Journal of Virology最新文献

{"title":"Enterovirus C recombination groups: RNA sequence similarity and the viral polymerase underpin sexual replication mechanisms.","authors":"Evan M Okolovitch, Vishnu Govindarajan, Refugio Robles-Sikisaka, Grace Campagnola, Brian J Kempf, Andrew L Routh, Olve B Peersen, David J Barton","doi":"10.1128/jvi.00434-25","DOIUrl":"10.1128/jvi.00434-25","url":null,"abstract":"<p><p>Enteroviruses frequently recombine with one another in nature; however, it is unclear how viral replication machinery can distinguish between related and unrelated partners during recombination. We hypothesize that viral RNA recombination involves two parental RNA templates, nascent RNA products, and their dynamic interactions with the viral polymerase-a sexual replication strategy. When nascent RNA products move from one parental RNA template to another, RNA sequence similarity may be an important factor underpinning the mechanism and efficiency of recombination. To test this hypothesis, we focused on recombination between two related group C enteroviruses, poliovirus and Coxsackievirus A21 (CVA21), using bioinformatic, biological, and biochemical approaches. Bioinformatic analyses comparing 22 prototypical group C enteroviruses delineated four recombination groups where viruses in each group exhibit high RNA sequence and amino acid similarity in their polymerase genes. ClickSeq and ViReMa methods detect recombinant forms of poliovirus with P3 genes from CVA21, analogous to recombinant circulating vaccine-derived polioviruses (cVDPV). Biochemical assays show that poliovirus and CVA21 polymerases can detect mismatched base pairs as they traverse an extended primer grip surface adjacent to the active site. Mismatched base pairs in the -2 and -3 positions destabilize polymerase elongation complexes, consistent with the predicted role of RNA sequence similarity in recombination. Two subgroup-specific genetic elements, upstream open-reading frames (uORFs) and RNase L competitive inhibitor RNAs (RNase L ciRNAs), reinforce the existence and biological relevance of enterovirus C recombination groups. Altogether, our observations suggest that enterovirus RNA replication machinery can distinguish between related and unrelated partners during recombination.</p><p><strong>Importance: </strong>Viral RNA recombination transforms live-attenuated polioviruses into neurovirulent circulating vaccine-derived polioviruses, complicating the planned eradication of poliovirus. When humans are co-infected with poliovirus and related non-polio enteroviruses, viral replication machinery can produce recombinant viruses. However, who recombines with whom? What factors determine whether two distinct viruses can produce recombinant progeny that are fit for transmission from person to person? In this study, we clarify which viruses recombine with one another in nature and further elucidate the mechanisms by which the viral polymerase distinguishes between related and unrelated RNA templates-a sexual form of replication. Understanding these mechanisms could lead to better strategies for virus control and/or eradication.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0043425"},"PeriodicalIF":4.0,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12282145/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144475773","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The SHFV nsp2 and nucleocapsid proteins recruit G3BP1 to sites of viral replication, but stress granules are not induced by the infection.","authors":"Ayisha A Lavender, Oreoluwa Solanke, Hsin-Yao Tang, Hannah E Peck, Daryll Vanover, Philip J Santangelo, Margo A Brinton","doi":"10.1128/jvi.00794-25","DOIUrl":"10.1128/jvi.00794-25","url":null,"abstract":"<p><p>Stress granules (SGs) are dynamic, cytoplasmic foci that form in response to environmental stresses, including viral infections, and function to restore cellular homeostasis by regulating mRNA translation, storage, and decay. To inhibit SG formation and subvert their antiviral effects, viruses from diverse families sequester or cleave G3BP1, the key SG nucleating protein. We found that an infection with simian hemorrhagic fever virus (SHFV), a member of the family <i>Arteriviridae</i>, does not induce the formation of <i>bona fide</i> SGs despite