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 infe cted cells. The replication is mediated by cellular factors containing an R NA polymerase It-like enzyme activity through a double rolling-circle mecha nism and is regulated by delta antigens. In this study, UV cross-linking ex periments were carried out to examine interactions between HDV RNA and prot eins 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 anti genomic strand mapped the interacting domain within a 79-nucleotide fragmen t but not at the ends of the rod-shaped viral RNA structure. The specificit y of the RNA-protein interactions was demonstrated by competition experimen ts and the specific HDV RNA-binding proteins were purified through column c hromatography. Electrophoresis mobility shift assay with the purified fract ions demonstrated that the interaction between p36 and HDV RNA was relative ly stable even in the presence of 0,5 M NaCl. Biochemical analysis includin g 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 ef fect of GAPDH on the ribozyme activity of HDV antigenomic RNA. In addition, in the presence of HDV RNA cellular GAPDH relocalized from the cytoplasm t o the nucleus where HDV replication occurs. These results suggest that GAPD H is involved in the replication of HDV. (C) 2000 Academic Press.