Journal of Virology最新文献

{"title":"GP2a I118 and GP4 D43 play critical roles in the attachment of PRRSV to the CD163 receptor: implications for anti-PRRSV infection targets.","authors":"Guoqing Liu, Xinyi Huang, Yongbo Yang, Meng Chen, Xiaoxiao Tian, Hao Song, Haojie Wang, Shujie Wang, Haiwei Wang, Xuehui Cai, Tongqing An","doi":"10.1128/jvi.00963-25","DOIUrl":"https://doi.org/10.1128/jvi.00963-25","url":null,"abstract":"<p><p>Porcine reproductive and respiratory syndrome virus (PRRSV) poses a significant threat to the global swine industry. Numerous modified live vaccines (MLVs) against PRRSV have been developed through the serial passage of wild-type parental strains in Marc-145 cells. However, the infectivity of these MLVs toward their primary target cell <i>in vivo</i>, porcine alveolar macrophage (PAM), is markedly reduced. The underlying mechanism for this decreased tropism remains unclear. In this study, we determined that the 118th residue in GP2a and the 43rd residue in GP4 play critical roles in determining viral tropism for PAM cells. Individual or combined mutations of GP2a-V118I and GP4-N43D significantly enhanced viral attachment to PAM cells. Furthermore, synthesized small peptides containing GP2a-I118 and GP4-D43 effectively blocked viral attachment to PAM cells in a dose-dependent manner, exhibiting broad-spectrum blocking effects against various PRRSV strains, including newly emerged NADC30- and NADC34-like PRRSVs. Coimmunoprecipitation (co-IP) results demonstrated that mutations or deletions at GP2a-V118 and GP4-N43 could significantly affect the binding affinity of GP2a and GP4 for the CD163 receptor, particularly the SRCR5 domain of CD163. Overall, for the first time, we identified two key residues associated with the reduced infectivity of PRRSV MLVs, which play essential roles in the attachment of PRRSV to the CD163 receptor. These findings provide novel insights into PRRSV tropism for PAM cells and reveal new targets for the development of peptide-based drugs or neutralizing antibodies against PRRSV infection.</p><p><strong>Importance: </strong>Currently, most modified live vaccines (MLVs) against animal diseases are derived from serial passages of parental virulent viruses in heterologous animal cells. This process enhances viral adaptation to heterologous cells while significantly reducing viral infectivity to host animal cells, thereby attenuating virulence in hosts. However, the mechanisms underlying the changes in tropism of many MLVs remain largely unknown. In this study, we identified and confirmed two key residues associated with changes in tropism. Importantly, we demonstrated that small peptides can block viral binding to receptors. These findings not only provide potential targets for the development of antiviral drugs or neutralizing antibodies but also offer valuable references for studying tropism changes in other viruses.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0096325"},"PeriodicalIF":3.8,"publicationDate":"2025-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144873810","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Ubiquitin-specific protease 15 interacts directly with the HSV-1 alkaline nuclease and facilitates viral recombination and replication fork stability.","authors":"Yee Vue, Patrick J Mullon, Alexander Leehangin, Emiliano Maldonado-Luevano, Tyler D Hasselmann, Kavi P M Mehta, Kareem N Mohni","doi":"10.1128/jvi.00893-25","DOIUrl":"https://doi.org/10.1128/jvi.00893-25","url":null,"abstract":"<p><p>Herpes simplex virus type 1 replicates in the nucleus of the host cell and utilizes many cellular proteins to facilitate DNA replication. HSV-1 DNA replication is closely linked with homologous recombination, and HSV-1 encodes a two-subunit recombinase consisting of an exonuclease (UL12) and a single-strand DNA-binding protein (ICP8). We identified the cellular deubiquitinating enzyme USP15 as a new UL12-binding partner. USP15 is one of the most enriched proteins on viral DNA and has recently been implicated in regulating cellular homologous recombination. Utilizing isolation of proteins on nascent DNA (iPOND) to compare the proteins on wild-type and UL12-deficient viruses, we observed that USP15 is not recruited to newly replicated DNA in the absence of UL12. We also observed that UL12 and USP15 interact directly <i>in vitro</i> using purified proteins. Together, these data suggest that UL12 directly recruits USP15 to viral DNA. UL12 