Koch J O, Bartenschlager R
Institute for Virology, Johannes-Gutenberg University, Obere Zahlbacher Strasse 67, Mainz, 55101, Germany.
Virology. 1997 Oct 13;237(1):78-88. doi: 10.1006/viro.1997.8760.
Processing of the nonstructural polyprotein of the hepatitis C virus (HCV) requires the serine-type proteinase located in the amino-terminal domain of NS3. To identify residues within NS3 determining substrate specificity, a mutation analysis was performed. Using sequence alignments and three-dimensional structure predictions, amino acids assumed to be important for specificity were replaced and the enzymes were tested in an intracellular trans-processing assay for their effects on cleavage of an NS4B-5B substrate. For some of the substitutions at positions 133, 134, 135, 136, 138, 152, 155, 157, and 169, slightly reduced processing efficiencies were observed but in no case was the substrate specificity altered. In contrast, substitutions of the phenylalanine at position 154 resulted in a modified cleavage pattern, suggesting an important role for this residue in substrate specificity. To substantiate this assumption, a panel of NS4B-5B substrates carrying different P1 residues at the NS4B/5A site were tested for cleavage by these altered proteinases. We found that substitution of Phe-154 by alanine, by valine, and particularly by threonine generated enzymes with the following affinities for aliphatic P1 residues: C > L > I > V for 154 F --> A, C = L > I > V for 154 F --> V and L > C > I > V for 154 F --> T. Neither leucine nor isoleucine nor valine was accepted by the parental NS3 proteinase, showing that Phe-154 is an important determinant for substrate specificity. Furthermore, we present evidence that Ala-157 plays an additional but minor role for this property.
丙型肝炎病毒（HCV）非结构多聚蛋白的加工需要位于NS3氨基末端结构域的丝氨酸型蛋白酶。为了确定NS3中决定底物特异性的残基，进行了突变分析。利用序列比对和三维结构预测，替换假定对特异性重要的氨基酸，并在细胞内转加工试验中测试这些酶对NS4B - 5B底物切割的影响。对于133、134、135、136、138、152、155、157和169位的一些替换，观察到加工效率略有降低，但底物特异性均未改变。相反，154位苯丙氨酸的替换导致切割模式改变，表明该残基在底物特异性中起重要作用。为了证实这一假设，测试了一组在NS4B/5A位点携带不同P1残基的NS4B - 5B底物被这些改变的蛋白酶切割的情况。我们发现，用丙氨酸、缬氨酸，特别是苏氨酸替换154位苯丙氨酸产生的酶对脂肪族P1残基具有以下亲和力：对于154F→A为C＞L＞I＞V，对于154F→V为C = L＞I＞V，对于154F→T为L＞C＞I＞V。亲本NS3蛋白酶不接受亮氨酸、异亮氨酸和缬氨酸，表明154位苯丙氨酸是底物特异性的重要决定因素。此外，我们提供证据表明157位丙氨酸对该特性起额外但次要的作用。
