Tripartite motif 2b (trim2b) restricts spring viremia of carp virus by degrading viral proteins and negative regulators NLRP12-like receptors.

Tripartite motif (TRIM) proteins are involved in different cellular functions, including regulating virus infection. In teleosts, two orthologous genes of mammalian TRIM2 are identified. However, the functions and molecular mechanisms of piscine TRIM2 remain unclear. Here, we show that trim2b-knockout zebrafish are more susceptible to spring viremia of carp virus (SVCV) infection than wild-type zebrafish. Transcriptomic analysis demonstrates that NOD-like receptor (NLR), but not RIG-I-like receptor (RLR), signaling pathway is significantly enriched in the trim2b-knockout zebrafish. In vitro, overexpression of Trim2b fails to degrade RLRs and those key proteins involved in the RLR signaling pathway but does for negative regulators NLRP12-like proteins. Zebrafish Trim2b degrades NLRP12-like proteins through its NHL_TRIM2_like and IG_FLMN domains in a ubiquitin-proteasome degradation pathway. SVCV-N and SVCV-G proteins are also degraded by NHL_TRIM2_like domains, and the degradation pathway is an autophagy lysosomal pathway. Moreover, zebrafish Trim2b can interfere with the binding between NLRP12-like protein and SVCV viral RNA and can completely block the negative regulation of NLRP12-like protein on SVCV infection. Taken together, our data demonstrate that the mechanism of action of zebrafish trim2b against SVCV infection is through targeting the degradation of host-negative regulators NLRP12-like receptors and viral SVCV-N/SVCV-G genes.IMPORTANCESpring viremia of carp virus (SVCV) is a lethal freshwater pathogen that causes high mortality in cyprinid fish. In the present study, we identified zebrafish trim2b, NLRP12-L1, and NLRP12-L2 as potential pattern recognition receptors (PRRs) for sensing and binding viral RNA. Zebrafish trim2b functions as a positive regulator; however, NLRP12-L1 and NLRP12-L2 function as negative regulators during SVCV infection. Furthermore, we find that zebrafish trim2b decreases host lethality in two manners. First, zebrafish Trim2b promotes protein degradations of negative regulators NLRP12-L1 and NLRP12-L2 by enhancing K48-linked ubiquitination and decreasing K63-linked ubiquitination. Second, zebrafish trim2b targets viral RNAs for degradation. Therefore, this study reveals a special antiviral mechanism in lower vertebrates.

Snakehead vesiculovirus hijacks SH3RF1 for replication via mediating K63-linked ubiquitination at K264 of the phosphoprotein.

Snakehead vesiculovirus (SHVV) is a type of rhabdovirus that causes serious economic losses in snakehead fish culture in China. However, no specific antiviral drugs or vaccines are currently available for SHVV infection. In this study, 4D label-free ubiquitome analysis of SHVV-infected cells revealed dozens of ubiquitinated sites on the five SHVV proteins. We focused on investigating the ubiquitination of phosphoprotein (P), a viral polymerase co-factor involved in viral replication. SHVV-P was proved to be ubiquitinated via K63-linked ubiquitination at lysine 264 (K264). Overexpression of wild-type P, but not its K264R mutant, facilitated SHVV replication, indicating that K264 ubiquitination of the P protein is critical for SHVV replication. RNAi screening of 26 cellular E3 ubiquitin ligases identified five pro-viral factors for SHVV replication, including macrophage erythroblast attacher (MAEA), TNF receptor-associated factor 7 (TRAF7), and SH3 domain-containing ring finger protein 1 (SH3RF1), which interacted with and mediated ubiquitination of SHVV P. TRAF7 and SH3RF1, but not MAEA, mediated K63-linked ubiquitination of SHVV P, while only SH3RF1 mediated K264 ubiquitination of SHVV P. Besides, overexpression of SH3RF1 promoted SHVV replication and maintained the stability of SHVV P. In summary, SH3RF1 mediated K63-linked ubiquitination of SHVV P at K264 to facilitate SHVV replication, providing targets for developing anti-SHVV drugs and live-attenuated SHVV vaccines. Our study provides novel insights into the role of P protein in the replication of single-stranded, negative-sense RNA viruses.