stimulates a type of homology-directed repair called single-strand annealing (SSA). We generated a USP15 knockout cell line with an integrated GFP-based SSA reporter and observed that UL12 and USP15 both contribute to the maximal induction of SSA. In addition to the reporter cell line, there is a 10-fold reduction in the frequency of marker rescue, and individual replication forks move slower in the absence of USP15. These data support a role for USP15 in facilitating recombination during the virus life cycle and the maintenance of replication fork stability.IMPORTANCEHSV-1 is a ubiquitous pathogen in the global population that causes lifelong latent infection with sporadic reactivation in the host. It has long been appreciated that HSV-1 DNA replication exhibits high rates of recombination and is linked to the host DNA damage response. In this study, we show a novel interaction between UL12, a member of the HSV recombinase, and the deubiquitinating host enzyme USP15. We show that USP15 physically interacts with UL12 and is required for UL12 to maximally stimulate recombination. In the absence of USP15, we also observe an overall reduction in virus growth. This work demonstrated that host proteins facilitate viral-induced recombination. The interaction of USP15 with UL12 represents a potential target for intervention against HSV-1 infection and associated disease.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0089325"},"PeriodicalIF":3.8,"publicationDate":"2025-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144873857","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Discovery and rescue of porcine bastroviruses associated with polioencephalomyelitis in domestic pigs.","authors":"Nicole Wildi, Stefano Bagatella, Xuanxuan Zhang, Mark C Hawes, Kara L D Dawson, Honglei Chen, Som Walker, Gemma Harvey, Brenda van der Heide, David T Williams, Andrew Hemphill, Corinne Gurtner, Jianning Wang, Torsten Seuberlich","doi":"10.1128/jvi.01130-25","DOIUrl":"https://doi.org/10.1128/jvi.01130-25","url":null,"abstract":"<p><p>Bastroviruses (BastV) are non-enveloped single-stranded positive-sense RNA viruses that have been discovered recently in feces samples of different animals and humans. The non-structural proteins of these viruses show similarities to those of hepevirids (<i>Hepeviridae</i>), and the structural proteins exhibit similarities to those of astrovirids (<i>Astroviridae</i>). BastVs have been found in fecal samples of mammals, amphibians, and invertebrates, but the association of infection and clinical disease manifestations has not been established. Here, we report the identification of porcine bastroviruses (PoBastV) in central nervous system (CNS) tissue samples of domestic pigs that presented fatal neurological disease in two unrelated disease outbreak scenarios in Australia and Switzerland. Viral metatranscriptomics of formalin-fixed paraffin-embedded (FFPE) CNS tissues identified genomic sequences of two genetically closely related PoBastV strains (PoBastV AUS/2015 and CHE/2022). Genomic RNA of both strains was readily detected by <i>in situ</i> RNA hybridization in neurons and glial cells of CNS tissues presenting histopathological lesions, thus supporting a plausible causal relationship between neurotropism and disease. We generated a molecular cDNA clone of PoBastV CHE/2022 and rescued infectious virus by reverse genetics in swine kidney cells (SK6) and further virus passage in intestinal porcine enterocytes (IPEC-J2). We used transmission electron microscopy to demonstrate PoBastV CHE/2022 virions in infected IPEC-J2 cells. These findings pave the way toward PoBastV as one of the etiologies of neurological disease outbreaks. Additionally, they allow studies further elucidating the molecular biology and pathogenesis of emerging BastV infections.IMPORTANCEBastroviruses (BastV) have been discovered recently in feces samples of different animals and humans. To date, BastV infections have not been associated with clinical diseases. Here, we report the identification of porcine BastV (PoBastV) in central nervous system tissue samples of domestic pigs that presented fatal neurological disease in two unrelated disease outbreak scenarios in Australia and Switzerland. This finding supports the hypothesis that PoBastV infections may cause clinical disease. We further rescued infectious PoBastV <i>in vitro</i> using the genome sequence data of one neuroinvasive PoBastV strain. With these tools, we can now start deciphering the molecular biology of BastV replication and the interaction of the virus with the host, which will lay the ground for future prophylactic and therapeutic strategies.