike (S) and membrane (M) and undergoes dynamic fusion/fission to maintain the optimal unprocessed-to-processed ratio of S on assembled virions. Disruption of 3DB formation resulted in virions assembled with an abnormal S processing rate, leading to a dramatic reduction in viral entry efficiency. Our study uncovers the crucial role of 3DB in maintaining maximal SARS-CoV-2 infectivity and highlights its potential as a target for COVID-19 prophylactics and therapeutics.

Head and neck cancer is the seventh most common cancer worldwide and its development is associated with viral infection. Human papillomavirus (HPV) is the major cause of oropharyngeal cancer and encodes three known oncoproteins, E5, E6, and E7. Epstein-Barr virus (EBV), which is the causative agent of most nasopharyngeal carcinoma, also employs several immunosuppressive mechanisms that contribute to the development of the disease. In this review, we synthesize and discuss several mechanisms used by these viruses to evade and escape the host immune system. In particular, we focus on the evasive tactics of HPV E5 which, we argue, is critical to establishing persistent infection and the development and progression of carcinomas. Importantly the mechanisms by which these viruses suppress immune responses may also play a key role in resistance to checkpoint blockade immunotherapies and thus impact patient outcomes.

Structural virology has emerged as the foundation for the development of effective antiviral therapeutics. It is pivotal in providing crucial insights into the three-dimensional frame of viruses and viral proteins at atomic-level or near-atomic-level resolution. Structure-based assessment of viral components, including capsids, envelope proteins, replication machinery, and host interaction interfaces, is instrumental in unraveling the multiplex mechanisms of viral infection, replication, and pathogenesis. The structural elucidation of viral enzymes, including proteases, polymerases, and integrases, has been essential in combating viruses like HIV-1 and HIV-2, SARS-CoV-2, and influenza. Techniques including X-ray crystallography, Nuclear Magnetic Resonance spectroscopy, Cryo-electron Microscopy, and Cryo-electron Tomography have revolutionized the field of virology and significantly aided in the discovery of antiviral therapeutics. The ubiquity of chronic viral infections, along with the emergence and reemergence of new viral threats necessitate the development of novel antiviral strategies and agents, while the extensive structural diversity of viruses and their high mutation rates further underscore the critical need for structural analysis of viral proteins to aid antiviral development. This review highlights the significance of structure-based investigations for bridging the gap between structure and function, thus facilitating the development of effective antiviral therapeutics, vaccines, and antibodies for tackling emerging viral threats.

A truncated form of the ATP release channel pannexin 1 (Panx1), Panx11-89, is enriched in metastatic breast cancer cells and has been proposed to mediate metastatic cell survival by increasing ATP release through mechanosensitive Panx1 channels. However, whether Panx11-89 on its own [without the presence of wild-type Panx1 (wtPanx1)] mediates ATP release has not been tested. Here, we show that Panx11-89 by itself can form a constitutively active membrane channel, capable of releasing ATP even in the absence of wtPanx1. Our biophysical characterization reveals that most basic structure-function features of the channel pore are conserved in the truncated Panx11-89 polypeptide. Thus, augmenting extracellular potassium ion concentrations enhances Panx11-89-mediated conductance. Moreover, despite the severe truncation, Panx11-89 retains sensitivity to most wtPanx1 channel inhibitors. Therefore, Panx1 blockers may be of therapeutic value to combat metastatic cell survival. Our study both provides a mechanism for ATP release from cancer cells and suggests that Panx11-89 might aid in the structure-function analysis of Panx1 channels.

Viruses are the most abundant biological entities on Earth, with an estimated 1031 viruses in the biosphere. These particles serve as the crucial link between viral replication cycles in different host cells, employing a variety of release mechanisms, such as cell lysis, exocytosis, and budding. Among the diverse viral groups, giant viruses have garnered significant scientific interest due to their complex particles and genomes. Giant viruses may infect amoebae and other unicellular protists, exhibiting remarkable variation in size, shape, and symmetry. They belong to the realm Varidnaviria, kingdom Bamfordvirae, and phylum Nucleocytoviricota. This review examines the diverse viral release strategies employed by giant viruses, highlighting the mechanisms they use to exit host cells. These include the induction of cell lysis, vesicle formation, and exocytosis, which vary not only between different species but also within individual viral groups. The diversity of release mechanisms reflects the complex evolutionary adaptations of giant viruses, providing information about their biology and life cycles.

SARS-CoV-2 E and 3a proteins are important for the assembly, budding, and release of viral particles. These two transmembrane proteins have been implicated in forming channels in the membrane that allow the transport of ions to favor viral replication. During an active infection, both proteins generally localize to the endoplasmic reticulum (ER), ER-Golgi intermediate compartment (ERGIC), and the Golgi where viral assembly occurs. The ER and Golgi are critical for the proper packaging and trafficking of cellular proteins along the secretory pathways which determine a protein's final destination inside or outside of the cell. The SARS-CoV-2 virus primarily infects epithelial cells that are highly secretory in nature such as those in the lung and gut. Here we quantified the distribution of SARS-CoV-2 E and 3a proteins along the secretory pathways in a human intestinal epithelial cell line. We used NaturePatternMatch to demonstrate that epitope-tagged E and 3a proteins expressed alone via transient transfection have a similar immunoreactivity pattern as E and 3a proteins expressed by wild-type viral infection. While E and 3a proteins localized with all selected cellular markers to varying degrees, 3a protein displayed a higher correlation coefficient with the Golgi, early/late endosome, lysosome, and plasma membrane when compared to E protein. This work is the first to provide quantification of the subcellular distribution of E and 3a proteins along the multiple components of the secretory pathway and serves as a basis to develop models for examining how E and 3a alter proteostasis within these structures and affect their function.

