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.