AIM: To examine the effect of hepatitis C virus (HCV) structural mimics of regulatory regions of the genome on HCV replication.METHODS: HCV RNA structural mimics were constructed and tested in a HCV genotype 1b aBB7 r...AIM: To examine the effect of hepatitis C virus (HCV) structural mimics of regulatory regions of the genome on HCV replication.METHODS: HCV RNA structural mimics were constructed and tested in a HCV genotype 1b aBB7 replicon,and a Japanese fulminant hepatitis-1 (JFH-1) HCV genotype 2a infection model.All sequences were computer-predicted to adopt stem-loop structures identical to the corresponding elements in full-length viral RNA.Huh7.5 cells bearing the BB7 replicon or infected with JFH-1 virus were transfected with expression vectors generating HCV mimics and controls.Cellular HCV RNA and protein levels were quantified by real-time polymerase chain reaction and Western blotting,respectively.To evaluate possible antisense effects,complementary RNAs spanning a mimic were prepared.RESULTS: In the BB7 genotype 1b replicon system,mimics of the polymerase (NS-5B),X and BA regions inhibited replication by more than 90%,50%,and 60%,respectively.In the JFH-1 genotype 2 infection system,mimics that were only 74% and 46% identical in sequence relative to the corresponding region in JFH-1 inhibited HCV replication by 91.5% and 91.2%,respectively,as effectively as a mimic with complete identity to HCV genotype 2a.The inhibitory effects were confirmed by NS3 protein levels.Antisense RNA molecules spanning the 74% identical mimic had no significant effects.CONCLUSION: HCV RNA structural mimics can inhibit HCV RNA replication in replicon and infectious HCV systems and do so independent of close sequence identity with the target.展开更多
Background and Aims:The noncoding regions in the 3'-untranslated region (UTR) of the hepatitis C virus (HCV)genome contain secondary structures that are important for replication.The aim of this study was to ident...Background and Aims:The noncoding regions in the 3'-untranslated region (UTR) of the hepatitis C virus (HCV)genome contain secondary structures that are important for replication.The aim of this study was to identify detailed conformational elements of the X-region involved in HCV replication.Methods:Ribonucleic acid (RNA) structural analogs X94,X12,and X12c were constructed to have identical conformation but 94%,12%,and 0% sequence identity,respectively,to the X region of HCV genotype 2a.Effects of structural analogs on replication of HCV genotypes 1b and 2a HCV RNA were studied by quantitative reverse transcriptase polymerase chain reaction.Results:In replicon BB7 cells,a constitutive replication model,HCV RNA levels decreased to 55%,52%,53%,and 54% after transfection with expression plasmids generating RNA structural analogs 5B-46,X-94,X-12,and X-12c,respectively (p<0.001 for all).In an HCV genotype 2a infection model,RNA analogs 5B-46,X-94,and X-12 in hepatic cells inhibited replication to 11%,9%,and 12%,respectively.Because the X-12 analog was only 12% identical to the corresponding sequence of HCV genotype 2a,the sequence per se,or antisense effects were unlikely to be involved.Conclusions:The data suggest that conformation of secondary structures in 3'-UTR of HCV RNA genome is required for HCV replication.Stable expression of RNA analogs predicted to have identical stem-loop structures might inhibit HCV infection of hepatocytes in liver and may represent a novel approach to design anti-HCV agents.展开更多
基金Supported by In part Grants from NIDDK DK042182 and the Herman Lopata Chair for Hepatitis Research (Wu GY)
文摘AIM: To examine the effect of hepatitis C virus (HCV) structural mimics of regulatory regions of the genome on HCV replication.METHODS: HCV RNA structural mimics were constructed and tested in a HCV genotype 1b aBB7 replicon,and a Japanese fulminant hepatitis-1 (JFH-1) HCV genotype 2a infection model.All sequences were computer-predicted to adopt stem-loop structures identical to the corresponding elements in full-length viral RNA.Huh7.5 cells bearing the BB7 replicon or infected with JFH-1 virus were transfected with expression vectors generating HCV mimics and controls.Cellular HCV RNA and protein levels were quantified by real-time polymerase chain reaction and Western blotting,respectively.To evaluate possible antisense effects,complementary RNAs spanning a mimic were prepared.RESULTS: In the BB7 genotype 1b replicon system,mimics of the polymerase (NS-5B),X and BA regions inhibited replication by more than 90%,50%,and 60%,respectively.In the JFH-1 genotype 2 infection system,mimics that were only 74% and 46% identical in sequence relative to the corresponding region in JFH-1 inhibited HCV replication by 91.5% and 91.2%,respectively,as effectively as a mimic with complete identity to HCV genotype 2a.The inhibitory effects were confirmed by NS3 protein levels.Antisense RNA molecules spanning the 74% identical mimic had no significant effects.CONCLUSION: HCV RNA structural mimics can inhibit HCV RNA replication in replicon and infectious HCV systems and do so independent of close sequence identity with the target.
文摘Background and Aims:The noncoding regions in the 3'-untranslated region (UTR) of the hepatitis C virus (HCV)genome contain secondary structures that are important for replication.The aim of this study was to identify detailed conformational elements of the X-region involved in HCV replication.Methods:Ribonucleic acid (RNA) structural analogs X94,X12,and X12c were constructed to have identical conformation but 94%,12%,and 0% sequence identity,respectively,to the X region of HCV genotype 2a.Effects of structural analogs on replication of HCV genotypes 1b and 2a HCV RNA were studied by quantitative reverse transcriptase polymerase chain reaction.Results:In replicon BB7 cells,a constitutive replication model,HCV RNA levels decreased to 55%,52%,53%,and 54% after transfection with expression plasmids generating RNA structural analogs 5B-46,X-94,X-12,and X-12c,respectively (p<0.001 for all).In an HCV genotype 2a infection model,RNA analogs 5B-46,X-94,and X-12 in hepatic cells inhibited replication to 11%,9%,and 12%,respectively.Because the X-12 analog was only 12% identical to the corresponding sequence of HCV genotype 2a,the sequence per se,or antisense effects were unlikely to be involved.Conclusions:The data suggest that conformation of secondary structures in 3'-UTR of HCV RNA genome is required for HCV replication.Stable expression of RNA analogs predicted to have identical stem-loop structures might inhibit HCV infection of hepatocytes in liver and may represent a novel approach to design anti-HCV agents.