Eukaryotic translation elongation factor 1 alpha (eEF1A) inhibits Siniperca chuatsi rhabdovirus (SCRV) infection through two distinct mechanisms.

IMPORTANCE: Although a virus can regulate many cellular responses to facilitate its replication by interacting with host proteins, the host can also restrict virus infection through these interactions. In the present study, we showed that the host eukaryotic translation elongation factor 1 alpha (eEF1A), an essential protein in the translation machinery, interacted with two proteins of a fish rhabdovirus, Siniperca chuatsi rhabdovirus (SCRV), and inhibited virus infection via two different mechanisms: (i) inhibiting the formation of crucial viral protein complexes required for virus transcription and replication and (ii) promoting the ubiquitin-proteasome degradation of viral protein. We also revealed the functional regions of eEF1A that are involved in the two processes. Such a host protein inhibiting a rhabdovirus infection in two ways is rarely reported. These findings provided new information for the interactions between host and fish rhabdovirus.

hnRNPA1 impedes snakehead vesiculovirus replication via competitively disrupting viral phosphoprotein-nucleoprotein interaction and degrading viral phosphoprotein.

Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) plays an important role in regulating the replication of many viruses. However, it remains elusive whether and how hnRNPA1 regulates fish virus replication. In this study, the effects of twelve hnRNPs on the replication of snakehead vesiculovirus (SHVV) were screened. Three hnRNPs, one of which was hnRNPA1, were identified as anti-SHVV factors. Further verification showed that knockdown of hnRNPA1 promoted, while overexpression of hnRNPA1 inhibited, SHVV replication. SHVV infection reduced the expression level of hnRNPA1 and induced the nucleocytoplasmic shuttling of hnRNPA1. Besides, we found that hnRNPA1 interacted with the viral phosphoprotein (P) via its glycine-rich domain, but not with the viral nucleoprotein (N) or large protein (L). The hnRNPA1-P interaction competitively disrupted the viral P-N interaction. Moreover, we found that overexpression of hnRNPA1 enhanced the polyubiquitination of the P protein and degraded it through proteasomal and lysosomal pathways. This study will help understanding the function of hnRNPA1 in the replication of single-stranded negative-sense RNA viruses and providing a novel antiviral target against fish rhabdoviruses.

FIP200 restricts RNA virus infection by facilitating RIG-I activation.

Retinoic acid-inducible gene I (RIG-I) senses viral RNA and instigates an innate immune signaling cascade to induce type I interferon expression. Currently, the regulatory mechanisms controlling RIG-I activation remain to be fully elucidated. Here we show that the FAK family kinase-interacting protein of 200 kDa (FIP200) facilitates RIG-I activation. FIP200 deficiency impaired RIG-I signaling and increased host susceptibility to RNA virus infection. In vivo studies further demonstrated FIP200 knockout mice were more susceptible to RNA virus infection due to the reduced innate immune response. Mechanistic studies revealed that FIP200 competed with the helicase domain of RIG-I for interaction with the two tandem caspase activation and recruitment domains (2CARD), thereby facilitating the release of 2CARD from the suppression status. Furthermore, FIP200 formed a dimer and facilitated 2CARD oligomerization, thereby promoting RIG-I activation. Taken together, our study defines FIP200 as an innate immune signaling molecule that positively regulates RIG-I activation.

Analysis of the dark proteome of Chandipura virus reveals maximum propensity for intrinsic disorder in phosphoprotein.

