The hepatitis C virus 5' untranslated region gene amplified by rapid amplification of cDNA ends and its secondary structure

Abstract

Objectives: To obtain very end full-length cDNA of hepatitis C virus (HCV) 5'untranslated region (5'UTR) and analyze its primary and secondary structure.

Methods: A patient infected genotype 2a HCV was identified by reverse transcription-nested polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP). Total RNA isolated from the serum was used as template, and the cDNA of the 5'untranslated region was amplified using rapid amplification of cDNA ends (RACE). The fragments were recombinated by A-T clone strategy, and the recombinants were confirmed by RFLP and PCR, and sequenced subsequently. Secondary structures were analysed by RNAdraw.

Results: Very end full-length cDNA of genotype 2a HCV 5'UTR was obtained by RACE. In five clones obtained, three contained full-length 5'UTR cDNA; A21G, G170A, T222C, T247C, C339T substitutions were found as compared to HC-J6. Homological results of HCV-1, HC-J6, HC-C2, HC-J8 were 93.6%-94.4%, 92.1%-93%, 98.8%-99.7%, 96.2%-96.5%, respectively; however, the substitutions did not alter secondary structure. Two of 5 clones were deletions of 53bp and 135bp at the 5'terminal of HCV 5'UTR, respectively.

Conclusions: RACE can be used to obtain the full-length cDNA of 2a genotype HCV 5'UTR. Genes deleted at the 5'terminal of HCV circulate in hepatitis C patients.