Mechanism Analysis Indicates that Recombination Events in HIV-1 Initiate and Complete Over Short Distances, Explaining Why Recombination Frequencies Are Similar in Different Sections of the Genome

Abstract

Strand transfer drives recombination between the co-packaged genomes of HIV-1, a process that allows rapid viral evolution. The proposed invasion-mediated mechanism of strand transfer during HIV-1 reverse transcription has three steps: (1) invasion of the initial or donor primer template by the second or acceptor template; (2) propagation of the primer-acceptor hybrid; and (3) primer terminus transfer. Invasion occurs at a site at which the reverse transcriptase ribonuclease H (RNase H) has created a nick or short gap in the donor template. We used biochemical reconstitution to determine the distance over which a single invasion site can promote transfer. The DNA-primed RNA donor template used had a single-stranded pre-created invasion site (PCIS). Results showed that the PCIS could influence transfer by 20 or more nucleotides in the direction of synthesis. This influence was augmented by viral nucleocapsid protein and additional reverse transcriptase-RNase H cleavage. Strand-exchange assays were performed specifically to assess the distance over which a hybrid interaction initiated at the PCIS could propagate to achieve transfer. Propagation by simple branch migration of strands was limited to 24-32 nt. Additional RNase H cuts in the donor RNA allowed propagation to a maximum distance of 32-64 nt. Overall, results indicate that a specific invasion site has a limited range of influence on strand transfer. Evidently, a series of invasion sites cannot collaborate over a long distance to promote transfer. This result explains why the frequency of recombination events does not increase with increasing distance from the start of synthesis, a characteristic that supports effective mixing of viral mutations.