Multiple enzymatic activities associated with recombinant NS3 protein of hepatitis C virus
Multiple enzymatic activities associated with recombinant NS3 protein of hepatitis C virus
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DOI：
10.1128/jvi.72.8.6758-6769.1998
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发表时间：
1998-08-01
影响因子：
5.4
通讯作者：
De Francesco, R
中科院分区：
文献类型：
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作者：
Gallinari, P;Brennan, D;De Francesco, R
丙型肝炎病毒（HCV）非结构3蛋白（NS3）包含至少两个与多种酶活性相关的结构域；丝氨酸蛋白酶活性位于蛋白质的n端三分之一，而RNA解旋酶活性和RNA刺激的核苷三磷酸酶活性与c端部分有关。为了研究这些酶活性可能的相互影响，在大肠杆菌中以非融合蛋白的形式表达了全长67 kDa的NS3多肽，纯化至均匀性，并显示保留了所有三种酶活性。全长NS3的蛋白酶活性强烈依赖于跨越NS4A辅因子中心疏水核心的合成肽的激活。一旦与ns4a衍生肽络合，全长NS3蛋白和分离的n端蛋白酶结构域以相当的效率切割合成肽底物。我们发现，与分离的蛋白酶结构域一样，全长NS3的蛋白酶活性受到NS4A-NS4B和NS5A-NS5B对应的底物肽的n端切割产物的抑制。我们还对NS3 atp酶，RNA解旋酶？优化反应条件下的rna结合活性。与分离的n端和c端结构域相比，重组全长NS3在独立体外分析中对三种酶的活性均无显著差异。我们通过分析多核苷酸对所有NS3酶功能调节的影响，进一步探索了NS3 n端和e端结构域可能的相互依赖性。我们的研究结果表明，单链RNA对NS3蛋白水解活性的抑制是通过与蛋白酶结构域的直接相互作用介导的，而不是与解旋酶RNA结合结构域的相互作用。
The hepatitis C virus (HCV) nonstructural 3 protein (NS3) contains at least two domains associated with multiple enzymatic activities; a serine protease activity resides in the N-terminal one-third of the protein, whereas RNA helicase activity and RNA-stimulated nucleoside triphosphatase activity are associated with the C-terminal portion. To study the possible mutual influence of these enzymatic activities, a full length NS3 polypeptide of 67 kDa was expressed as a nonfusion protein in Escherichia coli, purified to homogeneity, and shown to retain all three enzymatic activities. The protease activity of the full-length NS3 was strongly dependent on the activation by a synthetic peptide spanning the central hydrophobic core of the NS4A cofactor. Once complexed with the NS4A-derived peptide, the full-length NS3 protein and the isolated N-terminal protease domain cleaved synthetic peptide substrates with comparable efficiency. We show that, as in the case of the isolated protease domain, the protease activity of full-length NS3 undergoes inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A-NS4B and NS5A-NS5B. We have also characterized and quantified the NS3 ATPase, RNA helicase? and RNA-binding activities under optimized reaction conditions. Compared with the isolated N-terminal and C-terminal domains, recombinant full-length NS3 did not show significant differences in the three enzymatic activities analyzed in independent in vitro assays. We have further explored the possible interdependence of the NS3 N-terminal and e-terminal domains by analyzing the effect of polynucleotides on the modulation of all NS3 enzymatic functions. Our results demonstrated that the observed inhibition of the NS3 proteolytic activity by single-stranded RNA is mediated by direct interaction with the protease domain rather than with the helicase RNA-binding domain.