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0113025"},"PeriodicalIF":3.8,"publicationDate":"2025-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144873772","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>N</i>-linked glycans on the stalk of influenza virus neuraminidase promote functional tetramer formation by compensating for local hydrophobicity.","authors":"Soma Saeidi, Hongquan Wan, Hyeog Kang, Jin Gao, Wells W Wu, Tahir Malik, Robert Daniels","doi":"10.1128/jvi.00879-25","DOIUrl":"https://doi.org/10.1128/jvi.00879-25","url":null,"abstract":"<p><p>Enveloped virus surface antigens, such as influenza neuraminidase (NA), typically depend on <i>N-</i>linked glycans for assembly, trafficking in the host cell, and immune evasion. Here, we examined the function of the <i>N-</i>linked glycans on the NA stalk from H1N1 2009 pandemic (pdm09) viruses using reverse genetics coupled with a recombinant NA (rNA) analysis. Our results with the NA from A/Brisbane/02/2018 (H1N1) show that all five glycosylation sites in the stalk generally receive an <i>N-</i>linked glycan and that viral growth is largely unaffected by removing any of these sites individually. In contrast, viral growth decreased when we sequentially removed the stalk glycosylation sites, consistent with the observation that H1N1 pdm09 strains predominantly possess four or five stalk glycan sites in NA. Upon passage, the growth of the virus lacking the five stalk glycan sites significantly increased, and multiple substitutions were found that decreased the hydrophobicity near each mutated site. Reverse genetics experiments confirmed the polar stalk substitutions significantly improved viral growth and revealed an increase in the amount of NA dissociated from the virus, suggesting the stalk glycans or the hydrophobic regions protect against proteolysis. The polar substitutions also rescued the expression of a secreted recombinant NA (rNA) lacking the stalk glycan sites and significantly increased production of enzymatically active rNA that exceeded the wild-type version. These findings demonstrate that the stalk glycans promote functional NA tetramer formation by compensating for local hydrophobicity and that viral-based studies can be leveraged in the rational design of rNA antigens.IMPORTANCE<i>N-</i>linked glycans can play critical roles in viral glycoprotein maturation and immune evasion. The influenza virus glycoprotein neuraminidase (NA) possesses multiple <i>N-</i>linked glycan sites in the enzymatic head domain and stalk region that tether it to the viral surface. This study demonstrates that the stalk <i>N-</i>linked glycans contribute to viral fitness by compensating for local hydrophobicity to enable functional NA tetramer formation. Supporting this conclusion, our results show that sequential removal of the stalk glycan sites on a NA from a recent H1N1 strain leads to decreased viral growth that rapidly recovers following passage. The increased growth coincided with stalk mutations that reduced the hydrophobicity around the stalk glycan sites. We observed similar results with a secreted recombinant NA (rNA), and the amount of functional rNA produced with the polar substitutions exceeded the wild-type rNA, illustrating how viral-based studies can assist with the rational design of rNA antigens.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0087925"},"PeriodicalIF":3.8,"publicationDate":"2025-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144873769","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Assessing the structural boundaries of broadly reactive antibody interactions with diverse H3 influenza hemagglutinin proteins.","authors":"John V Dzimianski, Kaito A Nagashima, Joseph M Cruz, Giuseppe A Sautto, Sara M O'Rourke, Vitor H B Serrão, Ted M Ross, Jarrod J Mousa, Rebecca M DuBois","doi":"10.1128/jvi.00453-25","DOIUrl":"https://doi.org/10.1128/jvi.00453-25","url":null,"abstract":"<p><p>Influenza virus infections are an ongoing seasonal disease burden and a persistent pandemic threat. Formulating successful vaccines remains a challenge due to accumulating mutations in circulating strains, necessitating the development of innovative strategies to combat present and future viruses. One promising