Severe acute respiratory syndrome coronavirus (SARS-CoV), the virus responsible for COVID-19, interacts with the host immune system through complex mechanisms that significantly influence disease outcomes, affecting both innate and adaptive immunity. These interactions are crucial in determining the disease's severity and the host's ability to clear the virus. Given the virus's substantial socioeconomic impact, high morbidity and mortality rates, and public health importance, understanding these mechanisms is essential. This article examines the diverse innate immune responses triggered by SARS-CoV-2's structural proteins, including the spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins, along with nonstructural proteins (NSPs) and open reading frames. These proteins play pivotal roles in immune modulation, facilitating viral replication, evading immune detection, and contributing to severe inflammatory responses such as cytokine storms and acute respiratory distress syndrome (ARDS). The virus employs strategies like suppressing type I interferon production and disrupting key antiviral pathways, including MAVS, OAS-RNase-L, and PKR. This study also explores the immune pathways that govern the activation and suppression of immune responses throughout COVID-19. By analyzing immune sensing receptors and the responses initiated upon recognizing SARS-CoV-2 structural proteins, this review elucidates the complex pathways associated with the innate immune response in COVID-19. Understanding these mechanisms offers valuable insights for therapeutic interventions and informs public health strategies, contributing to a deeper understanding of COVID-19 immunopathogenesis.

Alphaviruses, a genus of vector-borne viruses in the Togaviridae family, encode a small ion-channel-forming protein, 6K, and its transframe variant (TF) during infections. Although 6K/TF have vital roles in glycoprotein transport, virus assembly, and budding, there is no mechanistic explanation for these functions. We investigated the distinct biochemical functionalities of 6K and TF from the mosquito-borne alphavirus, Chikungunya Virus. We show that like 6K, TF is also capable of forming ion channels in bilayer membranes. The assemblies formed by 6K in membranes are structurally more complex and potentially more ion-restrictive than those formed by TF. Both 6K and TF show strong affinity toward the ER membranes, indicating that the localization of these components at the plasma membrane, as previously reported, is either linked to post-translational modification or mediated through interaction with binding partners. These structural and functional insights may elucidate the distinct roles of 6K and TF in the alphavirus life cycle.

Viruses engage in a variety of processes to subvert host defenses and create an environment amenable to replication. Here, using rotavirus as a prototype, we show that calcium conductance out of the endoplasmic reticulum by the virus encoded ion channel, NSP4, induces intercellular calcium waves that extend beyond the infected cell and contribute to pathogenesis. Viruses that lack the ability to induce this signaling show diminished viral shedding and attenuated disease in a mouse model of rotavirus diarrhea. This implicates nonstructural protein 4 (NSP4) as a virulence factor and provides mechanistic insight into its mode of action. Critically, this signaling induces a transcriptional signature characteristic of interferon-independent innate immune activation, which is not observed in response to a mutant NSP4 that does not conduct calcium. This implicates calcium dysregulation as a means of pathogen recognition, a theme broadly applicable to calcium-altering pathogens beyond rotavirus.

The 'Viroporin' family comprises a number of mostly small-sized, integral membrane proteins encoded by animal and plant viruses. Despite their sequence and structural diversity, viroporins share a common functional trend: their capacity to assemble transmembrane channels during the replication cycle of the virus. Their selectivity spectrum ranges from low-pH-activated, unidirectional proton transporters, to size-limited permeating pores allowing passive diffusion of metabolites. Through mechanisms not fully understood, expression of viroporins facilitates virion assembly/release from infected cells, and subverts the cell physiology, contributing to cytopathogenicity. Compounds that interact with viroporins and interfere with their membrane-permeabilizing activity in vitro, are known to inhibit virus production. Moreover, viroporin-defective viruses comprise a source of live attenuated vaccines that prevent infection by notorious human and livestock pathogens. This review dives into the origin and evolution of the viroporin concept, summarizes some of the methodologies used to characterize the structure-function relationships of these important virulence factors, and attempts to classify them on biophysical grounds attending to their mechanisms of ion/solute transport across membranes.

Modern bioelectronic tools are rapidly advancing to detect electric potentials within networks of electrogenic cells, such as cardiomyocytes, neurons, and pancreatic beta cells. However, it is becoming evident that electrical signaling is not limited to the animal kingdom but may be a universal form of cell-cell communication. In this review, we discuss the existing evidence of, and tools used to collect, subcellular, single-cell and network-level electrical signals across kingdoms, including bacteria, plants, fungi, and even viruses. We discuss how cellular networks employ altered electrical "circuitry" and intercellular mechanisms across kingdoms, and we assess the functionality and scalability of cutting-edge nanobioelectronics to collect electrical signatures regardless of cell size, shape, or function. Researchers today aim to design micro- and nano-topographic structures which harness mechanosensitive membrane and cytoskeletal pathways that enable tight electrical coupling to subcellular compartments within high-throughput recording systems. Finally, we identify gaps in current knowledge of inter-species and inter-kingdom electrical signaling and propose critical milestones needed to create a central theory of electrical signaling across kingdoms. Our discussion demonstrates the need for high resolution, high throughput tools which can probe multiple, diverse cell types at once in their native or experimentally-modeled environments. These advancements will not only reveal the underlying biophysical laws governing the universal language of electrical communication, but can enable bidirectional electrical communication and manipulation of biological systems.

