The astrovirus N-terminal nonstructural protein anchors replication complexes to the perinuclear ER membranes.

An essential aspect of positive-sense RNA virus replication is anchoring the replication complex (RC) to cellular membranes. Positive-sense RNA viruses employ diverse strategies, including co-translational membrane targeting through signal peptides and co-opting cellular membrane trafficking components. Often, N-terminal nonstructural proteins play a crucial role in linking the RC to membranes, facilitating the early association of the replication machinery. Astroviruses utilize a polyprotein strategy to synthesize nonstructural proteins, relying on subsequent processing to form replication-competent complexes. This study provides evidence for the perinuclear ER membrane association of RCs in five distinct human astrovirus strains. Using tagged recombinant classical human astrovirus 1 and neurotropic MLB2 strains, we establish that the N-terminal domain guides the ER membrane association. We identified di-arginine motifs responsible for the perinuclear ER retention and formation of functional RCs through mutational analysis of the N-terminal domain in replicon and reverse genetics systems. In addition, we demonstrate the association of key components of the astrovirus replication complex: double-stranded RNA, RNA-dependent RNA polymerase, protease, and N-terminal protein. Our findings highlight the intricate virus-ER interaction mechanism employed by astroviruses, potentially leading to the development of novel antiviral intervention strategies.

Dual topology of equine arteritis virus GP3 protein and the role of arginine motif RXR in GP3 ER retention.

Glycoprotein 3 (GP3) serves as a structural protein in equine arteritis virus (EAV), forming a heterotrimeric complex that plays a pivotal role in virus tropism. In this study, we tested the membrane topology of GP3, both when expressed separately and during infection with recombinant tagged EAV GP3-HA. In our antibody accessibility experiment, we made a noteworthy discovery: GP3, when expressed separately, exhibits a dual topology. We introduced an additional N-glycosylation site, which was only partially used, providing further evidence for the dual topology of GP3. Intriguingly, this mutated GP3 was secreted into the medium, a result of the disruption of the ER retention motif RXR. The additional glycosylation site was not used when we examined the recombinant EAV virus with the same mutation. Despite the fact of higher expression levels of mutant GP3-HA, the protein was not secreted, and the recombinant mutant virus did not have growth delay compared to the EAV wild-type virus. This finding suggests that GP3 has a single type one membrane topology in virus infected cells, whereas the expression of GP3 in trans results in the dual topology of this protein. The RXR motif in the C-terminus is a co-factor of ER retention of the protein, but the main retention signal remains elusive.

Intracellular "In Silico Microscopes"-Comprehensive 3D Spatio-Temporal Virus Replication Model Simulations.

Despite their small and simple structure compared with their hosts, virus particles can cause severe harm and even mortality in highly evolved species such as humans. A comprehensive quantitative biophysical understanding of intracellular virus replication mechanisms could aid in preparing for future virus pandemics. By elucidating the relationship between the form and function of intracellular structures from the host cell and viral components, it is possible to identify possible targets for direct antiviral agents and potent vaccines. Biophysical investigations into the spatio-temporal dynamics of intracellular virus replication have thus far been limited. This study introduces a framework to enable simulations of these dynamics using partial differential equation (PDE) models, which are evaluated using advanced numerical mathematical methods on leading supercomputers. In particular, this study presents a model of the replication cycle of a specific RNA virus, the hepatitis C virus. The diffusion-reaction model mimics the interplay of the major components of the viral replication cycle, including non structural viral proteins, viral genomic RNA, and a generic host factor. Technically, surface partial differential equations (sufPDEs) are coupled on the 3D embedded 2D endoplasmic reticulum manifold with partial differential equations (PDEs) in the 3D membranous web and cytosol volume. The membranous web serves as a viral replication factory and is formed on the endoplasmic reticulum after infection and in the presence of nonstructural proteins. The coupled sufPDE/PDE model was evaluated using realistic cell geometries based on experimental data. The simulations incorporate the effects of non structural viral proteins, which are restricted to the endoplasmic reticulum surface, with effects appearing in the volume, such as host factor supply from the cytosol and membranous web dynamics. Because the spatial diffusion properties of genomic viral RNA are not yet fully understood, the model allows for viral RNA movement on the endoplasmic reticulum as well as within the cytosol. Visualizing the simulated intracellular viral replication dynamics provides insights similar to those obtained by microscopy, complementing data from in vitro/in vivo viral replication experiments. The output data demonstrate quantitative consistence with the experimental findings, prompting further advanced experimental studies to validate the model and refine our quantitative biophysical understanding.

