Co-evolution of the 5'-untranslated region, nonstructural proteins 2 and 3 of hepatitis C virus

• Main Authors: Nai-Ying Ou, 歐乃瑛
• Other Authors: Kung-Chia Young
• Format: Others
• Language: en_US
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/55963582344710648874

碩士 === 國立成功大學 === 醫學檢驗生物技術學系碩博士班 === 97 === Hepatitis C virus (HCV) has been recognized as one of the major causes of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC). The nonstructural (NS) protein 5B of HCV is a RNA-dependent RNA polymerase (RdRp) which lacks a proof-reading function, together with the high replicative activity, is the basis of high genetic variability of HCV. In this study, 217 full-length HCV sequences were downloaded from Los Alomas HCV database, analyzed with the use of data mining software Weka, and we found that there existed 9 intra-genotypic co-evolutionary pairs between NS2, NS3 and 5'UTR in naturally occurring HCV strains. In this study, co-evolutionary pairs of the 243rd nucleotide of 5'-untranslated region (5'UTR) and the 14th, 41st, 76th, 110th, 211th, 212th amino acids of NS2 as well as the 71st, 175th, 621st amino acids of NS3 were examined to find out if these co-evolutionary mutations will regulate HCV replication. The HCV subgenomic replicon carrying single or multiple mutations were electroporated into cured-Huh7 cells and luciferase reporter activities were analyzed. The results showed that NS2-I41L, NS2-I76V, NS2-I110L, NS2-G211S, NS3-I71V and NS3-M175L single mutation down-regulated HCV replication when compared with the wild-type HCV replicon, but as these mutations were combined with 5'UTR ntG243A, the replicative activities could be compensated. The NS2-F14L, NS2-Q212K, and NS3-A621T single mutation also attenuated HCV replicative activities and these impairment effects could not be compensated as combined with 5'UTR ntG243A. The cell-based assay thus supported that the 14th, 41st, 76th, 110th, 211th of NS2, and 71st, 175th of NS3 might modulate HCV replication in concert of 5'UTR nt243. Furthermore, the combination of NS2-I76V and NS3-I71V also has compensatory effect with 5'UTR-G243A. In the absence of NS2 protein, the NS3-I71V and NS3-M175L mutations abolished HCV replication. These results indicated that NS2 protein play a role in modulating the co-evolution between 5'UTR-nt243 and 71st, 175th amino acids of NS3. Besides, the 5'UTR-G243C downregulated HCV replication activities to about half and the substitution of 5'UTR-G243T almost eliminated the HCV replication activities in 2-3'sibgenomic replicon, but the substitutions of 5'UTR-G243A, T, C had no influences on 3-3'subgenomic replicon, indicating that the substitution of 5'UTR nt243 might interfere the interaction between 5'UTR and NS2 protein thus modulating HCV replication activities. Finally, RNA-immunoprecipitation assay was used to investigate whether there are direct interactions between 5'UTR-nt243 and NS2 wt and mutated proteins. The results indicated that there were interactions of HCV genome with NS2 mutated proteins (NS2-F14L, NS2-I41L, NS2-I76V, NS2-I110L, NS2-G211S, NS2-Q212K) but not NS2-wt proteins in Feo-3-3' wild-type stable cells. In Feo-3-3'-G243A stable cells, however, interactions of HCV genome with NS2-wt, NS2-F14L were much stronger than those with NS2-I41L, NS2-I76V, NS2-I110L, NS2-G211S and the stronger the interactions between HCV RNAs and NS2 mutated proteins, the lower the replication of HCV, suggesting that the interactions between the co-evolutionary pairs might impair HCV replication. In conclusion, amino acid residues at NS2 and NS3 might co-evolve with nucleotide of 5'UTR-nt243, thus entails reservation of HCV replication.