Journal of Virology最新文献

Hepatitis E virus (HEV) is a viral hepatitis pathogen that poses a significant threat to global human health, representing a serious yet long-overlooked public health concern. In this study, we identified glucose-regulated protein 75 (GRP75) as an interaction partner of HEV-ORF2 using recombinant ORF2 truncation as bait. The substrate-binding domain of GRP75 interacted with HEV-ORF2 and inhibited HEV replication by facilitating HEV-ORF2 degradation. Further analysis revealed that HEV-ORF2 contains three KFERQ-like motifs, the key signature sequence required for chaperone-mediated autophagy (CMA). Our data demonstrated that GRP75-mediated degradation of HEV-ORF2 was heat-shock cognate protein 70 (HSC70)-dependent, although no direct interaction between HSC70 and ORF2 was detected. Instead, GRP75, together with HEV-ORF2 and HSC70, formed a complex that mediated CMA-dependent degradation of HEV-ORF2, whereas deletion of all three KFERQ-like motifs from ORF2 conferred resistance to such processes. Additionally, GRP75 blocked mitochondrial transport of HEV-ORF2, potentially mitigating ORF2's function as an interferon (IFN) induction antagonist. Furthermore, GRP75 enhanced the interaction between mitochondrial antiviral signaling protein (MAVS) and TANK-binding kinase 1 (TBK1), promoting IFN-β production and ultimately inhibiting HEV infection. In conclusion, our findings identify GRP75 as a novel restriction factor for HEV infection and provide new insights into its role in CMA and antiviral innate immunity.

Importance: Due to the lack of an effective in vitro model, the viral-host interaction of HEV remains largely elusive. This study uncovers a novel mechanism by which GRP75 inhibits HEV infection. On one hand, the GRP75 protein facilitates the degradation of HEV-ORF2 through the lysosome-associated, chaperone-mediated autophagy by recognizing KFERQ-like motif presented on HEV-ORF2. On the other hand, GRP75 enhances the production of IFN-β by promoting interaction between MAVS and TBK1, thereby establishing an antiviral state and suppressing HEV infection. This research expands our current understanding of host resistance to HEV and provides a new function of GRP75, suggesting that GRP75 might be a novel antiviral factor against virus infection.

Rotavirus (RV) infection remains a leading cause of hospitalization and mortality among infants and young children. Despite global implementation of RV vaccines, hundreds of thousands of infants and young children still succumb to this disease each year due to ineffective treatment. In this study, we demonstrated that NVP-HSP990, a novel small-molecule heat shock protein 90 (HSP90) inhibitor, inhibited RV infection with a fascinatingly higher selectivity index compared to conventional HSP90 inhibitors like geldanamycin and its derivative tanespimycin (17-allylamino-17-demethoxygeldanamycin [17-AAG]). NVP-HSP990 effectively inhibited RV replication in vitro without blocking the initial establishment of infection. NVP-HSP990 restored host gene expression in most KEGG pathways disrupted by RV infection in Caco-2 cells, except some inflammatory pathways (such as IL-17 and TNF pathways). NVP-HSP990 significantly inhibited RV-induced activation of the MAPK pathway and prevented the disruption of tight junctions in Caco-2 cells. More importantly, NVP-HSP990 effectively suppressed RV infection in BALB/c suckling mice and significantly alleviated RV-induced diarrhea.IMPORTANCERotavirus (RV) infection poses a global health threat with an urgent need for targeted antiviral therapies. Here, we identified NVP-HSP990 as a next-generation HSP90 inhibitor with exceptional translational potential against RV infection. Compared to conventional HSP90 inhibitors, NVP-HSP990 demonstrated markedly enhanced anti-RV selectivity. NVP-HSP990 effectively reversed dysregulation of key host pathways in RV infection while selectively modulating pro-inflammatory responses, thereby balancing antiviral and immunopathological outcomes. NVP-HSP990 also blocked MAPK-driven tight junction disruption to preserve intestinal barrier integrity. As a result, NVP-HSP990 significantly alleviated the severity of RV-induced diarrhea. Given its excellent oral efficacy and systemic penetration previously reported, NVP-HSP990 emerges as a promising HSP90-targeted candidate capable of addressing both intestinal and possible extraintestinal RV infections, which also repositions HSP90 inhibition as a viable strategy in RV management.