The glycosylation deficiency of flavivirus NS1 attenuates virus replication through interfering with the formation of viral replication compartments.

BACKGROUND: Flavivirus is a challenge all over the world. The replication of flavivirus takes place within membranous replication compartments (RCs) derived from endoplasmic reticulum (ER). Flavivirus NS1 proteins have been proven essential for the formation of viral RCs by remodeling the ER. The glycosylation of flavivirus NS1 proteins is important for viral replication, yet the underlying mechanism remains unclear. METHODS: HeLa cells were used to visualize the ER remodeling effects induced by NS1 expression. ZIKV replicon luciferase assay was performed with BHK-21 cells. rZIKV was generated from BHK-21 cells and the plaque assay was done with Vero Cells. Liposome co-floating assay was performed with purified NS1 proteins from 293T cells. RESULTS: We found that the glycosylation of flavivirus NS1 contributes to its ER remodeling activity. Glycosylation deficiency of NS1, either through N-glycosylation sites mutations or tunicamycin treatment, compromises its ER remodeling activity and interferes with viral RCs formation. Disruption of NS1 glycosylation results in abnormal aggregation of NS1, rather than reducing its membrane-binding activity. Consequently, deficiency in NS1 glycosylation impairs virus replication. CONCLUSIONS: In summary, our results highlight the significance of NS1 glycosylation in flavivirus replication and elucidate the underlying mechanism. This provides a new strategy for combating flavivirus infections.

Enterovirus 3A protein disrupts endoplasmic reticulum homeostasis through interaction with GBF1.

Enteroviruses are single-stranded, positive-sense RNA viruses causing endoplasmic reticulum (ER) stress to induce or modulate downstream signaling pathways known as the unfolded protein responses (UPR). However, viral and host factors involved in the UPR related to viral pathogenesis remain unclear. In the present study, we aimed to identify the major regulator of enterovirus-induced UPR and elucidate the underlying molecular mechanisms. We showed that host Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1), which supports enteroviruses replication, was a major regulator of the UPR caused by infection with enteroviruses. In addition, we found that severe UPR was induced by the expression of 3A proteins encoded in human pathogenic enteroviruses, such as enterovirus A71, coxsackievirus B3, poliovirus, and enterovirus D68. The N-terminal-conserved residues of 3A protein interact with the GBF1 and induce UPR through inhibition of ADP-ribosylation factor 1 (ARF1) activation via GBF1 sequestration. Remodeling and expansion of ER and accumulation of ER-resident proteins were observed in cells infected with enteroviruses. Finally, 3A induced apoptosis in cells infected with enteroviruses via activation of the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/C/EBP homologous protein (CHOP) pathway of UPR. Pharmaceutical inhibition of PERK suppressed the cell death caused by infection with enteroviruses, suggesting the UPR pathway is a therapeutic target for treating diseases caused by infection with enteroviruses.IMPORTANCEInfection caused by several plus-stranded RNA viruses leads to dysregulated ER homeostasis in the host cells. The mechanisms underlying the disruption and impairment of ER homeostasis and its significance in pathogenesis upon enteroviral infection remain unclear. Our findings suggested that the 3A protein encoded in human pathogenic enteroviruses disrupts ER homeostasis by interacting with GBF1, a major regulator of UPR. Enterovirus-mediated infections drive ER into pathogenic conditions, where ER-resident proteins are accumulated. Furthermore, in such scenarios, the PERK/CHOP signaling pathway induced by an unresolved imbalance of ER homeostasis essentially drives apoptosis. Therefore, elucidating the mechanisms underlying the virus-induced disruption of ER homeostasis might be a potential target to mitigate the pathogenesis of enteroviruses.

Natural-Target-Mimicking Translocation-Based Fluorescent Sensor for Detection of SARS-CoV-2 PLpro Protease Activity and Virus Infection in Living Cells.