Chandipura virus (CHPV, a member of the Rhabdoviridae family) is an emerging pathogen that causes rapidly progressing influenza-like illness and acute encephalitis often leading to coma and death of the human host. Given several CHPV outbreaks in Indian sub-continent, recurring sporadic cases, neurological manifestation, and high mortality rate of this infection, CHPV is gaining global attention. The 'dark proteome' includes the whole proteome with special emphasis on intrinsically disordered proteins (IDP) and IDP regions (IDPR), which are proteins or protein regions that lack unique (or ordered) three-dimensional structures within the cellular milieu. These proteins/regions, however, play a number of vital roles in various biological processes, such as cell cycle regulation, control of signaling pathways, etc. and, therefore, are implicated in many human diseases. IDPs and IPPRs are also abundantly found in many viral proteins enabling their multifunctional roles in the viral life cycles and their capability to highjack various host systems. The unknown abundance of IDP and IDPR in CHPV, therefore, prompted us to analyze the dark proteome of this virus. Our analysis revealed a varying degree of disorder in all five CHPV proteins, with the maximum level of intrinsic disorder propensity being found in Phosphoprotein (P). We have also shown the flexibility of P protein using extensive molecular dynamics simulations up to 500 ns (ns). Furthermore, our analysis also showed the abundant presence of the disorder-based binding regions (also known as molecular recognition features, MoRFs) in CHPV proteins. The identification of IDPs/IDPRs in CHPV proteins suggests that their disordered regions may function as potential interacting domains and may also serve as novel targets for disorder-based drug designs.

Peripheral Injection of Tim-3 Antibody Attenuates VSV Encephalitis by Enhancing MHC-I Presentation.

Viral encephalitis is the most common cause of encephalitis. It is responsible for high morbidity rates, permanent neurological sequelae, and even high mortality rates. The host immune response plays a critical role in preventing or clearing invading pathogens, especially when effective antiviral treatment is lacking. However, due to blockade of the blood-brain barrier, it remains unclear how peripheral immune cells contribute to the fight against intracerebral viruses. Here, we report that peripheral injection of an antibody against human Tim-3, an immune checkpoint inhibitor widely expressed on immune cells, markedly attenuated vesicular stomatitis virus (VSV) encephalitis, marked by decreased mortality and improved neuroethology in mice. Peripheral injection of Tim-3 antibody enhanced the recruitment of immune cells to the brain, increased the expression of major histocompatibility complex-I (MHC-I) on macrophages, and as a result, promoted the activation of VSV-specific CD8+ T cells. Depletion of macrophages abolished the peripheral injection-mediated protection against VSV encephalitis. Notably, for the first time, we found a novel post-translational modification of MHC-I by Tim-3, wherein, by enhancing the expression of MARCH9, Tim-3 promoted the proteasome-dependent degradation of MHC-I via K48-linked ubiquitination in macrophages. These results provide insights into the immune response against intracranial infections; thus, manipulating the peripheral immune cells with Tim-3 antibody to fight viruses in the brain may have potential applications for combating viral encephalitis.

Transcriptome profiling in head kidney of rainbow trout (Oncorhynchus mykiss) after infection with the low-virulent Nagano genotype of infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) causes clinical diseases and mortality in a wide variety of salmonid species. Here, we studied transcriptional responses in rainbow trout infected by the IHNV-Nagano strain isolated in Korea. RNA-seq-based transcriptome analysis of head kidney tissues cataloged differentially expressed genes. Enrichment analysis of gene ontology annotations was performed, and a total of fifteen biological process terms were commonly identified at all time points. In the Kyoto Encyclopedia of Genes and Genomes pathway analysis, pathogen recognition receptor (PRR) signaling pathways such as the retinoic-acid-inducible gene-I-like receptor signaling pathway and the Toll-like receptor signaling pathway were identified at all time points. The nucleotide-binding oligomerization-domain-like receptor signaling pathway and cytosolic DNA-sensing pathway were identified at days 1 and 3. Protein-protein interaction network and centrality analyses revealed that the immune system, signaling molecules, and interaction pathways were upregulated at days 1 and 3, with the highest centrality of tumor necrosis factor. Cancer, cellular community, and endocrine system pathways were downregulated, with the highest centrality of fibronectin 1 at day 5. STAT1 was upregulated from days 1 to 5 with a high centrality. The reproducibility and repeatability of the transcriptome analysis were validated by RT-qPCR. IHNV-Nagano infection dynamically changed the transcriptome profiles in the head kidney of rainbow trout and induced a defense mechanism by regulating the immune and inflammatory pathways through PRR signaling at an early stage. Downregulated pathways involved in extracellular matrix formation and focal adhesion at day 5 indicated the possible failure of wound healing, which is important in the pathogenesis of IHNV infection.

A novel role of Zebrafish TMEM33 in negative regulation of interferon production by two distinct mechanisms.

