Pub Date : 2024-08-20Epub Date: 2024-07-09DOI: 10.1128/jvi.00231-24
Douglas P Gladue, Lidia Gomez-Lucas, Eneko Largo, Elizabeth Ramirez-Medina, Johana Torralba, Maria Queralt-Martín, Antonio Alcaraz, Lauro Velazquez-Salinas, Jose L Nieva, Manuel V Borca
African swine fever virus (ASFV) is the causative agent of a contagious disease affecting wild and domestic swine. The function of B169L protein, as a potential integral structural membrane protein, remains to be experimentally characterized. Using state-of-the-art bioinformatics tools, we confirm here earlier predictions indicating the presence of an integral membrane helical hairpin, and further suggest anchoring of this protein to the ER membrane, with both terminal ends facing the lumen of the organelle. Our evolutionary analysis confirmed the importance of purifying selection in the preservation of the identified domains during the evolution of B169L in nature. Also, we address the possible function of this hairpin transmembrane domain (HTMD) as a class IIA viroporin. Expression of GFP fusion proteins in the absence of a signal peptide supported B169L insertion into the ER as a Type III membrane protein and the formation of oligomers therein. Overlapping peptides that spanned the B169L HTMD were reconstituted into ER-like membranes and the adopted structures analyzed by infrared spectroscopy. Consistent with the predictions, B169L transmembrane sequences adopted α-helical conformations in lipid bilayers. Moreover, single vesicle permeability assays demonstrated the assembly of lytic pores in ER-like membranes by B169L transmembrane helices, a capacity confirmed by ion-channel activity measurements in planar bilayers. Emphasizing the relevance of these observations, pore-forming activities were not observed in the case of transmembrane helices derived from EP84R, another ASFV protein predicted to anchor to membranes through a α-helical HTMD. Overall, our results support predictions of viroporin-like function for the B169L HTMD.IMPORTANCEAfrican swine fever (ASF), a devastating disease affecting domestic swine, is widely spread in Eurasia, producing significant economic problems in the pork industry. Approaches to prevent/cure the disease are mainly restricted to the limited information concerning the role of most of the genes encoded by the large (160-170 kba) virus genome. In this report, we present the experimental data on the functional characterization of the African swine fever virus (ASFV) gene B169L. Data presented here indicates that the B169L gene encodes for an essential membrane-associated protein with a viroporin function.
非洲猪瘟病毒(ASFV)是一种影响野猪和家猪的传染性疾病的病原体。B169L 蛋白作为一种潜在的整体结构膜蛋白,其功能仍有待实验鉴定。利用最先进的生物信息学工具,我们在此证实了早先的预测,即存在一个整体膜螺旋发夹,并进一步表明该蛋白锚定在 ER 膜上,其两端面向细胞器的内腔。我们的进化分析证实了纯化选择对 B169L 在自然界进化过程中保留已确定结构域的重要性。此外,我们还探讨了这种发夹式跨膜结构域(HTMD)作为 IIA 类病毒蛋白可能具有的功能。在没有信号肽的情况下表达 GFP 融合蛋白支持 B169L 作为 III 型膜蛋白插入 ER 并在其中形成寡聚体。将跨越 B169L HTMD 的重叠肽重组为类似 ER 的膜,并通过红外光谱分析所采用的结构。与预测一致,B169L 跨膜序列在脂质双层膜中采用了 α-螺旋构象。此外,单个囊泡通透性试验表明,B169L 跨膜螺旋在类似 ER 的膜中组装了裂孔,平面双层膜中的离子通道活性测量也证实了这一能力。强调这些观察结果的相关性的是,在来自 EP84R 的跨膜螺旋的情况下没有观察到孔形成活性,EP84R 是另一种 ASFV 蛋白,预测其通过 α-helical HTMD 固定在膜上。重要意义非洲猪瘟(ASF)是一种影响家猪的毁灭性疾病,在欧亚大陆广泛传播,给猪肉行业带来了严重的经济问题。预防/治疗该疾病的方法主要局限于有关大型(160-170 kba)病毒基因组所编码的大多数基因的作用的有限信息。在本报告中,我们介绍了有关非洲猪瘟病毒(ASFV)基因 B169L 功能特征的实验数据。本文提供的数据表明,B169L 基因编码一种重要的膜相关蛋白,具有病毒蛋白功能。
{"title":"Viroporin-like activity of the hairpin transmembrane domain of African swine fever virus B169L protein.","authors":"Douglas P Gladue, Lidia Gomez-Lucas, Eneko Largo, Elizabeth Ramirez-Medina, Johana Torralba, Maria Queralt-Martín, Antonio Alcaraz, Lauro Velazquez-Salinas, Jose L Nieva, Manuel V Borca","doi":"10.1128/jvi.00231-24","DOIUrl":"10.1128/jvi.00231-24","url":null,"abstract":"<p><p>African swine fever virus (ASFV) is the causative agent of a contagious disease affecting wild and domestic swine. The function of B169L protein, as a potential integral structural membrane protein, remains to be experimentally characterized. Using state-of-the-art bioinformatics tools, we confirm here earlier predictions indicating the presence of an integral membrane helical hairpin, and further suggest anchoring of this protein to the ER membrane, with both terminal ends facing the lumen of the organelle. Our evolutionary analysis confirmed the importance of purifying selection in the preservation of the identified domains during the evolution of B169L in nature. Also, we address the possible function of this hairpin transmembrane domain (HTMD) as a class IIA viroporin. Expression of GFP fusion proteins in the absence of a signal peptide supported B169L insertion into the ER as a Type III membrane protein and the formation of oligomers therein. Overlapping peptides that spanned the B169L HTMD were reconstituted into ER-like membranes and the adopted structures analyzed by infrared spectroscopy. Consistent with the predictions, B169L transmembrane sequences adopted α-helical conformations in lipid bilayers. Moreover, single vesicle permeability assays demonstrated the assembly of lytic pores in ER-like membranes by B169L transmembrane helices, a capacity confirmed by ion-channel activity measurements in planar bilayers. Emphasizing the relevance of these observations, pore-forming activities were not observed in the case of transmembrane helices derived from EP84R, another ASFV protein predicted to anchor to membranes through a α-helical HTMD. Overall, our results support predictions of viroporin-like function for the B169L HTMD.IMPORTANCEAfrican swine fever (ASF), a devastating disease affecting domestic swine, is widely spread in Eurasia, producing significant economic problems in the pork industry. Approaches to prevent/cure the disease are mainly restricted to the limited information concerning the role of most of the genes encoded by the large (160-170 kba) virus genome. In this report, we present the experimental data on the functional characterization of the African swine fever virus (ASFV) gene B169L. Data presented here indicates that the B169L gene encodes for an essential membrane-associated protein with a viroporin function.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11334534/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141559014","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}
Pub Date : 2024-08-20Epub Date: 2024-07-17DOI: 10.1128/jvi.00983-24
