Genetic and Functional Characterization of the N-Terminal Region of the Hepatitis C Virus NS2 Protein
Genetic and Functional Characterization of the N-Terminal Region of the Hepatitis C Virus NS2 Protein
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DOI：
10.1128/jvi.03174-12
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发表时间：
2013-04-01
影响因子：
5.4
通讯作者：
Rice, Charles M.
中科院分区：
文献类型：
--
作者：
de la Fuente, Cynthia;Goodman, Zachary;Rice, Charles M.
丙型肝炎病毒NS2蛋白在丙型肝炎病毒的生命周期中具有双重作用。虽然NS2是一种裂解NS2/NS3连接的自身蛋白酶，但NS2主要通过其N-末端区域参与病毒粒子的形态发生。为了定位传染性病毒产生所必需的决定因素，并进一步了解NS2可能影响这一过程的多个点，对跨越氨基酸(AA)1至92的残基进行了详细的突变分析。在基于双顺反子和单顺反子的丙型肝炎病毒细胞培养(HCVcc)基因组中的初始区块突变(5或7个氨基酸残基)显示，除两个区块外，所有区块都有不同程度的感染病毒产生受损。这些突变都没有影响RNA复制，表明NS2的N末端区域不是NS2-3加工和复制酶组装所必需的。精细定位确定了29个关键残基，当突变时，传染性病毒滴度至少下降了1个对数。这些突变体在细胞外释放丙型肝炎病毒RNA和核心、细胞内的感染性、病毒颗粒的热稳定性和NS2相互作用方面进行了进一步的鉴定。虽然最严重衰弱的突变体在组装过程的早期就受到了损害，这与之前的报道一致，但其他突变体的目标是病毒产生的后期步骤，最明显的是出口。因此，除了参与病毒粒子组装的早期步骤外，这项全面的突变研究表明，NS2在病毒生产的后期步骤中还有另一个作用。
The hepatitis C virus (HCV) NS2 protein has dual roles within the HCV life cycle. While well characterized as an autoprotease that cleaves the NS2/NS3 junction, NS2, primarily via its N-terminal region, is also involved in virion morphogenesis. In order to map the determinants necessary for infectious virus production and gain further insight into the multiple points at which NS2 may impact this process, a detailed mutational analysis of residues spanning amino acids (aa) 1 to 92 was performed. Initial block mutagenesis (5 or 7 amino acid residues) in both bicistronic and monocistronic HCV cell culture-based (HCVcc) genomes revealed that all but two blocks had various levels of impaired infectious virus production. None of these mutations affected RNA replication, indicating that the N-terminal region of NS2 is not required for NS2-3 processing and replicase assembly. Fine mapping identified 29 critical residues that, when mutated, yielded at least a 1 log decrease in infectious virus titers. These mutants were characterized further with respect to release of extracellular HCV RNA and core, intracellular infectivity, thermal stability of virus particles, and NS2 interactions. While the most severely debilitated mutants were impaired early in the assembly process, which is in agreement with previous reports, others targeted later steps of virus production, most notably egress. Thus, in addition to participating in early steps in virion assembly, this comprehensive mutagenesis study suggests yet another role for NS2 in later steps in virus production.