Mutational analysis of hepatitis C virus nonstructural protein 5A: Potential role of differential phosphorylation in RNA replication and identification of a genetically flexible domain
Mutational analysis of hepatitis C virus nonstructural protein 5A: Potential role of differential phosphorylation in RNA replication and identification of a genetically flexible domain
复制标题
DOI：
10.1128/jvi.79.5.3187-3194.2005
复制
发表时间：
2005-03-01
影响因子：
5.4
通讯作者：
Bartenschlager, R
中科院分区：
文献类型：
--
作者：
Appel, N;Pietschmann, T;Bartenschlager, R
丙型肝炎病毒的非结构蛋白5A是一种高度磷酸化的分子，参与与宿主细胞的多种相互作用，很可能参与RNA的复制。已经描述了NS5A的两个磷酸化变体，根据它们的表观分子质量(以千道尔顿为单位)命名为p56和p58，分别对应于基础形式和过度磷酸化形式。为了确定NS5A磷酸化对RNA复制的可能作用，我们对参与NS5A磷酸化和过度磷酸化的三个丝氨酸簇进行了广泛的突变分析。在大多数情况下，中央簇I中丝氨酸残基的丙氨酸替代将RNA复制增强到最高水平，导致NS5A过度磷酸化减少。同样，NS4B(也是复制复合体的一部分)中的几个高度适应性突变也导致了NS5A过度磷酸化的减少，认为NS5A磷酸化模式的改变对RNA复制起着重要作用。另一方面，NS5A C-末端参与基础磷酸化的所有高度保守的丝氨酸残基的缺失并没有显著影响RNA的复制，但减少了p56的形成。研究发现，该区域甚至可以耐受较大的插入，但对复制只有适度的影响。基于这些结果，我们提出了NS5A磷酸化在病毒生命周期中的作用模型。
Nonstructural protein 5A of the hepatitis C virus (HCV) is a highly phosphorylated molecule implicated in multiple interactions with the host cell and most likely involved in RNA replication. Two phosphorylated variants of NS5A have been described, designated according to their apparent molecular masses (in kilodaltons) as p56 and p58, which correspond to the basal and hyperphosphorylated forms, respectively. With the aim of identifying a possible role of NS5A phosphorylation for RNA replication, we performed an extensive mutation analysis of three serine clusters that are involved in phosphorylation and hyperphosphorylation of NS5A. In most cases, alanine substitutions for serine residues in the central cluster I that enhanced RNA replication to the highest levels led to a reduction of NS5A hyperphosphorylation. Likewise, several highly adaptive mutations in NS4B, which is also part of the replication complex, resulted in a reduction of NS5A hyperphosphorylation too, arguing that alterations of the NS5A phosphorylation pattern play an important role for RNA replication. On the other hand, a deletion encompassing all highly conserved serine residues in the C-terminal region of NS5A that are involved in basal phosphorylation did not significantly affect RNA replication but reduced formation of p56. This region was found to tolerate even large insertions with only a moderate effect on replication. Based on these results, we propose a model of the role of NS5A phosphorylation in the viral life cycle.