Papain-like protease PLpro, a domain within a large polyfunctional protein, nsp3, plays key roles in the life cycle of SARS-CoV-2, being responsible for the first events of cleavage of a polyprotein into individual proteins (nsp1-4) as well as for the suppression of cellular immunity. Here, we developed a new genetically encoded fluorescent sensor, named PLpro-ERNuc, for detection of PLpro activity in living cells using a translocation-based readout. The sensor was designed as follows. A fragment of nsp3 protein was used to direct the sensor on the cytoplasmic surface of the endoplasmic reticulum (ER) membrane, thus closely mimicking the natural target of PLpro. The fluorescent part included two bright fluorescent proteins-red mScarlet I and green mNeonGreen-separated by a linker with the PLpro cleavage site. A nuclear localization signal (NLS) was attached to ensure accumulation of mNeonGreen into the nucleus upon cleavage. We tested PLpro-ERNuc in a model of recombinant PLpro expressed in HeLa cells. The sensor demonstrated the expected cytoplasmic reticular network in the red and green channels in the absence of protease, and efficient translocation of the green signal into nuclei in the PLpro-expressing cells (14-fold increase in the nucleus/cytoplasm ratio). Then, we used PLpro-ERNuc in a model of Huh7.5 cells infected with the SARS-CoV-2 virus, where it showed robust ER-to-nucleus translocation of the green signal in the infected cells 24 h post infection. We believe that PLpro-ERNuc represents a useful tool for screening PLpro inhibitors as well as for monitoring virus spread in a culture.

Mechanism, structural and functional insights into nidovirus-induced double-membrane vesicles.

During infection, positive-stranded RNA causes a rearrangement of the host cell membrane, resulting in specialized membrane structure formation aiding viral genome replication. Double-membrane vesicles (DMVs), typical structures produced by virus-induced membrane rearrangements, are platforms for viral replication. Nidoviruses, one of the most complex positive-strand RNA viruses, have the ability to infect not only mammals and a few birds but also invertebrates. Nidoviruses possess a distinctive replication mechanism, wherein their nonstructural proteins (nsps) play a crucial role in DMV biogenesis. With the participation of host factors related to autophagy and lipid synthesis pathways, several viral nsps hijack the membrane rearrangement process of host endoplasmic reticulum (ER), Golgi apparatus, and other organelles to induce DMV formation. An understanding of the mechanisms of DMV formation and its structure and function in the infectious cycle of nidovirus may be essential for the development of new and effective antiviral strategies in the future.

Japanese encephalitis virus hijacks ER-associated degradation regulators for its replication.

Flaviviruses target their replication on membranous structures derived from the ER, where both viral and host proteins play crucial structural and functional roles. Here, we have characterized the involvement of the ER-associated degradation (ERAD) pathway core E3 ligase complex (SEL1L-HRD1) regulator proteins in the replication of Japanese encephalitis virus (JEV). Through high-resolution immunofluorescence imaging of JEV-infected HeLa cells, we observe that the virus replication complexes marked by NS1 strongly colocalize with the ERAD adapter SEL1L, lectin OS9, ER-membrane shuttle factor HERPUD1, E3 ubiquitin ligase HRD1 and rhomboid superfamily member DERLIN1. NS5 positive structures also show strong overlap with SEL1L. While these effectors show significant transcriptional upregulation, their protein levels remain largely stable in infected cells. siRNA mediated depletion of OS9, SEL1L, HERPUD1 and HRD1 significantly inhibit viral RNA replication and titres, with SEL1L depletion showing the maximum attenuation of replication. By performing protein translation arrest experiments, we show that SEL1L, and OS9 are stabilised upon JEV infection. Overall results from this study suggest that these ERAD effector proteins are crucial host-factors for JEV replication.

Assembly and Evolution of Poxviruses.

Poxvirus assembly has been an intriguing area of research for several decades. While advancements in experimental techniques continue to yield fresh insights, many questions are still unresolved. Large genome sizes of up to 380 kbp, asymmetrical structure, an exterior lipid bilayer, and a cytoplasmic life cycle are some notable characteristics of these viruses. Inside the particle are two lateral bodies and a protein wall-bound-biconcave core containing the viral nucleocapsid. The assembly progresses through five major stages-endoplasmic reticulum (ER) membrane alteration and rupture, crescent formation, immature virion formation, genome encapsidation, virion maturation and in a subset of viruses, additional envelopment of the virion prior to its dissemination. Several large dsDNA viruses have been shown to follow a comparable sequence of events. In this chapter, we recapitulate our understanding of the poxvirus morphogenesis process while reviewing the most recent advances in the field. We also briefly discuss how virion assembly aids in our knowledge of the evolutionary links between poxviruses and other Nucleocytoplasmic Large DNA Viruses (NCLDVs).

Nanoscale cellular organization of viral RNA and proteins in SARS-CoV-2 replication organelles.

