Sub-cellular Localization Of APOBEC-3F And-3G And Their Interaction On Hepatitis B Core Antigen

Background and aimsAPOBECs, belong to innate immune molecules, were reported to have the anti-viraleffect on numerous viruses, such as HBV and HIV, during the process of DNA editing, RNAediting, and non-edited process. Among all the APOBEC members, A3F and A3G have beendemonstrated to inhibit the replication of HBV in different ways. For example, they can inhibitthe mature of HBV pre-RNA by interacting with HBcAg during the non-edited process, as wellas disturbing the HBV hypermutation during the process of editing. However, it is still unclearthat whether the interaction between A3F/A3G and HBcAg is directly or mediated by otherproteins. It is not clear that the subcellular localization of A3F and A3G and their function ofinhibiting viral replication by different ways. In this study,we cloned and expressed the A3F andA3G, and analysed their subcellular localization and interaction with HBcAg to reveal themechanism of A3F and A3G in inhibiting replication of HBV and packaging of HBVMaterials and Methods1. Firstly, We extracted RNA from human peripheral blood mononuclear cells(PBMC) whichstimulated by PHA, cloned the cDNA of human A3F and A3G, and constructed yeastexpression plasmid containing pGADT7-3G /-3F；cloned HBcAg of ayw subtype of HBV byPCR, and constructed yeast expression plasmid containing pGBKT7– HBcAg.2. After excluded self-activated effect, the constructed plasmid pGBKT7-HBcAg wastransformed into yeast cells AH109 with pGADT7-3G and pGADT7-3F respectively , thenplated on synthetic droput nutrient medium DDO and QDO containing X-α-gal to detect thedirect interaction of HBcAg with A3F and A3G .3. Constructed eukaryotic expression vector of multiple tags gene fusion which contains HBcAg,human A3F and A3G. HeLa cell which were transfected through the liposome method toparticipate in Co-Immunoprecipitation and western blotting detection of the recyclingproduct of Co-Immunoprecipitation. 4. A3F and A3G eukaryotic expression vector were transfected into MDCK cells, and theirsub-cellular localization were indirectly detected by immunofluorescence fluorescence.5. Constructed a green fluorescent protein-expressing carrier containing nuclear localizationsignal(NLS); transfected MDCK cells, and observe the locate functional of nuclearlocalization signal; and fusion expression between human A3F or A3G and nuclearlocalization signal.ResultResults and conclusions1. The results from Enzyme cut and DNA sequencing analysis showed that we had successfullycloned HBcAg, A3F and A3G. And successfully constructing yeast double hybrid, immunetotal precipitation, eukaryotic expression and sub-cellular localization related expressionplasmid.2. Yeast double hybrid experiment with alpha half lactose glucoside enzyme qualitative andquantitative detection displayed that there may not exist or not occur direct interactionbetween A3F /A3G and HBcAg.3. The constructed pcFlag-3F, pcFlag-3G, pcMyc-HBcAg plasmids were respectivelytransfected into HeLa cells, after 48hr, Western blot showed that A3F/A3G protein andHBcAg protein were well expressed in HeLa cells; we further use the transfected HeLa celllysate to perform the Co-IP experiments that A3F/A3G protein and HBcAg proteins could beprecipitated, but the quantity of the protein have declined.4. Fluorescence microscope was used to monitor the MDCK cells showed that A3F and A3Gprotein mainly presetented some dot-like distirbution within the cytoplasm, without obviousfluorescent signals in the nucleus.Subcellular localization in this study showed A3F and A3Gprotein mainly distributed in the cytoplasm.5. By using the fluorescence microscope, the MDCK cells which is tranfected pEGFP-C1vector as a control have showed that GFP protein dispersed in the cells, but co-expressingGFP and NLS(BNLS, MNLS) protein mainly distributed in the nucleus. Subcellularlocalization in this study showed BNLS and MNLS have the biological activity of thenuclear localization.6. Fluorescence microscope was used to monitor the MDCK cells showed that the MDCK cells were co-express A3F/A3G and NLS (BNLS, MNLS), We found that A3F/A3G proteinmainly distributed in the cytoplasm. These results suggest that MNLS and BNLS doesn'taffect A3F/A3G, so we speculate that A3F/A3G could exist more stronger cytoplasmiclocation signals or combine with other cytoplasmic protein.