Majeau Nathalie, Bolduc Marilène, Duvignaud Jean-Baptiste, Fromentin Rémi, Leclerc Denis
Centre de Recherche en Infectiologie, Pav. CHUL, Université Laval, 2705 boul. Laurier, QC G1V 4G2, Canada.
Biochem Cell Biol. 2007 Feb;85(1):78-87. doi: 10.1139/o06-195.
The primary function of the hepatitis C virus (HCV) core protein is genome encapsidation. Core protein is also subject to post-translational modifications that can impact on the assembly process. In this report, we have studied the effect of cAMP-dependent protein kinase A (PKA) phosphorylation on its assembly and stability in a yeast Pichia pastoris expression system. We have recently shown that co-expression of the human signal peptide peptidase and core protein (amino acids 1-191) in yeast leads to the formation of nucleocapsid-like particles (NLPs) that are morphologically similar to the wild-type HCV capsid. In this system, we expressed mutants S53A and S116A and mutants S53D and S116D to abolish or mimic PKA phosphorylation, respectively. None of these mutations affected HCV assembly, but S116D led to the degradation of core protein. We also showed that nonenveloped NLPs were labelled in vitro by PKA, suggesting that the phosphorylation sites are available at the surface of the NLPs. The co-expression of human PKA with core and human signal peptide peptidase in yeast did not produce phosphorylated NLPs and led to a decreased accumulation of nonenveloped particles. Mutation S116A restored the core protein content. These results suggest that PKA phosphorylation can modulate HCV core levels in infected cells.
丙型肝炎病毒（HCV）核心蛋白的主要功能是基因组衣壳化。核心蛋白还会经历可影响组装过程的翻译后修饰。在本报告中，我们在酵母毕赤酵母表达系统中研究了环磷酸腺苷依赖性蛋白激酶A（PKA）磷酸化对其组装和稳定性的影响。我们最近发现，在酵母中共表达人信号肽肽酶和核心蛋白（氨基酸1 - 191）会导致形成形态上与野生型HCV衣壳相似的核衣壳样颗粒（NLP）。在这个系统中，我们分别表达了突变体S53A和S116A以及突变体S53D和S116D，以分别消除或模拟PKA磷酸化。这些突变均未影响HCV组装，但S116D导致核心蛋白降解。我们还表明，无包膜的NLP在体外被PKA标记，这表明磷酸化位点在NLP表面是可及的。在酵母中共表达人PKA与核心蛋白和人信号肽肽酶不会产生磷酸化的NLP，并导致无包膜颗粒的积累减少。突变S116A恢复了核心蛋白含量。这些结果表明，PKA磷酸化可调节受感染细胞中的HCV核心水平。