Infection by Human Papillomaviruses accounts for the most widespread sexually transmitted infection worldwide. Clinical presentation of these infections can range from subclinical and asymptomatic to anogenital cancers, with the latter associated with persistent infection over a significant period of time. Of the over 200 isotypes of the human virus identified, a subset of these has been characterized as high-risk due to their ability to induce oncogenesis. At the core of Papillomavirus pathogenesis sits three virally encoded oncoproteins: E5, E6, and E7. In this review we will discuss the respective roles of these proteins and how they contribute to carcinogenesis, evaluating key distinguishing features that separate them from their low-risk counterparts. Furthermore, we will consider the complex relationship between this trio and how their interwoven functional networks underpin the development of cancer.

Molecular mimicry has been proposed to be a possible mechanism of induction of autoimmunity. In some cases, it is believed that such events could lead to a disease such as Type 1 diabetes (T1D). One of the primary MHC-I epitopes in the non-obese diabetic (NOD) mouse model of T1D has been identified as a peptide from the islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) protein. In humans, the most common MHC-I model allele is HLA-A02; based on this, the study here identified a potential HLA-A0201-restricted human IGRP epitope as YLKTNLFLFL and also found a homologous A0201-restricted peptide in an Enterococcal protein. Using cells obtained from healthy human donors, it was seen that after a 2-week incubation with the synthetic bacterial protein, healthy A0201+ donor CD8+ cells displayed increased staining for human IGRP-peptide-dextramer. On the other hand, in control cultures, no significant levels of dextramer-staining CD8+ T-cells were detectable. From these outcomes, it is possible to conclude that certain bacterial proteins may initiate CD8+ T-cell-mediated immune reaction toward homologous human antigens.

Influenza B viruses have cocirculated during most seasonal flu epidemics and can cause significant human morbidity and mortality due to their rapid mutation, emerging drug resistance, and severe impact on vulnerable populations. The influenza B M2 proton channel (BM2) plays an essential role in viral replication, but the mechanisms behind its symmetric proton conductance and the involvement of a second histidine (His27) cluster remain unclear. Here we performed membrane-enabled continuous constant-pH molecular dynamics simulations on wildtype BM2 and a key H27A mutant channel to explore its pH-dependent conformational switch. Simulations captured the activation as the first histidine (His19) protonates and revealed the transition at lower pH values compared to AM2 is a result of electrostatic repulsions between His19 and preprotonated His27. Crucially, we provided an atomic-level understanding of the symmetric proton conduction by identifying preactivating channel hydration in the C-terminal portion. This research advances our understanding of the function of BM2 function and lays the groundwork for further chemically reactive modeling of the explicit proton transport process as well as possible antiflu drug design efforts.

The Ebola delta peptide is an amphipathic, 40-residue peptide encoded by the Ebola virus, referred to as E40. The membrane-permeabilising activity of the E40 delta peptide has been demonstrated in cells and lipid vesicles suggesting the E40 delta peptide likely acts as a viroporin. The lytic activity of the peptide increases in the presence of anionic lipids and a disulphide bond in the C-terminal part of the peptide. Previous in silico work predicts the peptide to show a partially helical structure, but there is no experimental information on the structure of E40. Here, we use circular dichroism spectroscopy to report the secondary structure propensities of the reduced and oxidised forms of the E40 peptide in water, detergent micelles, and lipid vesicles composed of neutral and anionic lipids (POPC and POPG, respectively). Results indicate that the peptide is predominately a random coil in solution, and the disulphide bond has a small but measurable effect on peptide conformation. Secondary structure analysis shows large uncertainties and dependence on the reference data set and, in our system, cannot be used to accurately determine the secondary structure motifs of the peptide in membrane environments. Nevertheless, the spectra can be used to assess the relative changes in secondary structure propensities of the peptide depending on the solvent environment and disulphide bond. In POPC-POPG vesicles, the peptide transitions from a random coil towards a more structured conformation, which is even more pronounced in negatively charged SDS micelles. In vesicles, the effect depends on the peptide-lipid ratio, likely resulting from vesicle surface saturation. Further experiments with zwitterionic POPC vesicles and DPC micelles show that both curvature and negatively charged lipids can induce a change in conformation, with the two effects being cumulative. Electrostatic screening from Na+ ions reduced this effect. The oxidised form of the peptide shows a slightly lower propensity for secondary structure and retains a more random coil conformation even in the presence of PG-PC vesicles.