Canine distemper virus (CDV, species Morbillivirus canis) is a highly contagious pathogen with a broad host range among carnivores. In common with measles virus, alveolar macrophages (AMs) are among the first target cells of infection in the respiratory tract. Therefore, in vitro infections of primary canine AMs were performed with the attenuated Onderstepoort (Ond) and field R252 strain of CDV over a period of 6 days. This showed that AMs are permissive to CDV infection and that such infections are productive with respect to the release of new virus particles. Phenotypic differences were observed over the entire course of the experiment, as higher levels of infection and virus production were observed in CDV R252-infected AMs, while infection with CDV Ond resulted in more prominent cytopathic effects, including syncytium formation. Transcriptome analyses of samples from 1 day post-infection via total RNA sequencing demonstrated further marked differences with respect to the pro-inflammatory response and cell death pathways. CDV Ond-infected AMs exhibited robust induction of pro-inflammatory mediators including type I interferon-related signaling pathways, whereas CDV R252-infected cells showed much weaker expression of these pathways. These transcriptomic differences were further highlighted by the detection of the highest rates of cell apoptosis and lactate dehydrogenase activity in the supernatants of CDV Ond-infected AM cultures over the entire course of the experiment. In addition, transcriptome differences indicate disturbances of homeostatic AM functions associated with CDV infection. These results provide insights into early events in the pathogenesis of CDV infection and mechanisms underlying vaccine strain attenuation.IMPORTANCEMorbilliviruses, including canine distemper virus (CDV) and human measles virus, cause severe systemic disease with respiratory distress, immunosuppression, and neurologic signs. While natural infection in dogs has become rare due to efficient vaccination, outbreaks in wildlife populations can be devastating, and concerns about zoonotic potential of CDV have been raised. The impact of CDV infection on the transcriptome of alveolar macrophages has not been elucidated thus far. Knowledge about early events in CDV pathogenesis and phenotypic consequences of vaccine attenuation is therefore necessary to protect endangered wildlife populations and might furthermore serve as a model for human measles. This study presents the first transcriptomic analyses of primary AMs during the initial phase of morbillivirus infection. These results provide insights into early events in the pathogenesis of CDV infection and mechanisms serving to restrict the spread of an attenuated virus strain.

The HIV-1 envelope glycoprotein (Env) is expressed at the surface of infected cells and, as such, can be targeted by non-neutralizing antibodies (nnAbs) that mediate antibody-dependent cellular cytotoxicity (ADCC). Previous single-molecule Förster resonance energy transfer (smFRET) studies demonstrated that Envs from clinical isolates predominantly adopt a "closed" conformation (State 1), which is resistant to nnAbs. After interacting with the cellular receptor CD4, the conformational equilibrium of Env shifts toward States 2 and 3, exposing the coreceptor-binding site (CoRBS) and permitting targeting by CD4-induced (CD4i) antibodies. We showed that the binding of anti-CoRBS Abs enables the engagement of other nnAbs that target the cluster A epitopes on Env. Anti-cluster A nnAbs stabilize an asymmetric Env conformation, State 2A, and have potent ADCC activity. CRF01_AE strains were suggested to be intrinsically susceptible to ADCC mediated by nnAbs. This may be due to the presence of a histidine at position 375, known to shift Env toward more "open" conformations. In this work, through adaptation of an established smFRET imaging approach, we report that native, unliganded CRF01_AE HIV-1 Envs frequently sample the State 2A conformation. This is in striking contrast with Envs from clades A and B, for example HIV-1_JR-FL, which do not transition to State 2A in the absence of ligands. These findings inform on the conformational dynamics of CRF01_AE Env, which are relevant for structure-based design of both synthetic inhibitors of receptor binding and enhancers of ADCC as therapeutic alternatives.IMPORTANCEA concerning increase in infections with HIV-1 from CRF01_AE has occurred globally and regionally in recent years, especially in Southeast Asia. Despite the advances made in understanding HIV-1 envelope glycoprotein (Env) conformational dynamics, the knowledge about Env from CRF01_AE HIV-1 is limited. Here, we demonstrate that the unliganded CRF01_AE Env readily samples an "open" conformation (State 2A), which is susceptible to antibody-dependent cellular cytotoxicity (ADCC). This is in contrast with the subtypes previously studied from HIV-1 group M that rely on anti-cluster A antibodies to adopt State 2A. These findings are relevant for the structure-based design of novel synthetic inhibitors of CD4 binding and enhancers of ADCC for the elimination of infected cells.