Cynthia M McMillen, Christina Megli, Rebecca Radisic, Lauren B Skvarca, Ryan M Hoehl, Devin A Boyles, Jackson J McGaughey, Brian H Bird, Anita K McElroy, Amy L Hartman
Rift Valley fever virus (RVFV) infection causes abortions in ruminant livestock and is associated with an increased likelihood of miscarriages in women. Using sheep and human placenta explant cultures, we sought to identify tissues at the maternal-fetal interface targeted by RVFV. Sheep villi and fetal membranes were highly permissive to RVFV infection resulting in markedly higher virus titers than human cultures. Sheep cultures were most permissive to wild-type RVFV and ΔNSm infection, while live-attenuated RVFV vaccines (LAVs; MP-12, ΔNSs, and ΔNSs/ΔNSm) exhibited reduced replication. The human fetal membrane restricted wild-type and LAV replication, and when infection occurred, it was prominent on the maternal-facing side. Type I and type III interferons were induced in human villi exposed to LAVs lacking the NSs protein. This study supports the use of sheep and human placenta explants to understand vertical transmission of RVFV in mammals and whether LAVs are attenuated at the maternal-fetal interface.IMPORTANCEA direct comparison of replication of Rift Valley fever virus (RVFV) in sheep and human placental explants reveals comparative efficiencies and permissivity to infection and replication. Vaccine strains of RVFV demonstrated reduced infection and replication capacity in the mammalian placenta. This study represents the first direct cross-host comparison of the vertical transmission capacity of this high-priority emerging mosquito-transmitted virus.
{"title":"Vaccine strains of Rift Valley fever virus exhibit attenuation at the maternal-fetal placental interface.","authors":"Cynthia M McMillen, Christina Megli, Rebecca Radisic, Lauren B Skvarca, Ryan M Hoehl, Devin A Boyles, Jackson J McGaughey, Brian H Bird, Anita K McElroy, Amy L Hartman","doi":"10.1128/jvi.00983-24","DOIUrl":"10.1128/jvi.00983-24","url":null,"abstract":"<p><p>Rift Valley fever virus (RVFV) infection causes abortions in ruminant livestock and is associated with an increased likelihood of miscarriages in women. Using sheep and human placenta explant cultures, we sought to identify tissues at the maternal-fetal interface targeted by RVFV. Sheep villi and fetal membranes were highly permissive to RVFV infection resulting in markedly higher virus titers than human cultures. Sheep cultures were most permissive to wild-type RVFV and ΔNSm infection, while live-attenuated RVFV vaccines (LAVs; MP-12, ΔNSs, and ΔNSs/ΔNSm) exhibited reduced replication. The human fetal membrane restricted wild-type and LAV replication, and when infection occurred, it was prominent on the maternal-facing side. Type I and type III interferons were induced in human villi exposed to LAVs lacking the NSs protein. This study supports the use of sheep and human placenta explants to understand vertical transmission of RVFV in mammals and whether LAVs are attenuated at the maternal-fetal interface.IMPORTANCEA direct comparison of replication of Rift Valley fever virus (RVFV) in sheep and human placental explants reveals comparative efficiencies and permissivity to infection and replication. Vaccine strains of RVFV demonstrated reduced infection and replication capacity in the mammalian placenta. This study represents the first direct cross-host comparison of the vertical transmission capacity of this high-priority emerging mosquito-transmitted virus.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11334480/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141627114","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}
Pub Date : 2024-08-20Epub Date: 2024-07-19DOI: 10.1128/jvi.00342-24
Shuyuan Qiao, Xinquan Wang
The recurrent spillovers of coronaviruses (CoVs) have posed severe threats to public health and the global economy. Bat severe acute respiratory syndrome (SARS)-like CoVs RsSHC014 and WIV1, currently circulating in bat populations, are poised for human emergence. The trimeric spike (S) glycoprotein, responsible for receptor recognition and membrane fusion, plays a critical role in cross-species transmission and infection. Here, we determined the cryo-electron microscopy (EM) structures of the RsSHC014 S protein in the closed state at 2.9 Å, the WIV1 S protein in the closed state at 2.8 Å, and the intermediate state at 4.0 Å. In the intermediate state, one receptor-binding domain (RBD) is in the "down" position, while the other two RBDs exhibit poor density. We also resolved the complex structure of the WIV1 S protein bound to human ACE2 (hACE2) at 4.5 Å, which provides structural basis for the future emergence of WIV1 in humans. Through biochemical experiments, we found that despite strong binding affinities between the RBDs and both human and civet ACE2, the pseudoviruses of RsSHC014, but not WIV1, failed to infect 293T cells overexpressing either human or civet ACE2. Mutagenesis analysis revealed that the Y623H substitution, located in the SD2 region, significantly improved the cell entry efficiency of RsSHC014 pseudoviruses, which is likely accomplished by promoting the open conformation of spike glycoproteins. Our findings emphasize the necessity of both efficient RBD lifting and tight RBD-hACE2 binding for viral infection and underscore the significance of the 623 site of the spike glycoprotein for the infectivity of bat SARS-like CoVs.
Importance: The bat SARS-like CoVs RsSHC014 and WIV1 can use hACE2 for cell entry without further adaptation, indicating their potential risk of emergence in human populations. The S glycoprotein, responsible for receptor recognition and membrane fusion, plays a crucial role in cross-species transmission and infection. In this study, we determined the cryo-EM structures of the S glycoproteins of RsSHC014 and WIV1. Detailed comparisons revealed dynamic structural variations within spike proteins. We also elucidated the complex structure of WIV1 S-hACE2, providing structural evidence for the potential emergence of WIV1 in humans. Although RsSHC014 and WIV1 had similar hACE2-binding affinities, they exhibited distinct pseudovirus cell entry behavior. Through mutagenesis and cryo-EM analysis, we revealed that besides the structural variations, the 623 site in the SD2 region is another important structural determinant of spike infectivity.