strategy for attaining greater vaccine effectiveness and longer-lasting protection is the use of computationally optimized broadly reactive antigens (COBRAs). The COBRA approach involves <i>in silico</i> antigen design by generating iterative, layered consensus sequences based on current and historic viruses. Antigens designed by this process show a greater breadth of antibody-mediated protection compared to wild-type antigens, with effectiveness that often extends beyond the sequence design space of the COBRA. In particular, the use of COBRA hemagglutinin (HA) proteins has led to the discovery of broadly reactive antibodies that are suggestive of their therapeutic potential. Understanding the extent to which these antibodies are effective is key to assessing the resilience of vaccine-induced immunity to diverging influenza strains. To investigate this, we tested the binding of broadly reactive antibodies with a diverse panel of H3 HA proteins. Using cryo-electron microscopy, we defined the molecular characteristics of binding for these antibodies at the paratope-epitope interface. Through sequence and structural comparisons, we observed the correlative patterns between antibody affinity and antigen structure. These data shed light on the breadth and limitations of broadly reactive antibody responses in the context of an ever-changing landscape of influenza virus strains, yielding insights into strategies for universal vaccine design.IMPORTANCEFormulating effective influenza vaccines remains a challenge due to a constantly changing landscape of circulating viruses. This is particularly true for H3N2 viruses that undergo a high degree of antigenic drift. Several new vaccine designs can elicit broadly neutralizing antibodies that are effective against a range of influenza strains. More insight is needed, however, into how resilient these antibodies will be to future strains that evolve in the context of this selective pressure. Here, we measured the precise binding characteristics of three broadly neutralizing antibodies to 18 different hemagglutinin (HA) proteins representing almost 50 years of virus evolution. Using single-particle cryo-electron microscopy and X-ray crystallography, we determined the structural characteristics of the epitopes bound by these antibodies and identified specific amino acids that greatly impact the effectiveness of these antibodies. This provides important insights into the longevity of antibody efficacy that can help guide design choices in next-generation vaccines.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0045325"},"PeriodicalIF":3.8,"publicationDate":"2025-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144855693","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Self-assembled cyanidin-3-O-glucoside nanoparticles alleviate inflammation and ferroptosis induced by PRRSV infection.","authors":"Xiaohan Chen, Yipeng Pang, Fructueux Modeste Amona, Zilu Liu, Fang Wang, Yuan Liang, Jiachen Yang, Wanhan Zhang, Xingtang Fang, Xi Chen","doi":"10.1128/jvi.00954-25","DOIUrl":"https://doi.org/10.1128/jvi.00954-25","url":null,"abstract":"<p><p>Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly contagious pathogen that causes substantial economic losses in the global swine industry, primarily by impairing reproductive performance and respiratory health. However, current therapeutic approaches remain limited in effectively controlling PRRSV infection. Here, we present self-assembled cyanidin-3-O-glucoside (C3G)-based nanoparticles (C3G-Cs-SeNPs) as a promising antiviral agent against PRRSV infection. These nanoparticles demonstrated excellent stability and biocompatibility, with minimal cytotoxicity in Marc-145 cells. C3G-Cs-SeNPs significantly reduced ROS levels in PRRSV-infected cells, enhancing the antioxidant capacity and mitigating oxidative stress. In addition, they effectively inhibited key stages of the PRRSV lifecycle, including viral internalization and replication, leading to a marked decrease in viral proliferation. Moreover, C3G-Cs-SeNPs alleviated ferroptosis by restoring redox homeostasis through enhanced antioxidant enzyme activity. Mechanistically, these nanoparticles potentially activate the SIRT1/Nrf2/HO-1 signaling pathway, which plays a critical role in regulating inflammation and ferroptosis in PRRSV-infected cells. These findings suggest that C3G-Cs-SeNPs could serve as a novel therapeutic approach to modulate inflammation, ferroptosis, and viral replication in respiratory viral infections. Our study highlights the therapeutic potential of C3G-Cs-SeNPs in combating PRRSV infection, with important implications for viral infection management and respiratory disease treatment.