Journal of Virology最新文献

{"title":"Characterization of quasispecies of severe fever with thrombocytopenia syndrome virus.","authors":"Sithumini M W Lokupathirage, Devinda S Muthusinghe, Rakiiya S Sarii, Olusola A Akanbi, Kenta Shimizu, Yoshimi Tsuda, Kumiko Yoshimatsu","doi":"10.1128/jvi.01794-24","DOIUrl":"https://doi.org/10.1128/jvi.01794-24","url":null,"abstract":"<p><p>Three specific amino acid variations have been identified in the quasispecies of the isolated YG1 strain of severe fever with thrombocytopenia syndrome virus (SFTSV): Gn (Y328H), Gc (R624W), and L (N1891K). The Gn (Y328H) accounted for 26.9% of the viruses in the patient's blood. The other two mutations are less frequent, indicating that these mutations appeared during propagation in Vero E6 cells. To investigate the effects of each mutation on viral properties, we evaluated viruses with one to three mutations. Mutations Y328H and R624W in glycoprotein (GP) resulted in increased plaque size and cell fusion activity. Viruses with the N1891K mutation in L showed a notable cytopathic effect (CPE), which was inhibited by a pan-caspase inhibitor, suggesting that caspase-dependent cell death occurred. Programmed cell death-associated caspases were induced in both CPE-inducing and wild-type virus-infected cells. Furthermore, infection with the wild-type virus suppressed actinomycin D-induced cell death. These results suggest that SFTSV-infected cells initiate programmed cell death, whereas the wild-type virus inhibits cell death. Additionally, the recombinant single mutant virus outcompeted by a 10-fold lower amount of the wild-type virus in Vero E6 cells, indicating that the mutations were not advantageous for viral propagation in Vero E6 cells. These findings suggest that the quasispecies composition of SFTSV is influenced by the propagative environment.IMPORTANCEThis study presents findings on viral pathogenesis by analyzing quasispecies derived from a fatal case of severe fever with thrombocytopenia syndrome virus (SFTSV) infection. Analysis of recombinant SFTSV with mutations in Gn and Gc suggested that combinations of mutations may enhance the viability of mutant viruses, thereby selecting viruses to create a suitable population for propagation. The N1891K mutation in the L protein of SFTSV is associated with promoting cytopathic effects (CPE). Conversely, the wild-type virus, which is the predominant virus in infected patients, suppresses cell death. It has been suggested that SFTSV possesses a mechanism to evade cell death for prolonged viral propagation within the infected cells. Although the precise mechanism remains unknown, RNA virus polymerase may be involved in regulating cell death. This study contributes to our understanding of the mechanisms underlying the adaptation and survival of viruses as quasispecies.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0179424"},"PeriodicalIF":4.0,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143811624","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":"CPSF6 promotes HIV-1 preintegration complex function.","authors":"Evan Chaudhuri, Sooin Jang, Rajasree Chakraborty, Rajalingam Radhakrishnan, Bjarki Arnarson, Prem Prakash, Daphne Cornish, Nicholas Rohlfes, Parmit K Singh, Jiong Shi, Christopher Aiken, Edward Campbell, Judd Hultquist, Muthukumar Balsubramaniam, Alan N Engelman, Chandravanu Dash","doi":"10.1128/jvi.00490-25","DOIUrl":"https://doi.org/10.1128/jvi.00490-25","url":null,"abstract":"<p><p>Cleavage and polyadenylation specificity factor 6 (CPSF6) is part of the cellular cleavage factor I mammalian (CFIm) complex that regulates mRNA processing and polyadenylation. CPSF6 also functions as an HIV-1 capsid (CA) binding host factor to promote viral DNA integration targeting into gene-dense regions of the host genome. However, the effects of CPSF6 on the activity of the HIV-1 preintegration complex (PIC)-the sub-viral machinery that carries out viral DNA integration-are unknown. To study CPSF6's role in HIV-1 PIC function, we extracted PICs from cells that are either depleted of CPSF6 or express a mutant form that cannot bind to CA. These PICs exhibited significantly lower viral DNA integration activity when compared to the control PICs. The addition of purified recombinant CPSF6 restored the integration activity of PICs extracted from the CPSF6-mutant cells, suggesting a direct role of CPSF6 in PIC function. To solidify CPSF6's role in PIC function, we inoculated CPSF6-depleted and CPSF6-mutant cells with HIV-1 particles and