[Construction of point mutation plasmids expressing HCV NS3/4A with different secondary structures at amino-terminal and their expressions in Huh 7 cells].
OBJECTIVE
To construct point mutation plasmids expressing HCV NS3/4A with different secondary structures at amino-terminal, and express the constructs in Huh 7 cells.


METHODS
Using pSG5/M-H05-5/4A as the template (A1-1) and primers designed according to the typing criteria, 4 single point mutation plasmids, namely pSG5/M-H05-5(A1-2)/4A(A1-2) (Y56F), pSG5/M-H05-5(B1-1)/4A(B1-1) (L80Q), pSG5/M-H05-5(B2-1)/4A(B2-1) (V51A), and pSG5/M-H05-5(B2-2)/4A(B2-2) (S61A), were constructed. With A1-2, B2-1, and B2-2 as the templates, the leucine to glutamine mutation at position 80 (L80Q) was induced to construct another 3 double point mutation plasmids pSG5/M-H05-5(B1-2)/4A(B1-2), pSG5/M-H05-5(A2-1)/4A(A2-1), and pSG5/M-H05-5(A2-2)/4A(A2-2), respectively. DNA sequencing was performed for confirmation of the mutations. Huh 7 cells were transfected with the constructs using FuGene 6 transfection reagents. Indirect immunofluorescence staining and Western blotting were used to detect the expression of the constructs.


RESULTS
Indirect immunofluorescence assay revealed 4 subcellular localization patterns of NS3 protein, including dot-like staining, diffuse staining, doughnut-like staining, and rod-shape staining. Western blotting also demonstrated successful expression of the constructs and weak in cis and in trans NS3 serine protease activities of subtypes A2-1 and B2-1 in comparison with other subtypes.


CONCLUSIONS
The point mutation plasmids expressing HCV NS3/4A with different secondary structures at amino-terminal are constructed successfully, which provides the basis for further study of different subtypes of HCV.
Wang
Xue-ping
XP
Editorial Department of Journal of Fourth Military Medical University, Xi'an 710033, China. wangxp@fmmu.edu.cn
Li
Fu-jun
FJ
Motoko
Nagano-fujii
NF
Kikumi
Kitayama
K
Hotta
Hak
H
D000595:Amino Acid Sequence; D002460:Cell Line; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D016174:Hepacivirus / Q000201:enzymology*; D007150:Immunohistochemistry; D042541:Intracellular Space / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D017354:Point Mutation; D017433:Protein Structure, Secondary; D021381:Protein Transport; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral 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