The transmembrane protein 33 (TMEM33) was originally identified as an endoplasmic reticulum (ER) protein that influences the tubular structure of the ER and modulates intracellular calcium homeostasis. However, the role of TMEM33 in antiviral immunity in vertebrates has not been elucidated. In this article, we demonstrate that zebrafish TMEM33 is a negative regulator of virus-triggered interferon (IFN) induction via two mechanisms: mitochondrial antiviral signaling protein (MAVS) ubiquitination and a decrease in the kinase activity of TANK binding kinase 1 (TBK1). Upon stimulation with viral components, tmem33 was remarkably upregulated in the zebrafish liver cell line. The IFNφ1 promoter (IFNφ1pro) activity and mRNA level induced by retinoic acid-inducible gene (RIG)-I-like receptors (RLRs) were significantly inhibited by TMEM33. Knockdown of TMEM33 increased host ifn transcription. Subsequently, we found that TMEM33 was colocalized in the ER and interacted with the RLR cascades, whereas MAVS was degraded by TMEM33 during the K48-linked ubiquitination. On the other hand, TMEM33 reduced the phosphorylation of mediator of IFN regulatory factor 3 (IRF3) activation (MITA)/IRF3 by acting as a decoy substrate of TBK1, which was also phosphorylated. A functional domain assay revealed that the N-terminal transmembrane domain 1 (TM1) and TM2 regions of TMEM33 were necessary for IFN suppression. Finally, TMEM33 significantly attenuated the host cellular antiviral capacity by blocking the IFN response. Taken together, our findings provide insight into the different mechanisms employed by TMEM33 in cellular IFN-mediated antiviral process.

TRK-Fused Gene (TFG), a protein involved in protein secretion pathways, is an essential component of the antiviral innate immune response.

Antiviral innate immune response to RNA virus infection is supported by Pattern-Recognition Receptors (PRR) including RIG-I-Like Receptors (RLR), which lead to type I interferons (IFNs) and IFN-stimulated genes (ISG) production. Upon sensing of viral RNA, the E3 ubiquitin ligase TNF Receptor-Associated Factor-3 (TRAF3) is recruited along with its substrate TANK-Binding Kinase (TBK1), to MAVS-containing subcellular compartments, including mitochondria, peroxisomes, and the mitochondria-associated endoplasmic reticulum membrane (MAM). However, the regulation of such events remains largely unresolved. Here, we identify TRK-Fused Gene (TFG), a protein involved in the transport of newly synthesized proteins to the endomembrane system via the Coat Protein complex II (COPII) transport vesicles, as a new TRAF3-interacting protein allowing the efficient recruitment of TRAF3 to MAVS and TBK1 following Sendai virus (SeV) infection. Using siRNA and shRNA approaches, we show that TFG is required for virus-induced TBK1 activation resulting in C-terminal IRF3 phosphorylation and dimerization. We further show that the ability of the TRAF3-TFG complex to engage mTOR following SeV infection allows TBK1 to phosphorylate mTOR on serine 2159, a post-translational modification shown to promote mTORC1 signaling. We demonstrate that the activation of mTORC1 signaling during SeV infection plays a positive role in the expression of Viperin, IRF7 and IFN-induced proteins with tetratricopeptide repeats (IFITs) proteins, and that depleting TFG resulted in a compromised antiviral state. Our study, therefore, identifies TFG as an essential component of the RLR-dependent type I IFN antiviral response.

A virus-induced conformational switch of STAT1-STAT2 dimers boosts antiviral defenses.

Type I interferons (IFN-I) protect us from viral infections. Signal transducer and activator of transcription 2 (STAT2) is a key component of interferon-stimulated gene factor 3 (ISGF3), which drives gene expression in response to IFN-I. Using electron microscopy, we found that, in naive cells, U-STAT2, lacking the activating tyrosine phosphorylation, forms a heterodimer with U-STAT1 in an inactive, anti-parallel conformation. A novel phosphorylation of STAT2 on T404 promotes IFN-I signaling by disrupting the U-STAT1-U-STAT2 dimer, facilitating the tyrosine phosphorylation of STATs 1 and 2 and enhancing the DNA-binding ability of ISGF3. IKK-ε, activated by virus infection, phosphorylates T404 directly. Mice with a T-A mutation at the corresponding residue (T403) are highly susceptible to virus infections. We conclude that T404 phosphorylation drives a critical conformational switch that, by boosting the response to IFN-I in infected cells, enables a swift and efficient antiviral defense.

Murine GFP-Mx1 forms nuclear condensates and associates with cytoplasmic intermediate filaments: Novel antiviral activity against VSV.

Type I and III interferons induce expression of the "myxovirus resistance proteins" MxA in human cells and its ortholog Mx1 in murine cells. Human MxA forms cytoplasmic structures, whereas murine Mx1 forms nuclear bodies. Whereas both HuMxA and MuMx1 are antiviral toward influenza A virus (FLUAV) (an orthomyxovirus), only HuMxA is considered antiviral toward vesicular stomatitis virus (VSV) (a rhabdovirus). We previously reported that the cytoplasmic human GFP-MxA structures were phase-separated membraneless organelles ("biomolecular condensates"). In the present study, we investigated whether nuclear murine Mx1 structures might also represent phase-separated biomolecular condensates. The transient expression of murine GFP-Mx1 in human Huh7 hepatoma, human Mich-2H6 melanoma, and murine NIH 3T3 cells led to the appearance of Mx1 nuclear bodies. These GFP-MuMx1 nuclear bodies were rapidly disassembled by exposing cells to 1,6-hexanediol (5%, w/v), or to hypotonic buffer (40-50 mosm), consistent with properties of membraneless phase-separated condensates. Fluorescence recovery after photobleaching (FRAP) assays revealed that the GFP-MuMx1 nuclear bodies upon photobleaching showed a slow partial recovery (mobile fraction: ∼18%) suggestive of a gel-like consistency. Surprisingly, expression of GFP-MuMx1 in Huh7 cells also led to the appearance of GFP-MuMx1 in 20-30% of transfected cells in a novel cytoplasmic giantin-based intermediate filament meshwork and in cytoplasmic bodies. Remarkably, Huh7 cells with cytoplasmic murine GFP-MuMx1 filaments, but not those with only nuclear bodies, showed antiviral activity toward VSV. Thus, GFP-MuMx1 nuclear bodies comprised phase-separated condensates. Unexpectedly, GFP-MuMx1 in Huh7 cells also associated with cytoplasmic giantin-based intermediate filaments, and such cells showed antiviral activity toward VSV.

Lyssavirus P-protein selectively targets STAT3-STAT1 heterodimers to modulate cytokine signalling.

Many viruses target signal transducer and activator of transcription (STAT) 1 to antagonise antiviral interferon signalling, but targeting of STAT3, a pleiotropic molecule that mediates signalling by diverse cytokines, is poorly understood. Here, using lyssavirus infection, quantitative live cell imaging, innate immune signalling and protein interaction assays, and complementation/depletion of STAT expression, we show that STAT3 antagonism is conserved among P-proteins of diverse pathogenic lyssaviruses and correlates with pathogenesis. Importantly, P-protein targeting of STAT3 involves a highly selective mechanism whereby P-protein antagonises cytokine-activated STAT3-STAT1 heterodimers, but not STAT3 homodimers. RT-qPCR and reporter gene assays indicate that this results in specific modulation of interleukin-6-dependent pathways, effecting differential antagonism of target genes. These data provide novel insights into mechanisms by which viruses can modulate cellular function to support infection through discriminatory targeting of immune signalling complexes. The findings also highlight the potential application of selective interferon-antagonists as tools to delineate signalling by particular STAT complexes, significant not only to pathogen-host interactions but also cell physiology, development and cancer.

Effect of the Viral Hemorrhagic Septicemia Virus Nonvirion Protein on Translation via PERK-eIF2α Pathway.

