Hepatitis D virus RNA editing is inhibited by a GFP fusion protein containing a C-terminally deleted delta antigen.
During its life cycle, hepatitis D virus (HDV) produces two forms of delta antigen (HDAg), small delta antigen (SDAg) and large delta antigen (LDAg), which differ in their C-terminal 19 amino acids. Host enzymes termed ADARs (adenosine deaminases that act on double-stranded RNA) are required for LDAg production. These enzymes change the stop codon (UAG) of SDAg to a tryptophan codon (UGG). However, the temporal and spatial regulation of HDV RNA editing is largely unknown. In this study, we constructed three GFP fusion proteins containing different lengths of SDAg and characterized their cellular localization and effects on HDV replication. One of these fusion proteins, designated D(1-88)-GFP, inhibited LDAg but not SDAg production, suggesting that D(1-88)-GFP inhibits HDV RNA editing. Two experiments further supported this supposition: (i). RT-PCR analysis combined with NcoI restriction enzyme digestion revealed that HDV RNA editing was reduced by 42% in HeLa-D(1-88)-GFP when compared with HeLa cells; and (ii). the ratio of SDAg/LDAg production from the reporter RNAs was reduced in cells co-transfected with ADAR-expressing and reporter plasmids in the presence of D(1-88)-GFP. Double fluorescence microscopy found that D(1-88)-GFP was either associated with SC-35 or was adjacent to PML (premyelocytic leukaemia antigen) at nuclear speckles, but D(1-88)-GFP was not co-localized with ADAR, which was mainly located in the nucleolus. In situ hybridization showing co-localization of HDV RNA with D(1-88)-GFP at nuclear speckles suggested that HDV RNA editing might occur in the nuclear speckles and require other nuclear factor(s), in addition to ADAR.
Shih
Ko-Nien
KN
Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan 112.
Chuang
Ya-Ting
YT
Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan 112.
Liu
Hsuan
H
Department of Life Science, National Yang-Ming University, Taipei, Taiwan 112.
Lo
Szecheng J
SJ
Department of Life Science, School of Medicine, Chang Gung University, TaoYun, Taiwan 333.|Department of Life Science, National Yang-Ming University, Taipei, Taiwan 112.|Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan 112.
D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000821:virology; D004279:DNA, Viral / Q000235:genetics; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D003698:Hepatitis Delta Virus / Q000235:genetics* / Q000502:physiology*; D035923:Hepatitis delta Antigens / Q000235:genetics* / Q000502:physiology*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D017393:RNA Editing; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection
D004279:DNA, Viral; D035923:Hepatitis delta Antigens; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins