The proposed research is directed at the characterization of the mechanism of template switching during retroviral reverse transcription. Preliminary evidence indicates that the first strand transfer step is an intermolecular reaction involving both RNAs of the virion while the second transfer step involves only a single DNA template. Additional preliminary evidence indicates that the presence of heterologous r regions in the viral RNA still allows efficient transfer during reverse transcription. Experiments are planned to characterize the LTRs in the proviruses that arise from infection by such composite viruses. To determine whether strand switching plays a significant role in the generation of diverse retroviral species, related experiments will be carried out to see whether intermolecular template switching might occur in heterozygous particles containing RNAs from two different retrovirus species. A genetic analysis of mutations in the terminal nucleic acid sequences of the virus through the use of a novel in vivo assay to determine strand switching. It is hoped that this will reveal specific segments of the viral genome required for template switching. Mutants defective in switching will be further characterized by determining whether efficient RNA dimerization occurs. Other related experiments are designed to determine, by several diverse approaches, whether the viral nucleocapsid protein or any other viral proteins are required for template switching. This includes the characterization of a mutation in the NC protein, studies to determine whether antiserum against various viral proteins blocks in vitro reverse transcription, and the construction of second site revertants of primary cis-acting sites required for strand switching. Finally, an attempt will be made to construct a high fidelity in vitro intermolecular strand transfer assay using a variety of potential substrates and reaction conditions. The construction of this system will make use of the information that results from the complementary in vivo analysis.
| Geraghty, R J; Talbot, K J; Callahan, M et al. (1994) Cell type-dependence for Vpu function. J Med Primatol 23:146-50 |
| Ramsey, C A; Panganiban, A T (1993) Replication of the retroviral terminal repeat sequence during in vivo reverse transcription. J Virol 67:4114-21 |