measured viral DNA integration into the host genome. A significant reduction in integration in these cells was detected, and this reduction was not a consequence of lower reverse transcription or nuclear entry. Additionally, mutant viruses deficient in CA-CPSF6 binding showed no integration defect in CPSF6-mutant cells. Finally, sequencing analysis revealed that HIV-1 integration into CPSF6-mutant cell genomes was significantly redirected away from gene-dense regions of chromatin compared to the control cells. Collectively, these results suggest that the CPSF6-CA interaction promotes PIC function both <i>in vitro</i> and in infected cells.IMPORTANCEHIV-1 infection is dependent on the interaction of the virus with cellular host factors. However, the molecular details of HIV-host factor interactions are not fully understood. For instance, the HIV-1 capsid provides binding interfaces for several host factors. CPSF6 is one such capsid-binding host factor, whose cellular function is to regulate mRNA processing and polyadenylation. Initial work identified a truncated cytosolic form of CPSF6 to restrict HIV infection by blocking viral nuclear entry. However, it is now established that the full-length CPSF6 primarily promotes HIV-1 integration targeting into gene-dense regions of the host genome. Here, we provide evidence that CPSF6-CA interaction stimulates the activity of HIV-1 preintegration complexes (PICs). We also describe that disruption of CPSF6-CA binding in target cells significantly reduces viral DNA integration and redirects integration targeting away from gene-dense regions into regions of low transcriptional activity. These findings identify a critical role for the CPSF6-CA interaction in PIC function and integration targeting.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0049025"},"PeriodicalIF":4.0,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143810926","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":"Molecular cloning and host range analysis of three cytomegaloviruses from <i>Mastomys natalensis</i>.","authors":"Laura Staliunaite, Olha Puhach, Eleonore Ostermann, Kyle Rosenke, Jenna Nichols, Lisa Oestereich, Nafomon Sogoba, Heinz Feldmann, Andrew J Davison, Michael A Jarvis, Wolfram Brune","doi":"10.1128/jvi.02147-24","DOIUrl":"https://doi.org/10.1128/jvi.02147-24","url":null,"abstract":"<p><p>Herpesvirus-based vectors are attractive for use as conventional or transmissible vaccines against emerging zoonoses in inaccessible animal populations. In both cases, cytomegaloviruses (CMVs) as members of the subfamily <i>Betaherpesvirinae</i> are particularly suitable for vaccine development as they are highly specific for their natural host species, infect a large proportion of their host population, and cause mild infections in healthy individuals. The Natal multimammate mouse (<i>Mastomys natalensis</i>) is the natural reservoir of Lassa virus, which causes deadly hemorrhagic fever in humans. <i>M. natalensis</i> was recently reported to harbor at least three different cytomegaloviruses (MnatCMV1, MnatCMV2, and MnatCMV3). Herein, we report the molecular cloning of three complete MnatCMV genomes in a yeast and bacterial artificial chromosome (YAC-BAC) hybrid vector. Purified viral genomes were cloned in yeast by single-step transformation-associated recombination (STAR cloning) and subsequently transferred to <i>Escherichia coli</i> for further genetic manipulation. The integrity of the complete cloned viral genomes was verified by sequencing, and the replication fitness of viruses reconstituted from these clones was analyzed by replication kinetics in <i>M. natalensis</i> fibroblasts and kidney epithelial cells. We also found that neither parental nor cloned MnatCMVs replicated in mouse and rat fibroblasts, nor did they show sustained replication in baby hamster kidney cells, consistent with the expected narrow host range for these viruses. We further demonstrated that an exogenous sequence can be inserted by BAC-based mutagenesis between open reading frames M25 and m25.1 of MnatCMV2 without affecting replication fitness <i>in vitro</i>, identifying this site as potentially suitable for the insertion of vaccine target antigen genes.IMPORTANCECytomegaloviruses (CMVs) recently discovered in the Natal multimammate mouse (<i>Mastomys natalensis</i>) are widespread within the <i>M. natalensis</i> population. Since these rodents also serve as natural hosts of the human pathogen Lassa virus (LASV), we investigated the potential suitability of <i>M. natalensis</i> CMVs (MnatCMVs) as vaccine vectors. We describe the cloning of three different MnatCMV genomes as bacterial artificial chromosomes (BACs). The replicative capacity and species specificity of these BAC-derived MnatCMVs were analyzed in multiple cell types. We also identified a transgene insertion site within one of the MnatCMV genomes suitable for the incorporation of vaccine target antigens. Together, this study provides a foundation for the development of MnatCMVs as transmissible MnatCMV-based LASV vaccines to reduce LASV prevalence in hard-to-reach <i>M. natalensis</i> populations and, thereby, zoonotic transmission to humans.