Specific interaction between the hepatitis delta virus RNA and glyceraldehyde 3-phosphate dehydrogenase: an enhancement on ribozyme catalysis.
Replication of hepatitis delta virus (HDV) RNA occurs in the nuclei of infected cells. The replication is mediated by cellular factors containing an RNA polymerase II-like enzyme activity through a double rolling-circle mechanism and is regulated by delta antigens. In this study, UV cross-linking experiments were carried out to examine interactions between HDV RNA and proteins present in HeLa nuclear extract. Cellular proteins with molecular mass of 23 (p23), 36 (p36), 38 (p38), and 58 (p58) kDa bound to full-length HDV RNA of both genomic and antigenomic strands. Deletion analysis on the antigenomic strand mapped the interacting domain within a 79-nucleotide fragment but not at the ends of the rod-shaped viral RNA structure. The specificity of the RNA-protein interactions was demonstrated by competition experiments and the specific HDV RNA-binding proteins were purified through column chromatography. Electrophoresis mobility shift assay with the purified fractions demonstrated that the interaction between p36 and HDV RNA was relatively stable even in the presence of 0.5 M NaCl. Biochemical analysis including protein microsequencing identified the p36 as glyceraldehyde 3-phosphate dehydrogenase (GAPDH). RNase footprinting indicated that the UC-rich domain between nucleotides 379 and 414 of the HDV antigenomic RNA was involved in the GAPDH binding. Functional studies further demonstrated an enhancing effect of GAPDH on the ribozyme activity of HDV antigenomic RNA. In addition, in the presence of HDV RNA cellular GAPDH relocalized from the cytoplasm to the nucleus where HDV replication occurs. These results suggest that GAPDH is involved in the replication of HDV.
Lin
S S
SS
Institute of Biochemistry, National Taiwan University, Taipei, Taiwan, Republic of China.
Chang
S C
SC
Wang
Y H
YH
Sun
C Y
CY
Chang
M F
MF
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D006367:HeLa Cells; D003698:Hepatitis Delta Virus / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008969:Molecular Sequence Data; D016337:RNA, Catalytic / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000302:isolation & purification; D012260:Ribonucleases / Q000378:metabolism
D016337:RNA, Catalytic; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D012260:Ribonucleases