The HDV large-delta antigen fused with GFP remains functional and provides for studying its dynamic distribution.
Hepatitis D virus (HDV) requires the isoprenylated large delta antigen (LDAg) for interaction with hepatitis B surface antigen (HBsAg) to allow packaging and secretion out of the host cell. Phosphorylated LDAg has been found but, as yet, neither localization of LDAg within the nucleus nor any other function has been correlated with modification. In this study, we transfected HuH-7 or HeLa cells with plasmids encoding various lengths of LDAg [designated GFP-LD and GFP-LD(31-214) for full length and a deletion, respectively] or non-isoprenylated mutants of these [designated GFP-LDM and GFP-LD(31-214)M] fused to the green fluorescent protein (GFP). These fusion proteins were then characterized and it was found that: (i) the addition of the GFP did not interfere with the functioning of the full-length or N-terminally deleted LDAgs when interacting with HBsAg for secretion; (ii) the HDV small antigen (SDAg) together with the GFP-LD, but not the GFP-LD(31-214), could be cosecreted by HBsAg; and (iii) the GFP-LD, but not the GFP-LD(31-214), exerted a dominant-negative role on HDV genome replication. Analyses of transiently transfected cells and postmitotic permanent cells revealed the sequential appearance of GFP-LD in the nucleoplasm, then in the nucleolus, and finally in nuclear speckles (NS). Isoprenylation of LDAg seems to be important for targeting to and accumulating in the NS, which was evident from the dynamic and static localization of the non-isoprenylation mutant (GFP-LDM) and the distribution of wild-type (GFP-LD) when treated with an isoprenylation inhibitor, lovastatin, for more than 48 h. Permanently expressing GFP-LD cells allowed us to show the dynamic redistribution of dephosphorylated GFP-LD from the nucleolus to the SC-35 containing NS in the presence of dichlororibofuranosyl benzimidazole (DRB) and then the translocation back of the GFP-LD to the nucleolus within 2 h after removal of DRB. Our studies thus suggest that the various versions of the GFP-LD fusion protein, having the same function as their nonfusion counterparts, can be a powerful tool for the study of the dynamic localization of LDAg when correlated with the functional modification of this protein.
Shih
K N
KN
Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, 11221, ROC.
Lo
S J
SJ
D002466:Cell Nucleolus / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D018963:Hepatitis Antigens / Q000235:genetics / Q000378:metabolism*; D006514:Hepatitis B Surface Antigens / Q000378:metabolism; D003698:Hepatitis Delta Virus / Q000235:genetics / Q000276:immunology*; D035923:Hepatitis delta Antigens; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology; D007202:Indicators and Reagents; D008148:Lovastatin / Q000494:pharmacology; D008164:Luminescent Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins; D012367:RNA, Viral / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012261:Ribonucleoproteins; D000068103:Serine-Arginine Splicing Factors; D013997:Time Factors; D014162:Transfection
D004791:Enzyme Inhibitors; D018963:Hepatitis Antigens; D006514:Hepatitis B Surface Antigens; D035923:Hepatitis delta Antigens; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; C442244:hepatitis delta virus large antigen; C073288:SRSF2 protein, human; D049452:Green Fluorescent Proteins; D000068103:Serine-Arginine Splicing Factors; D004004:Dichlororibofuranosylbenzimidazole; D008148:Lovastatin