冠状病毒(CoVs)的反复蔓延对公共卫生和全球经济构成了严重威胁。类似于蝙蝠严重急性呼吸系统综合征(SARS)的冠状病毒 RsSHC014 和 WIV1 目前在蝙蝠种群中流行,并有望在人类中出现。三聚体尖峰(S)糖蛋白负责受体识别和膜融合,在跨物种传播和感染中起着关键作用。在这里,我们测定了 RsSHC014 S 蛋白在封闭状态下 2.9 Å 的低温电子显微镜(EM)结构、WIV1 S 蛋白在封闭状态下 2.8 Å 的低温电子显微镜(EM)结构以及中间状态下 4.0 Å 的低温电子显微镜(EM)结构。我们还解析了 WIV1 S 蛋白与人类 ACE2(hACE2)结合的复合物结构,该结构达到 4.5 Å,为将来 WIV1 在人类中的出现提供了结构依据。通过生化实验,我们发现尽管 RBD 与人类和果子狸 ACE2 都有很强的结合亲和力,但 RsSHC014 的假病毒却不能感染过表达人类或果子狸 ACE2 的 293T 细胞,而 WIV1 则不能。突变分析表明,位于SD2区的Y623H取代显著提高了RsSHC014假病毒进入细胞的效率,这可能是通过促进尖峰糖蛋白的开放构象实现的。我们的发现强调了病毒感染需要有效的 RBD 提升和 RBD-hACE2 紧密结合,并突出了尖峰糖蛋白的 623 位点对蝙蝠 SARS 样 CoVs 感染性的重要性:蝙蝠SARS样CoVs RsSHC014和WIV1可利用hACE2进入细胞而无需进一步适应,这表明它们有可能出现在人类群体中。S糖蛋白负责受体识别和膜融合,在跨物种传播和感染中起着至关重要的作用。在这项研究中,我们测定了 RsSHC014 和 WIV1 的 S 糖蛋白的冷冻电镜结构。详细比较显示了尖峰蛋白内部的动态结构变化。我们还阐明了 WIV1 S-hACE2 的复合结构,为 WIV1 可能在人类中出现提供了结构证据。虽然 RsSHC014 和 WIV1 具有相似的 hACE2 结合亲和力,但它们却表现出不同的伪病毒细胞进入行为。通过诱变和低温电子显微镜分析,我们发现除了结构变异外,SD2区的623位点是决定尖峰感染性的另一个重要结构因素。
{"title":"Structural determinants of spike infectivity in bat SARS-like coronaviruses RsSHC014 and WIV1.","authors":"Shuyuan Qiao, Xinquan Wang","doi":"10.1128/jvi.00342-24","DOIUrl":"10.1128/jvi.00342-24","url":null,"abstract":"<p><p>The recurrent spillovers of coronaviruses (CoVs) have posed severe threats to public health and the global economy. Bat severe acute respiratory syndrome (SARS)-like CoVs RsSHC014 and WIV1, currently circulating in bat populations, are poised for human emergence. The trimeric spike (S) glycoprotein, responsible for receptor recognition and membrane fusion, plays a critical role in cross-species transmission and infection. Here, we determined the cryo-electron microscopy (EM) structures of the RsSHC014 S protein in the closed state at 2.9 Å, the WIV1 S protein in the closed state at 2.8 Å, and the intermediate state at 4.0 Å. In the intermediate state, one receptor-binding domain (RBD) is in the \"down\" position, while the other two RBDs exhibit poor density. We also resolved the complex structure of the WIV1 S protein bound to human ACE2 (hACE2) at 4.5 Å, which provides structural basis for the future emergence of WIV1 in humans. Through biochemical experiments, we found that despite strong binding affinities between the RBDs and both human and civet ACE2, the pseudoviruses of RsSHC014, but not WIV1, failed to infect 293T cells overexpressing either human or civet ACE2. Mutagenesis analysis revealed that the Y623H substitution, located in the SD2 region, significantly improved the cell entry efficiency of RsSHC014 pseudoviruses, which is likely accomplished by promoting the open conformation of spike glycoproteins. Our findings emphasize the necessity of both efficient RBD lifting and tight RBD-hACE2 binding for viral infection and underscore the significance of the 623 site of the spike glycoprotein for the infectivity of bat SARS-like CoVs.</p><p><strong>Importance: </strong>The bat SARS-like CoVs RsSHC014 and WIV1 can use hACE2 for cell entry without further adaptation, indicating their potential risk of emergence in human populations. The S glycoprotein, responsible for receptor recognition and membrane fusion, plays a crucial role in cross-species transmission and infection. In this study, we determined the cryo-EM structures of the S glycoproteins of RsSHC014 and WIV1. Detailed comparisons revealed dynamic structural variations within spike proteins. We also elucidated the complex structure of WIV1 S-hACE2, providing structural evidence for the potential emergence of WIV1 in humans. Although RsSHC014 and WIV1 had similar hACE2-binding affinities, they exhibited distinct pseudovirus cell entry behavior. Through mutagenesis and cryo-EM analysis, we revealed that besides the structural variations, the 623 site in the SD2 region is another important structural determinant of spike infectivity.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11334503/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141723825","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}
Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, is a serious threat to piglets and has zoonotic potential. Here, we aimed to further explore the role of aminopeptidase N (APN) as a receptor for PDCoV and test the inhibitory effect of a chimeric APN protein strategy on PDCoV infection. PK-15 cells and LLC-PK1 cells expressing chimeric APN were selected and infected with PDCoV. Viral replication was significantly decreased in these chimeric APN cells compared with that in control group cells. To further characterize the effect of the chimeric APN strategy on PDCoV infection in vitro, primary intestinal epithelial cells isolated from chimeric APN pigs were inoculated with PDCoV. Viral challenge of these cells led to decreased PDCoV infection. More importantly, virally challenged chimeric APN neonatal piglets displayed reduced viral load, significantly fewer microscopic lesions in the intestinal tissue, and no diarrhea. Taken together, these findings deepen our understanding of the mechanism of PDCoV infection and provide a valuable model for the production of disease-resistant animals.
Importance: Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhea in piglets and possesses the potential to infect humans. However, there are currently no effective measures for the prevention or control of PDCoV infection. Here, we have developed PK-15 cells, LLC-PK1 cells, and primary intestinal epithelial cells expressing chimeric APN, and viral challenge of these cells led to decreased PDCoV infection. Furthermore, virally challenged chimeric APN neonatal piglets displayed reduced viral load, significantly fewer microscopic lesions in the intestinal tissue, and no diarrhea. These data show that chimeric APN is a promising strategy to combat PDCoV infection.
