Longitudinal Quasispecies Analysis of Viral Variants in HIV Type 1 Dually Infected Individuals Highlights the Importance of Sequence Identity in Viral Recombination

Abstract

Little is known regarding the likelihood of recombination between any given pair of nonidentical HIV-1 viruses in vivo. The present study analyzes the HIV-1 quasispecies in the C1C2 region of env, the vif-vpr-vpu accessory gene region, and the reverse transcriptase region of pol. These sequences were amplified from samples obtained sequentially over a 12-to 33-month period from five dually HIV-1-infected subjects. Analysis of an average of 248 clones amplified from each subject revealed no recombinants within the three loci studied of the subtypediscordant infecting strains, whose genetic diversity was >11% in env. In contrast, two subjects who were initially coinfected by two subtype-concordant variants with genetic diversity of 7.4% in env were found to harbor 10 unique recombinants of these strains, as exhibited by analysis of the env gene. The frequent recombination observed among the subtype-concordant strains studied herein correlates with prior sequence analyses that have commonly found higher rates of recombination at loci bearing the most conserved sequences, demonstrating an important role for sequence identity in HIV-1 recombination. Viral load analysis revealed that the samples studied contained an average of 8125 virus copies=ml (range, 882-31,626 copies=ml), signifying that the amount of viral RNA in the samples was not limiting for studying virus diversity. These data reveal that recombination between genetically distant strains may not be an immediate or common outcome to dual infection in vivo and suggest critical roles for viral and host factors such as viral fitness, virus diversity, and host immune responses that may contribute to limiting the frequency of intersubtype recombination during in vivo dual infection.

Sequence Analysis of the Dimerization Initiation Site of Concordant and Discordant Viral Variants Superinfecting HIV Type 1 Patients

“…10 Thus, the single nucleotide changes we observed in the DIS loop, the flanking nucleotides, and the stem region might prevent dimerization of the two superinfecting discordant HIV-1 strains and might be responsible for the lack of recombination in these study subjects in vivo. 6 These data confirm that sequence identity plays an important role in HIV-1 recombination (Fig. 3).…”

“…4,5 We recently conducted a longitudinal study in which we analyzed three genetic loci (the C1C2 region of env, RT region of pol, and vvv accessory gene region) of HIV-1 in superinfected subjects. 6 No recombinants or recombination breakpoints were detected in the sequences analyzed of the subtype-discordant infecting strains. On the contrary, several recombinants with characteristic breakpoints were detected in subjects superinfected with subtypeconcordant strains.…”

“…On the contrary, several recombinants with characteristic breakpoints were detected in subjects superinfected with subtypeconcordant strains. 6 An important factor that affects HIV recombination is the extent of sequence similarity among two viral strains, especially compatibility of the sequences at the DIS. 7,8 Indeed, it was demonstrated in vitro that intersubtype recombination events occur at a much lower rate than intrasubtype recombinations.…”

“…Viral RNA was extracted from patient plasma and the region from position 536 bp to 1200 bp (Hxb2 numbering engine) of each of the viral strains was amplified by nested polymerase chain reaction (PCR) (outer primers: 5'-GACCAGATCTGAGCCTGGGAGC-3' and 5'-TGAAAGCCTTTTCTTCTA-3'; inner primers: 5'-CAATAAA GCTTGCCTTGAG-3' and 5'-ATTTTGCACTATAGGGTAAT TT-3'), reverse transcribed into cDNA, and cloned, as described previously. 6 Sequence alignment and phylogenetic analysis of the 664-bp sequence (536-1200 bp) by the neighbor-joining method (1000 bootstrap replicas), with genetic distances evaluated with the Kimura two-parameter method, were performed using MAFFT, Seaview, and MEGA4.…”

An in vitro Model to Mimic Selection of Replication-Competent HIV-1 Intersubtype Recombination in Dual or Superinfected Patients

“…For example, increased recombination frequency at 5’ and 3’ ends of the env gene, regions corresponding to the gp120 C1 and gp41, respectively, may be related to nucleotide and amino acid conservation [28-34]. However, with the exception of the study by Simon-Loriere et al [28], functionality of the Env glycoproteins derived from in vitro recombination events has not been thoroughly investigated.…”