Demonstration of the presence of protease-cutting site in the spacer of hepatitis B viral Pol protein

Ching Gong Lin, Shun Jyun Yang, Wen Ling Hwang, Tsung Sheng Su, Szecheng J. Lo*

*此作品的通信作者

研究成果: 期刊稿件文章同行評審

7 引文 斯高帕斯(Scopus)

摘要

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 β-galactosidase of E. coli, exhibited a lower percentage (~ 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 (~ 78%) or with a bipartite protein of SV40 NLS-β-galactosidase (~ 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 β-galactosidase and rendering a lower frequency of X-gal staining in the nucleus.

原文英語
頁(從 - 到)61-73
頁數13
期刊Journal of Virological Methods
51
發行號1
DOIs
出版狀態已出版 - 01 1995
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