Abstract:

OBJECTIVE: APOBEC-3F (A3F) and -3G (A3G) inhibit viral replication of HBV and participate in hypermutation of HBV genome. HBcAg is considered as a potential interaction site. Therefore, we studied the interactions between A3F/A3G and HBcAg proteins. METHODS:The cDNAs of human A3F and A3G were cloned by RT-PCR from PHA-stimulated PBMC, and DNA of HBcAg was cloned by PCR from ayw subtype HBV. Then the yeast two hybrid expression plasmids pGADT7-A3F/-A3G and pGBKT7-HBcAg were constructed. To ascertain the direct interactions between HBcAg and A3F or A3G, pGBKT7-HBcAg was co-transformed into yeast AH109 with pGADT7-A3F or pGADT7-A3G, then the transformed yeast cells were cultured in synthetic double drop-out medium (DDO) and quadruple drop-out medium (QDO) containing X-α-gal, and Alpha galactose glucoside enzyme activity tests were carried out. The eukaryotic expression plasmids, containing HBcAg or A3F or A3G respectively, were constructed and transfected into HeLa cells, then co-immunoprecipiation (Co-IP) and western blot were performed to ensure the indirect interactions between HBcAg and A3F or A3G. RESULTS:The results of restriction endonuclease analysis and DNA sequencing showed that the plasmids involved were constructed successfully. Yeast two hybrid experiments with alpha galactosidase qualitative and quantitative assays displayed that A3F and A3G had no evident direct interaction with HBcAg. The results of Co-IP and western blot indicated A3F and A3G had interactions with HBcAg. CONCLUSION:We demonstrated the interactions between A3F/A3G and HBcAg, but the interactions might not be realized by direct bind of the proteins.

Key words: APOBEC-3F, APOBEC-3G, HBcAg, yeast two hybrid, co-immunoprecipitation