inducing phosphorylation of PKR and eIF2α. The SG proteins, G3BP1, G3BP2, TIA-1, Caprin-1, and USP10, but not the translation initiation proteins, eIF3A, eIF4G, and small ribosomal protein S6 (rpS6), were redistributed into foci located in the same intracellular region as the viral dsRNA foci. However, SGs could be induced in infected cells by exogenous inducers. LC-MS/MS analysis of the proteins co-immunoprecipitating with endogenous G3BP1 from SHFV-infected cell lysates detected multiple viral replication/transcription complex proteins. Interaction between G3BP1 and the nsp2 and N proteins of SHFV was observed in reciprocal co-immunoprecipitation assays, and colocalization was detected by IFA. A conserved FGAP motif in nsp2 and a FAEP motif in the N protein were shown to be required for interaction with G3BP1. We also detected G3BP cleavage products in the SHFV-infected cell lysates and hypothesize that cleavage is mediated by a viral protease. These findings suggest that SG formation is not induced by an SHFV infection due to recruitment of G3BP to sites of viral replication and cleavage of G3BP by viral proteins.IMPORTANCEEukaryotic cells shut down translation by assembling stress granules (SGs) in response to environmental stresses, including viral infections. Viruses require cellular translation machinery for protein synthesis and have developed mechanisms to subvert SG assembly. Simian hemorrhagic fever virus (SHFV), a simian arterivirus, causes asymptomatic infections in African cercopithecoid monkeys but fatal hemorrhagic fever disease in Asian macaques. Even though intracellular production of SHFV RNA activates the stress sensor, PKR, SGs are not induced. G3BP1, the main nucleating protein of SGs, is recruited to foci located near viral replication complexes through interaction with the viral proteins nsp2 and N. An FGAP motif in nsp2 and an FAEP motif in the N protein are required for interaction with G3BP1. Cleavage of G3BP1 was identified as an additional mechanism of viral counteraction of SG formation.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0079425"},"PeriodicalIF":4.0,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12282152/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144475776","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"TRIM56 enhances adenoviral E1A steady state to improve oncolytic adenovirus therapy efficacy.","authors":"Nan Sun, Jikai Zhang, Chen Zhang, Hang Yin, Botao Zou, Yunfeng Geng, Wanjun Gao, Yuxin Zhang, Gang Wang, Junnian Zheng, Lin Fang","doi":"10.1128/jvi.00041-25","DOIUrl":"10.1128/jvi.00041-25","url":null,"abstract":"<p><p>Oncolytic adenoviruses (OAVs) engineered to carry antitumor or immune-activating transgenes are being actively explored for cancer immunotherapy. While many efforts have focused on modifying OAVs to counteract the immunosuppressive tumor microenvironment, comparatively less attention has been given to enhancing OAV replication by targeting host factors to increase oncolytic activity. Here, we report that TRIM56 is strongly upregulated during HAdV-C5 infection, with its expression correlating with increasing levels of the viral E1A protein, ultimately promoting HAdV-C5 replication. Mechanistically, TRIM56 stabilizes the viral E1A protein and enhances viral genome transcription. To leverage this effect, we engineered a recombinant oncolytic adenovirus expressing TRIM56 (OAV-TRIM56) and found that it achieved significantly higher replication titers <i>in vitro</i> compared to conventional OAVs, leading to superior antitumor efficacy <i>in vivo</i>. Our study presents a novel strategy to enhance OAV replication by targeting host factors, offering a promising approach for improving oncolytic virotherapy.IMPORTANCEAdenoviruses (Ads) can be engineered into replication-defective adenoviral (Adv) vectors and replication-competent oncolytic adenovirus (OAd), both of which are widely used in gene therapy and virotherapy. Understanding the mechanisms regulating adenoviral infection is crucial for optimizing the therapeutic potential of Adv and OAd. In this study, we demonstrate for the first time that TRIM56, a host protein broadly expressed in various cell types, stabilizes the adenoviral E1A protein and assists E1A in antagonizing STING, thereby significantly enhancing adenoviral replication. Our findings provide new insights into strategies for improving the efficacy of Adv and OAd in gene therapy.