Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe, Japan.
Kobe J Med Sci. 2021 Sep 30;67(2):E38-E47.
We previously reported that hepatitis C virus (HCV) NS5A (1b, Con1) protein accepts covalent ISG15 conjugation at specific lysine (Lys) residues (K44, K68, K166, K215 and K308), exhibiting proviral effects on HCV RNA replication. Here we investigated a role of NS5A-ISGylation via Lys residues in HCV propagation using HCV infectious clone. The alignment of amino acid sequences revealed that 5 Lys residues (K20, K26, K44, K139, and K166) of the 13 Lys residues within NS5A (genotype 2a, JFH1 strain) were conserved compared to those of HCV (genotype 1b, Con1 strain). The cell-based ISGylation assay revealed that the K26 residue in the amphipathic helix (AH) domain and the K139 residue in domain I of NS5A (2a, JFH1) had the potential to accept ISGylation. Use of the HCV replicon carrying luciferase gene revealed that the K26 residue but not K139 residue of NS5A (2a, JFH1) was important for HCV RNA replication. Furthermore, cell culture HCV revealed that the mutation with the K26 residue in combination with K139 or K166 on NS5A (2a, JFH1) resulted in complete abolishment of viral propagation, suggesting that the K26 residue collaborates with either the K139 residue or K166 residue for efficient HCV propagation. Taken together, these results suggest that HCV NS5A protein has the potential to accept ISGylation via specific Lys residues, involving efficient viral propagation in a genotype-specific manner.
我们之前报道称，丙型肝炎病毒 (HCV) NS5A（1b、Con1）蛋白在特定赖氨酸（Lys）残基（K44、K68、K166、K215 和 K308）上接受共价 ISG15 缀合，对 HCV RNA 复制表现出前病毒效应。在这里，我们使用 HCV 感染性克隆研究了 NS5A 通过赖氨酸残基的 ISGylation 在 HCV 传播中的作用。氨基酸序列比对表明，与 HCV（1b、Con1 株）相比，NS5A（基因型 2a、JFH1 株）中的 13 个赖氨酸残基中的 5 个赖氨酸残基（K20、K26、K44、K139 和 K166）是保守的。基于细胞的 ISGylation 测定显示，NS5A （2a，JFH1）AH 结构域中的 K26 残基和 I 结构域中的 K139 残基具有接受 ISGylation 的潜力。使用携带荧光素酶基因的 HCV 复制子发现，NS5A（2a，JFH1）的 K26 残基而不是 K139 残基对于 HCV RNA 复制很重要。此外，细胞培养 HCV 表明，NS5A（2a，JFH1）上的 K26 残基与 K139 或 K166 残基的突变导致病毒繁殖完全被消除，表明 K26 残基与 K139 残基或 K166 残基共同作用，以有效地促进 HCV 繁殖。总之，这些结果表明 HCV NS5A 蛋白具有通过特定赖氨酸残基接受 ISGylation 的潜力，以基因型特异性的方式参与有效的病毒繁殖。