Viroporins possess important potential as antiviral targets due to their critical roles during virus life cycles, spanning from virus entry to egress. Although the antiviral amantadine targets the M2 viroporin of influenza A virus, successful progression of other viroporin inhibitors into clinical use remains challenging. These challenges relate in varying proportions to a lack of reliable full-length 3D-structures, difficulties in functionally characterising individual viroporins, and absence of verifiable direct binding between inhibitor and viroporin. This review offers perspectives to help overcome these challenges. We provide a comprehensive overview of the viroporin family, including their structural and functional features, highlighting the moldability of their energy landscapes and actions. To advance the field, we suggest a list of best practices to aspire towards unambiguous viroporin identification and characterisation, along with considerations of potential pitfalls. Finally, we present current and future scenarios of, and prospects for, viroporin targeting drugs.

Recent advances in studies exploring the roles of extracellular vesicles (EVs) in viral transmission and replication have illuminated hepatotropic viruses, such as hepatitis A (HAV), hepatitis B (HBV), hepatitis C (HCV), hepatitis D (HDV), and hepatitis E (HEV). While previous investigations have uncovered these viruses' ability to exploit cellular EV pathways for replication and transmission, most have focused on the impacts of exosomal pathways. With an improved understanding of EVs, four main subtypes, including exosomes, microvesicles, large oncosomes, and apoptotic bodies, have been categorized based on size and biogenic pathways. However, there remains a noticeable gap in comprehensive reviews summarizing recent findings and outlining future perspectives for EV studies related to hepatotropic viruses. This review aims to consolidate insights into EV pathways utilized by hepatotropic viruses, offering guidance for the future research direction in this field. By comprehending the diverse range of hepatotropic virus-associated EVs and their role in cellular communication during productive viral infections, this review may offer valuable insights for targeting therapeutics and devising strategies to combat virulent hepatotropic virus infections and the associated incidence of liver cancer.

Evolution from precellular supramolecular assemblies to cellular world originated from the ability to make a barrier between the interior of the cell and the outer environment. This step resulted from the possibility to form a membrane, which preserves the cell like a wall of the castle. However, every castle needs gates for trading, i.e. in the case of cell, for controlled exchange of substances. These 'gates' should have the mechanism of opening and closing, guards, entry rules, and so on. Different structures are known to be able to make membrane permeable to various substances, from ions to macromolecules. They are amphipathic peptides, their assemblies, sophisticated membrane channels with numerous transmembrane domains, etc. Upon evolving, cellular world preserved and selected many variants, which, finally, have provided both prokaryotes and eukaryotes with highly selective and regulated ion channels. However, various simpler variants of ion channels are found in viruses. Despite the origin of viruses is still under debates, they have evolved parallelly with the cellular forms of life. Being initial form of the enveloped organisms, reduction of protocells or their escaped parts, viruses might be fingerprints of the evolutionary steps of cellular structures like ion channels. Therefore, viroporins may provide us a necessary information about selection between high functionality and less complex structure in supporting all the requirements for controlled membrane permeability. In this review we tried to elucidate these compromises and show the possible way of the evolution of ion channels, from peptides to complex multi-subunit structures, basing on viral examples.

Plant-virus interaction models propose that a virus's ability to infect a host genotype depends on the compatibility between virulence and resistance genes. Recently, we conducted an evolution experiment in which lineages of turnip mosaic virus (TuMV) were passaged in Arabidopsis thaliana genotypes carrying mutations in components of the DNA methylation and the histone demethylation epigenetic pathways. All evolved lineages increased infectivity, virulence and viral load in a host genotype-dependent manner.

To better understand the underlying reasons for these evolved relationships, we delved into the transcriptomic responses of mutant and WT plant genotypes in mock conditions and infected with either the ancestral or evolved viruses. Such a comparison allowed us to classify every gene into nine basic expression profiles. Regarding the targets of viral adaptation, our analyses allowed the identification of common viral targets as well as host genotype-specific genes and categories of biological processes. As expected, immune response-related genes were found to be altered upon infection. However, we also noticed the pervasive over-representation of other functional groups, suggesting that viral adaptation was not solely driven by the level of expression of plant resistance genes. In addition, a significant association between the presence of transposable elements within or upstream the differentially expressed genes was observed. Finally, integration of transcriptomic data into a virus-host protein-protein interaction network highlighted the most impactful interactions.

These findings shed extra light on the complex dynamics between plants and viruses, indicating that viral infectivity depends on various factors beyond just the plant's resistance genes.

One of the actively developing areas of drug development is the creation of chimeric toxins, recombinant bifunctional molecules designed to affect target cells selectively. The prevalent approach involves fusing bacterial and plant toxins with molecules that facilitate targeted delivery. However, the therapeutic use of such toxins often encounters challenges associated with negative side effects. Concurrently, viruses encode proteins possessing toxin-like properties, exerting multiple effects on the vital activity of cells. In contrast to bacterial and plant toxins, the impact of viral proteins is typically milder, presenting a significant advantage by potentially reducing the likelihood of side effects. This review delineates the characteristics of extensively studied viral proteins with toxic and immunomodulatory properties and explores the prospects of incorporating them into chimeric toxins.