Hepatitis E virus (HEV) causes roughly 20 million yearly global infections and is associated with chronic hepatitis, neurological sequelae, and pregnancy-related adverse outcomes that require antiviral intervention. While there are no approved HEV-specific therapeutics, ribavirin and pegylated interferon, prescribed off-label, remain the current standard of care. However, ribavirin resistance and toxicity highlight the unmet clinical need to identify safer, HEV-specific antivirals. Here, we identify reactive oxygen species (ROS) promotion as a previously unrecognized host-directed antiviral mechanism against HEV, revealed through the activity of the FDA-approved drug auranofin. Auranofin, which is known to elevate intracellular ROS, displays antiviral activity against several viruses. We revealed here that auranofin exhibits robust, dose-dependent antiviral activity against two clinically relevant HEV genotypes and a ribavirin treatment failure-associated mutant. ROS inhibition reversed auranofin-mediated ROS promotion and antiviral activity, establishing a mechanistic link between ROS promotion and antiviral activity. Treatment with D-amino acid oxidase, which breaks down D-amino acids producing the ROS H₂O₂, exerted dose-dependent anti-HEV activity. This effect was reversed by ROS inhibition, demonstrating that ROS accumulation alone is sufficient for antiviral activity. We also revealed that ROS promotion by auranofin drives activation of antioxidant, ER stress, and interferon-stimulated gene expressions, further supporting induction of ROS-dependent antiviral signaling. Lastly, we demonstrated that combined treatment with auranofin and ribavirin exhibits synergistic antiviral activity in vitro. These findings highlight the promotion of ROS as a previously underappreciated host-directed antiviral mechanism and support the repurposing of auranofin-alone or in combination with ribavirin-as a therapeutic strategy against HEV.

Importance: Hepatitis E virus (HEV) lacks approved virus-specific antiviral therapies, and off-label treatments with ribavirin and pegylated interferon are limited by toxicity and emerging resistance mutants. This study identifies reactive oxygen species (ROS) promotion mediated by the FDA-approved drug auranofin and D-amino acid oxidase as an effective antiviral strategy against multiple genotypes of HEV, including two globally relevant human-associated genotypes and a ribavirin treatment failure-associated HEV mutant. The observed synergistic anti-HEV activity in vitro for combined treatment with both auranofin and ribavirin suggests a potential clinically effective combinational therapeutic approach. ROS promotion through auranofin or other means represents an underexplored antiviral strategy with potential for broad-spectrum activity against a range of viral diseases.

Defective viral genomes (DVGs) are byproducts of replication that arise during infection with diverse RNA viruses and can impact virus infection and disease outcome. To gain insight into DVG generation during arenavirus infection, we serially passaged Tacaribe virus at a high multiplicity of infection, which led to the generation of both deletion DVGs (del-DVGs) and copyback DVGs (cb-DVGs). Interestingly, specific combinations of start/stop breakpoints were highly overrepresented, resulting in certain DVGs being highly enriched within the population. Functional characterization of the most prevalent del-DVGs suggests that they are able to compete for interaction with the viral RNA synthesis machinery and that this ability is length-dependent. A closer analysis of the DVG breakpoints used to generate highly abundant DVGs revealed a role for local sequence identity in the formation of cb-DVGs, while del-DVG formation was associated with the presence of specific nucleotide triplets (i.e., TAG, AGA, and GAA). Taken together with similar findings from other virus families, this then supports the idea that DVG formation is not a random process, but rather that specific mechanisms promote their formation at certain positions. The characterization of these individual arenavirus DVG sequences, and also the identification of sequence elements associated with their production, will facilitate future work examining their impact on arenavirus biology, and also opens up the possibility of using such sequences as a part of antiviral approaches and/or of modulating their production as a part of virus attenuation strategies.

Importance: Infection with diverse RNA viruses can generate defective viral genomes (DVGs) that, while unable to support productive virus infection on their own, appear to play a crucial role in determining infection outcome. In light of this apparent biological importance, there is an urgent need to better understand the sequence characteristics of individual DVGs and the molecular mechanisms that regulate their formation to study their biological functions. We have now characterized several DVGs that are highly enriched during infection with the arenavirus Tacaribe virus. Functional analysis of a subset of these DVGs showed length-dependent competition for the viral RNA synthesis machinery, while detailed sequence analysis revealed that DVG formation involves either regions of sequence identity within the genome or the presence of specific nucleotide sequences. Understanding these mechanisms opens up the possibility to leverage DVG generation in support of antiviral and/or vaccine attenuation approaches.

