Demonstration of the presence of protease-cutting site in the spacer of hepatitis B viral Pol protein.
Molecular genetic studies have revealed that the human hepatitis B viral (HBV) Pol protein, a polypeptide of about 94 kDa, contains four domains. These are the 5'-terminal protein, spacer, RNA reverse transcriptase/DNA polymerase, and RNase H, respectively, from the amino (N) to carboxy (C) terminus. No evidence indicates as yet the involvement of a specific protease in cleaving the Pol protein or the presence of protease-cutting sites in the Pol protein. An in vitro-translated Pol protein was shown to be cleaved by purified thrombin but not in the presence of its inhibitor, hirudin. Two thrombin-cutting sites, spanning 194 amino acids, were then deduced by thrombin digestion of Pol protein with various lengths of C-terminal deletion. These two putative cutting sites, one located in the spacer region and the other in the beginning of the polymerase region, were found to be conserved at similar positions in the Pol protein of all hepadnaviruses. By using a novel method called the LacZ localization assay (LLA), it was demonstrated that a tripartite fusion protein containing the nucleus localization sequence (NLS) of SV40 large T Ag, the putative thrombin cutting sequence (Ile-Arg-Ile-Pro-Arg320-Thr) of HBV Pol protein and the full length beta-galactosidase of E. coli, exhibited a lower percentage (approximately 53%) of targeting into the nucleus of transfected hepatoma cells when compared with a similar tripartite protein containing a single mutation (Arg320 residue into Trp320) of HBV Pol protein (approximately 78%) or with a bipartite protein of SV40 NLS-beta-galactosidase (approximately 90%). These results indicate that the putative thrombin-cutting site in the spacer region of HBV Pol protein could be cleaved by a cellular protease resulting in the separation of NLS sequence from the beta-galactosidase and rendering a lower frequency of X-gal staining in the nucleus.
Lin
C G
CG
Graduate Institute of Microbiology and Immunology, National Yang-Ming Medical College, Taipei, Taiwan, R.O.C.
Yang
S J
SJ
Hwang
W L
WL
Su
T S
TS
Lo
S J
SJ
D000595:Amino Acid Sequence; D000957:Antigens, Viral, Tumor / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002874:Chromosome Mapping; D004279:DNA, Viral / Q000235:genetics; D010450:Endopeptidases; D004926:Escherichia coli / Q000235:genetics; D015684:Gene Products, pol / Q000235:genetics*; D005814:Genes, Viral; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D007763:Lac Operon; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013917:Thrombin; D014162:Transfection; D014407:Tumor Cells, Cultured; D014773:Virology / Q000379:methods
D000957:Antigens, Viral, Tumor; D004279:DNA, Viral; D015684:Gene Products, pol; D011993:Recombinant Fusion Proteins; D010450:Endopeptidases; D013917:Thrombin