ROLE OF NONSTRUCTURAL PROTEINS IN PESTIVIRUS ASSEMBLY

非结构蛋白在瘟病毒组装中的作用

• 批准号: 7715843
• 负责人: Arash Grakoui
• 金额: $ 3.56万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7715843
• 关键词: Affect; Apoptosis; Cell Survival; Cells; Chimera organism; Code; Computer Retrieval of Information on Scientific Projects Database; Cultured Cells; Engineering; Funding; Genes; Genome; Genotype; Grant; Hepatitis C virus; Infection; Infectious hepatitides; Institution; Length; Mutation; Nonstructural Protein; Nucleotides; Pestivirus; Pliability; Process; Production; Proteins; Recombinants; Reporting; Research; Research Personnel; Resources; Role; Source; Translations; United States National Institutes of Health; Viral; Virion; Virus; Virus Replication; cell growth; particle; viral RNA

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The full-length hepatitis C virus (HCV) JFH1 genome (genotype 2a) has been shown to produce moderate titers of infectious particles in cell culture but the optimal determinants required for virion production are unclear. It has been previously shown that intragenotypic recombinants that encode from core up to NS2 from J6CF in the context of JFH1 are more robust in the release of viral particles. To understand the contributions of structural and nonstructural genes to HCV replication potential and infectivity, we have characterized several intragenotypic recombinant genotype 2a viruses with different portions of the J6 isolate engineered into the JFH1 infectious clone. All genomes produced high levels of intracellular HCV RNA and NS3 protein in Huh7.5 transfected cells. However, JFH1 genomes containing J6 sequences from C to E2 (CE2) or C to p7 (Cp7) secreted up to 100-fold more infectious HCV particles than the parental JFH1 clone. Subsequent infection of na¿ve Huh7.5 cells with each of the J6/JFH1 recombinants at a multiplicity of infection of 0.0003 resulted in high viral titers only for CE2 and Cp7 viruses. Comparison of virion production by the Cp7 J6/JFH1 recombinant to previously described J6/JFH1 recombinants showed flexibility of the chimeric junction. Moreover NTRNS2 chimeric virus, which is equivalent to the previously reported FL-J6/JFH chimera, showed a 10- fold enhancement of virus titers compared to CNS2. Since there were no significant differences in terms of translation and replication between these two clones, it is possible that these three mutations may affect post-translational processes leading to an increase in virion production. Sequence comparisons between these two clones showed that NTRNS2 had three nucleotide differences that differed from CNS2. These mutations residing in the 5'NTR and core coding sequence had no effect on virion production. Importantly, we demonstrated that cells replicating and producing Cp7 virus showed decreased cell growth and increased apoptosis compared with JFH1, an effect that correlated with robust virion production. These studies begin to unravel the requirements for robust virus replication and the relationship between increased virion production and host cell viability.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 全长丙型肝炎病毒（HCV）JFH1基因组（基因型2A）已显示出在细胞培养中产生中等的传染性颗粒滴度，但病毒产生所需的最佳认同尚不清楚。以前已经表明，在JFH1的背景下，从核心到NS2编码的内部型重组因素在病毒颗粒的释放中更强大。 为了了解结构和非结构基因对HCV复制潜力和感染的贡献，我们表征了几种基因内型重组基因型2A病毒，其中具有不同部分的J6分离株，该病毒被JFH1感染的克隆。所有基因组在HUH7.5翻译细胞中产生高水平的细胞内HCV RNA和NS3蛋白。但是，与亲属JFH1克隆相比，含有从C到E2或C到P7（CP7）的J6序列的JFH1基因组分泌高达100倍。随后在每种J6/JFH1重组剂中以多种感染为0.0003的J6/JFH1重组者中的Na�VeHuh7.5细胞的后续感染导致CE2和CP7病毒的高病毒滴度。将CP7 J6/JFH1重组因子与先前描述的J6/JFH1重组剂的病毒产生比较显示了嵌合结的灵活性。 此外，与CNS2相比，NTRNS2嵌合病毒相当于先前报道的FL-J6/JFH嵌合体，病毒滴度的增强10倍。由于这两个克隆之间的翻译和复制方面没有显着差异，因此这三个突变可能会影响翻译后过程，从而导致病毒产生增加。这两个克隆之间的序列比较表明，NTRNS2具有与CNS2不同的三个核苷酸差异。这些驻留在5'NTR和核心编码序列中的突变对病毒体的产生没有影响。 重要的是，我们证明了与JFH1相比，复制和产生CP7病毒的细胞降低了细胞的生长和凋亡的增加，这种作用与可稳健的病毒产生相关。这些研究开始揭示对鲁棒病毒复制的要求以及病毒体产生增加与宿主细胞活力之间的关系。