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0004125"},"PeriodicalIF":4.0,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12282078/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144208886","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"METTL3 regulates PRRSV replication by suppressing interferon beta through autophagy-mediated IKKε degradation.","authors":"Yunyun Zhai, Lucai Wang, Lijie Lv, Xuyang Zhao, Mengjie Li, Jiajing Tian, Xiangqi Qiu, Lulu Yao, Wenhui Zhu, Yunzhe Kang, Angke Zhang, Guoqing Zhuang, Aijun Sun","doi":"10.1128/jvi.00098-25","DOIUrl":"10.1128/jvi.00098-25","url":null,"abstract":"<p><p>Methyltransferase-like-3 (METTL3)-mediated N6-methyladenosine (m⁶A) modification of messenger RNAs plays a pivotal role in regulating innate immune responses, either promoting or combating virus replication. However, the biological function of METTL3 during porcine reproductive and respiratory syndrome virus (PRRSV) infection remains unclear. In this study, we found that PRRSV infection reprograms m⁶A modifications in cellular transcripts, enhances METTL3 expression, and alters its subcellular distribution. Intriguingly, METTL3 overexpression facilitates PRRSV replication, whereas its deficiency suppresses it, primarily through the negative regulation of type I interferon (IFN-I) production. Further investigation revealed that METTL3 interacts with and promotes the degradation of IκB kinase-ε (IKKε) during PRRSV infection. Mechanistically, METTL3-mediated m⁶A modification of SQSTM1 (sequestosome 1) enhances <i>SQSTM1</i> messenger RNA (mRNA) expression, increasing autophagy levels. Moreover, METTL3 facilitates the formation of K63-linked ubiquitin chains on IKKε, targeting it for degradation via SQSTM1-dependent selective autophagy. Collectively, our findings unveil a novel mechanism whereby METTL3 facilitates PRRSV replication by suppressing antiviral innate immunity, thereby offering potential targets for antiviral therapy.IMPORTANCEPorcine reproductive and respiratory syndrome (PRRS), induced by the porcine reproductive and respiratory syndrome virus (PRRSV), poses a highly contagious threat to the global swine industry, leading to substantial economic losses. The genetic variability and immune evasion capabilities of PRRSV complicate the development of effective vaccines and control strategies. Thus, a comprehensive understanding of PRRSV's immune evasion mechanisms is imperative. In this study, we reveal that METTL3 plays a pivotal role in PRRSV's evasion of interferon (IFN) immunity. Specifically, METTL3 targets IKKε, inducing its autophagy degradation and subsequently inhibiting the expression of interferon beta 1 (IFNB1). Furthermore, PRRSV infection alters the N6-methyladenosine (m⁶A) modification of various host genes, with notable changes observed in the m⁶A modification and transcriptional levels of SQSTM1, which are regulated by METTL3. This regulation is crucial for SQSTM1-mediated autophagy degradation of IKKε. Our findings offer novel insights into the mechanisms underlying host protein involvement in PRRSV's immune evasion.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0009825"},"PeriodicalIF":4.0,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12282061/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144475774","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Zika virus non-structural protein NS2A mediated endoplasmic reticulum stress through interacting with Sarco/endoplasmic reticulum Ca²⁺-ATPase 2.","authors":"Shan Wang, Shanshan Tang, Yuxin Zhou, Qifei An, Mengxing Du, Xiujuan Yang, Peng Zou, Li Tang, Yufeng Yu","doi":"10.1128/jvi.00405-25","DOIUrl":"10.1128/jvi.00405-25","url":null,"abstract":"<p><p>Zika virus (ZIKV) infection of neuronal cells leads to endoplasmic reticulum (ER) stress, which is one of the key causes of neuronal damage. However, how ZIKV mediates ER stress has not been fully understood. Here, we observed that ZIKV infection of astrocytes elevated Sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) expression, increased intracellular Ca²⁺ concentration, and upregulated ER stress-related genes. SERCA2 was identified to