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) remains a major challenge in the swine industry, causing significant economic losses due to its high mutation rate and ability to evade host immunity. Current antiviral treatments and vaccines offer limited efficacy, necessitating the development of novel therapeutic strategies. This study introduces self-assembled cyanidin-3-O-glucoside-based chitosan-selenium nanoparticles (C3G-Cs-SeNPs) as a promising antiviral agent. These nanoparticles effectively inhibit PRRSV replication, reduce oxidative stress, and alleviate inflammation and ferroptosis by activating the SIRT1/Nrf2/HO-1 signaling pathway. By mitigating virus-induced cellular damage, C3G-Cs-SeNPs offer a potential therapeutic approach for PRRSV and other respiratory viral infections. This study highlights the role of ferroptosis in PRRSV pathogenesis and presents an innovative nanotechnology-based solution to combat viral infections, contributing to the development of more effective antiviral strategies.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0095425"},"PeriodicalIF":3.8,"publicationDate":"2025-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144855695","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An African swine fever virus-specific antibody reactome reveals antigens as potential candidates for vaccine development.","authors":"Songxin Guo, Yi Ru, Hui Zhang, Junbiao Xue, Huanan Liu, Dong Men, Zongqiang Cui, Chaochao Shen, Hong Tian, Chun Ma, Jun Gong, Jintian Xu, Dianbing Wang, Rui Gong, Xiaowei Zhang, Heng Rong, Yan-Yi Wang, Chenli Liu, Zhuojun Dai, Shengce Tao, Jiaoyu Deng, Haixue Zheng, Feng Li, Xian-En Zhang","doi":"10.1128/jvi.00478-25","DOIUrl":"https://doi.org/10.1128/jvi.00478-25","url":null,"abstract":"<p><p>Developing an efficient and safe vaccine for African swine fever (ASF), a devastating disease of pigs, remains a significant challenge mainly due to limited knowledge of the immune correlates of protection. Identifying protective determinants is difficult because ASF virus (ASFV) is a large and complex DNA virus encoding over 160 proteins. Here, we constructed an ASFV proteome microarray containing 160 full-length proteins for profiling ASFV-specific antibodies. An antibody reactome containing 46 ASFV proteins (including 12 newly recognized B-cell antigens) was established by analyzing several cohorts of serum samples from pigs protected with different live-attenuated vaccines (LAVs). A proteome-wide study of antibody dynamics over a 26-day period provided a multi-dimensional landscape of the host humoral response against ASFV after acute infection, LAV immunization, and post-vaccination challenge. This study provides a comprehensive understanding of ASFV-induced humoral immune responses, highlights B-cell antigen candidates for vaccine design, supports the investigation of LAV protection mechanisms, and would accelerate vaccine development.IMPORTANCEAfrican swine fever (ASF) poses a severe threat to global swine industries, with vaccine development hindered by limited understanding of immune protection. A comprehensive understanding of antibody responses against ASF virus (ASFV) and the discovery of protective antigens are fundamental to vaccine development. This study constructed an ASFV proteome microarray to profile antibody responses against 160 viral proteins and established the antibody spectra against ASFV with dynamic features. The proteome microarray offers a high-throughput platform for understanding ASFV immunology and pathogenicity and will contribute to ASF vaccine development and diagnosis.