The envelope (E) protein of SARS-CoV-2 is the smallest of the three structural membrane proteins of the virus. E mediates budding of the progeny virus in the endoplasmic reticulum Golgi intermediate compartment of the cell. It also conducts ions, and this channel activity is associated with the pathogenicity of SARS-CoV-2. The structural basis for these functions is still poorly understood. Biochemical studies of E in detergent micelles found a variety of oligomeric states, but recent 19F solid-state NMR data indicated that the transmembrane domain (ETM, residues 8-38) forms pentamers in lipid bilayers. Hexamethylene amiloride (HMA), an E inhibitor, binds the pentameric ETM at the lipid-exposed helix-helix interface. Here, we investigate the oligomeric structure and drug interaction of an ectodomain-containing E construct, ENTM (residues 1-41). Unexpectedly, 19F spin diffusion NMR data reveal that ENTM adopts an average oligomeric state of dimers instead of pentamers in lipid bilayers. A new amiloride inhibitor, AV-352, shows stronger inhibitory activity than HMA in virus-like particle assays. Distance measurements between 13C-labeled protein and a trifluoromethyl group of AV-352 indicate that the drug binds ENTM with a higher stoichiometry than ETM. We measured protein-drug contacts using a sensitivity-enhanced two-dimensional 13C-19F distance NMR technique. The results indicate that AV-352 binds the C-terminal half of the TM domain, similar to the binding region of HMA. These data provide evidence for the existence of multiple oligomeric states of E in lipid bilayers, which may carry out distinct functions and may be differentially targeted by antiviral drugs.

Potassium (K+) plays crucial roles in both plant development and immunity. However, the function of K+ in plant-virus interactions remains largely unknown. Here, we utilized Barley yellow striate mosaic virus (BYSMV), an insect-transmitted plant cytorhabdovirus, to investigate the interplay between viral infection and plant K+ homeostasis. The BYSMV accessory P9 protein exhibits viroporin activity by enhancing membrane permeability in Escherichia coli. Additionally, P9 increases K+ uptake in yeast (Saccharomyces cerevisiae) cells, which is disrupted by a point mutation of glycine 14 to threonine (P9G14T). Furthermore, BYSMV P9 forms oligomers and targets to both the viral envelope and the plant membrane. Based on the recombinant BYSMV-GFP (BYGFP) virus, a P9-deleted mutant (BYGFPΔP9) was rescued and demonstrated infectivity within individual plant cells of Nicotiana benthamiana and insect vectors. However, BYGFPΔP9 failed to infect barley plants after transmission by insect vectors. Furthermore, infection of barley plants was severely impaired for BYGFP-P9G14T lacking P9 K+ channel activity. In vitro assays demonstrate that K+ facilitates virion disassembly and the release of genome RNA for viral mRNA transcription. Altogether, our results show that the K+ channel activity of viroporins is conserved in plant cytorhabdoviruses and plays crucial roles in insect-mediated virus transmission.

Classical swine fever virus (CSFV) p7 viroporin plays crucial roles in cellular ion balance and permeabilization. The antiviral drug amantadine effectively inhibits viral replication by blocking the activity of CSFV p7 viroporin. However, little information is available for the binding mode of amantadine with CSFV p7 viroporin, due to the lack of a known polymer structure for CSFV p7. In this study, we employed AlphaFold2 to predict CSFV p7 structures. Subsequently, we conducted a docking study to investigate the binding sites of amantadine to CSFV p7. Computational analysis showed that CSFV p7 forms a pore channel in a hexameric structure. Furthermore, molecular dynamics (MD) simulations and mutant analyses further suggest that CSFV p7 likely exists as a hexamer. Docking studies and MD simulations showed that amantadine interacts with the hydrophibic regions of tetramer and pentamer, as well as with the hydrophobic pore channel of the hexamer. Considering the potential hexameric assembly of CSFV p7, along with docking results, MD simulations, and the characteristics of the gated ion channels, we propose a model of CSFV p7 ion channel based on its hexameric configuration. In this model, residues E21, Y25, and R34 are suggested to selectively recruit and dehydrate ions, while residues L28 and L31 likely act as hydrophobic constrictors, thereby restricting the free movement of water. The binding of amantadine to residues I20, E21, V24 and Y25 effectively blocks ion transport. However, this proposed molecular model requires experimental validation. Our findings give a structural insight into the models of CSFV p7 as an ion channel and provide a molecular explanation for the inhibition effects of amantadine on CSFV p7-mediated ion channel conductance.

Viroporins are small, hydrophobic viral proteins that modify cellular membranes to form tiny pores for influx of ions and small molecules. Previously, viroporins were identified exclusively in vertebrate viruses. Recent studies have shown that both plant-infecting positive-sense single-stranded (+ss) and negative-sense single-stranded (-ss) RNA viruses also encode functional viroporins. These seminal discoveries not only advance our understanding of the distribution and evolution of viroporins, but also open up a new field of plant virus research.

