School of Medicine, National Taiwan University, Taipei, Taiwan.
Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.
J Virol. 2020 Sep 15;94(19). doi: 10.1128/JVI.00420-20.
Replication of the genotype 2 hepatitis C virus (HCV) requires hyperphosphorylation of the nonstructural protein NS5A. It has been known that NS5A hyperphosphorylation results from the phosphorylation of a cluster of highly conserved serine residues (S2201, S2208, S2211, and S2214) in a sequential manner. It has also been known that NS5A hyperphosphorylation requires an NS3 protease encoded on one single NS3-5A polyprotein. It was unknown whether NS3 protease participates in this sequential phosphorylation process. Using an inventory of antibodies specific to S2201, S2208, S2211, and S2214 phosphorylation, we found that protease-dead S1169A mutation abrogated NS5A hyperphosphorylation and phosphorylation at all serine residues measured, consistent with the role of NS3 in NS5A sequential phosphorylation. These effects were not rescued by a wild-type NS3 protease provided in by another molecule. Mutations (T1661R, T1661Y, or T1661D) that prohibited proper cleavage at the NS3-4A junction also abolished NS5A hyperphosphorylation and phosphorylation at all serine residues, whereas mutations at the other cleavage sites, NS4A-4B (C1715S) or NS4B-5A (C1976F), did not. In fact, any combinatory mutations that prohibited NS3-4A cleavage (T1661Y/C1715S or T1661Y/C1976F) abrogated NS5A hyperphosphorylation and phosphorylation at all serine residues. In the C1715S/C1976F double mutant, which resulted in an NS4A-NS4B-NS5A fusion polyprotein, a hyperphosphorylated band was observed and was phosphorylated at all serine residues. We conclude that NS3-mediated autocleavage at the NS3-4A junction is critical to NS5A hyperphosphorylation at S2201, S2208, S2211, and S2214 and that NS5A hyperphosphorylation could occur in an NS4A-NS4B-NS5A polyprotein. For ca. 20 years, the HCV protease NS3 has been implicated in NS5A hyperphosphorylation. We now show that it is the NS3-mediated cleavage at the NS3-4A junction that permits NS5A phosphorylation at serines 2201, 2208, 2211, and 2214, leading to hyperphosphorylation, which is a necessary condition for genotype 2 HCV replication. We further show that NS5A may already be phosphorylated at these serine residues right after NS3-4A cleavage and before NS5A is released from the NS4A-5A polyprotein. Our data suggest that the dual-functional NS3, a protease and an ATP-binding RNA helicase, could have a direct or indirect role in NS5A hyperphosphorylation.
复制基因型 2 丙型肝炎病毒 (HCV) 需要非结构蛋白 NS5A 的高度磷酸化。已知 NS5A 的高度磷酸化是由于一个高度保守的丝氨酸残基簇 (S2201、S2208、S2211 和 S2214) 的顺序磷酸化所致。也已知 NS5A 的高度磷酸化需要一个单一的 NS3-5A 多蛋白上编码的 NS3 蛋白酶。尚不清楚 NS3 蛋白酶是否参与此顺序磷酸化过程。使用针对 S2201、S2208、S2211 和 S2214 磷酸化的特异性抗体库，我们发现蛋白酶失活的 S1169A 突变消除了 NS5A 的高度磷酸化和所有测量的丝氨酸残基的磷酸化，这与 NS3 在 NS5A 顺序磷酸化中的作用一致。这些影响不能通过另一种分子提供的野生型 NS3 蛋白酶来挽救。禁止 NS3-4A 连接处适当切割的突变 (T1661R、T1661Y 或 T1661D) 也消除了 NS5A 的高度磷酸化和所有丝氨酸残基的磷酸化，而在其他切割位点 NS4A-4B (C1715S) 或 NS4B-5A (C1976F) 处的突变则不会。事实上，任何禁止 NS3-4A 切割的组合突变 (T1661Y/C1715S 或 T1661Y/C1976F) 均消除了 NS5A 的高度磷酸化和所有丝氨酸残基的磷酸化。在导致 NS4A-NS4B-NS5A 融合多蛋白的 C1715S/C1976F 双突变体中，观察到一个高度磷酸化的条带，并在所有丝氨酸残基上发生磷酸化。我们得出结论，NS3 介导的 NS3-4A 连接处的自身切割对于 NS5A 在 S2201、S2208、S2211 和 S2214 的高度磷酸化至关重要，并且 NS5A 的高度磷酸化可能发生在 NS4A-NS4B-NS5A 多蛋白中。大约 20 年来，丙型肝炎病毒蛋白酶 NS3 一直与 NS5A 的高度磷酸化有关。我们现在表明，正是 NS3 介导的 NS3-4A 连接处的切割允许 NS5A 在丝氨酸 2201、2208、2211 和 2214 处磷酸化，导致高度磷酸化，这是基因型 2 丙型肝炎病毒复制的必要条件。我们进一步表明，NS5A 可能在 NS3-4A 切割后立即在这些丝氨酸残基上发生磷酸化，然后再从 NS4A-5A 多蛋白中释放出来。我们的数据表明，双重功能的 NS3，一种蛋白酶和一种 ATP 结合 RNA 解旋酶，可能在 NS5A 的高度磷酸化中发挥直接或间接作用。