Singapore grouper iridovirus (SGIV), a novel member of the genus Ranavirus, family Iridoviridae, frequently causes a severe disease with high mortality in grouper aquaculture. Although previous findings have demonstrated that SGIV envelope protein VP088 was crucial for its infectivity, the underlying mechanism still remained uncertain. Here, we screened the potential viral proteins that interacted with VP088 during SGIV infection using GFP pull-down assay. Co-immunoprecipitation (Co-IP) assays verified the interactions between VP088 and VP018, VP068, or VP156 in vitro. Moreover, confocal microscopy analysis showed that VP088 markedly altered the cellular distribution of exogenously expressed VP018 and VP068 and ultimately translocated into virus assembly sites together upon SGIV infection. Differently, VP088 mostly co-localized with exogenous VP156 in co-transfected cells and almost simultaneously translocated into the virus assembly sites, suggesting that VP088 participated in SGIV replication through interactions with other viral proteins in different ways. Interestingly, VP088 also abrogated IFN response induced by grouper (Epinephelus coioides) EccGAS-EcSTING and EcTBK1 in vitro. Co-IP assays showed that VP088 interacted with EccGAS, EcSTING, EcTBK1, or EcIRF3, while only degraded EcTBK1 via Ecp62-mediated autophagic degradation. Furthermore, VP088 decreased EcTBK1-induced EcIRF3 phosphorylation and nuclear translocation. In addition, the ectopic expression of VP088 attenuated the antiviral function of EcSTING/EcTBK1/EcIRF3 against red-spotted grouper nervous necrosis virus (RGNNV) infection. Thus, our results not only identified the association between SGIV VP088 and other viral proteins during replication, but also for the first time demonstrated that an iridoviral envelope protein could function as an immune evasion protein via abrogating EcTBK1-induced interferon response.IMPORTANCEIridovirus infection frequently causes high levels of morbidity and mortality among commercially and ecologically important fish, crustaceans, amphibians, and reptiles. However, the molecular mechanism of iridovirus pathogenesis still remains largely unknown, and few effective countermeasures have been developed to date. Using the Singapore grouper iridovirus (SGIV) infection model in vitro, we identified the potential viral proteins that interacted with envelope protein VP088 during virus replication. Moreover, for the first time, we demonstrated that VP088 interacted with EccGAS, EcSTING, EcTBK1, and EcIRF3, but only degraded EcTBK1 via Ecp62-mediated autophagic degradation, thereby inhibiting the host IFN response. Thus, our results not only contribute to elucidating the mechanism of SGIV pathogenesis but also provide a novel molecular target for the construction of immunogenic live vaccines against iridoviral diseases in the future.

Short-chain fatty acids (SCFAs) are gut microbial metabolites produced by gut microbiota from dietary fiber. SCFAs have shown both pro- and anti-viral roles among different viruses and are known to regulate immune functions during infections. However, their role against the Zika virus (ZIKV), in general, and ocular infection, in particular, has never been investigated. In the present study, we aimed to examine the role of three SCFA derivatives: phenylbutyrate (PBA), sodium butyrate (NaB), and sodium acetate (NaAC), on ZIKV replication and associated ocular complications using primary human trabecular meshwork cells (HTMCs) and an IFNAR1-deficient mouse model of ocular infection. Our findings reveal that PBA and NaAc treatment dramatically suppressed the ZIKV replication in HTMCs. NaB showed a slightly less effect than PBA and NaAc. PBA and NaAc treatment significantly attenuated the ZIKV-induced inflammatory cytokine, interferons, and interferon-stimulated genes response via antagonizing the RIG-I/NFκB/MAPKs/STAT1-3 signaling pathways. We discovered that ZIKV induces the expression of free fatty acid receptor 2 (FFAR2)/GPR43 in HTMCs, which is further potentiated by PBA/NaAc. Pharmacological inhibition of FFAR2 abrogated the protective abilities of PBA/NaAc and significantly increased viral replication. Blocking FFAR2 receptors promoted ZIKV-induced cell death, which was suppressed by PBA and NaAc. Butyrate and acetate also inhibited ZIKV binding and cellular entry and inactivated the virus before internalization. PBA and NaAc treatment in mice attenuated the ZIKV-induced ocular manifestations (intraocular pressure, RPE/retinal atrophy, and TM/anterior segment inflammation), which was abrogated by FFAR2 inhibition by 4-CMTB, a selective pharmacological inhibitor of FFAR2. Collectively, our findings indicate that SCFA treatment is an effective approach to limit ZIKV replication and associated ocular damage and may be worth exploring as a means to treat or prevent ZIKV-induced ocular complications/glaucoma in humans.IMPORTANCEZIKV is known to cause severe ocular manifestations in in-utero exposed infants; however, the molecular mechanisms of ZIKV-induced ocular complications remain unknown. SCFAs have demonstrated both pro- and anti-viral roles against different viruses; however, their role against ZIKV is unknown. We showed that SCFAs butyrate and acetate suppress ZIKV transmission and associated ocular complications. The anti-ZIKV activity of these SFACs is mediated via FFAR2, and pharmacological inhibition of FFAR2 promotes ZIKV-induced inflammatory and cell death responses, as well as ocular malformations.

