9th Annual Workshop, October 28-31, 1999
Co-sponsored by the U.S. Department of Energy
Identification of a novel cellular protein that binds to the HBV RNA pregenome
S. Kreft and M. Nassal
Department of Internal Medicine II, University Hospital, Freiburg, Germany
Due to its small genome of only 3,2 kb, the replication of the hepatitis B virus (HBV) is expected to be tightly linked to the exploitation of host cell functions. The viral RNA pregenome (pgRNA) serves several functions in the viral life-cycle. It functions as mRNA for the capsid and the polymerase (P protein), and it is the substrate for encapsidation and reverse transcription. On its 5' region the structured encapsidation signal is present, which upon binding of P protein, mediates specific RNA packaging into capsids and initiation of reverse transcription. Previous UV-crosslinking data provided direct evidence for the existence of cellular factors that bind close to .
A North-Western (NW) screening procedure with DIG-labeled RNA encompassing HBV as a probe was employed to identify cellular binding proteins. Thereby, a 2.1 kb cDNA from a human liver cDNA expression library was isolated, whose gene product, NIII, consistently bound to RNA in the presence of excess nonspecific nucleic acid competitors. It contained a 3' terminally incomplete ORF encoding a protein of 666 aa with no strong homology to any known protein or RNA-binding motif in the database. Using deletion variants of a bacterially expressed MBP fusion protein in NW-experiments, we mapped the RNA-binding domain to a lysine-rich region close to the C terminus of NIII. In a search for a full-length cDNA, several additional libraries were screened for homology to the central part of NIII (service provided by RZPD, Heidelberg, Germany). Four independent clones with differing 3'-ends were isolated. Two of them encode proteins with 95 and 542 additional amino acids at their C terminus compared to NIII, and hence contain the putative RNA-binding domain as an integral part of the peptide chain. The longest form was termed RBP138 for RNA-binding protein of 138kD. The two other clones both encode the same "truncated" form of RBP138, corresponding to the first 303aa of RBP138, consequently lacking the RNA-binding domain. Both carry a 14nt insertion that generates a STOP codon shortly thereafter. Although the genomic sequence of RBP138 is not yet characterized, this insertion is most likely due to an alternative splicing event. Taken all that together, different isoforms of RBP138 protein seem to exist. Currently, we study the RNA-binding specificity as well as the subcellular localization of some isoforms to unravel the biological role of these new proteins.