Molecular Mechanism of RNA Recombination of Hepatitis Delta Virus---Role of RNA Secondary Structure and Host Rna Polymerases

Recombination is an important means of generating genetic diversity in RNA viruses. Recently, homologous inter-genotypic RNA recombination of hepatitis delta virus (HDV) has been identified in mixed genotype infection and cultured cells co-transfected with genotype I and IIb HDV RNAs（Wang and Chao. 2005. J. Virol. 79:2221-2229). Furthermore, I found that the frequency of HDV RNA recombination could be determined by the degree of nucleotide sequence homology and the replication levels between two HDV strains. The higher degree of sequence homology and the higher levels of HDV RNA replication, the higher frequency of HDV RNA recombination was observed. These data also suggest that HDV RNA recombination is preformed via a replication-dependent mechanism. The replicating template-switching recombination is a well-accepted mechanism for virus RNA recombination. Since HDV replication is carried out by host RNA polymerases, I thereby propose that HDV RNA recombination is possibly performed via a cellular RNA polymerase-driven template-switching mechanism. Based on these findings, the following Specific Aims are proposed in this grant proposal to elucidate the molecular mechanism of HDV RNA recombination: First Year Based on the template-switching mechanism of RNA recombination, the polymerase has to be dissociated from the donor template and jump to the acceptor template to perform the RNA recombination. I am interested in the identification of the factor which can promote the template-switching during HDV RNA replication. The RNA secondary structure is a frequently-mentioned factor contributing to this event. To elucidate the role of RNA structure in HDV RNA recombination, HDV mutants with different RNA secondary structures are designed. The effect of these mutants on HDV RNA recombination will be investigated to elucidate the possible role of HDV RNA structure on the distribution of recombination junctions on HDV RNA. Second Year HDV replication is performed by host RNA polymerases; specifically, HDV genomic and antigenomic RNAs are synthesized by polymerase II and I, respectively. Therefore, analysis of RNA recombination of HDV mutants, which genomic RNA synthesis is preferentially inhibited, in the presence of different concentration of α-amanitin should provide insights into the molecular mechanism of HDV RNA recombination. Third Year The HDV recombinant RNA with rod-like structure has the abilities to replicate to a level similar to those of parental RNAs. Therefore, I propose that HDV recombinants with survival advantage could accumulate in cells. The interactions between parental HDV clones and the accumulation of HDV recombinants in cells stably expressing two genotype HDV RNAs will be examined to elucidate the role of RNA recombination in HDV evolution.

Project ID:PC9801-1452
External Project ID:NSC96-2320-B182-011-MY3