The hepatitis C virus 5' untranslated region gene amplified by rapid amplification of cDNA ends and its secondary structure.
OBJECTIVE
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.
Qin
Zhao-Xi
ZX
Institute of Hepatology, People's Hospital, Beijing University, Beijing 100044, China.
Cong
Xu
X
Jiang
Dong
D
Ha
Ming-Hao
MH
Chen
Hong-Song
HS
Wei
Lai
L
D020121:5' Untranslated Regions / Q000235:genetics*; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D017433:Protein Structure, Secondary; D020133:Reverse Transcriptase Polymerase Chain Reaction
D020121:5' Untranslated Regions; D018076:DNA, Complementary; D004279:DNA, Viral