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High-frequency recombination is a hallmark of HIV-1 replication. Recombination can occur between two members of the same subtype or between viruses from two different subtypes, generating intra- or intersubtype recombinants, respectively. Many intersubtype recombinants have been shown to circulate in human populations. We hypothesize that sequence diversity affects the emergence of viable recombinants by decreasing recombination events and reducing the ability of the recombinants to replicate. To test our hypothesis, we compared recombination between two viruses containing subtype B pol genes (B/B) and between viruses with pol genes from subtype B or F (B/F). Recombination events generated during a single cycle of infection without selection pressure on pol gene function were analyzed by single-genome sequencing. We found that recombination occurred slightly ( approximately 30%) less frequently in B/F than in B/B viruses, and the overall distribution of crossover junctions in pol was similar for the two classes of recombinants. We then examined the emergence of recombinants in a multiple cycle assay, so that functional pol gene products were selected. We found that the emerging B/B recombinants had complex patterns, and the crossover junctions were distributed throughout the pol gene. In contrast, selected B/F recombinants had limited recombination patterns and restricted crossover junction distribution. These results provide evidence for the evolved coadapted sites in variants from different subtypes; these sites may be segregated by recombination events, causing the newly generated intersubtype recombinants to undergo purifying selection. Therefore, the ability of the recombinants to replicate is the major barrier for many of these viruses.

The generation of genetic diversity is a fundamental characteristic of HIV-1 replication, allowing the virus to successfully evade the immune response and antiviral therapies. Although mutations are the first step towards diversity, mixing of the mutations through the process of recombination increases the variation and allows for the faster establishment of advantageous strains within the viral population. Therefore, studying recombination of HIV-1 provides insights into not only the mechanisms of HIV-1 replication but also into the potential for spread of antiviral drug resistance mutations within and across viral subtypes. This chapter describes, in detail, a highly sensitive recombination assay designed to measure the frequency of recombination between two viruses. This assay allows us to investigate the requirements, mechanisms, and final products of recombination. Additionally, software-based phylogenetic tools are described in this chapter, which allow for the identification of specific recombination events within patient samples or viral progeny from the recombination assay.

HIV-1 intersubtype recombinants have an increasingly important role in shaping the AIDS pandemic. We sought to understand the molecular mechanisms that generate intersubtype HIV-1 recombinants. We analyzed recombinants of HIV-1 subtypes B and C, and identified their crossover junctions in the viral genome from the 5' long terminal repeat (LTR) to the end of pol. We identified 56 recombination events in 56 proviruses; the distribution of these events indicated an apparent recombination gradient: there were significantly more crossover junctions in the 3' half than in the 5' half of the region analyzed. HIV-1 subtypes B and C have different dimerization initiation signal (DIS). We hypothesized that the inability of subtype B and C RNAs to form perfect base-pairing of the DIS affects the dimeric RNA structure and causes a decrease in recombination events at the 5' end of the viral genome. To test this hypothesis, we examined recombinants generated from a subtype C virus and a modified subtype B virus containing a subtype C DIS. In the 56 proviruses analyzed, we identified 96 recombination events, which are significantly more frequent than in the B/C recombinants. Furthermore, these crossover junctions were distributed evenly throughout the region analyzed, indicating that the recombination gradient was corrected by matching the DIS. Therefore, base-pairing at the DIS has an important function during HIV-1 reverse transcription, most likely in maintaining nucleic-acid structure in the complex. These findings reveal elements important to retroviral recombination and provide insights into the generation of HIV-1 intersubtype recombinants that are important to the AIDS epidemic.

Genetic recombination increases diversity in HIV-1 populations, thereby allowing variants to escape from host immunity or antiviral therapies. In addition to the currently described nine subtypes of HIV-1, many of the circulating strains are intersubtype recombinants. In this study, we determined the recombination rate between two HIV-1 subtype C viruses and between a subtype B virus and a subtype C virus during a single round of virus replication. Although HIV-1 subtype C recombines at a high rate, similar to that of HIV-1 subtype B, the recombination rate between a subtype B virus and a subtype C virus is much lower than the intrasubtype recombination rate. A 3-nt sequence difference in the dimerization initiation signal (DIS) region between HIV-1 subtypes B and C accounts for most of the reduction of intersubtype recombination. By matching the DIS sequences, the B/C intersub-type recombination rate was elevated 4-fold; by introducing mismatches in the 3-nt sequences, the B/B intrasubtype recombination rate was reduced 4-fold. Further analyses showed that the intermolecular template-switching frequency was unaffected by the sequence identity of the DIS region. These results support the hypothesis that mismatched sequences in the DIS region alter the formation of heterozygous virions, thereby lowering the observable recombination rate. Here, we present the discovery of a major restriction in HIV-1 intersubtype recombination. These results have important implications for virus evolution, the mechanism of HIV-1 RNA packaging, high negative interference in recombination, and the generation of circulating intersubtype recombinants within the infected population.