The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelles, the sites of replication of viral genomic RNA (vgRNA). To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain numerous vgRNA molecules along with the replication enzymes and clusters of viral double-stranded RNA (dsRNA). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of endoplasmic reticulum (ER) markers and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are encapsulated into DMVs, which have membranes derived from the host ER. These organelles merge into larger vesicle packets as infection advances. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.

P3N-PIPO but not P3 is the avirulence determinant in melon carrying the Wmr resistance against watermelon mosaic virus, although they contain a common genetic determinant.

Viruses employ a series of diverse translational strategies to expand their coding capacity, which produces viral proteins with common domains and entangles virus-host interactions. P3N-PIPO, which is a transcriptional slippage product from the P3 cistron, is a potyviral protein dedicated to intercellular movement. Here, we show that P3N-PIPO from watermelon mosaic virus (WMV) triggers cell death when transiently expressed in Cucumis melo accession PI 414723 carrying the Wmr resistance gene. Surprisingly, expression of the P3N domain, shared by both P3N-PIPO and P3, can alone induce cell death, whereas expression of P3 fails to activate cell death in PI 414723. Confocal microscopy analysis revealed that P3N-PIPO targets plasmodesmata (PD) and P3N associates with PD, while P3 localizes in endoplasmic reticulum in melon cells. We also found that mutations in residues L35, L38, P41, and I43 of the P3N domain individually disrupt the cell death induced by P3N-PIPO, but do not affect the PD localization of P3N-PIPO. Furthermore, WMV mutants with L35A or I43A can systemically infect PI 414723 plants. These key residues guide us to discover some WMV isolates potentially breaking the Wmr resistance. Through searching the NCBI database, we discovered some WMV isolates with variations in these key sites, and one naturally occurring I43V variation enables WMV to systemically infect PI 414723 plants. Taken together, these results demonstrate that P3N-PIPO, but not P3, is the avirulence determinant recognized by Wmr, although the shared N terminal P3N domain can alone trigger cell death.IMPORTANCEThis work reveals a novel viral avirulence (Avr) gene recognized by a resistance (R) gene. This novel viral Avr gene is special because it is a transcriptional slippage product from another virus gene, which means that their encoding proteins share the common N-terminal domain but have distinct C-terminal domains. Amazingly, we found that it is the common N-terminal domain that determines the Avr-R recognition, but only one of the viral proteins can be recognized by the R protein to induce cell death. Next, we found that these two viral proteins target different subcellular compartments. In addition, we discovered some virus isolates with variations in the common N-terminal domain and one naturally occurring variation that enables the virus to overcome the resistance. These results show how viral proteins with common domains interact with a host resistance protein and provide new evidence for the arms race between plants and viruses.

Human coronavirus OC43 infection remodels connexin 43-mediated gap junction intercellular communication in vitro.

ß-coronaviruses cause acute infection in the upper respiratory tract, resulting in various symptoms and clinical manifestations. OC43 is a human ß-coronavirus that induces mild clinical symptoms and can be safely studied in the BSL2 laboratory. Due to its low risk, OC43 can be a valuable and accessible model for understanding ß-coronavirus pathogenesis. One potential target for limiting virus infectivity could be gap junction-mediated communication. This study aims to unveil the status of cell-to-cell communications through gap junctions in human ß-coronavirus infection. Infection with OC43 leads to reduced expression of Cx43 in A549, a lung epithelial carcinoma cell line. Infection with this virus also shows a significant ER and oxidative stress increase. Internal localization of Cx43 is observed post-OC43 infection in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) region, which impairs the gap junction communication between two adjacent cells, confirmed by Lucifer yellow dye transfer assay. It also affects hemichannel formation, as depicted by the EtBr uptake assay. Impairment of Cx43 trafficking and the ability to form hemichannels and functional GJIC are hampered by virus-induced Golgi apparatus disruption. Altogether, these results suggest that several physiological changes accompany OC43 infection in A549 cells and can be considered an appropriate model system for understanding the differences in gap junction communication post-viral infections. This model system can provide valuable insights for developing therapies against human ß-coronavirus infections.IMPORTANCEThe enduring impact of the recent SARS-CoV-2 pandemic underscores the importance of studying human ß-coronaviruses, advancing our preparedness for future coronavirus infections. As SARS-CoV-2 is highly infectious, another human ß-coronavirus OC43 can be considered an experimental model. One of the crucial pathways that can be considered is gap junction communication, as it is vital for cellular homeostasis. Our study seeks to understand the changes in Cx43-mediated cell-to-cell communication during human ß-coronavirus OC43 infection. In vitro studies showed downregulation of the gap junction protein Cx43 and upregulation of the endoplasmic reticulum and oxidative stress markers post-OC43 infection. Furthermore, HCoV-OC43 infection causes reduced Cx43 trafficking, causing impairment of functional hemichannel and GJIC formation by virus-mediated Golgi apparatus disruption. Overall, this study infers that OC43 infection reshapes intercellular communication, suggesting that this pathway may be a promising target for designing highly effective therapeutics against human coronaviruses by regulating Cx43 expression.