Viral hemorrhagic septicemia virus (VHSV) is one of the most deadly infectious fish pathogens, posing a serious threat to the aquaculture industry and freshwater ecosystems worldwide. Previous work showed that VHSV sub-genotype IVb suppresses host innate immune responses, but the exact mechanism by which VHSV IVb inhibits antiviral response remains incompletely characterized. As with other novirhabdoviruses, VHSV IVb contains a unique and highly variable nonvirion (NV) gene, which is implicated in viral replication, virus-induced apoptosis and regulating interferon (IFN) production. However, the molecular mechanisms underlying the role of IVb NV gene in regulating viral or cellular processes is poorly understood. Compared to the wild-type recombinant (rWT) VHSV, mutant VHSV lacking a functional IVb NV reduced IFN expression and compromised innate immune response of the host cells by inhibiting translation. VHSV IVb infection increased phosphorylated eukaryotic initiation factor 2α (p-eIF2α), resulting in host translation shutoff. However, VHSV IVb protein synthesis proceeds despite increasing phosphorylation of eIF2α. During VHSV IVb infection, eIF2α phosphorylation was mediated via PKR-like endoplasmic reticulum kinase (PERK) and was required for efficient viral protein synthesis, but shutoff of host translation and IFN signaling was independent of p-eIF2α. Similarly, IVb NV null VHSV infection induced less p-eIF2α, but exhibited decreased viral protein synthesis despite increased levels of viral mRNA. These findings show a role for IVb NV in VHSV pathogenesis by utilizing the PERK-eIF2α pathway for viral-mediated host shutoff and interferon signaling to regulate host cell response.

Snakehead vesiculovirus (SHVV) infection alters striped snakehead (Ophicephalus striatus) cells (SSN-1) glutamine metabolism and apoptosis pathways.

Snakehead vesiculovirus (SHVV) causes enormous economic losses in snakehead fish (Ophicephalus striatus) culture. Understanding replication mechanisms of virus is considerable significance in preventing and treating viral disease. In our previous studies, we have reported that glutamine starvation could significant inhibit the replication of SHVV. Furthermore, we also showed that SHVV infection could cause apoptosis of striped snakehead fish cells (SSN-1). However, the underlying mechanisms remain enigmatic. To decipher the relationships among the viral infection, glutamine starvation and apoptosis, SSN-1 cells transcriptomic profilings of SSN-1 cells infected with or without SHVV under glutamine deprived condition were analyzed. RNA-seq was used to identify differentially expressed genes (DEGs). Our data revealed that 1215 up-regulated and 226 down-regulated genes at 24 h post-infection were involved in MAPK, apoptosis, RIG-1-like and toll-like receptors pathways and glutamine metabolism. Subsequently, DEGs of glutamine metabolism and apoptosis pathways were selected to validate the sequencing data by quantitative real-time PCR (qRT-PCR). The expression patterns of both transcriptomic data and qRT-PCR were consistent. We observed that lack of glutamine alone could cause mild cellular apoptosis. However, lack of glutamine together with SHVV infection could synergistically enhance cellular apoptosis. When the cells were cultured in complete medium with glutamine, overexpression of glutaminase (GLS), an essential enzyme for glutamine metabolism, could significantly enhance the SHVV replication. While, SHVV replication was decreased in cells when GLS was knocked down by specific siRNA, indicating that glutamine metabolism was essential for viral replication. Furthermore, the expression level of caspase-3 and Bax was significantly decreased in SHVV infected cells with GLS overexpression. By contrast, they were significantly increased in SHVV infected cells with GLS silence by SiRNA, indicating that SHVV infection activated the Bax and caspase-3 pathways to induce apoptosis independent of glutamine. Our results reveal that SHVV replication and starvation of glutamine could synergistically promote the cellular apoptosis, which will pave a new way for developing strategies against the vial infection.

Zebrafish C-reactive protein isoforms inhibit SVCV replication by blocking autophagy through interactions with cell membrane cholesterol.

