Recombination contributes to the structural diversity that occurs during the rapid evolution of HIV. This evolutionary process allows the virus to escape immune response and efforts at therapy. Two parental RNA molecules are copackaged in HIV. Frequent recombination is thought to occur during viral replication in the step where the reverse transcriptase copies the parental RNA into DNA. The mechanism involves transfer of the growing DNA strand from one RNA template to the other. Our group is investigating this process by two complementary approaches. In one we are mapping the positions of recombination hot spots during viral replication in vivo. In the other we are probing the exact steps in the crossover process using viral RNA and proteins in vitro. The latter effort is the focus of this application. Examining recombination using viral RNA and reverse transcriptase we found that hairpin stem-loop structures strongly promoted crossovers. Our system allowed us to determine the exact distribution of crossover positions. With these results we proposed a model for recombination in which the two RNAs interact at hairpin sites, facilitating primer transfer. We now propose experiments to determine the exact features of hairpin loop and stem structure that lead to this important RNA-RNA interaction. We also propose to determine the role of the reverse transcriptase in the primer transfer. The viral nucleocapsid protein has been found to promote recombination. Its contribution to the transfer mechanism will also be determined.
|Roda, Ricardo H; Balakrishnan, Mini; Hanson, Mark N et al. (2003) Role of the Reverse Transcriptase, Nucleocapsid Protein, and Template Structure in the Two-step Transfer Mechanism in Retroviral Recombination. J Biol Chem 278:31536-46|
|Roda, Ricardo H; Balakrishnan, Mini; Kim, Jin K et al. (2002) Strand transfer occurs in retroviruses by a pause-initiated two-step mechanism. J Biol Chem 277:46900-11|