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0214724"},"PeriodicalIF":4.0,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143811099","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":"Human cytomegalovirus gH/gL/gO binding to PDGFRα provides a regulatory signal activating the fusion protein gB that can be blocked by neutralizing antibodies.","authors":"Eric P Schultz, Lars Ponsness, Jean-Marc Lanchy, Matthias Zehner, Florian Klein, Brent J Ryckman","doi":"10.1128/jvi.00035-25","DOIUrl":"https://doi.org/10.1128/jvi.00035-25","url":null,"abstract":"<p><p>Herpesviruses require membrane fusion for entry and spread, a process facilitated by the fusion glycoprotein B (gB) and the regulatory factor gH/gL. The human cytomegalovirus (HCMV) gH/gL can be modified by the accessory protein gO, or the set of proteins UL128, UL130, and UL131. While the binding of the gH/gL/gO and gH/gL/UL128-131 complexes to cellular receptors, including PDGFRα and NRP2, has been well-characterized structurally, the specific role of receptor engagements by the gH/gL/gO and gH/gL/UL128-131 in regulation of fusion has remained unclear. We describe a cell-cell fusion assay that can quantitatively measure fusion on a timescale of minutes and demonstrate that binding of gH/gL/gO to PDGFRα dramatically enhances gB-mediated cell-cell fusion. In contrast, gH/gL/pUL128-131-regulated fusion is significantly slower, and gH/gL alone cannot promote gB fusion activity within this timescale. The genetic diversity of gO influenced the observed cell-cell fusion rates, correlating with previously reported effects on HCMV infectivity. Mutations in gL that had no effect on the formation of gH/gL/gO or binding to PDGFRa dramatically reduced the cell-cell fusion rate, suggesting that gL plays a critical role in linking the gH/gL/gO-PDGFRa receptor binding to activation of gB. Several neutralizing human monoclonal antibodies were found to potently block gH/gL/gO-PDGFRa-regulated cell-cell fusion, suggesting this mechanism as a therapeutic target.</p><p><strong>Importance: </strong>Development of vaccines and therapeutics targeting the fusion apparatus of human cytomegalovirus (HCMV) has been limited by the lack of an <i>in vitro</i> cell-cell fusion assay that faithfully models the receptor-dependent fusion characteristic of HCMV entry. The cell-cell fusion assay described here demonstrated that the binding of gH/gL/gO to its receptor, PDGFRα, serves to regulate the activity of the fusion protein gB, and this is specifically vulnerable to inhibition by neutralizing antibodies. Moreover, the measurement of fusion kinetics allows for mutational studies of the fusion mechanism, assessing the influence of genetic diversity among the viral glycoproteins and studying the mechanism of neutralizing antibodies.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0003525"},"PeriodicalIF":4.0,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143810936","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":"Structure and function of an unusual R452-dependent monoclonal antibody against SARS-CoV-2.","authors":"Bing Zhou, Qi Gui, Congcong Liu, Huimin Guo, Haiyan Wang, Lin Cheng, Qing Fan, Xiangyang Ge, Zheng Zhang, Bin Ju","doi":"10.1128/jvi.01844-24","DOIUrl":"https://doi.org/10.1128/jvi.01844-24","url":null,"abstract":"<p><p>The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants is still a major public health concern worldwide. Currently, SARS-CoV-2 variants have been widely used to develop the updated vaccine. However, whether these mutated residues still have good immunogenicity remains elusive. In particular, we know little about what kind of antibodies can be induced by the infection or vaccination of SARS-CoV-2 variants and their biological characteristics. Here, we identified an R452-dependent monoclonal neutralizing antibody, ConD-852, from a