The SARS-CoV-2 E protein is an enigmatic viral structural protein with reported viroporin activity associated with the acute respiratory symptoms of COVID-19, as well as the ability to deform cell membranes for viral budding. Like many viroporins, the E protein is thought to oligomerize with a well-defined stoichiometry. However, attempts to determine the structure of the protein complex have yielded inconclusive results, suggesting several possible oligomers, ranging from dimers to pentamers. Here, we combined patch-clamp, confocal fluorescence microscopy on giant unilamellar vesicles, and atomic force microscopy to show that E protein can exhibit two modes of membrane activity depending on membrane lipid composition. In the absence or the presence of a low content of cholesterol, the protein forms short-living transient pores, which are seen as semi-transmembrane defects in a membrane by atomic force microscopy. Approximately 30 mol% cholesterol is a threshold for the transition to the second mode of conductance, which could be a stable pentameric channel penetrating the entire lipid bilayer. Therefore, the E-protein has at least two different types of activity on membrane permeabilization, which are regulated by the amount of cholesterol in the membrane lipid composition and could be associated with different types of protein oligomers.

Flaviviruses, including Zika virus (ZIKV), are a significant global health concern, yet no licensed antivirals exist to treat disease. The small membrane (M) protein plays well-defined roles during viral egress and remains within virion membranes following release and maturation. However, it is unclear whether M plays a functional role in this setting. Here, we show that M forms oligomeric membrane-permeabilising channels in vitro, with increased activity at acidic pH and sensitivity to the prototypic channel-blocker, rimantadine. Accordingly, rimantadine blocked an early stage of ZIKV cell culture infection. Structure-based channel models, comprising hexameric arrangements of two trans-membrane domain protomers were shown to comprise more stable assemblages than other oligomers using molecular dynamics simulations. Models contained a predicted lumenal rimantadine-binding site, as well as a second druggable target region on the membrane-exposed periphery. In silico screening enriched for repurposed drugs/compounds predicted to bind to either one site or the other. Hits displayed superior potency in vitro and in cell culture compared with rimantadine, with efficacy demonstrably linked to virion-resident channels. Finally, rimantadine effectively blocked ZIKV viraemia in preclinical models, supporting that M constitutes a physiologically relevant target. This could be explored by repurposing rimantadine, or development of new M-targeted therapies.

African swine fever virus (ASFV) is the causative agent of a contagious disease affecting wild and domestic swine. The function of B169L protein, as a potential integral structural membrane protein, remains to be experimentally characterized. Using state-of-the-art bioinformatics tools, we confirm here earlier predictions indicating the presence of an integral membrane helical hairpin, and further suggest anchoring of this protein to the ER membrane, with both terminal ends facing the lumen of the organelle. Our evolutionary analysis confirmed the importance of purifying selection in the preservation of the identified domains during the evolution of B169L in nature. Also, we address the possible function of this hairpin transmembrane domain (HTMD) as a class IIA viroporin. Expression of GFP fusion proteins in the absence of a signal peptide supported B169L insertion into the ER as a Type III membrane protein and the formation of oligomers therein. Overlapping peptides that spanned the B169L HTMD were reconstituted into ER-like membranes and the adopted structures analyzed by infrared spectroscopy. Consistent with the predictions, B169L transmembrane sequences adopted α-helical conformations in lipid bilayers. Moreover, single vesicle permeability assays demonstrated the assembly of lytic pores in ER-like membranes by B169L transmembrane helices, a capacity confirmed by ion-channel activity measurements in planar bilayers. Emphasizing the relevance of these observations, pore-forming activities were not observed in the case of transmembrane helices derived from EP84R, another ASFV protein predicted to anchor to membranes through a α-helical HTMD. Overall, our results support predictions of viroporin-like function for the B169L HTMD.IMPORTANCEAfrican swine fever (ASF), a devastating disease affecting domestic swine, is widely spread in Eurasia, producing significant economic problems in the pork industry. Approaches to prevent/cure the disease are mainly restricted to the limited information concerning the role of most of the genes encoded by the large (160-170 kba) virus genome. In this report, we present the experimental data on the functional characterization of the African swine fever virus (ASFV) gene B169L. Data presented here indicates that the B169L gene encodes for an essential membrane-associated protein with a viroporin function.

The Alphavirus genus includes viruses that cause encephalitis due to neuroinvasion and viruses that cause arthritis due to acute and chronic inflammation. There is no approved therapeutic for alphavirus infections, but significant efforts are ongoing, more so in recent years, to develop vaccines and therapeutics for alphavirus infections. This review article highlights some of the major advances made so far to identify small molecules that can selectively target the structural and the nonstructural proteins in alphaviruses with the expectation that persistent investigation of an increasingly expanding chemical space through a variety of structure-based design and high-throughput screening strategies will yield candidate drugs for clinical studies. While most of the works discussed are still in the early discovery to lead optimization stages, promising avenues remain for drug development against this family of viruses.

Autophagy refers to a set of degradative mechanisms whereby cytoplasmic contents are targeted to the lysosome. This is best described for macroautophagy, where a double-membrane compartment (autophagosome) is generated to engulf cytoplasmic contents. Autophagosomes are decorated with ubiquitin-like ATG8 molecules (ATG8s), which are recruited through covalent lipidation, catalysed by the E3-ligase-like ATG16L1 complex. LC3 proteins are ATG8 family members that are often used as a marker for autophagosomes. In contrast to canonical macroautophagy, conjugation of ATG8s to single membranes (CASM) describes a group of non-canonical autophagy processes in which ATG8s are targeted to pre-existing single-membrane compartments. CASM occurs in response to disrupted intracellular pH gradients, when the V-ATPase proton pump recruits ATG16L1 in a process called V-ATPase-ATG16L1-induced LC3 lipidation (VAIL). Recent work has demonstrated a parallel, alternative axis for CASM induction, triggered when the membrane recruitment factor TECPR1 recognises sphingomyelin exposed on the cytosolic face of a membrane and forms an alternative E3-ligase-like complex. This sphingomyelin-TECPR1-induced LC3 lipidation (STIL) is independent of the V-ATPase and ATG16L1. In light of these discoveries, this Cell Science at a Glance article summarises these two mechanisms of CASM to highlight how they differ from canonical macroautophagy, and from each other.