{"title":"Effective inhibition of PDCoV infection in chimeric APN gene-edited neonatal pigs.","authors":"Jizong Li, Jian Zhou, Tianyi Zhang, Heyong Wu, Feng Li, Chunyun Qi, Liyuan Fan, Xuesong Yuan, Wei Wang, Rongli Guo, Baochao Fan, Xiaochun Tang, Daxin Pang, Hongsheng Ouyang, Zicong Xie, Bin Li","doi":"10.1128/jvi.00611-24","DOIUrl":"10.1128/jvi.00611-24","url":null,"abstract":"<p><p>Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, is a serious threat to piglets and has zoonotic potential. Here, we aimed to further explore the role of aminopeptidase N (APN) as a receptor for PDCoV and test the inhibitory effect of a chimeric APN protein strategy on PDCoV infection. PK-15 cells and LLC-PK1 cells expressing chimeric APN were selected and infected with PDCoV. Viral replication was significantly decreased in these chimeric APN cells compared with that in control group cells. To further characterize the effect of the chimeric APN strategy on PDCoV infection <i>in vitro</i>, primary intestinal epithelial cells isolated from chimeric APN pigs were inoculated with PDCoV. Viral challenge of these cells led to decreased PDCoV infection. More importantly, virally challenged chimeric APN neonatal piglets displayed reduced viral load, significantly fewer microscopic lesions in the intestinal tissue, and no diarrhea. Taken together, these findings deepen our understanding of the mechanism of PDCoV infection and provide a valuable model for the production of disease-resistant animals.</p><p><strong>Importance: </strong>Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhea in piglets and possesses the potential to infect humans. However, there are currently no effective measures for the prevention or control of PDCoV infection. Here, we have developed PK-15 cells, LLC-PK1 cells, and primary intestinal epithelial cells expressing chimeric APN, and viral challenge of these cells led to decreased PDCoV infection. Furthermore, virally challenged chimeric APN neonatal piglets displayed reduced viral load, significantly fewer microscopic lesions in the intestinal tissue, and no diarrhea. These data show that chimeric APN is a promising strategy to combat PDCoV infection.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11334500/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141792790","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}
Pub Date : 2024-08-20Epub Date: 2024-07-30DOI: 10.1128/jvi.00781-24
Kristin B Wiggins, Stephen M Winston, Isaiah L Reeves, Jessica Gaevert, Yunyu Spence, Mark A Brimble, Brandi Livingston, Christopher L Morton, Paul G Thomas, Andrea J Sant, Ted M Ross, Andrew M Davidoff, Stacey Schultz-Cherry
Influenza remains a worldwide public health threat. Although seasonal influenza vaccines are currently the best means of preventing severe disease, the standard-of-care vaccines require frequent updating due to antigenic drift and can have low efficacy, particularly in vulnerable populations. Here, we demonstrate that a single administration of a recombinant adenovirus-associated virus (rAAV) vector expressing a computationally optimized broadly reactive antigen (COBRA)-derived influenza H1 hemagglutinin (HA) induces strongly neutralizing and broadly protective antibodies in naïve mice and ferrets with pre-existing influenza immunity. Following a lethal viral challenge, the rAAV-COBRA vaccine allowed for significantly reduced viral loads in the upper and lower respiratory tracts and complete protection from morbidity and mortality that lasted for at least 5 months post-vaccination. We observed no signs of antibody waning during this study. CpG motif enrichment of the antigen can act as an internal adjuvant to further enhance the immune responses to allow for lower vaccine dosages with the induction of unique interferon-producing CD4+ and CD8+ T cells specific to HA head and stem peptide sequences. Our studies highlight the utility of rAAV as an effective platform to improve seasonal influenza vaccines.
Importance: Developing an improved seasonal influenza vaccine remains an ambitious goal of researchers and clinicians alike. With influenza routinely causing severe epidemics with the potential to rise to pandemic levels, it is critical to create an effective, broadly protective, and durable vaccine to improve public health worldwide. As a potential solution, we created a rAAV viral vector expressing a COBRA-optimized influenza hemagglutinin antigen with modestly enriched CpG motifs to evoke a robust and long-lasting immune response after a single intramuscular dose without needing boosts or adjuvants. Importantly, the rAAV vaccine boosted antibody breadth to future strains in ferrets with pre-existing influenza immunity. Together, our data support further investigation into the utility of viral vectors as a potential avenue to improve our seasonal influenza vaccines.