primarily Delta variant-infected individual, indicating that the mutated R452 residue has good immunogenicity. We determined the high-resolution cryo-electron microscopy (cryo-EM) structure of ConD-852 complexed with the Delta receptor-binding domain (RBD), revealing how it binds to the R452-related epitopes and their detailed interactions. Interestingly, ConD-852 could only bind to the amino acid residue \"R\" at the 452 position on RBD, displaying a strict restriction to recognize SARS-CoV-2. Overall, our findings regarding ConD-852 confirmed the good immunogenicity of SARS-CoV-2 variants carrying the L452R mutation and enriched our knowledge of the binding model involving the neutralizing antibody and the mutated virus.IMPORTANCEAlthough SARS-CoV-2 variants have been widely used to update the COVID-19 vaccine candidate, whether these mutations still have good immunogenicity is unknown. This study demonstrates that the mutated R452 residue can induce potent neutralizing antibodies and reports a high-resolution cryo-EM structure of an R452-dependent monoclonal antibody binding to the epitopes around the R452 residue on SARS-CoV-2 RBD.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0184424"},"PeriodicalIF":4.0,"publicationDate":"2025-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143803719","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":"Peste des petits ruminants virus (PPRV) induces ferroptosis via LONP1-mediated mitochondrial GPX4 degradation in cell culture.","authors":"Qiaodi Hou, Shuijin Cheng, Zhijun Li, Congshang Lei, Yan Chen, Mingzhuo Ma, Jinming Liu, Xiwen Chen, Lizhen Wang, Qinghong Xue, Xuefeng Qi","doi":"10.1128/jvi.02310-24","DOIUrl":"https://doi.org/10.1128/jvi.02310-24","url":null,"abstract":"<p><p>Peste des petits ruminants virus (PPRV) is an important pathogen that seriously affects the productivity of small ruminants worldwide. Ferroptosis is a programmed cell death characterized by iron-dependent lipid peroxidation and the accumulation of reactive oxygen species (ROS). Emerging evidence has demonstrated that mitochondria play diverse roles in the process of ferroptosis, but the interaction between mitochondria and ferroptosis during virus infection remains largely unknown. Here, we demonstrate that PPRV induces ferroptosis, including Fe²⁺ overload, accumulation of lipid peroxidation, and shrinkage of mitochondria. Importantly, mitochondria play a crucial role in the process of PPRV-induced ferroptosis characterized by decreased mitochondrial GPX4 and lipid peroxidation in mitochondria. Mechanistically, PPRV infection downregulates mitochondrial Lon protease-1 (LONP1) expression, an important multifaceted enzyme that is essential for maintaining mitochondrial homeostasis and function, which leads to mitochondrial GPX4 degradation through the Nrf2/Keap pathway and accumulation of ROS in mitochondria. More importantly, PPRV-induced ferroptosis is tightly associated with inflammatory responses and enhanced virus replication. Overall, this study is the first to show that LONP1-mediated ferroptosis is involved in the inflammatory responses during PPRV infection.</p><p><strong>Importance: </strong>Peste des petits ruminants virus (PPRV) infection induces a transient but severe immunosuppression in the host, which threatens both small livestock and endangered susceptible wildlife populations in many countries. Despite extensive research, it is unknown whether PPRV causes ferroptosis and what the mechanism of regulation is. Our data provide the first direct evidence that the relationship between Lon protease-1 (LONP1)-mediated dysfunctional mitochondria and the consequent induction of ferroptosis is involved in PPRV-induced pathogenesis. Importantly, we demonstrate that PPRV infection induces ferroptosis via the LONP1-mediated GPX4 degradation and ROS accumulation in mitochondria, and PPRV-induced ferroptosis is tightly associated with inflammatory responses and enhanced virus replication levels. Taken together, our research has provided new insight into understanding the effect of ferroptosis on PPRV replication and pathogenesis and revealed a potential therapeutic target for antiviral intervention.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0231024"},"PeriodicalIF":4.0,"publicationDate":"2025-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143803718","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":"Safety, pharmacokinetics, and biological activity of CD4-mimetic BNM-III-170 in SHIV-infected rhesus macaques.","authors":"Elise G Viox, Jonathan Richard, Andres G Grandea, Kevin Nguyen, Justin Harper, James Auger, Shilei Ding, Romain Gasser, Jérémie Prévost, Lorie Marchitto, Halima Medjahed, Catherine Bourassa, Fleur Gaudette, Amélie Pagliuzza, Cesar Ariel Trifone, Christina Gavegnano, Selwyn J Hurwitz, Jun Park, Natasha M Clark, Iman Hammad, Saverio Capuano, Malcolm A Martin, Raymond F Schinazi, Guido Silvestri, Deanna A Kulpa, Priti Kumar, Nicolas Chomont, Marzena Pazgier, Amos B Smith, Joseph Sodroski, David T Evans, Andrés Finzi, Mirko Paiardini","doi":"10.1128/jvi.00062-25","DOIUrl":"https://doi.org/10.1128/jvi.00062-25","url":null,"abstract":"<p><p>Anti-HIV-1 antibodies capable of mediating ADCC are elicited by the majority of people with HIV-1 and preferentially target the \"open,\" CD4-bound conformation of HIV-1 envelope glycoproteins (Env). However, due to the \"closed\" conformation sampled by unliganded HIV-1-Envs, these antibodies are ineffective at eliminating infected cells. BNM-III-170 is a small-molecule CD4-mimetic compound that binds the Phe43 cavity of the gp120 subunit of Env, forcing Env to \"open up,\" thus exposing epitopes targeted by CD4-induced (CD4i), ADCC-mediating antibodies. Here, we assessed the safety, pharmacokinetics, and biological activity of BNM-III-170 in uninfected and SHIV-AD8-EO-infected rhesus macaques (RMs). In uninfected RMs, single subcutaneous administrations of 3-36 mg/kg BNM-III-170 were well-tolerated, with serum half-lives ranging from 3 to 6 h. In SHIV-infected RMs, four different regimens were evaluated: 2 × 36 mg/kg daily, 1 × 24 mg/kg, 3 × 36 mg/kg every 7 days, and 3 × 36 mg/kg every 3 days. While toxicity was observed with daily doses, all other regimens demonstrated reasonable safety profiles. No changes in plasma viral loads were observed in SHIV-infected RMs following any of the evaluated BNM-III-170 dosing regimens. However, plasma collected following BNM-III-170 administration was shown to have increased binding to infected cells and to sensitize SHIV AD8-EO virions to neutralization by otherwise non-neutralizing antibodies. In addition, the plasma of treated animals mediated ADCC in the presence of BNM-III-170. These results establish a well-tolerated BNM-III-170 dosing regimen in SHIV-infected RMs and serve as proof of concept for its biological activity in promoting the targeting of infected cells by CD4i ADCC-mediating antibodies. Thus, they inform future studies evaluating CD4mc treatment in ART-treated animals.IMPORTANCEA therapeutic regimen able to eradicate or functionally cure HIV-1 remains elusive and may require a \"shock-and-kill\" approach to reactivate and then purge the latent HIV-1 reservoir. The small-molecule CD4-mimetic compound BNM-III-170 has previously been shown to (i) sensitize HIV-1-infected cells to ADCC mediated by plasma from people with HIV-1 (PWH) <i>in vitro</i> and (ii) significantly delay the time to viral rebound following ART interruption when combined with anti-CoRBS + anti-cluster A Abs or plasma from PWH in humanized mice. To evaluate the use of BNM-III-170 as part of a kill approach, we characterized the safety, pharmacokinetics, and biological activity of BNM-III-170 in uninfected and SHIV-infected RMs. Our study identifies a tolerable BNM-III-170 dosing regimen in SHIV-infected RMs and provides insights into its antiviral activities; as such, it informs future studies evaluating the efficacy of BNM-III-170 in reducing the viral reservoir.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0006225"},"PeriodicalIF":4.0,"publicationDate":"2025-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143795695","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":"Identification and characterization of a broadly neutralizing and protective nanobody against the HA1 domain of H5 avian influenza virus hemagglutinin.","authors":"Siqi Xu, Yutong Liu, Chenying Luo, Mengruo Zhou, Ke Wang, Qianmei Xie, Qi Zhang, Qinying Zhang, Qianyu Li, Zhichao Pan, Saixiang Feng, Ming Liao","doi":"10.1128/jvi.02090-24","DOIUrl":"https://doi.org/10.1128/jvi.02090-24","url":null,"abstract":"<p><p>The highly pathogenic avian influenza viruses (HPAIVs) of subtype H5, particularly those of the currently circulating clades 2.3.2.1 and 2.3.4.4, are largely responsible for the sporadic human infections that frequently present with a high case fatality rate. Consequently, there is an urgent necessity for the development of advanced antiviral therapeutic options against the H5 HPAIVs. Herein, the yeast two-hybrid system was employed for identifying seven nanobodies that bind the HA1 domain of hemagglutinin (HA). Among these nanobodies, Nb10 was found to exhibit high-affinity and broad-spectrum neutralization capacity against viruses of clades 2.3.2.1 and 2.3.4.4 under both <i>in vitro</i> and <i>in vivo</i> conditions. Surprisingly, Nb10 exhibited excellent efficacy against the recombinant viruses Re6/PR8, Re8/PR8, Re10/PR8, Re11/PR8, and Re14/PR8 of the subtype H5, with average half-maximal inhibitory concentrations ranging from 0.01 to 0.42 µg/mL in a microneutralization assay. Furthermore, the intratracheal administration of Nb10 resulted in remarkable prophylactic and therapeutic efficacy in mice. The findings herein reveal that the virus-neutralizing effect of Nb10 is achieved by obstructing viral entrance into host cells. Moreover, Western blot analysis and enzyme-linked immunosorbent assay revealed that Nb10 recognizes a conformational epitope located in the region spanning amino acid residues 50-271 of the protein HA1 displayed on the surface of yeast cells. The predicted structure of the binding pocket indicates that Nb10 recognizes the highly conserved receptor-binding site of HA1. Taken together, the current study offers valuable insights for the development of protective therapeutics with broad-spectrum activity and the design of broadly protective influenza vaccines.IMPORTANCEHPAIVs of subtype H5 have raised substantial public health concerns regarding the potential for viral adaptation and sustained human-to-human transmission. The prevention and treatment of the disease are replete with numerous challenges due to frequent antigenic alterations in the virus. Nanobodies have significant potential for clinical applications and therapies owing to their small size and robust tissue-penetrating capabilities. Herein, we describe the identification of Nb10, a broad-spectrum virus-neutralizing and protective nanobody that is effective against the currently circulating H5 HPAIVs of clades 2.3.2.1 and 2.3.4.4. The intratracheal administration of Nb10 afforded significant protection in mice infected with the H5 virus. This result provides novel insights for the rational design of antiviral pharmaceuticals. Furthermore, an analysis of the binding site of the target protein HA1 may be useful for the development of more effective vaccinations against influenza viruses of the subtype H5.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0209024"},"PeriodicalIF":4.0,"publicationDate":"2025-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143795689","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":"Amino acid changes accumulated in the fusion protein allow neuropathogenic measles viruses to use a broad repertoire of host factors for cell fusion triggering.","authors":"Yuichi Hirai, Ryuichi Takemoto, Yusuke Yanagi, Yuta Shirogane","doi":"10.1128/jvi.02307-24","DOIUrl":"https://doi.org/10.1128/jvi.02307-24","url":null,"abstract":"<p><p>Measles virus (MeV), an enveloped RNA virus belonging to the genus <i>Morbillivirus</i> of the family <i>Paramyxoviridae</i>, is the causative agent of measles, an acute febrile illness with skin rash. MeV has two types of envelope glycoproteins: the hemagglutinin (H) and fusion (F) protein. The H protein initially binds to its receptors, signaling lymphocytic activation molecule family member 1 (SLAMF1) and nectin-4, triggering conformational changes in the F protein that result in virus-to-cell or cell-to-cell membrane fusion. MeV may persist in the brain, which does not express SLAMF1 and nectin-4, leading to subacute sclerosing panencephalitis (SSPE) several years after acute infection. Recently, we have reported that MeV isolates from SSPE patients have hyperfusogenic amino acid changes (e.g., T461I) in the F protein, which enable MeV to spread in the brain by using cell adhesion molecule 1 (CADM1) and CADM2 as <i>cis</i>-acting fusion-triggering molecules. However, F proteins of SSPE isolates, such as the Patient B and OSA-3/Bs/B strains, carry additional changes besides T461I. In this study, we show that specific combinations of amino acid changes in the F proteins from SSPE isolates enable the induction of membrane fusion without CADM1/2 expression. We further demonstrate that these cumulative changes in the F protein allow the virus to exploit other fusion-triggering molecules than CADM1/2. These changes also promote efficient neuronal cell fusion. Our findings suggest that cumulative changes in the F protein may