regulate Ca²⁺ homeostasis and ER stress during ZIKV infection through both knockdown and overexpression of SERCA2 in astrocytes. Furthermore, ZIKV NS2A interacted with SERCA2 and increased the expression of SERCA2, disrupted Ca²⁺ homeostasis, and induced ER stress in astrocytes. After the knockdown of SERCA2 expression, Ca²⁺ homeostasis and ER stress were significantly mitigated in astrocytes expressing NS2A. Additionally, pTMS1-2 and pTMS4-5 of NS2A interacted with SERCA2 and regulated Ca²⁺ homeostasis and ER stress. ZIKV infection of the brains of BALB/c neonatal mice also elevated expression of SERCA2 and ER stress-related genes. Furthermore, SERCA2 expression facilitated ZIKV replication. These results suggested that ZIKV NS2A mediates ER stress through its interaction with SERCA2, providing new insights into the pathogenic mechanism of ZIKV and the development of anti-ZIKV therapies.</p><p><strong>Importance: </strong>Zika virus (ZIKV) infection induces intracellular Ca²⁺ imbalance and endoplasmic reticulum (ER) stress. However, the molecular mechanisms involved in it remain unknown. Here we reported, for the first time, that ZIKV infection increased the expression of Sarco/endoplasmic reticulum Ca²⁺-ATPase 2 (SERCA2), which plays a crucial role in regulating Ca²⁺ homeostasis and ER stress. Furthermore, ZIKV NS2A was found to interact with SERCA2, contributing to the regulation of Ca²⁺ homeostasis and ER stress during ZIKV infection. And ZIKV NS2A pTMS1-pTMS2 and pTMS4-pTMS5 were the specific sites that interacted with SERCA2. These findings elucidate that the interaction between NS2A and SERCA2 is responsible for the regulation of the upstream signaling pathway of ER stress mediated by ZIKV infection. Additionally, the expression of SERCA2 promoted ZIKV proliferation, indicating that SERCA2 may serve as a potential target for anti-ZIKV therapies.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0040525"},"PeriodicalIF":4.0,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12282174/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144475777","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Genetic resilience or resistance in poultry against avian influenza virus: mirage or reality?","authors":"Paula R Chen, Stephen N White, Lianna R Walker, Darrell R Kapczynski, David L Suarez","doi":"10.1128/jvi.00820-25","DOIUrl":"10.1128/jvi.00820-25","url":null,"abstract":"<p><p>The unprecedented global spread of the highly pathogenic avian influenza (HPAI) virus in wild birds, poultry, and mammalian species has challenged our control efforts. Alternative approaches to limit avian influenza viruses (AIV) include the development of resilient or resistant chickens. Genetically resilient birds may become infected but can overcome disease, whereas resistant birds prevent virus attachment or entry and do not become infected. The most intensively studied host gene is myxovirus-resistance (<i>Mx</i>), which is expressed via the interferon pathway. Both sensitive and resistant chicken <i>Mx</i> genotypes have been described, but this only provides limited resilience. Acidic nuclear phosphoprotein 32 family member A (<i>ANP32A</i>) has been demonstrated as a host cofactor for AIV replication via interaction with the polymerase. Small nucleotide changes within this gene have demonstrated some promise for the establishment of disease resilience. Certain MHC-defined genetic chicken lines have demonstrated increased resilience with higher innate immune responses, but HPAI-infected birds still have high morbidity and mortality. Alternatively, gene-edited or -transgenic chickens have had some success in increasing resilience. This strategy allows flexibility to include foreign genes, modification of existing genes, or combined approaches to block critical steps in the viral life cycle. Some candidate genes include solute carrier 35A1 (<i>SLC35A1</i>), retinoic acid-inducible gene I (<i>RIG-I</i>), and toll-like receptors 3 and 7 (<i>TLR3/7</i>), but animal testing needs to be conducted. Furthermore, existing hurdles for technology transfer to commercial application from either naturally occurring resistance genes or foreign genes remain high and will require acceptance by both the poultry industry and consumers.