{"title":"rAAV expressing a COBRA-designed influenza hemagglutinin generates a protective and durable adaptive immune response with a single dose.","authors":"Kristin B Wiggins, Stephen M Winston, Isaiah L Reeves, Jessica Gaevert, Yunyu Spence, Mark A Brimble, Brandi Livingston, Christopher L Morton, Paul G Thomas, Andrea J Sant, Ted M Ross, Andrew M Davidoff, Stacey Schultz-Cherry","doi":"10.1128/jvi.00781-24","DOIUrl":"10.1128/jvi.00781-24","url":null,"abstract":"<p><p>Influenza remains a worldwide public health threat. Although seasonal influenza vaccines are currently the best means of preventing severe disease, the standard-of-care vaccines require frequent updating due to antigenic drift and can have low efficacy, particularly in vulnerable populations. Here, we demonstrate that a single administration of a recombinant adenovirus-associated virus (rAAV) vector expressing a computationally optimized broadly reactive antigen (COBRA)-derived influenza H1 hemagglutinin (HA) induces strongly neutralizing and broadly protective antibodies in naïve mice and ferrets with pre-existing influenza immunity. Following a lethal viral challenge, the rAAV-COBRA vaccine allowed for significantly reduced viral loads in the upper and lower respiratory tracts and complete protection from morbidity and mortality that lasted for at least 5 months post-vaccination. We observed no signs of antibody waning during this study. CpG motif enrichment of the antigen can act as an internal adjuvant to further enhance the immune responses to allow for lower vaccine dosages with the induction of unique interferon-producing CD4+ and CD8+ T cells specific to HA head and stem peptide sequences. Our studies highlight the utility of rAAV as an effective platform to improve seasonal influenza vaccines.</p><p><strong>Importance: </strong>Developing an improved seasonal influenza vaccine remains an ambitious goal of researchers and clinicians alike. With influenza routinely causing severe epidemics with the potential to rise to pandemic levels, it is critical to create an effective, broadly protective, and durable vaccine to improve public health worldwide. As a potential solution, we created a rAAV viral vector expressing a COBRA-optimized influenza hemagglutinin antigen with modestly enriched CpG motifs to evoke a robust and long-lasting immune response after a single intramuscular dose without needing boosts or adjuvants. Importantly, the rAAV vaccine boosted antibody breadth to future strains in ferrets with pre-existing influenza immunity. Together, our data support further investigation into the utility of viral vectors as a potential avenue to improve our seasonal influenza vaccines.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11338075/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141792832","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}
Pub Date : 2024-08-20Epub Date: 2024-07-15DOI: 10.1128/jvi.00775-24
Judith M Reyes Ballista, Ashley J Hoover, Joseph T Noble, Marissa D Acciani, Kerri L Miazgowicz, Sarah A Harrison, Grace Andrea L Tabscott, Avery Duncan, Don N Barnes, Ariana R Jimenez, Melinda A Brindley
T-cell immunoglobin and mucin domain protein-1 (TIM-1) mediates entry of chikungunya virus (CHIKV) into some mammalian cells through the interaction with envelope phospholipids. While this interaction enhances entry, TIM-1 has been shown to tether newly formed HIV and Ebola virus particles, limiting their efficient release. In this study, we investigate the ability of surface receptors such as TIM-1 to sequester newly budded virions on the surface of infected cells. We established a luminescence reporter system to produce chikungunya viral particles that integrate nano-luciferase and easily quantify viral particles. We found that TIM-1 on the surface of host cells significantly reduced CHIKV release efficiency in comparison to other entry factors. Removal of cell surface TIM-1 through direct cellular knock-out or altering the cellular lipid distribution enhanced CHIKV release. Over the course of infection, CHIKV was able to counteract the tethering effect by gradually decreasing the surface levels of TIM-1 in a process mediated by the nonstructural protein 2. This study highlights the importance of phosphatidylserine receptors in mediating not only the entry of CHIKV but also its release and could aid in developing cell lines capable of enhanced vaccine production.
Importance: Chikungunya virus (CHIKV) is an enveloped alphavirus transmitted by the bites of infectious mosquitoes. Infection with CHIKV results in the development of fever, joint pain, and arthralgia that can become chronic and last for months after infection. Prevention of this disease is still highly focused on vector control strategies. In December 2023, a new live attenuated vaccine against CHIKV was approved by the FDA. We aimed to study the cellular factors involved in CHIKV release, to better understand CHIKV's ability to efficiently infect and spread among a wide variety of cell lines. We found that TIM-1 receptors can significantly abrogate CHIKV's ability to efficiently exit infected cells. This information can be beneficial for maximizing viral particle production in laboratory settings and during vaccine manufacturing.
{"title":"Chikungunya virus release is reduced by TIM-1 receptors through binding of envelope phosphatidylserine.","authors":"Judith M Reyes Ballista, Ashley J Hoover, Joseph T Noble, Marissa D Acciani, Kerri L Miazgowicz, Sarah A Harrison, Grace Andrea L Tabscott, Avery Duncan, Don N Barnes, Ariana R Jimenez, Melinda A Brindley","doi":"10.1128/jvi.00775-24","DOIUrl":"10.1128/jvi.00775-24","url":null,"abstract":"<p><p>T-cell immunoglobin and mucin domain protein-1 (TIM-1) mediates entry of chikungunya virus (CHIKV) into some mammalian cells through the interaction with envelope phospholipids. While this interaction enhances entry, TIM-1 has been shown to tether newly formed HIV and Ebola virus particles, limiting their efficient release. In this study, we investigate the ability of surface receptors such as TIM-1 to sequester newly budded virions on the surface of infected cells. We established a luminescence reporter system to produce chikungunya viral particles that integrate nano-luciferase and easily quantify viral particles. We found that TIM-1 on the surface of host cells significantly reduced CHIKV release efficiency in comparison to other entry factors. Removal of cell surface TIM-1 through direct cellular knock-out or altering the cellular lipid distribution enhanced CHIKV release. Over the course of infection, CHIKV was able to counteract the tethering effect by gradually decreasing the surface levels of TIM-1 in a process mediated by the nonstructural protein 2. This study highlights the importance of phosphatidylserine receptors in mediating not only the entry of CHIKV but also its release and could aid in developing cell lines capable of enhanced vaccine production.</p><p><strong>Importance: </strong>Chikungunya virus (CHIKV) is an enveloped alphavirus transmitted by the bites of infectious mosquitoes. Infection with CHIKV results in the development of fever, joint pain, and arthralgia that can become chronic and last for months after infection. Prevention of this disease is still highly focused on vector control strategies. In December 2023, a new live attenuated vaccine against CHIKV was approved by the FDA. We aimed to study the cellular factors involved in CHIKV release, to better understand CHIKV's ability to efficiently infect and spread among a wide variety of cell lines. We found that TIM-1 receptors can significantly abrogate CHIKV's ability to efficiently exit infected cells. This information can be beneficial for maximizing viral particle production in laboratory settings and during vaccine manufacturing.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11334481/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141616750","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}
Pub Date : 2024-08-20Epub Date: 2024-07-17DOI: 10.1128/jvi.01046-24
Yunrong Gao, Anirudh Raghavan, Sara Andrea Espinosa Garcia, Bowei Deng, Diego Hurtado de Mendoza, Bo Liang
The respiratory syncytial virus (RSV) M2-1 protein is a transcriptional antitermination factor crucial for efficiently synthesizing multiple full-length viral mRNAs. During RSV infection, M2-1 exists in a complex with mRNA within cytoplasmic compartments called inclusion body-associated granules (IBAGs). Prior studies showed that M2-1 can bind along the entire length of viral mRNAs instead of just gene-end (GE) sequences, suggesting that M2-1 has more sophisticated RNA recognition and binding characteristics. Here, we analyzed the higher oligomeric complexes formed by M2-1 and RNAs in vitro using size exclusion chromatography (SEC), electrophoretic mobility shift assays (EMSA), negative stain electron microscopy (EM), and mutagenesis. We observed that the minimal RNA length for such higher oligomeric assembly is about 14 nucleotides for polyadenine sequences, and longer RNAs exhibit distinct RNA-induced binding modality to M2-1, leading to enhanced particle formation frequency and particle homogeneity as the local RNA concentration increases. We showed that particular cysteine residues of the M2-1 cysteine-cysteine-cystine-histidine (CCCH) zinc-binding motif are essential for higher oligomeric assembly. Furthermore, complexes assembled with long polyadenine sequences remain unaffected when co-incubated with ribonucleases or a zinc chelation agent. Our study provided new insights into the higher oligomeric assembly of M2-1 with longer RNA.IMPORTANCERespiratory syncytial virus (RSV) causes significant respiratory infections in infants, the elderly, and immunocompromised individuals. The virus forms specialized compartments to produce genetic material, with the M2-1 protein playing a pivotal role. M2-1 acts as an anti-terminator in viral transcription, ensuring the creation of complete viral mRNA and associating with both viral and cellular mRNA. Our research focuses on understanding M2-1's function in viral mRNA synthesis by modeling interactions in a controlled environment. This approach is crucial due to the challenges of studying these compartments in vivo. Reconstructing the system in vitro uncovers structural and biochemical aspects and reveals the potential functions of M2-1 and its homologs in related viruses. Our work may contribute to identifying targets for antiviral inhibitors and advancing RSV infection treatment.
