Lin C, Thomson J A, Rice C M
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093, USA.
J Virol. 1995 Jul;69(7):4373-80. doi: 10.1128/JVI.69.7.4373-4380.1995.
A virus-encoded serine proteinase mediates four site-specific cleavages in the hepatitis C virus polyprotein. In addition to the catalytic domain, which is located in the N-terminal one-third of nonstructural protein NS3, the 54-residue NS4A protein is required for cleavage at some but not all sites. Here, we provide evidence for a non-ionic detergent-stable interaction between NS4A and the NS3 serine proteinase domain and demonstrate that the central region of NS4A plays a key role in NS4A-dependent processing. Hydrophobic residues, in particular Ile-29, were shown to be important for NS4A activity, and a synthetic peptide, spanning NS4A residues 22 to 34, could substitute for intact NS4A in a cell-free trans cleavage assay. Furthermore, NS4A mutations, which abolished or inhibited processing, correlated with destabilization of the NS3-NS4A complex. These results suggest that a stable interaction exists between the central region of NS4A and the NS3 catalytic domain which is required for NS4A-dependent processing. Since NS4A is required for processing at certain serine proteinase-dependent cleavage sites, this interaction may represent a new target for development of antiviral compounds.
一种病毒编码的丝氨酸蛋白酶介导丙型肝炎病毒多聚蛋白中的四个位点特异性切割。除了位于非结构蛋白NS3 N端三分之一区域的催化结构域外，54个氨基酸残基的NS4A蛋白对于部分而非所有位点的切割是必需的。在此，我们提供了NS4A与NS3丝氨酸蛋白酶结构域之间存在非离子去污剂稳定相互作用的证据，并证明NS4A的中央区域在NS4A依赖性加工过程中起关键作用。疏水性残基，特别是异亮氨酸-29，被证明对NS4A活性很重要，并且在无细胞反式切割试验中，一段跨越NS4A残基22至34的合成肽可以替代完整的NS4A。此外，消除或抑制加工的NS4A突变与NS3-NS4A复合物的不稳定相关。这些结果表明，NS4A的中央区域与NS3催化结构域之间存在稳定的相互作用，这是NS4A依赖性加工所必需的。由于NS4A是某些丝氨酸蛋白酶依赖性切割位点加工所必需的，这种相互作用可能代表了开发抗病毒化合物的新靶点。