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0047825"},"PeriodicalIF":3.8,"publicationDate":"2025-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144855692","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Activation of the HPV16 late promoter by viral E2 and cellular BRD4 and ZC3H4 proteins.","authors":"Janis Renner, Karsten Boldt, Andreas Wieland, Adam Grundhoff, Thomas Guenther, Patrick Bluemke, Frank Stubenrauch, Thomas Iftner","doi":"10.1128/jvi.00762-25","DOIUrl":"https://doi.org/10.1128/jvi.00762-25","url":null,"abstract":"<p><p>High-risk human papillomaviruses (HPV), particularly HPV16, are major causes of anogenital and oropharyngeal cancers. The HPV late promoter, P670 in the case of HPV16, is activated upon host cell differentiation and drives the expression of viral capsid proteins. While differentiation-specific host transcription factors have been implicated in regulating this promoter, the mechanism remains incompletely understood. HPV E2 proteins activate transcription by interacting with the host protein BRD4 (Bromodomain-containing protein 4). A biotin proximity ligation screen identified several novel E2 interactors, of which many overlap with the BRD4 interactome, suggesting BRD4 mediates a large fraction of these interactions. One such interactor, ZC3H4 (Zinc finger CCCH domain-containing protein 4), is known to restrict the expression of long non-coding RNAs, including enhancer and promoter upstream antisense RNAs (uaRNAs). E2 recruits ZC3H4 in a BRD4-dependent manner to specifically activate the P670 promoter in reporter assays. Supporting this, E2 and ZC3H4 co-localize in cells with high P670 activity. ZC3H4 is upregulated during differentiation, and its knockdown in differentiated HPV16- or HPV31-positive cells reduces late viral transcripts in an E2-BRD4-dependent manner. Interestingly, knockdown of ZC3H4 does not increase viral uaRNAs, suggesting that ZC3H4 does not enhance HPV late transcription by regulating viral antisense transcription.</p><p><strong>Importance: </strong>High-risk human papillomaviruses (HPVs), particularly HPV16, can cause anogenital and oropharyngeal cancers. HPV16 relies on the differentiation-dependent activation of its late promoter, P670, to produce capsid proteins. While host transcription factors contribute to this regulation, the mechanisms remain incompletely defined. Our findings reveal that the viral E2 protein collaborates with the host protein BRD4-a critical transcriptional regulator-to recruit other cellular partners, such as ZC3H4. Normally, ZC3H4 suppresses non-coding RNAs in cells, but HPV16 repurposes it via BRD4 to activate P670. This interaction intensifies in differentiated cells, where ZC3H4 levels rise, and disrupting ZC3H4 specifically blocks late viral gene expression without affecting antisense viral transcription. This highlights a unique, differentiation-dependent strategy HPV16 uses to hijack host machinery for its replication.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0076225"},"PeriodicalIF":3.8,"publicationDate":"2025-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144835482","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Deciphering the membrane topology of the pestiviral non-structural protein 4B (NS4B).","authors":"S Höppner, O Isken, N Tautz","doi":"10.1128/jvi.00825-25","DOIUrl":"https://doi.org/10.1128/jvi.00825-25","url":null,"abstract":"<p><p>Pestiviruses like bovine viral diarrhea virus and other members of the family <i>Flaviviridae</i> form replication complexes consisting of viral non-structural and cellular proteins at rearranged intracellular membranes. Despite the pivotal roles of non-structural protein 4B (NS4B) throughout the pestiviral life cycle, little is known about how this protein exerts its multiple functions. It is assumed that pestiviral NS4B promotes replication complex assembly and virion morphogenesis by interacting with defined sets of viral and host proteins. The membrane topology of the protein dictates the availability of individual protein structures and interfaces for such interactions. Thus, the knowledge of the NS4B membrane topology is required for a detailed functional understanding of this protein. Therefore, we experimentally determined the membrane topology for NS4B <i>in cellulo</i> by using the substituted cysteine accessibility method (SCAM) in combination with computational secondary structure and transmembrane domain (TMD) predictions. Our model indicates the formation of two TMDs in the N-terminal region of NS4B (TMD2-3) followed by nine putative membrane-associated α-helices. Furthermore, a dual topology of the N-terminal amphipathic α-helix AH1 was detected by applying a Split-GFP assay, exposing similarities to hepatitis C virus NS4B. The translocation of AH1 across the membrane and the luminal orientation of the proposed loop connecting TMD2-3 was further confirmed by glycosylation acceptor site recognition analysis. Together, our model will assist further studies on the diverse functions of pestiviral NS4B throughout the viral life