Phosphoacceptors Threonine 162 and Serines 170 and 178 within the Carboxyl-Terminal RRRS/T Motif of the Hepatitis B Virus Core Protein Make Multiple Contributions to Hepatitis B Virus Replication
Phosphoacceptors Threonine 162 and Serines 170 and 178 within the Carboxyl-Terminal RRRS/T Motif of the Hepatitis B Virus Core Protein Make Multiple Contributions to Hepatitis B Virus Replication
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DOI：
10.1128/jvi.01343-14
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发表时间：
2014-08-01
影响因子：
5.4
通讯作者：
Kim, Kyongmin
中科院分区：
文献类型：
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作者：
Jung, Jaesung;Hwang, Seong Gyu;Kim, Kyongmin
B型肝炎病毒（HBV）核心（C）蛋白羧基末端SPRRR基序内丝氨酸157、164和172的磷酸化在多个阶段调节HBV复制。位于HBV C蛋白羧基端保守RRRS/T基序内的苏氨酸162和丝氨酸170、178被认为是蛋白激酶A磷酸化位点。然而，这些残基的体内磷酸化从未被观察到，它们对HBV复制的贡献仍然未知。在这项研究中，[P-32]正磷酸盐标记的细胞表达C蛋白，然后与抗HBc抗体免疫沉淀显示，苏氨酸162和丝氨酸170和178是磷酸受体残基。一个三丙氨酸取代的突变体，模仿所有三个残基的去磷酸化，大大减少前基因组RNA（pgRNA）的磷酸化，从而减少HBV DNA的合成。相比之下，一个三重谷氨酸取代的突变体，模仿这些残基的磷酸化，减少DNA的合成，而不显着减少的磷酸化。三重突变体均不影响C蛋白表达或核心颗粒组装。苏氨酸162位的丙氨酸取代显著降低负链、正链和松弛环状DNA合成，表明该残基在HBV DNA合成中起多种作用。丝氨酸170和178的双丙氨酸取代在多个阶段降低HBV复制，表明这些残基也有助于HBV复制。因此，除了丝氨酸157、164和172之外，HBV C蛋白的苏氨酸162和丝氨酸170和178也在细胞中被磷酸化，并且这些残基的磷酸化和去磷酸化在HBV复制的调节中起多种作用。
Phosphorylation of serines 157, 164, and 172 within the carboxyl-terminal SPRRR motif of the hepatitis B virus (HBV) core (C) protein modulates HBV replication at multiple stages. Threonine 162 and serines 170 and 178, located within the carboxyl-terminal conserved RRRS/T motif of HBV C protein, have been proposed to be protein kinase A phosphorylation sites. However, in vivo phosphorylation of these residues has never been observed, and their contribution to HBV replication remains unknown. In this study, [P-32] orthophosphate labeling of cells expressing C proteins followed by immunoprecipitation with anti-HBc antibody revealed that threonine 162 and serines 170 and 178 are phosphoacceptor residues. A triple-alanine-substituted mutant, mimicking dephosphorylation of all three residues, drastically decreased pregenomic RNA (pgRNA) encapsidation, thereby decreasing HBV DNA synthesis. In contrast, a triple-glutamate-substituted mutant, mimicking phosphorylation of these residues, decreased DNA synthesis without significantly decreasing encapsidation. Neither triple mutant affected C protein expression or core particle assembly. Individual alanine substitution of threonine 162 significantly decreased minus-strand, plus-strand, and relaxed-circular DNA synthesis, demonstrating that this residue plays multiple roles in HBV DNA synthesis. Double-alanine substitution of serines 170 and 178 reduced HBV replication at multiple stages, indicating that these residues also contribute to HBV replication. Thus, in addition to serines 157, 164, and 172, threonine 162 and serines 170 and 178 of HBV C protein are also phosphorylated in cells, and phosphorylation and dephosphorylation of these residues play multiple roles in modulation of HBV replication.