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0082025"},"PeriodicalIF":4.0,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12282110/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144528595","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Highly variable antigenic site located at the apex of GII.4 norovirus capsid protein induces cross-reactive blocking antibodies in a variant-specific manner.","authors":"Michael Landivar, Kentaro Tohma, Kelsey A Pilewski, Lauren A Ford-Siltz, Joseph Kendra, Yamei Gao, Gabriel I Parra","doi":"10.1128/jvi.00652-25","DOIUrl":"10.1128/jvi.00652-25","url":null,"abstract":"<p><p>GII.4 norovirus is responsible for most cases of viral gastroenteritis worldwide. The complex epidemiology and extreme GII.4 antigenic diversity could challenge the development of a vaccine. Most antibody responses to GII.4 norovirus target five antigenic sites mapping to the major viral capsid, whose variability has been associated with the emergence of antigenically distinct variants. Bioinformatics analyses of amino acid sequences from these antigenic sites demonstrated that antigenic site A is the most variable and relevant site for antigenic diversification. Despite this complex diversity, cross-blocking was demonstrated in the profiling of site A-mapping mouse monoclonal antibodies generated against two antigenically distinct GII.4 variants. Using a novel immunoassay, we determined the contribution of specific epitopes to blockade function in polyclonal sera and observed extensive cross-blocking in which 36% was attributed to antibodies mapping to antigenic site A. However, cross-reactivity was virus-dependent, with the greatest breadth exhibited by contemporary variants that emerged after 2006. Notably, cross-blocking titers associated with antibodies mapping to antigenic site A are differentially affected by an increasing number of mutations on the capsid. Since the elicitation of blocking antibodies to conserved regions of the GII.4 norovirus capsid is infrequent, a better understanding of how to elicit cross-protective responses to immunodominant variable antigenic sites could guide norovirus vaccine development.IMPORTANCEGII.4 noroviruses exhibit an accumulation of mutations on their capsid protein, leading to the continuous emergence and turnover of new variants that can escape herd immunity. Despite the fact that most antibodies mapping to the variable antigenic sites of GII.4 norovirus show exquisite specificity, cross-neutralizing antibodies mapping to these variable sites have also been described. In this study, we systematically evaluate the antigenicity of a panel of different GII.4 antigens to demonstrate that cross-reactive responses are elicited in a virus-dependent manner in naïve mice. Notably, one wild-type virus demonstrated multiple instances of potent cross-blocking responses, providing new hopes for the development of cross-protective vaccines against human norovirus.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0065225"},"PeriodicalIF":4.0,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12282187/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144187298","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Discovery of a non-nucleoside inhibitor that binds to a novel site in the palm domain of the respiratory syncytial virus RNA-dependent RNA polymerase.","authors":"Jay H Kalin, Yanting Yin, Minh T Tran, Madison Piassek, Amy Fung, Sandrine Grosse, Edgar Jacoby, Anusarka Bhaumik, Suraj Adhikary, Robyn Miller, Cynthia Lemmens, Ferdinand H Lutter, Serge Pieters, Ludwig Cooymans, Geert Rombouts, Daniel Oehlrich, Sonia Tomaso, Kate Lozada, Miguel Osorio Garcia, Brandon Anson, Suzanne De Bruyn, Constance Smith-Monroy, Jean-Marc Neefs, Nádia Conceição-Neto, Bart Stoops, Herman van Vlijmen, Aaron Patrick, Xiaodi Yu, Victoria Wong, Daniel Krosky, Pravien Abeywickrema, Stephen Mason, Zhinan Jin, Tim H M Jonckers, Sujata Sharma","doi":"10.1128/jvi.00178-25","DOIUrl":"10.1128/jvi.00178-25","url":null,"abstract":"<p><p>Respiratory syncytial virus (RSV) is a major cause of severe respiratory tract infections in infants, young children, and the elderly. We report herein the discovery and characterization of a novel RSV polymerase (RSVpol) non-nucleoside inhibitor (NNI) chemotype that binds to a previously undescribed, highly conserved