Transcriptional activation function of hepatitis B virus Pre S1 protein in yeast.
OBJECTIVE
To explore the feasibility of cloning of the hepatocyte receptor interacting with the Pre S1 protein of HBV by two-hybrid system.


METHODS
Yeast expression plasmids encoding fusion proteins of full length or portions of Pre S1 of HBV and DNA binding domain of yeast protein GAL4 were constructed and used to transform yeast reporter strain SFY526. Reporter gene product beta-galactosidase activity was assayed as a measure of transcriptional activation in yeast. Mammalian expression plasmid encoding fusion proteins of full length Pre S1 and DNA binding domain of GAL4 was constructed and used to cotransfect hepatoma cell line Huh-7 together with CAT reporter plasmid. Cell extracts were assayed for CAT activity by thin-layer chromatography.


RESULTS
The fusion proteins of full length Pre S1 protein and GAL4 DNA binding domain presented transcriptional activation function in yeast. The transcription activating sequence was localized to the 21 to 47 amino acids of Pre S1 protein. Fusion proteins of full length Pre S1 and GAL4 DNA binding domain did not show transcriptional activation function in mammalian cells.


CONCLUSIONS
The transcription activating sequence of HBV Pre S1 protein in yeast overlaps the hepatocyte receptor binding site. The transcriptional activation function of HBV Pre S1 protein in yeast may prevent researchers from using yeast two-hybrid system to clone HBV receptor interacting with Pre S1 protein. However, the Pre S1 protein does not show transcriptional activation function in mammalian cells. Mammalian two-hybrid system may be a practical method to clone the HBV hepatocyte receptor interacting with Pre S1 protein.
Xiao
Sheng-Xiang
SX
Laboratory of Molecular Biology, Institute of Dermatology, Second Hospital, Xi'an Jiaotong University, Xi'an 710004, China. xiaosx@163.com
Peng
Xuan-Xian
XX
Chu
Yong-Lie
YL
Xiao
Sheng-Bin
SB
Wu
Yan-Hong
YH
Cao
Zhen-Ping
ZP
D000818:Animals; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins; D006514:Hepatitis B Surface Antigens / Q000737:chemistry / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics*; D008113:Liver Neoplasms; D008322:Mammals; D010957:Plasmids; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D015533:Transcriptional Activation / Q000502:physiology*; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics
D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C050806:presurface protein 1, hepatitis B surface antigen