Kolykhalov A A, Agapov E V, Rice C M
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093.
J Virol. 1994 Nov;68(11):7525-33. doi: 10.1128/JVI.68.11.7525-7533.1994.
Cleavage at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the hepatitis C virus polyprotein requires a viral serine protease activity residing in the N-terminal one-third of the NS3 protein. Sequence comparison of the residues flanking these cleavage sites reveals conserved features including an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position. In this study, we used site-directed mutagenesis to assess the importance of these and other residues for NS3 protease-dependent cleavages. Substitutions at the P7 to P2' positions of the 4A/4B site had varied effects on cleavage efficiency. Only Arg at the P1 position or Pro at P1' substantially blocked processing at this site. Leu was tolerated at the P1 position, whereas five other substitutions allowed various degrees of cleavage. Substitutions with positively charged or other hydrophilic residues at the P7, P3, P2, and P2' positions did not reduce cleavage efficiency. Five substitutions examined at the P6 position allowed complete cleavage, demonstrating that an acidic residue at this position is not essential. Parallel results were obtained with substrates containing an active NS3 protease domain in cis or when the protease domain was supplied in trans. Selected substitutions blocking or inhibiting cleavage at the 4A/4B site were also examined at the 3/4A, 4B/5A, and 5A/5B sites. For a given substitution, a site-dependent gradient in the degree of inhibition was observed, with a 3/4A site being least sensitive to mutagenesis, followed by the 4A/4B, 4B/5A, and 5A/5B sites. In most cases, mutations abolishing cleavage at one site did not affect processing at the other serine protease-dependent sites. However, mutations at the 3/4A site which inhibited cleavage also interfered with processing at the 4B/5A site. Finally, during the course of these studies an additional NS3 protease-dependent cleavage site has been identified in the NS4B region.
丙型肝炎病毒多聚蛋白中四个位点（3/4A、4A/4B、4B/5A和5A/5B）的切割需要一种存在于NS3蛋白N端三分之一区域的病毒丝氨酸蛋白酶活性。对这些切割位点两侧残基的序列比较揭示了保守特征，包括P6位置的酸性残基（天冬氨酸或谷氨酸）、P1位置的半胱氨酸或苏氨酸残基以及P1'位置的丝氨酸或丙氨酸残基。在本研究中，我们使用定点诱变来评估这些及其他残基对NS3蛋白酶依赖性切割的重要性。4A/4B位点P7至P2'位置的取代对切割效率有不同影响。只有P1位置的精氨酸或P1'位置的脯氨酸会显著阻断该位点的加工。P1位置可耐受亮氨酸，而其他五个取代允许不同程度的切割。P7、P3、P2和P2'位置用带正电荷或其他亲水性残基进行取代不会降低切割效率。在P6位置检测的五个取代允许完全切割，表明该位置的酸性残基并非必需。当底物在顺式中含有活性NS3蛋白酶结构域或蛋白酶结构域以反式提供时，获得了平行结果。在3/4A、4B/5A和5A/5B位点也检测了在4A/4B位点阻断或抑制切割的选定取代。对于给定的取代，观察到了抑制程度的位点依赖性梯度，3/4A位点对诱变最不敏感，其次是4A/4B、4B/5A和5A/5B位点。在大多数情况下，消除一个位点切割的突变不会影响其他丝氨酸蛋白酶依赖性位点的加工。然而，3/4A位点抑制切割的突变也会干扰4B/5A位点的加工。最后，在这些研究过程中，在NS4B区域鉴定出了一个额外的NS3蛋白酶依赖性切割位点。