In the present work, the mechanisms involved in the recently reported antiviral activity of zebrafish C-reactive protein-like protein (CRP1-7) against the spring viraemia of carp rhabdovirus (SVCV) in fish are explored. The results neither indicate blocking of the attachment or the binding step of the viral replication cycle nor suggest the direct inhibition of G protein fusion activity or the stimulation of the host's interferon system. However, an antiviral state in the host is induced. Further results showed that the antiviral protection conferred by CRP1-7 was mainly due to the inhibition of autophagic processes. Thus, given the high affinity of CRPs for cholesterol and the recently described influence of the cholesterol balance in lipid rafts on autophagy, both methyl-ß-cyclodextrin (a cholesterol-complexing agent) and 25-hydroxycholesterol (a cholesterol molecule with antiviral properties) were used to further describe CRP activity. All the tested compounds exerted antiviral activity by affecting autophagy in a similar manner. Further assays indicate that CRP reduces autophagy activity by initially disturbing the cholesterol ratios in the host cellular membranes, which in turn negatively affects the intracellular regulation of reactive oxygen species (ROS) and increases lysosomal pH as a consequence. Ultimately, here we propose that such pH changes exert an inhibitory direct effect on SVCV replication by disrupting the pH-dependent membrane-fusogenic ability of the viral glycoprotein G, which allows the release of the virus from endosomes into cytoplasm during its entry phase.

Zebrafish RPZ5 Degrades Phosphorylated IRF7 To Repress Interferon Production.

Interferon (IFN) production activated by phosphorylated interferon regulatory factor 7 (IRF7) is a pivotal process during host antiviral infection. For viruses, suppressing the host IFN response is beneficial for viral proliferation; in such cases, evoking host-derived IFN negative regulators would be very useful for viruses. Here, we report that the zebrafish rapunzel 5 (RPZ5) protein which activated by virus degraded phosphorylated IRF7 is activated by TANK-binding kinase 1 (TBK1), leading to a reduction in IFN production. Upon viral infection, zebrafish rpz5 was significantly upregulated, as was ifn, in response to the stimulation. Overexpression of RPZ5 blunted the IFN expression induced by both viral and retinoic acid-inducible gene I (RIG-I) like-receptor (RLR) factors. Subsequently, RPZ5 interacted with RLRs but did not affect the stabilization of the proteins in the normal state. Interestingly, RPZ5 degraded the phosphorylated IRF7 under TBK1 activation through K48-linked ubiquitination. Finally, the overexpression of RPZ5 remarkably reduced the host cell antiviral capacity. These findings suggest that zebrafish RPZ5 is a negative regulator of phosphorylated IRF7 and attenuates IFN expression during viral infection, providing insight into the IFN balance mechanism in fish.IMPORTANCE The phosphorylation of IRF7 is helpful for host IFN production to defend against viral infection; thus, it is a potential target for viruses to mitigate the antiviral response. We report that the fish RPZ5 is an IFN negative regulator induced by fish viruses and degrades the phosphorylated IRF7 activated by TBK1, leading to IFN suppression and promotion of viral proliferation. These findings reveal a novel mechanism for interactions between the host cell and viruses in the lower vertebrate.

Chandipura Virus Utilizes the Prosurvival Function of RelA NF-κB for Its Propagation.

Chandipura virus (CHPV), a cytoplasmic RNA virus, has been implicated in several outbreaks of acute encephalitis in India. Despite the relevance of CHPV to human health, how the virus interacts with the host signaling machinery remains obscure. In response to viral infections, mammalian cells activate RelA/NF-κB heterodimers, which induce genes encoding interferon beta (IFN-ß) and other immune mediators. Therefore, RelA is generally considered to be an antiviral transcription factor. However, RelA activates a wide spectrum of genes in physiological settings, and there is a paucity of direct genetic evidence substantiating antiviral RelA functions. Using mouse embryonic fibroblasts, we genetically dissected the role of RelA in CHPV pathogenesis. We found that CHPV indeed activated RelA and that RelA deficiency abrogated the expression of IFN-ß in response to virus infections. Unexpectedly, infection of Rela-/- fibroblasts led to a decreased CHPV yield. Our investigation clarified that RelA-dependent synthesis of prosurvival factors restrained infection-inflicted cell death and that exacerbated cell death processes prevented multiplication of CHPV in RelA-deficient cells. Chikungunya virus, a cytopathic RNA virus associated also with epidemics, required RelA, and Japanese encephalitis virus, which produced relatively minor cytopathic effects in fibroblasts, circumvented the need of RelA for their propagation. In sum, we documented a proviral function of the pleiotropic factor RelA linked to its prosurvival properties. RelA promoted the growth of cytopathic RNA viruses by extending the life span of infected cells, which serve as the replicative niche of intracellular pathogens. We argue that our finding bears significance for understanding host-virus interactions and may have implications for antiviral therapeutic regimes.IMPORTANCE RelA/NF-κB participates in a wide spectrum of physiological processes, including shaping immune responses against invading pathogens. In virus-infected cells, RelA typically induces the expression of IFN-ß, which restrains viral propagation in neighboring cells involving paracrine mechanisms. Our study suggested that RelA might also play a proviral role. A cell-autonomous RelA activity amplified the yield of Chandipura virus, a cytopathic RNA virus associated with human epidemics, by extending the life span of infected cells. Our finding necessitates a substantial revision of our understanding of host-virus interactions and indicates a dual role of NF-κB signaling during the course of RNA virus infections. Our study also bears significance for therapeutic regimes which alter NF-κB activities while alleviating the viral load.