Influenza B viruses have co-circulated during most seasonal flu epidemics and can cause significant human morbidity and mortality due to their rapid mutation, emerging drug resistance, and severe impact on vulnerable populations. The influenza B M2 proton channel (BM2) plays an essential role in viral replication, but the mechanisms behind its symmetric proton conductance and the involvement of a second histidine (His27) cluster remain unclear. Here we perform the membrane-enabled continuous constant-pH molecular dynamics simulations on wildtype BM2 and a key H27A mutant to explore its pH-dependent conformational switch. Simulations capture the activation as the first histidine (His19) protonates and reveal the transition at lower pH values compared to AM2 is a result of electrostatic repulsions between His19 and pre-protonated His27. Crucially, we provide an atomic-level understanding of the symmetric proton conduction by identifying pre-activating channel hydration in the C-terminal portion. This research advances our understanding of the function of BM2 function and lays the groundwork for further chemically reactive modeling of the explicit proton transport process as well as possible anti-flu drug design efforts.

The picornavirus genome encodes a large, single polyprotein that is processed by viral proteases to form an active replication complex. The replication complex is formed with the viral genome, host proteins, and viral proteins that are produced/translated directly from each of the viral genomes (viral proteins provided in cis). Efficient complementation in vivo of replication complex formation by viral proteins provided in trans, thus exogenous or ectopically expressed viral proteins, remains to be demonstrated. Here, we report an efficient trans complementation system for the replication of defective poliovirus (PV) mutants by a viral polyprotein precursor in HEK293 cells. Viral 3AB in the polyprotein, but not 2BC, was processed exclusively in cis. Replication of a defective PV replicon mutant, with a disrupted cleavage site for viral 3Cpro protease between 3Cpro and 3Dpol (3C/D[A/G] mutant) could be rescued by a viral polyprotein provided in trans. Only a defect of 3Dpol activity of the replicon could be rescued in trans; inactivating mutations in 2CATPase/hel, 3B, and 3Cpro of the replicon completely abrogated the trans-rescued replication. An intact N-terminus of the 3Cpro domain of the 3CDpro provided in trans was essential for the trans-active function. By using this trans complementation system, a high-titer defective PV pseudovirus (PVpv) (>107 infectious units per mL) could be produced with the defective mutants, whose replication was completely dependent on trans complementation. This work reveals potential roles of exogenous viral proteins in PV replication and offers insights into protein/protein interaction during picornavirus infection.

Viral polyprotein processing is an elaborately controlled step by viral proteases encoded in the polyprotein; fully processed proteins and processing intermediates need to be correctly produced for replication, which can be detrimentally affected even by a small modification of the polyprotein. Purified/isolated viral proteins can retain their enzymatic activities required for viral replication, such as protease, helicase, polymerase, etc. However, when these proteins of picornavirus are exogenously provided (provided in trans) to the viral replication complex with a defective viral genome, replication is generally not rescued/complemented, suggesting the importance of viral proteins endogenously provided (provided in cis) to the replication complex. In this study, I discovered that only the viral polymerase activity of poliovirus (PV) (the typical member of picornavirus family) could be efficiently rescued by exogenously expressed viral proteins. The current study reveals potential roles for exogenous viral proteins in viral replication and offers insights into interactions during picornavirus infection.

Infections and neurodegenerative diseases induce neuroinflammation, but affected individuals often show nonneural symptoms including muscle pain and muscle fatigue. The molecular pathways by which neuroinflammation causes pathologies outside the central nervous system (CNS) are poorly understood. We developed multiple models to investigate the impact of CNS stressors on motor function and found that Escherichia coli infections and SARS-CoV-2 protein expression caused reactive oxygen species (ROS) to accumulate in the brain. ROS induced expression of the cytokine Unpaired 3 (Upd3) in Drosophila and its ortholog, IL-6, in mice. CNS-derived Upd3/IL-6 activated the JAK-STAT pathway in skeletal muscle, which caused muscle mitochondrial dysfunction and impaired motor function. We observed similar phenotypes after expressing toxic amyloid-β (Aβ42) in the CNS. Infection and chronic disease therefore activate a systemic brain-muscle signaling axis in which CNS-derived cytokines bypass the connectome and directly regulate muscle physiology, highlighting IL-6 as a therapeutic target to treat disease-associated muscle dysfunction.