The 6-kilodalton peptide 1 in plant viruses of the family Potyviridae is a viroporin.

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.

HCMV US2 co-opts TRC8 to degrade the endoplasmic reticulum-resident protein LMAN2L.

The human cytomegalovirus (HCMV) pUS2 glycoprotein exploits the host's endoplasmic reticulum (ER)-associated degradation (ERAD) pathway to degrade major histocompatibility complex class I (MHC-I) and prevent antigen presentation. Beyond MHC-I, pUS2 has been shown to target a range of cellular proteins for degradation, preventing their cell surface expression. Here we have identified a novel pUS2 target, ER-resident protein lectin mannose binding 2 like (LMAN2L). pUS2 expression was both necessary and sufficient for the downregulation of LMAN2L, which was dependent on the cellular E3 ligase TRC8. Given the hypothesized role of LMAN2L in the trafficking of glycoproteins, we employed proteomic plasma membrane profiling to measure LMAN2L-dependent changes at the cell surface. A known pUS2 target, integrin alpha-6 (ITGA6), was downregulated from the surface of LMAN2L-deficient cells, but not other integrins. Overall, these results suggest a novel strategy of pUS2-mediated protein degradation whereby pUS2 targets LMAN2L to impair trafficking of ITGA6. Given that pUS2 can directly target other integrins, we propose that this single viral protein may exhibit both direct and indirect mechanisms to downregulate key cell surface molecules.

Endoplasmic reticulum-associated SARS-CoV-2 ORF3a elicits heightened cytopathic effects despite robust ER-associated degradation.

IMPORTANCE: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has tragically claimed millions of lives through coronavirus disease 2019 (COVID-19), and there remains a critical gap in our understanding of the precise molecular mechanisms responsible for the associated fatality. One key viral factor of interest is the SARS-CoV-2 ORF3a protein, which has been identified as a potent inducer of host cellular proinflammatory responses capable of triggering the catastrophic cytokine storm, a primary contributor to COVID-19-related deaths. Moreover, ORF3a, much like the spike protein, exhibits a propensity for frequent mutations, with certain variants linked to the severity of COVID-19. Our previous research unveiled two distinct types of ORF3a mutant proteins, categorized by their subcellular localizations, setting the stage for a comparative investigation into the functional and mechanistic disparities between these two types of ORF3a variants. Given the clinical significance and functional implications of the natural ORF3a mutations, the findings of this study promise to provide invaluable insights into the potential roles undertaken by these mutant ORF3a proteins in the pathogenesis of COVID-19.

Palmitoylation of the hemagglutinin of influenza B virus by ER-localized DHHC enzymes 1, 2, 4, and 6 is required for efficient virus replication.

IMPORTANCE: Influenza viruses are a public health concern since they cause seasonal outbreaks and occasionally pandemics. Our study investigates the importance of a protein modification called "palmitoylation" in the replication of influenza B virus. Palmitoylation involves attaching fatty acids to the viral protein hemagglutinin and has previously been studied for influenza A virus. We found that this modification is important for the influenza B virus to replicate, as mutating the sites where palmitate is attached prevented the virus from generating viable particles. Our experiments also showed that this modification occurs in the endoplasmic reticulum. We identified the specific enzymes responsible for this modification, which are different from those involved in palmitoylation of HA of influenza A virus. Overall, our research illuminates the similarities and differences in fatty acid attachment to HA of influenza A and B viruses and identifies the responsible enzymes, which might be promising targets for anti-viral therapy.

SARS-CoV-2 protein ORF8 limits expression levels of Spike antigen and facilitates immune evasion of infected host cells.