Long noncoding RNAs (lncRNAs) have been found to play significant regulatory roles within antiviral and immune responses. We previously identified the novel lncRNA virus-inducible lncRNA modulator of interferon response (VILMIR), which was found to broadly regulate the host transcriptional response to interferon-beta (IFN-β) treatment in A549 human lung epithelial cells. Here, we investigated the mechanism by which VILMIR regulates the host interferon response in trans by identifying interacting proteins and gene regulatory networks of VILMIR. Through an RNA pull-down assay, we found that VILMIR interacted with both nuclear and cytoplasmic proteins in vitro, including the transcriptional regulators FUBP1 and PUF60 in the nucleus, as well as the antiviral proteins IFIT1 and IFIT3 and the aminoacyl-tRNA synthetases QARS1 and KARS1 in the cytoplasm. In addition, we found that the overexpression of VILMIR in A549 cells resulted in an overall enhancement of host interferon response genes and identified a core set of interferon-stimulated genes that were consistently regulated by VILMIR knockdown and overexpression. Finally, we proposed several possible mechanisms by which VILMIR may interact with the identified proteins to regulate the interferon response, such as by interacting with FUBP1 and PUF60 in the nucleus to regulate host transcription in trans or by interacting with the IFIT proteins and aminoacyl-tRNA synthetases in the cytoplasm to regulate translation.IMPORTANCEDespite thousands of long noncoding RNAs (lncRNAs) being differentially expressed after immune responses and viral infections, there is limited knowledge on their individual functions in these contexts. We previously identified a novel lncRNA, VILMIR, that was found to be an interferon-stimulated gene that regulated the host transcriptional response to interferon-beta treatment in human epithelial cells. Here, we investigated the mechanism by which VILMIR regulates the interferon response. Through in vitro studies, we identified several nuclear and cytoplasmic proteins that interact with VILMIR, including proteins involved in transcriptional and translational regulation. In addition, we demonstrated that the overexpression of VILMIR results in an enhancement of host interferon response genes, supporting our hypothesis that VILMIR plays an activating role in the host interferon response. Finally, we propose several potential models for the mechanism of VILMIR, providing a foundation for the investigation of VILMIR as a novel therapeutic target in antiviral immunity.

Multicellular organisms rely on multilayered immune systems to defend against pathogen invasion. In Drosophila melanogaster, the primary antiviral barrier is RNA interference (RNAi). However, many viruses encode suppressors that disable RNAi, forcing hosts to activate complementary defense strategies. One such strategy involves the circular RNA circZfh1, which encodes the protein CRAV. CRAV is essential for activating the JAK-STAT pathway and providing antiviral protection when RNAi is neutralized. However, the molecular mechanism linking CRAV expression to upd3 induction and JAK-STAT activation remains unclear. Here, we show that CRAV directly interacts with the Ca²⁺-binding domain of the NADPH oxidase Nox, enhancing its enzymatic activity. This interaction promotes the generation of moderate reactive oxygen species (ROS) that act as signaling intermediates rather than stress inducers. CRAV-induced ROS selectively activate the ASK1-p38 mitogen-activated protein kinase cascade, which in turn triggers JAK-STAT signaling and the expressions of antiviral cytokines and effectors. Loss of Nox or inhibition of ASK1-p38 abolishes CRAV-mediated protection, underscoring the necessity of finely tuned redox signaling. These findings reveal a direct mechanistic link between a circRNA-derived protein and conserved innate immune pathways, highlighting the pivotal role of controlled ROS signaling in antiviral defense.

Importance: Antiviral immunity depends on the balance between host defenses and viral countermeasures. In fruit flies, RNA interference (RNAi) represents the primary barrier to viral infection, but viruses often disable this pathway. We show that the circRNA-encoded protein CRAV provides a backup defense by directly binding the NADPH oxidase Nox to generate moderate reactive oxygen species (ROS). Unlike damaging oxidative stress, these ROS serve as signaling cues that activate p38 and JAK-STAT pathways, which in turn drive antiviral cytokine production. This study uncovers how a circRNA-derived protein engages conserved redox-sensitive immune signaling, illustrating an adaptive strategy that ensures protection when RNAi is compromised. The results provide fundamental insights into the evolutionary diversification of circRNA-encoded proteins and broaden our understanding of how finely tuned ROS signaling contributes to innate antiviral immunity.