In contrast to selenoprotein Ps (SeIPs) from other animal species, bovine selenoprotein P-like-protein (SeIPLP) was found to contain a tandem repeat of (CAYYCC)(11). During an investigation into whether SeIPLP was a bovine substitute for SeIP or uniquely bovine, its mRNA was found to consist of multiple variants with different length tandem repeat, namely p(0) with (CAYYCC)(11), p(-4) lacking (CAYYCC)(4), p(-8) lacking (CAYYCC)(8), and p(-9) lacking (CAYYCC)(9). Although they were encoded on a single gene locus, neither classicalGT-AG: nor minor classAT-AC: donator-acceptor sequences for alternative splicing were identified. A subsequent S1 protection assay using oligonucleotides, whose sequence may occur as variants, performed against bovine poly(A)(+)RNA identified a total of nine variants. Judging from the sequence of these variants and the branch point mapping, the consensus sequence for recognition of the donator was CACCCCCAC: and of the acceptor and the branch point A nucleotide,ACCCC: CAT orACCCC: CATCCCCAT. Furthermore, when the p(0) insert mRNA was expressed in COS-7 cells derived from an African green monkey kidney, cDNAs corresponding to p(-8) and p(-9) could be isolated. Therefore, the bovine SeIPLP mRNAs consisted of multiple variants probably due to a novel splicing mechanism which was not bovine-specific but common to other mammals.

OBJECTIVETo construct point mutation plasmids expressing HCV NS3/4A with different secondary structures at amino-terminal, and express the constructs in Huh 7 cells.

METHODSUsing 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.

RESULTSIndirect 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.

CONCLUSIONThe 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.

Amino Acid Sequence ; Cell Line ; Gene Expression ; Genetic Engineering ; methods ; Hepacivirus ; enzymology ; Immunohistochemistry ; Intracellular Space ; metabolism ; Molecular Sequence Data ; Plasmids ; genetics ; Point Mutation ; Protein Structure, Secondary ; Protein Transport ; Viral Nonstructural Proteins ; chemistry ; genetics ; metabolism