{"title":"<i>In vitro</i> higher-order oligomeric assembly of the respiratory syncytial virus M2-1 protein with longer RNAs.","authors":"Yunrong Gao, Anirudh Raghavan, Sara Andrea Espinosa Garcia, Bowei Deng, Diego Hurtado de Mendoza, Bo Liang","doi":"10.1128/jvi.01046-24","DOIUrl":"10.1128/jvi.01046-24","url":null,"abstract":"<p><p>The respiratory syncytial virus (RSV) M2-1 protein is a transcriptional antitermination factor crucial for efficiently synthesizing multiple full-length viral mRNAs. During RSV infection, M2-1 exists in a complex with mRNA within cytoplasmic compartments called inclusion body-associated granules (IBAGs). Prior studies showed that M2-1 can bind along the entire length of viral mRNAs instead of just gene-end (GE) sequences, suggesting that M2-1 has more sophisticated RNA recognition and binding characteristics. Here, we analyzed the higher oligomeric complexes formed by M2-1 and RNAs <i>in vitro</i> using size exclusion chromatography (SEC), electrophoretic mobility shift assays (EMSA), negative stain electron microscopy (EM), and mutagenesis. We observed that the minimal RNA length for such higher oligomeric assembly is about 14 nucleotides for polyadenine sequences, and longer RNAs exhibit distinct RNA-induced binding modality to M2-1, leading to enhanced particle formation frequency and particle homogeneity as the local RNA concentration increases. We showed that particular cysteine residues of the M2-1 cysteine-cysteine-cystine-histidine (CCCH) zinc-binding motif are essential for higher oligomeric assembly. Furthermore, complexes assembled with long polyadenine sequences remain unaffected when co-incubated with ribonucleases or a zinc chelation agent. Our study provided new insights into the higher oligomeric assembly of M2-1 with longer RNA.IMPORTANCERespiratory syncytial virus (RSV) causes significant respiratory infections in infants, the elderly, and immunocompromised individuals. The virus forms specialized compartments to produce genetic material, with the M2-1 protein playing a pivotal role. M2-1 acts as an anti-terminator in viral transcription, ensuring the creation of complete viral mRNA and associating with both viral and cellular mRNA. Our research focuses on understanding M2-1's function in viral mRNA synthesis by modeling interactions in a controlled environment. This approach is crucial due to the challenges of studying these compartments <i>in vivo</i>. Reconstructing the system <i>in vitro</i> uncovers structural and biochemical aspects and reveals the potential functions of M2-1 and its homologs in related viruses. Our work may contribute to identifying targets for antiviral inhibitors and advancing RSV infection treatment.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11334520/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141627112","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}
Pub Date : 2024-08-20Epub Date: 2024-07-18DOI: 10.1128/jvi.00570-24
Xinghua Yu, Tingkai Teng, Zhuowen Duan, Jinwen Wang
Virus-encoded microRNAs (miRNAs) exert diverse regulatory roles in the biological processes of both viruses and hosts. This study delves into the functions of AcMNPV-miR-2, an early miRNA encoded by Autographa californica multiple nucleopolyhedrovirus (AcMNPV). AcMNPV-miR-2 targets viral early genes ac28 (lef-6), ac37 (lef-11), ac49, and ac63. Overexpression of AcMNPV-miR-2 leads to reduced production of infectious budded virions (BVs) and diminished viral DNA replication. Delayed polyhedron formation was observed through light and transmission electron microscopy, and the larval lifespan extended in oral infection assays. Moreover, the mRNA expression levels of two Lepidoptera-specific immune-related proteins, Gloverin and Spod-11-tox, significantly decreased. These findings indicate that AcMNPV-miR-2 restrains viral load, reducing host immune sensitivity. This beneficial effect enables the virus to combat host defense mechanisms and reside within the host for an extended duration.
Importance: Virus-encoded miRNAs have been extensively studied for their pivotal roles in finetuning viral infections. Baculoviruses, highly pathogenic in insects, remain underexplored concerning their encoded miRNAs. Previous reports outlined three AcMNPV-encoded miRNAs, AcMNPV-miR-1, -miR-3, and -miR-4. This study delves into the functions of another AcMNPV-encoded miRNA, AcMNPV-miR-2 (Ac-miR-2). Through a comprehensive analysis of target gene expression, the impact on larvae, and variations in host immune-related gene expression, we elucidate a functional pathway for Ac-miR-2. This miRNA suppresses viral load and infectivity and prolongs lifespans of infected larva by downregulating specific viral early genes and host immune-related genes. These mechanisms ultimately serve the virus's primary goal of enhanced propagation. Our study significantly contributes to understanding of the intricate regulatory mechanisms of virus-encoded miRNAs in baculovirus infections.
