NUST Center of Virology and Immunology, National University of Sciences and Technology, Sector H-12, Islamabad, Pakistan.
Virus Res. 2011 Sep;160(1-2):230-7. doi: 10.1016/j.virusres.2011.06.020. Epub 2011 Jul 2.
Hepatitis C virus (HCV) infection is a serious cause of chronic liver disease worldwide with more than 170 million infected individuals at risk of developing significant morbidity and mortality. Current interferon-based therapies are suboptimal especially in patients infected with HCV genotype 3 (predominant genotype in Pakistan) and they are poorly tolerated, highlighting the requirement of new therapeutics. HCV non-structural protein-3 (NS3) protease and helicase domains are essential for viral replication; they are highly conserved among various HCV strains. In the current study, we enrolled 56 HCV infected patients from various regions of Pakistan and determined their genotypes, ALT level and virus titer. We have cloned and sequenced NS3/NS4A from 4 of the HCV Serum samples. Nucleotide sequence alignment showed high level of identities among 3a genotypes. One of the samples (NCVI 01) showed unique amino acids substitutions, including R9Q, L332P, L354I, I605V and S622C. Three dimensional structures were determined and analyzed effect of substitutions on amino acids interactions. We further established fluorescence resonance energy transfer (FRET) based assays for detecting proteolytic activity of (NS3-4A) serine protease, using AnaSpec peptide, for high throughput screening (HTS) inhibitors against HCV. In future, this study could be of great interest in the development of HCV NS3 cell-based HTS FRET assay for genotype 3a and subsequent antiviral testing of drugs.
丙型肝炎病毒 (HCV) 感染是全球慢性肝病的一个严重病因，全球有超过 1.7 亿人感染 HCV，面临着严重发病和死亡的风险。目前基于干扰素的治疗方法并不理想，尤其是在感染丙型肝炎病毒基因型 3（巴基斯坦的主要基因型）的患者中，而且它们的耐受性较差，这突显了对新疗法的需求。HCV 非结构蛋白-3 (NS3) 蛋白酶和螺旋酶结构域是病毒复制所必需的；它们在各种 HCV 株之间高度保守。在本研究中，我们从巴基斯坦的不同地区招募了 56 名 HCV 感染患者，确定了他们的基因型、ALT 水平和病毒滴度。我们已经从 4 份 HCV 血清样本中克隆和测序了 NS3/NS4A。核苷酸序列比对显示 3a 基因型之间具有高度的同一性。其中一个样本（NCVI 01）显示出独特的氨基酸取代，包括 R9Q、L332P、L354I、I605V 和 S622C。我们确定了三维结构并分析了取代对氨基酸相互作用的影响。我们进一步建立了荧光共振能量转移 (FRET) 测定法，使用 AnaSpec 肽，用于检测 HCV 的 NS3-4A 丝氨酸蛋白酶的酶切活性，进行高通量筛选 (HTS) 抑制剂。在未来，这项研究对于开发针对 3a 基因型的 HCV NS3 基于细胞的 HTS FRET 测定法以及随后的抗病毒药物测试可能具有重要意义。