site in the palm domain of the L protein. Consistent with the observed mode of inhibition, cryogenic electron microscopy (cryo-EM) revealed the site to be adjacent to the nucleotide binding site. Minireplicon assays confirmed on-target activity against RSVpol, and cell-based antiviral assays showed that the lead compound effectively inhibited viral mRNA transcription and replication in clinically relevant A and B strains. Together, our data provides valuable insights into the molecular basis of inhibition for a novel mechanism of action and paves the way for structure-based design to deliver effective therapeutics against RSV.IMPORTANCERespiratory syncytial virus (RSV) is a negative-sense, single-stranded RNA virus belonging to the family <i>Pneumoviridae</i> of the order <i>Mononegavirales</i>. Currently, monoclonal antibody treatments are only approved for infants, and vaccines are reserved for pregnant women and adults aged 60 years and older. Prophylaxis is also limited to the pediatric patient population, and there are currently no direct antiviral therapies for post-exposure treatment. Viral polymerases are considered well-validated drug targets due to their critical role in transcription and genome replication. Herein, we disclose the discovery of a spiro-indolinone series as polymerase inhibitors and describe the preliminary structure-activity relationship (SAR). A cryogenic electron microscopy (cryo-EM) structure obtained with an optimized lead revealed a novel binding site located in the palm domain, which will enable future structure-based drug design efforts. Novel RSV antivirals could be beneficial both as therapeutics following diagnosis and as a prophylactic in patients less likely to respond to vaccines.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0017825"},"PeriodicalIF":4.0,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12282092/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144199543","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"COP I vesicles facilitate classical swine fever virus proliferation by transporting fatty acid synthase from the Golgi apparatus to the endoplasmic reticulum.","authors":"Liang Zhang, Tao Wang, Chen Chen, Mengzhao Song, Ning Li, Bihao Luo, Yuehan Quan, Kangkang Guo, Yanming Zhang","doi":"10.1128/jvi.00305-25","DOIUrl":"10.1128/jvi.00305-25","url":null,"abstract":"<p><p>Classical swine fever virus (CSFV) is an enveloped, positive-sense, single-stranded RNA virus in the <i>Flaviviridae</i> family that remodels the cell's endomembrane for its own propagation. The early secretory pathway is exploited by viruses for their lifecycle, but the mechanism underlying this hijacking of the early secretory pathway in CSFV infection remains unknown. Here, we observed that disrupting the functions of the early secretory pathway organelles, the Golgi apparatus, the endoplasmic reticulum (ER), and coatomer protein I (COP I) vesicles and coatomer protein II (COP II) vesicles resulted in a significant inhibition of CSFV propagation. Further, we revealed that COP I vesicles were required for CSFV RNA replication, but not for the formation of viral replication complexes. The results support the hypothesis that participation of COP I vesicles in viral RNA replication involves their capacity for cargo trafficking. Intact COP I vesicles were isolated and subjected to data-independent acquisition quantitative proteomics analysis to identify the differences in the proteomes of COP I vesicles. This analysis revealed an increase in fatty acid synthase (FASN), a critical factor for CSFV RNA replication, within COP I vesicles, while its presence in COP II vesicles decreased in CSFV-infected cells. Meanwhile, blocking COP I vesicle formation resulted in decreased levels of FASN in the ER, impairing CSFV RNA replication. Collectively, we provide evidence that COP I vesicles mediate FASN trafficking from the Golgi apparatus to the ER to facilitate CSFV RNA replication, which advances our understanding of the role of the early secretory pathway in CSFV proliferation.IMPORTANCEClassical swine fever is a highly contagious disease caused by the classical swine fever virus (CSFV) that infects domestic pigs and wild boars and results in significant economic losses to the swine industry. The early secretory