Recovery from COVID-19 depends on the ability of the host to effectively neutralize virions and infected cells, a process largely driven by antibody-mediated immunity. However, with the newly emerging variants that evade Spike-targeting antibodies, re-infections and breakthrough infections are increasingly common. A full characterization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mechanisms counteracting antibody-mediated immunity is therefore needed. Here, we report that ORF8 is a virally encoded SARS-CoV-2 factor that controls cellular Spike antigen levels. We show that ORF8 limits the availability of mature Spike by inhibiting host protein synthesis and retaining Spike at the endoplasmic reticulum, reducing cell-surface Spike levels and recognition by anti-SARS-CoV-2 antibodies. In conditions of limited Spike availability, we found ORF8 restricts Spike incorporation during viral assembly, reducing Spike levels in virions. Cell entry of these virions then leaves fewer Spike molecules at the cell surface, limiting antibody recognition of infected cells. Based on these findings, we propose that SARS-CoV-2 variants may adopt an ORF8-dependent strategy that facilitates immune evasion of infected cells for extended viral production.

Reticulons promote formation of ER-derived double-membrane vesicles that facilitate SARS-CoV-2 replication.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the etiologic agent for the global COVID-19 pandemic, triggers the formation of endoplasmic reticulum (ER)-derived replication organelles, including double-membrane vesicles (DMVs), in the host cell to support viral replication. Here, we clarify how SARS-CoV-2 hijacks host factors to construct the DMVs. We show that the ER morphogenic proteins reticulon-3 (RTN3) and RTN4 help drive DMV formation, enabling viral replication, which leads to productive infection. Different SARS-CoV-2 variants, including the delta variant, use the RTN-dependent pathway to promote infection. Mechanistically, our results reveal that the membrane-embedded reticulon homology domain (RHD) of the RTNs is sufficient to functionally support viral replication and physically engage NSP3 and NSP4, two viral non-structural membrane proteins known to induce DMV formation. Our findings thus identify the ER morphogenic RTN3 and RTN4 membrane proteins as host factors that help promote the biogenesis of SARS-CoV-2-induced DMVs, which can act as viral replication platforms.

The role of NSP6 in the biogenesis of the SARS-CoV-2 replication organelle.

SARS-CoV-2, like other coronaviruses, builds a membrane-bound replication organelle to enable RNA replication1. The SARS-CoV-2 replication organelle is composed of double-membrane vesicles (DMVs) that are tethered to the endoplasmic reticulum (ER) by thin membrane connectors2, but the viral proteins and the host factors involved remain unknown. Here we identify the viral non-structural proteins (NSPs) that generate the SARS-CoV-2 replication organelle. NSP3 and NSP4 generate the DMVs, whereas NSP6, through oligomerization and an amphipathic helix, zippers ER membranes and establishes the connectors. The NSP6(ΔSGF) mutant, which arose independently in the Alpha, Beta, Gamma, Eta, Iota and Lambda variants of SARS-CoV-2, behaves as a gain-of-function mutant with a higher ER-zippering activity. We identified three main roles for NSP6: first, to act as a filter in communication between the replication organelle and the ER, by allowing lipid flow but restricting the access of ER luminal proteins to the DMVs; second, to position and organize DMV clusters; and third, to mediate contact with lipid droplets (LDs) through the LD-tethering complex DFCP1-RAB18. NSP6 thus acts as an organizer of DMV clusters and can provide a selective means of refurbishing them with LD-derived lipids. Notably, both properly formed NSP6 connectors and LDs are required for the replication of SARS-CoV-2. Our findings provide insight into the biological activity of NSP6 of SARS-CoV-2 and of other coronaviruses, and have the potential to fuel the search for broad antiviral agents.

Gain-of-function genetic screening identifies the antiviral function of TMEM120A via STING activation.

Zika virus (ZIKV) infection can be associated with neurological pathologies, such as microcephaly in newborns and Guillain-Barre syndrome in adults. Effective therapeutics are currently not available. As such, a comprehensive understanding of virus-host interactions may guide the development of medications for ZIKV. Here we report a human genome-wide overexpression screen to identify host factors that regulate ZIKV infection and find TMEM120A as a ZIKV restriction factor. TMEM120A overexpression significantly inhibits ZIKV replication, while TMEM120A knockdown increases ZIKV infection in cell lines. Moreover, Tmem120a knockout in mice facilitates ZIKV infection in primary mouse embryonic fibroblasts (MEF) cells. Mechanistically, the antiviral activity of TMEM120A is dependent on STING, as TMEM120A interacts with STING, promotes the translocation of STING from the endoplasmic reticulum (ER) to ER-Golgi intermediate compartment (ERGIC) and enhances the phosphorylation of downstream TBK1 and IRF3, resulting in the expression of multiple antiviral cytokines and interferon-stimulated genes. In summary, our gain-of-function screening identifies TMEM120A as a key activator of the antiviral signaling of STING.