{"title":"AcMNPV-miR-2 affects <i>Autographa californica</i> nucleopolyhedrovirus infection by regulating the expression of <i>ac28</i> and several other viral early genes.","authors":"Xinghua Yu, Tingkai Teng, Zhuowen Duan, Jinwen Wang","doi":"10.1128/jvi.00570-24","DOIUrl":"10.1128/jvi.00570-24","url":null,"abstract":"<p><p>Virus-encoded microRNAs (miRNAs) exert diverse regulatory roles in the biological processes of both viruses and hosts. This study delves into the functions of AcMNPV-miR-2, an early miRNA encoded by <i>Autographa californica</i> multiple nucleopolyhedrovirus (AcMNPV). AcMNPV-miR-2 targets viral early genes <i>ac28</i> (<i>lef-6</i>), <i>ac37</i> (<i>lef-11</i>), <i>ac49</i>, and <i>ac63</i>. Overexpression of AcMNPV-miR-2 leads to reduced production of infectious budded virions (BVs) and diminished viral DNA replication. Delayed polyhedron formation was observed through light and transmission electron microscopy, and the larval lifespan extended in oral infection assays. Moreover, the mRNA expression levels of two Lepidoptera-specific immune-related proteins, Gloverin and Spod-11-tox, significantly decreased. These findings indicate that AcMNPV-miR-2 restrains viral load, reducing host immune sensitivity. This beneficial effect enables the virus to combat host defense mechanisms and reside within the host for an extended duration.</p><p><strong>Importance: </strong>Virus-encoded miRNAs have been extensively studied for their pivotal roles in finetuning viral infections. Baculoviruses, highly pathogenic in insects, remain underexplored concerning their encoded miRNAs. Previous reports outlined three AcMNPV-encoded miRNAs, AcMNPV-miR-1, -miR-3, and -miR-4. This study delves into the functions of another AcMNPV-encoded miRNA, AcMNPV-miR-2 (Ac-miR-2). Through a comprehensive analysis of target gene expression, the impact on larvae, and variations in host immune-related gene expression, we elucidate a functional pathway for Ac-miR-2. This miRNA suppresses viral load and infectivity and prolongs lifespans of infected larva by downregulating specific viral early genes and host immune-related genes. These mechanisms ultimately serve the virus's primary goal of enhanced propagation. Our study significantly contributes to understanding of the intricate regulatory mechanisms of virus-encoded miRNAs in baculovirus infections.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11334470/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141633823","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}
Pub Date : 2024-08-20Epub Date: 2024-07-30DOI: 10.1128/jvi.00858-24
Arundhati Deb, Shilpi Nagpal, Rajnesh Kumari Yadav, Harsh Thakur, Deepak Nair, Vengadesan Krishnan, Sudhanshu Vrati
Japanese encephalitis virus (JEV) is an arthropod-borne, plus-strand flavivirus causing viral encephalitis in humans with a high case fatality rate. The JEV non-structural protein 5 (NS5) with the RNA-dependent RNA polymerase activity interacts with the viral and host proteins to constitute the replication complex. We have identified the multifunctional protein Nucleolin (NCL) as one of the several NS5-interacting host proteins. We demonstrate the interaction and colocalization of JEV NS5 with NCL in the virus-infected HeLa cells. The siRNA-mediated knockdown of NCL indicated that it was required for efficient viral replication. Importantly, JEV grew to higher titers in cells over-expressing exogenous NCL, demonstrating its pro-viral role. We demonstrated that NS5 interacted with the RRM and GAR domains of NCL. We show that the NCL-binding aptamer AS1411 containing the G-quadruplex (GQ) structure and the GQ ligand BRACO-19 caused significant inhibition of JEV replication. The antiviral effect of AS1411 and BRACO-19 could be overcome in HeLa cells by the overexpression of exogenous NCL. We demonstrated that the synthetic RNAs derived from the 3'-NCR of JEV genomic RNA containing the GQ sequence could bind NCL in vitro. The replication complex binding to the 3'-NCR is required for the viral RNA synthesis. It is likely that NCL present in the replication complex destabilizes the GQ structures in the genomic RNA, thus facilitating the movement of the replication complex resulting in efficient virus replication.IMPORTANCEJapanese encephalitis virus (JEV) is endemic in most parts of South-East Asia and the Western Pacific region, causing epidemics of encephalitis with a high case fatality rate. While a tissue culture-derived JEV vaccine is available, no antiviral therapy exists. The JEV NS5 protein has RNA-dependent RNA polymerase activity. Together with several host and viral proteins, it constitutes the replication complex necessary for virus replication. Understanding the interaction of NS5 with the host proteins could help design novel antivirals. We identified Nucleolin (NCL) as a crucial host protein interactor of JEV NS5 having a pro-viral role in virus replication. The NS5-interacting NCL binds to the G-quadruplex (GQ) structure sequence in the 3'-NCR of JEV RNA. This may smoothen the movement of the replication complex along the genomic RNA, thereby facilitating the virus replication. This study is the first report on how NCL, a host protein, helps in JEV replication through GQ-binding.