broaden the range of host factors capable of triggering cell fusion, facilitating MeV spread in the brain of SSPE patients.IMPORTANCESubacute sclerosing panencephalitis (SSPE) is a fatal disease caused by persistent infection of measles virus (MeV) in the brain. There is no effective therapy for the disease. MeV isolates from SSPE patients accumulate multiple amino acid changes in the F protein, including hyperfusogenic changes such as the T461I substitution, which allow MeV to spread in the brain by utilizing cell adhesion molecule 1 (CADM1) and CADM2 as <i>cis</i>-acting fusion-triggering molecules. In this study, we show that F proteins of SSPE isolates harboring additional changes besides T461I can induce membrane fusion independently of CADM1 and CADM2. The data also indicate that cumulative changes in the F protein may enable MeV to use other fusion-triggering host molecules than CADM1 and CADM2, facilitating its spread in the brain of SSPE patients. The findings deepen our understanding of the molecular mechanism underlying MeV neuropathogenicity in SSPE.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0230724"},"PeriodicalIF":4.0,"publicationDate":"2025-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143795217","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 intra-family conserved high-order RNA structure within the M ORF is important for arterivirus subgenomic RNA accumulation and infectious virus production.","authors":"Pengcheng Shang, Yanhua Li, Sawsan Napthine, Chi Chen, Ian Brierley, Andrew E Firth, Ying Fang","doi":"10.1128/jvi.02167-24","DOIUrl":"https://doi.org/10.1128/jvi.02167-24","url":null,"abstract":"<p><p>Synthesis of subgenomic RNAs is a strategy commonly used by polycistronic positive-sense single-stranded RNA viruses to express 3'-proximal genes. Members of the order <i>Nidovirales</i>, including coronaviruses and arteriviruses, use a unique discontinuous transcription strategy to synthesize subgenomic RNAs. In this study, <i>in silico</i> synonymous site conservation analysis and RNA structure folding predicted the existence of intra-family conserved high-order RNA structure within the M ORF of arteriviral genomes, which was further confirmed by RNA secondary structure probing. This RNA structure was determined to be important for the transcription/accumulation of subgenomic RNAs and the production of infectious viral particles. Mutations disrupting the stability of the RNA structures significantly decreased the accumulation of multiple subgenomic RNAs. In contrast, the impact of mutagenesis on full-length genomic RNA accumulation was limited. The degree to which wild-type levels of subgenomic RNA accumulation were maintained was found to correlate with the efficiency of infectious virus production. Moreover, the thermo-stability of stems within the high-order RNA structure is also well correlated with viral replication capacity and the maintenance of subgenomic RNA accumulation. This study is the first to report an intra-<i>Arteriviridae</i> conserved high-order RNA structure that is located in a protein-coding region and functions as an important <i>cis</i>-acting element to control the accumulation/transcription of arteriviral subgenomic RNAs. This work suggests a complex regulation mechanism between genome replication and discontinuous transcription in nidoviruses.IMPORTANCEArteriviruses are a group of RNA viruses that infect different animal species. They can cause diseases associated with respiratory/reproductive syndromes, abortion, or hemorrhagic fever. Among arteriviruses, porcine reproductive and respiratory syndrome virus (PRRSV) and equine arteritis virus (EAV) are economically important veterinary pathogens. The challenge in control of arterivirus infection reflects our limited knowledge of viral biology. In this study, we conducted a comprehensive analysis of arteriviral genomes and discovered intra-family conserved regions in the M ORF with a high-order RNA structure. The thermo-stability of the RNA structure influences sgRNA transcription/accumulation and correlates with the level of infectious virus production. Our studies provide new insight into arterivirus replication mechanisms, which may have implications for developing disease control and prevention strategies.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":" ","pages":"e0216724"},"PeriodicalIF":4.0,"publicationDate":"2025-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143795422","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}