The COVID-19 pandemic was caused by infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which may lead to serious respiratory, vascular and neurological dysfunctions. The SARS-CoV-2 envelope protein (E protein) is a structural viroporin able to form ion channels in cell membranes, which is critical for viral replication. However, its effects in primary neurons have not been addressed. Here we used fluorescence microscopy and calcium imaging to study SARS-CoV-2 viroporin E localization and the effects on neuron damage and intracellular Ca2+ homeostasis in a model of rat hippocampal neurons aged in vitro. We found that the E protein quickly enters hippocampal neurons and colocalizes with the endoplasmic reticulum (ER) in both short-term (6-8 days in vitro, DIV) and long-term (20-22 DIV) cultures resembling young and aged neurons, respectively. Strikingly, E protein treatment induces apoptosis in aged neurons but not in young neurons. The E protein induces variable increases in cytosolic Ca2+ concentration in hippocampal neurons. Ca2+ responses to the E protein are due to Ca2+ release from intracellular stores at the ER. Moreover, E protein-induced Ca2+ release is very small in young neurons and increases dramatically in aged neurons, consistent with the enhanced Ca2+ store content in aged neurons. We conclude that the SARS-CoV-2 E protein quickly translocates to ER endomembranes of rat hippocampal neurons where it releases Ca2+, probably acting like a viroporin, thus producing Ca2+ store depletion and neuron apoptosis in aged neurons and likely contributing to neurological damage in COVID-19 patients.

Potyviridae, the largest family of plant RNA viruses, includes many important pathogens that significantly reduce the yields of many crops worldwide. In this study, we report that the 6-kilodalton peptide 1 (6K1), one of the least characterized potyviral proteins, is an endoplasmic reticulum-localized protein. AI-assisted structure modeling and biochemical assays suggest that 6K1 forms pentamers with a central hydrophobic tunnel, can increase the cell membrane permeability of Escherichia coli and Nicotiana benthamiana, and can conduct potassium in Saccharomyces cerevisiae. An infectivity assay showed that viral proliferation is inhibited by mutations that affect 6K1 multimerization. Moreover, the 6K1 or its homologous 7K proteins from other viruses of the Potyviridae family also have the ability to increase cell membrane permeability and transmembrane potassium conductance. Taken together, these data reveal that 6K1 and its homologous 7K proteins function as viroporins in viral infected cells.

SARS-CoV-2 uses the double-membrane vesicles as replication organelles. However, how virion assembly occurs has not been fully understood. Here we identified a SARS-CoV-2-driven membrane structure named the 3a dense body (3DB). 3DBs have unusual electron-dense and dynamic inner structures, and their formation is driven by the accessory protein ORF3a via hijacking a specific subset of the trans-Golgi network (TGN) and early endosomal membranes. 3DB formation is conserved in related bat and pangolin coronaviruses yet lost during the evolution to SARS-CoV. 3DBs recruit the viral structural proteins spike (S) and membrane (M) and undergo dynamic fusion/fission to facilitate efficient virion assembly. A recombinant SARS-CoV-2 virus with an ORF3a mutant specifically defective in 3DB formation showed dramatically reduced infectivity for both extracellular and cell-associated virions. Our study uncovers the crucial role of 3DB in optimal SARS-CoV-2 infectivity and highlights its potential as a target for COVID-19 prophylactics and therapeutics.

Acute gastroenteritis remains the second leading cause of death among children under the age of 5 worldwide. While enteric viruses are the most common etiology, the drivers of their virulence remain incompletely understood. We recently found that cells infected with rotavirus, the most prevalent enteric virus in infants and young children, initiate hundreds of intercellular calcium waves that enhance both fluid secretion and viral spread. Understanding how rotavirus triggers intercellular calcium waves may allow us to design safer, more effective vaccines and therapeutics, but we still lack a mechanistic understanding of this process. In this study, we used existing virulent and attenuated rotavirus strains, as well as reverse engineered recombinants, to investigate the role of rotavirus nonstructural protein 4 (NSP4) in intercellular calcium wave induction using in vitro , organoid, and in vivo model systems. We found that the capacity to induce purinergic intercellular calcium waves (ICWs) segregated with NSP4 in both simian and murine-like rotavirus backgrounds, and NSP4 expression alone was sufficient to induce ICWs. NSP4's ability to function as a viroporin, which conducts calcium out of the endoplasmic reticulum, was necessary for ICW induction. Furthermore, viroporin activity and the resulting ICWs drove transcriptional changes indicative of innate immune activation, which were lost upon attenuation of viroporin function. Multiple aspects of RV disease severity in vivo correlated with the generation of ICWs, identifying a critical link between viroporin function, intercellular calcium waves, and enteric viral virulence.

Herein, we fabricate host-directed virus-mimicking particles (VMPs) to block the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into host cells through competitive inhibition enabled by their interactions with the angiotensin-converting enzyme 2 (ACE2) receptor. A microfluidic platform is developed to fabricate a lipid core of the VMPs with a narrow size distribution and a low level of batch-to-batch variation. The resultant solid lipid nanoparticles are decorated with an average of 231 or 444 Spike S1 RBD protrusions mimicking either the original SARS-CoV-2 or its delta variant, respectively. Compared with that of the nonfunctionalized core, the cell uptake of the functionalized VMPs is enhanced with ACE2-expressing cells due to their strong interactions with the ACE2 receptor. The fabricated VMPs efficiently block the entry of SARS-CoV-2 pseudovirions into host cells and suppress viral infection. Overall, this study provides potential strategies for preventing the spread of SARS-CoV-2 or other coronaviruses employing the ACE2 receptor to enter into host cells.