[Construction of full-length complementary DNA of hepatitis C virus genome from an HCV infected patient].
BACKGROUND
To construct the full-length complementary DNA of HCV genome from an HCV infected patient.


METHODS
Four HCV gene fragments (1.6, 3.5, 2.4 kb and 2.6 kb) were amplified by RT-PCR from serum of a Chinese patient and fused and connected together to produce a 9.2 kb subgenomic fragment, which was further cloned into a cassette vector with fixed 5-prime and 3-prime termini of HCV to make the full-length cDNA. The cDNA heterogeneity was analyzed by comparing the sequences of 4 isolated HVR1 regions. The prokaryotic expressed Core, NS3 protease, NS3 helicase were detected for their specific reactivities with patient serum by Western blot analysis. And the protease activity of NS3 was evaluated in a cell-based NS3/4A-SEAP expression system.


RESULTS
The cDNA covered the near full-length of HCV genome from the patient's serum. The difference among HVR1 regions indicated no selection of HCV variants during RT-PCR and the quasi-species characteristic of the amplified cDNA. The prokaryotic expressed viral proteins could be identified by patient serum. In the NS3/4A-SEAP system, NS3 could cleave the 4A-4B site between NS4A and SEAP proteins and resulted in the secretion of SEAP in culture media.


CONCLUSIONS
These results suggest that the cloned HCV cDNA encodes a complete and functional open reading frame and will be useful for further construction of infectious cDNA clone.
Mao
Hong-xia
HX
Key Lab of Medical Molecular Virology, Ministries of Education and Health, Shanghai Medical College, Fudan University, Shanghai 200032, China.
Hu
Yun-wen
YW
Wu
Ying
Y
Lan
Shui-yun
SY
Yuan
Zheng-hong
ZH
D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics*; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary