ROLE OF NONSTRUCTURAL PROTEINS IN PESTIVIRUS ASSEMBLY

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 全长丙型肝炎病毒（HCV）JFH 1基因组（基因型2a）已被证明在细胞培养中产生中等滴度的感染性颗粒，但病毒体产生所需的最佳决定因素尚不清楚。 先前已经表明，在JFH 1的背景下，从J6 CF编码核心直到NS 2的基因型内重组体在病毒颗粒的释放中更稳健。 为了了解结构和非结构基因对HCV复制潜力和感染性的贡献，我们已经表征了几种基因型内重组基因型2a病毒，其中J6分离株的不同部分被工程化为JFH 1感染性克隆。所有基因组在Huh7.5转染的细胞中产生高水平的细胞内HCV RNA和NS 3蛋白。然而，JFH 1基因组含有J6序列从C到E2（CE 2）或C到p7（Cp 7）分泌高达100倍以上的感染性HCV颗粒比亲本JFH 1克隆。 继发性感染用每种J6/JFH 1重组体以0.0003的感染复数感染Huh7.5细胞，仅对CE 2和Cp 7病毒产生高病毒滴度。通过Cp 7 J6/JFH 1重组体与先前描述的J6/JFH 1重组体的病毒体产生的比较显示嵌合连接的灵活性。 此外，NTRNS 2嵌合病毒，这是相当于以前报道的FL-J6/JFH嵌合体，显示了10倍的病毒滴度相比，CNS 2增强。由于这两个克隆在翻译和复制方面没有显著差异，因此这三个突变可能影响翻译后过程，导致病毒体产量增加。这两个克隆之间的序列比较表明，NTRNS 2有三个核苷酸的差异，从CNS 2不同。这些位于5 'NTR和核心编码序列的突变对病毒体产生没有影响。 重要的是，我们证明了与JFH 1相比，复制和产生Cp 7病毒的细胞显示出细胞生长减少和细胞凋亡增加，这一效应与强大的病毒体产生相关。这些研究开始揭示了稳健病毒复制的要求以及增加的病毒体产生与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.