[Hepatitis C virus NS3/4A with different secondary structures at amino-terminus has different serine protease activities and inhibitory activities on host cell].
OBJECTIVE
To construct the point mutation plasmids expressing NS3/4A with different secondary structure of the amino-terminal 120 residues of NS3, and to investigate the differences in serine protease and inhibitory effects on host cells between each subgroup.


METHODS
The point mutation plasmids were constructed, which expressed NS3/4A with the corresponding secondary structures of subgroup, and were named as A1-2, A2-1, A2-2, B1-1, B1-2, B2-1, and B2-2, with the backbone of M-H05-5 (A1-1). Western blot was performed to detect the expression of NS3/4A and the difference in in cis and in trans NS3 serine protease activity between each subgroup. The inhibitory effects of HCV NS3/4A with different amino-terminal secondary structures on IFN-beta production and p53-dependent transcriptional activation were revealed by Luciferase reporter assay.


RESULTS
Western blot revealed the successful expression of the constructs and the incomplete cleavage of NS3/4A in subgroup A2-1 and B2-1, indicating that the in cis NS3 serine protease activities of subgroup A2-1 and B2-1 were weaker compared with that of the other subgroups. By using NS5A/5BDeltaC as a substrate for NS3/4A serine protease, it was also found that the in trans NS3 serine protease activities of subgroup A2-1 and B2-1 were also weaker compared with that of the other subgroups. Differences in inhibitory effects of HCV NS3 on IFN-beta promoter activity and on p53-dependent luciferase gene transcriptional activation were also observed between subgroup A2-1, B2-1 and the other subgroups.


CONCLUSIONS
HCV NS3/4A with different secondary structures at amino-terminus has different serine protease activities and inhibitory activities on host cell functions.
Wang
Xue-ping
XP
Central Laboratory of Lanzhou General Hospital, Lanzhou Military Command, Lanzhou 730050, China. xueping127@hotmail.com
Li
Fu-jun
FJ
Deng
Lin
L
Nagano-Fujii
Motoko
M
An
Chunying
C
Jiang
Dapeng
D
Kitayama
Kikumi
K
Hotta
Hak
H
D000595:Amino Acid Sequence; D002460:Cell Line; D016174:Hepacivirus / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D010957:Plasmids; D017354:Point Mutation; D017433:Protein Structure, Secondary; D057057:Serine Proteases / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D057057:Serine Proteases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases