Lodrini S, Bagaglio S, Canducci F, De Mitri M S, Andreone P, Loggi E, Lazzarin A, Clementi M, Morsica G
Division of Infectious Diseases, San Raffaele Vita-Salute University, San Raffaele Scientific Institute IRCCS, Milan, Italy.
J Biol Regul Homeost Agents. 2003 Apr-Jun;17(2):198-204.
The amino terminal region of the non structural gene 3 (NS3) of hepatitis C virus (HCV) is a chymotripsinlike serine-protease responsible for cleavage of the non structural proteins of Hepatitis C virus (HCV). In order to investigate the genetic variation of this region, we developed a nested PCR to obtain NS3 protease sequences from 54 patients chronically infected with HCV genotypes 1a, 1b and 3, respectively. Comparison of nucleotide and amino acids sequences of NS3 protease domain with consensus sequence obtained within the same genotype, showed 3.73% nucleotide divergence and 1.64% amino acid divergence in isolates of genotype 3a, whereas isolates 1a exhibited 4.45% nucleotide and 4% amino acid change, respectively. Finally, NS3 sequence from 1b isolates revealed 6.47% nucleotide and 3.5 % aa changes. Comparison of consensus amino acid sequences derived from isolates 1a, 1b and 3, with the HCV prototypes showed a low amino acid sequence diversity. However, the consensus sequence of HCV genotype 3 isolates showed an amino acid changed from the prototype, that was located within a region important for enzyme structure and activity. These results indicated that the NS3 protease gene is highly conserved within the same HCV genotype. The domains involved in enzyme function were highly conserved in 1a and 1b strains, whereas consensus sequence of isolates 3a showed that the majority of these strains were not perfectly conserved in one of such regions. These findings altogether suggested that the NS3 protease enzyme of HCV may constitute an important target for antiviral therapy, but the NS3 protease variability of isolates 3 within a region that is a potential target for antiviral therapy could pose a problem for structure based drug development.
丙型肝炎病毒（HCV）非结构基因3（NS3）的氨基末端区域是一种类胰凝乳蛋白酶丝氨酸蛋白酶，负责切割丙型肝炎病毒（HCV）的非结构蛋白。为了研究该区域的基因变异，我们开发了一种巢式PCR，以分别从54例慢性感染HCV 1a、1b和3基因型的患者中获取NS3蛋白酶序列。将NS3蛋白酶结构域的核苷酸和氨基酸序列与同一基因型内获得的共有序列进行比较，结果显示3a基因型分离株的核苷酸差异为3.73%，氨基酸差异为1.64%，而1a基因型分离株的核苷酸和氨基酸变化分别为4.45%和4%。最后，1b基因型分离株的NS3序列显示核苷酸变化为6.47%，氨基酸变化为3.5%。将1a、1b和3基因型分离株的共有氨基酸序列与HCV原型进行比较，结果显示氨基酸序列多样性较低。然而，HCV 3基因型分离株的共有序列显示，其氨基酸与原型相比发生了变化，该变化位于对酶结构和活性至关重要的区域内。这些结果表明，NS3蛋白酶基因在同一HCV基因型内高度保守。1a和1b基因型毒株中参与酶功能的结构域高度保守，而3a基因型分离株的共有序列显示，这些毒株中的大多数在其中一个区域并不完全保守。这些发现共同表明，HCV的NS3蛋白酶可能构成抗病毒治疗的重要靶点，但3基因型分离株的NS3蛋白酶在作为抗病毒治疗潜在靶点的区域内存在变异性，这可能给基于结构的药物开发带来问题。