{"title":"Japanese encephalitis virus NS5 protein interacts with nucleolin to enhance the virus replication.","authors":"Arundhati Deb, Shilpi Nagpal, Rajnesh Kumari Yadav, Harsh Thakur, Deepak Nair, Vengadesan Krishnan, Sudhanshu Vrati","doi":"10.1128/jvi.00858-24","DOIUrl":"10.1128/jvi.00858-24","url":null,"abstract":"<p><p>Japanese encephalitis virus (JEV) is an arthropod-borne, plus-strand flavivirus causing viral encephalitis in humans with a high case fatality rate. The JEV non-structural protein 5 (NS5) with the RNA-dependent RNA polymerase activity interacts with the viral and host proteins to constitute the replication complex. We have identified the multifunctional protein Nucleolin (NCL) as one of the several NS5-interacting host proteins. We demonstrate the interaction and colocalization of JEV NS5 with NCL in the virus-infected HeLa cells. The siRNA-mediated knockdown of NCL indicated that it was required for efficient viral replication. Importantly, JEV grew to higher titers in cells over-expressing exogenous NCL, demonstrating its pro-viral role. We demonstrated that NS5 interacted with the RRM and GAR domains of NCL. We show that the NCL-binding aptamer AS1411 containing the G-quadruplex (GQ) structure and the GQ ligand BRACO-19 caused significant inhibition of JEV replication. The antiviral effect of AS1411 and BRACO-19 could be overcome in HeLa cells by the overexpression of exogenous NCL. We demonstrated that the synthetic RNAs derived from the 3'-NCR of JEV genomic RNA containing the GQ sequence could bind NCL <i>in vitro</i>. The replication complex binding to the 3'-NCR is required for the viral RNA synthesis. It is likely that NCL present in the replication complex destabilizes the GQ structures in the genomic RNA, thus facilitating the movement of the replication complex resulting in efficient virus replication.IMPORTANCEJapanese encephalitis virus (JEV) is endemic in most parts of South-East Asia and the Western Pacific region, causing epidemics of encephalitis with a high case fatality rate. While a tissue culture-derived JEV vaccine is available, no antiviral therapy exists. The JEV NS5 protein has RNA-dependent RNA polymerase activity. Together with several host and viral proteins, it constitutes the replication complex necessary for virus replication. Understanding the interaction of NS5 with the host proteins could help design novel antivirals. We identified Nucleolin (NCL) as a crucial host protein interactor of JEV NS5 having a pro-viral role in virus replication. The NS5-interacting NCL binds to the G-quadruplex (GQ) structure sequence in the 3'-NCR of JEV RNA. This may smoothen the movement of the replication complex along the genomic RNA, thereby facilitating the virus replication. This study is the first report on how NCL, a host protein, helps in JEV replication through GQ-binding.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11334521/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141792830","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}
Pub Date : 2024-08-20Epub Date: 2024-07-16DOI: 10.1128/jvi.01242-23
Mable Chan, Bryce M Warner, Jonathan Audet, Douglas Barker, Nikesh Tailor, Robert Vendramelli, Thang Truong, Kevin Tierney, Amrit S Boese, Honguy Qiu, Frederick W Holtsberg, Javad Aman, Shantha Kodihalli, Darwyn Kobasa
Sudan ebolavirus (SUDV) is a member of the genus Ebolavirus (Family Filoviridae) and has caused sporadic outbreaks of Ebola disease (EBOD), or more specifically Sudan virus disease (SVD), with high mortality rates in Africa. Current vaccines and therapies that have been developed for filoviruses are almost all specific for Ebola virus (EBOV; of the species Zaire ebolavirus), and there is a current lack of therapeutics specific for SUDV. The recent SUDV outbreak in Uganda, which was distributed across multiple districts, including Kampala, a densely populated urban center, highlights the critical need for the development of novel SUDV-specific or pan-Ebola virus therapeutics. Previous work has characterized two monoclonal antibodies, FVM04 and CA45, which have neutralization capabilities against both EBOV and SUDV and have shown protective efficacy in animal challenge studies. Here, we expand upon this work, showing that treatment with a monoclonal antibody cocktail consisting of FVM04 and CA45 provides full protection against lethal SUDV infection in cynomolgus macaques. Studies that evaluate outcomes at late time points after infection, once clinical signs of illness are apparent, are vital for assessing the therapeutic efficacy of antibody therapeutics. We have shown that when treatment is initiated as late as 5 days after infection, with a second dose given on day 8, that treated groups showed few clinical signs or morbidity, with complete survival. This work provides further evidence that FVM04 and CA45 have strong therapeutic potential against SUDV and their development as a pan-Ebola virus therapeutic should be pursued.
Importance: There are currently no approved vaccines or therapeutics for Sudan virus, a filovirus which is highly related to Ebola virus and causes similar disease and outbreaks. In this study, a cocktail of two potent monoclonal antibodies that effectively neutralize Sudan virus was tested in a nonhuman primate model of Sudan virus disease. Treatment was highly effective, even when initiated as late as 5 days after infection, when clinical signs of infection were already evident. All treated animals showed complete recovery from infection, with little evidence of disease, while all animals that received a control treatment succumbed to infection within 8 days. The study further demonstrated the strong therapeutic potential of the antibody treatment and supported further development for use in Sudan virus outbreaks.
{"title":"Delayed treatment of cynomolgus macaques with a FVM04/CA45 monoclonal antibody cocktail provides complete protection against lethal Sudan virus infection.","authors":"Mable Chan, Bryce M Warner, Jonathan Audet, Douglas Barker, Nikesh Tailor, Robert Vendramelli, Thang Truong, Kevin Tierney, Amrit S Boese, Honguy Qiu, Frederick W Holtsberg, Javad Aman, Shantha Kodihalli, Darwyn Kobasa","doi":"10.1128/jvi.01242-23","DOIUrl":"10.1128/jvi.01242-23","url":null,"abstract":"<p><p>Sudan ebolavirus (SUDV) is a member of the genus <i>Ebolavirus</i> (Family <i>Filoviridae</i>) and has caused sporadic outbreaks of Ebola disease (EBOD), or more specifically Sudan virus disease (SVD), with high mortality rates in Africa. Current vaccines and therapies that have been developed for filoviruses are almost all specific for Ebola virus (EBOV; of the species <i>Zaire ebolavirus</i>), and there is a current lack of therapeutics specific for SUDV. The recent SUDV outbreak in Uganda, which was distributed across multiple districts, including Kampala, a densely populated urban center, highlights the critical need for the development of novel SUDV-specific or pan-Ebola virus therapeutics. Previous work has characterized two monoclonal antibodies, FVM04 and CA45, which have neutralization capabilities against both EBOV and SUDV and have shown protective efficacy in animal challenge studies. Here, we expand upon this work, showing that treatment with a monoclonal antibody cocktail consisting of FVM04 and CA45 provides full protection against lethal SUDV infection in cynomolgus macaques. Studies that evaluate outcomes at late time points after infection, once clinical signs of illness are apparent, are vital for assessing the therapeutic efficacy of antibody therapeutics. We have shown that when treatment is initiated as late as 5 days after infection, with a second dose given on day 8, that treated groups showed few clinical signs or morbidity, with complete survival. This work provides further evidence that FVM04 and CA45 have strong therapeutic potential against SUDV and their development as a pan-Ebola virus therapeutic should be pursued.</p><p><strong>Importance: </strong>There are currently no approved vaccines or therapeutics for Sudan virus, a filovirus which is highly related to Ebola virus and causes similar disease and outbreaks. In this study, a cocktail of two potent monoclonal antibodies that effectively neutralize Sudan virus was tested in a nonhuman primate model of Sudan virus disease. Treatment was highly effective, even when initiated as late as 5 days after infection, when clinical signs of infection were already evident. All treated animals showed complete recovery from infection, with little evidence of disease, while all animals that received a control treatment succumbed to infection within 8 days. The study further demonstrated the strong therapeutic potential of the antibody treatment and supported further development for use in Sudan virus outbreaks.</p>","PeriodicalId":17583,"journal":{"name":"Journal of Virology","volume":null,"pages":null},"PeriodicalIF":4.0,"publicationDate":"2024-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11334508/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141620281","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}