The stress granule protein G3BP1 binds viral dsRNA and RIG-I to enhance interferon-ß response.

RIG-I senses viral RNA in the cytosol and initiates host innate immune response by triggering the production of type 1 interferon. A recent RNAi knockdown screen yielded close to hundred host genes whose products affected viral RNA-induced IFN-ß production and highlighted the complexity of the antiviral response. The stress granule protein G3BP1, known to arrest mRNA translation, was identified as a regulator of RIG-I-induced IFN-ß production. How G3BP1 functions in RIG-I signaling is not known, however. Here, we overexpress G3BP1 with RIG-I in HEK293T cells and found that G3BP1 significantly enhances RIG-I-induced ifn-b mRNA synthesis. More importantly, we demonstrate that G3BP1 binds RIG-I and that this interaction involves the C-terminal RGG domain of G3BP1. Confocal microscopy studies also show G3BP1 co-localization with RIG-I and with infecting vesicular stomatitis virus in Cos-7 cells. Interestingly, immunoprecipitation studies using biotin-labeled viral dsRNA or poly(I·C) and cell lysate-derived or in vitro translated G3BP1 indicated that G3BP1 could directly bind these substrates and again via its RGG domain. Computational modeling further revealed a juxtaposed interaction between G3BP1 RGG and RIG-I RNA-binding domains. Together, our data reveal G3BP1 as a critical component of RIG-I signaling and possibly acting as a co-sensor to promote RIG-I recognition of pathogenic RNA.

Glutamine starvation inhibits snakehead vesiculovirus replication via inducing autophagy associated with the disturbance of endogenous glutathione pool.

Autophagy is a degradation cellular process which also plays an important role in virus infection. Glutamine is an essential substrate for the synthesis of glutathione which is the most abundant thiol-containing compound within the cells and plays a key role in the antioxidant defense and intracellular signaling. There is an endogenous cellular glutathione pool which consists of two forms of glutathione, i.e. the reduced form (GSH) and the oxidized form (GSSG). GSH serves as an intracellular antioxidant to maintain cellular redox homeostasis by scavenging free radicals and other reactive oxygen species (ROS) which can lead to autophagy. Under physiological conditions, the concentration of GSSG is only about 1% of total glutathione, while stress condition can result in a transient increase of GSSG. In our previous report, we showed that the replication of snakehead fish vesiculovirus (SHVV) was significant inhibited in SSN-1 cells cultured in the glutamine-starvation medium, however the underlying mechanism remains enigmatic. Here, we revealed that the addition of L-Buthionine-sulfoximine (BSO), a specific inhibitor of the GSH synthesis, could decrease the γ-glutamate-cysteine ligase (GCL) activity and GSH levels, resulting in autophagy and significantly inhibition of the replication of SHVV in SSN-1 cells cultured in the complete medium. On the other hand, the replication of SHVV was rescued and the autophagy was inhibited in the SSN-1 cells cultured in the glutamine-starvation medium supplemented with additional GSH. Furthermore, the inhibition of the synthesis of GSH had not significantly affected the generation of reactive oxygen species (ROS). However, it significantly decreased level of GSH and enhanced the level of GSSG, resulting in the decrease of the value of GSH/GSSG, indicating that it promoted the cellular oxidative stress. Overall, the present study demonstrated that glutamine starvation impaired the replication of SHVV in SSN-1 cells via inducing autophagy associated with the disturbance of the endogenous glutathione pool.