ROLE OF NONSTRUCTURAL PROTEINS IN PESTIVIRUS ASSEMBLY

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 丙型肝炎病毒(HCV)全长JFH1基因组(基因2a)在细胞培养中能产生中等滴度的感染性颗粒，但产生病毒粒子所需的最佳决定因素尚不清楚。以前已经证明，在JFH1的背景下，从J6CF编码从核心到NS2的基因内型内重组体在释放病毒颗粒方面更强大。 为了了解结构和非结构基因对丙型肝炎病毒复制潜力和传染性的贡献，我们鉴定了几种基因内型重组2a病毒，其中J6分离株的不同部分被工程化为JFH1感染性克隆。在Huh7.5转基因细胞中，所有基因组均产生高水平的细胞内丙型肝炎病毒RNA和NS3蛋白。然而，包含从C到E2(CE2)或C到p7(CP7)的J6序列的JFH1基因组分泌的感染性丙型肝炎病毒颗粒比亲本克隆JFH1多100倍。随后用J6/JFH1重组体以0.0003的复数感染NAéve Huh7.5细胞，仅对CE2和CP7病毒产生高滴度的病毒。将CP7J6/JFH1重组体与先前描述的J6/JFH1重组体产生的病毒粒子进行比较，表明嵌合连接的灵活性。 此外，与先前报道的FL-J6/JFH嵌合病毒相当的NTRNS2嵌合病毒，其病毒滴度比CNS2高10倍。由于这两个克隆在翻译和复制方面没有显著差异，这三个突变可能会影响翻译后过程，导致病毒粒子产量增加。这两个克隆的序列比较表明，NTRNS2有三个与CNS2不同的核苷酸差异。这些位于5‘NTR和核心编码序列的突变对病毒粒子的产生没有影响。 重要的是，我们证明了与JFH1相比，复制和生产CP7病毒的细胞显示出细胞生长减少和细胞凋亡增加，这一效果与强劲的病毒粒子产生相关。这些研究开始揭示强健病毒复制的要求，以及增加的病毒粒子产量和宿主细胞活力之间的关系。

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.