cycle.IMPORTANCEMembrane proteins are of special importance for positive-strand RNA viruses due to their replication at remodeled intracellular membranes. Moreover, the multi-functionality of these proteins can rely on alternative topologies. Studying their membrane topologies is challenging since protein purification can induce misfolding. Similarly, random insertions of large N-glycosylation acceptor sites may disturb transmembrane domains and thus the topology, while minimal glycosylation motifs (NXT/S) are often inefficiently glycosylated. Therefore, we used the SCAM assay utilizing single cysteine substitutions to analyze the membrane topology of BVDV-1 NS4B. A dual topology of the N-terminal region was demonstrated by a Split-GFP assay. Glycosylation acceptor site insertions at pre-analyzed positions further corroborated the model. In sum, BVDV-1 NS4B topology shows similarities but also remarkable differences to the NS4B membrane topologies of other <i>Flaviviridae</i> orthologues. This new information will allow further studies to clarify the molecular basis of the multi-functionality of this critical viral component.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0082525"},"PeriodicalIF":3.8,"publicationDate":"2025-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144835483","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Proliferative proteome induced by HPV 16E6 and NFX1-123 partnership in longitudinal keratinocyte cultures.","authors":"C L Billingsley, E H Doud, W Smith-Kinnaman, A L Mosley, S Chintala, R A Katzenellenbogen","doi":"10.1128/jvi.00967-25","DOIUrl":"10.1128/jvi.00967-25","url":null,"abstract":"<p><p>Human papillomaviruses (HPVs) cause cervical, anogenital, and oropharyngeal cancers. Despite the availability of preventive HPV vaccines, their poor uptake leaves individuals at risk for these cancers, many of which remain common globally and others that are increasing domestically. The HPV 16 E6 (16E6) protein plays a significant role in inducing and maintaining cellular transformation of its infected host cell; however, 16E6 itself has no enzymatic activity and executes its functions through partnerships with host proteins. Previously, we demonstrated that 16E6 binds to the host protein NFX1-123, and NFX1-123 expression is increased in HPV 16-positive cervical cancer cell lines and primary cancers compared to normal tissues. In this study, we quantify growth patterns of 16E6-expressing keratinocytes with endogenous or overexpressed NFX1-123 (16E6/vec and 16E6/FN123, respectively) levels and interrogate proteome expression changes early and late in longitudinal growth cultures. Early-passage 16E6/vec and 16E6/FN123 cells showed similar growth rates; however, late-passage 16E6/FN123 cells had accelerated growth, greater population doublings, increased telomerase activity, and a dysplastic phenotype when compared to 16E6/vec cells. Matching this, mass spectrometry revealed only 22 differentially expressed proteins at early passage; meanwhile, late-passaged cells had 827 differentially expressed proteins among 16E6/FN123 and 16E6/vec cells. In that, DNA maintenance and repair, proteins, namely, MCM2 and MCM4, were highly expressed in the 16E6/FN123 compared to 16E6/vec cells. These findings indicate that the 16E6 and NFX1-123 partnership, especially with sustained higher levels of NFX1-123, alters the longitudinal cellular environment in a manner that may initiate a preneoplastic phenotype.</p><p><strong>Importance: </strong>The high-risk HPV 16E6 protein plays a significant role in inducing and maintaining cellular transformation of its infected host cell; however, 16E6 has no enzymatic activity and carries out most of its functions through partnerships with host endogenous proteins, one being NFX1-123. That NFX1-123 expression is increased in HPV 16-positive cervical cancer and primary cancers compared to normal tissues, and NFX1-123 binds directly to 16E6. This study leverages proteomics to reinforce the synergistic actions of HPV16E6 and NFX1-123 and identifies cellular pathways impacted by this protein partnership over time. These findings encourage further investigation of NFX1-123 as a potential therapeutic target of HPV 16-positive tissues.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0096725"},"PeriodicalIF":3.8,"publicationDate":"2025-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144835485","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}