pathway in host cells has often been hijacked by viruses for viral genome replication, assembly, and release of virions. Here, our data revealed that the function of early secretory pathway organelles such as the endoplasmic reticulum (ER) and the Golgi apparatus, and the membrane-bound transport intermediates, COP I vesicles and COP II vesicles, that facilitate transport, were involved in CSFV proliferation in PK-15 cells. Our findings demonstrate that COP I vesicles significantly promote CSFV RNA replication by trafficking fatty acid synthase from the Golgi apparatus to the ER. Our data suggest that manipulation of early secretory pathway function in target host cells could provide a promising strategy for a novel anti-CSFV therapeutic.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0030525"},"PeriodicalIF":4.0,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12282108/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144208884","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rapid reconstruction of infectious bronchitis virus expressing fluorescent protein from its nsp2 gene based on transformation-associated recombination platform.","authors":"Yingfei Li, Linqing Duan, Lihua Tang, Min Huang, Ye Zhao, Guozhong Zhang, Jing Zhao","doi":"10.1128/jvi.00535-25","DOIUrl":"10.1128/jvi.00535-25","url":null,"abstract":"<p><p>Traditional reverse genetics strategies for large-genome RNA viruses are hindered by multiple technical bottlenecks, including dependence on specific restriction enzyme sites, cumbersome multi-step cloning, and genetic instability of oversized DNA in bacterial systems. Herein, we established a universal reverse genetics platform for infectious bronchitis virus (IBV) through systematic optimization of the transformation-associated recombination (TAR) technology. By dividing the IBV genome into seven overlapping fragments and employing <i>Saccharomyces cerevisiae</i> for seamless assembly, we obtained a stable full-length genome clone with an efficiency exceeding 95%. Optimizing cultivation temperature and selecting appropriate <i>Escherichia coli</i> strains were key improvements that minimized mutagenesis during bacterial replication, ensuring fidelity of the constructs. The rescued QX-type IBV strain replicated and induced pathogenicity in chicken embryos comparably to clinical isolates, while retaining engineered markers without additional mutations. The platform's feasibility was further confirmed by successfully rescuing the Mass-type IBV strain, demonstrating its broad applicability. Notably, we pioneeringly rescued a reporter virus expressing the mNeonGreen fluorescent protein, linked via the porcine teschovirus 1 2A proteolytic cleavage site, immediately upstream of the IBV nsp2 gene. This design enabled autonomous separation of the reporter from viral polyproteins without the deletion of any viral gene. The recombinant virus stably maintained this insertion for at least 10 passages, marking the nsp2 site as a flexible locus for foreign gene accommodation in IBV. This study not only establishes a universal TAR-based reverse genetics platform for gamma-coronaviruses but also provides a powerful tool for visualization-based high-throughput antiviral drug screening.</p><p><strong>Importance: </strong>Traditional reverse genetics systems for infectious bronchitis virus (IBV) are often hindered by assembly difficulties <i>in vitro</i> and viral genome instability during bacterial propagation. Here, we developed a transformation-associated recombination-based platform for seamless IBV genome assembly and rapid virus rescue within 12 days. Additionally, we identified a novel foreign gene insertion site between the 5' UTR and nsp2 in the viral genome, enabling stable fluorescent protein expression without deleting any viral genes, ensuring that virus replication is not affected. This system provides a powerful tool for tracking IBV infection, studying viral tropism, and screening antivirals, thereby advancing coronavirus research and poultry disease control.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0053525"},"PeriodicalIF":4.0,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12282139/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144225799","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}