Influence of sequence identity and unique breakpoints on the frequency of intersubtype HIV-1 recombination

BACKGROUND: HIV-1 recombination between different subtypes has a major impact on the global epidemic. The generation of these intersubtype recombinants follows a defined set of events starting with dual infection of a host cell, heterodiploid virus production, strand transfers during reverse transcription, and then selection. In this study, recombination frequencies were measured in the C1-C4 regions of the envelope gene in the presence (using a multiple cycle infection system) and absence (in vitro reverse transcription and single cycle infection systems) of selection for replication-competent virus. Ugandan subtypes A and D HIV-1 env sequences (115-A, 120-A, 89-D, 122-D, 126-D) were employed in all three assay systems. These subtypes co-circulate in East Africa and frequently recombine in this human population. RESULTS: Increased sequence identity between viruses or RNA templates resulted in increased recombination frequencies, with the exception of the 115-A virus or RNA template. Analyses of the recombination breakpoints and mechanistic studies revealed that the presence of a recombination hotspot in the C3/V4 env region, unique to 115-A as donor RNA, could account for the higher recombination frequencies with the 115-A virus/template. Single-cycle infections supported proportionally less recombination than the in vitro reverse transcription assay but both systems still had significantly higher recombination frequencies than observed in the multiple-cycle virus replication system. In the multiple cycle assay, increased replicative fitness of one HIV-1 over the other in a dual infection dramatically decreased recombination frequencies. CONCLUSION: Sequence variation at specific sites between HIV-1 isolates can introduce unique recombination hotspots, which increase recombination frequencies and skew the general observation that decreased HIV-1 sequence identity reduces recombination rates. These findings also suggest that the majority of intra- or intersubtype A/D HIV-1 recombinants, generated with each round of infection, are not replication-competent and do not survive in the multiple-cycle system. Ability of one HIV-1 isolate to outgrow the other leads to reduced co-infections, heterozygous virus production, and recombination frequencies

Sequence determinants of breakpoint location during HIV-1 intersubtype recombination

Retroviral recombination results from strand switching, during reverse transcription, between the two copies of genomic RNA present in the virus. We analysed recombination in part of the envelope gene, between HIV-1 subtype A and D strains. After a single infection cycle, breakpoints clustered in regions corresponding to the constant portions of Env. With some exceptions, a similar distribution was observed after multiple infection cycles, and among recombinant sequences in the HIV Sequence Database. We compared the experimental data with computer simulations made using a program that only allows recombination to occur whenever an identical base is present in the aligned parental RNAs. Experimental recombination was more frequent than expected on the basis of simulated recombination when, in a region spanning 40 nt from the 5′ border of a breakpoint, no more than two discordant bases between the parental RNAs were present. When these requirements were not fulfilled, breakpoints were distributed randomly along the RNA, closer to the distribution predicted by computer simulation. A significant preference for recombination was also observed for regions containing homopolymeric stretches. These results define, for the first time, local sequence determinants for recombination between divergent HIV-1 isolates