High recombination potential of subtype A HIV-1

Abstract

Recombination can assort polymorphic alleles to increase diversity in the HIV-1 population. To better understand the recombination potential of subtype A HIV-1, we generated viruses containing sequences from two variants circulating in Russia and analyzed the polymerase gene (pol) of the recombinants after one round of HIV-1 replication using single-genome sequencing. We observed that recombination occurred throughout pol and could easily assort alleles containing mutations that conferred resistance to currently approved antivirals. We measured the recombination rate in various regions of pol including a G-rich region that has been previously proposed to be a recombination hot spot. Our study does not support a recombination hot spot in this G-rich region. Importantly, of the 58 proviral sequences containing crossover event(s) in pol, we found that each sequence was a unique genotype indicating that recombination is a powerful genetic mechanism in assorting the genomes of subtype A HIV-1 variants.

Keywords: HIV-1; Recombination; Subtype A.

Fig. 1

Experimental system used to study HIV-1 recombination. A. General structures of the HIV-1 constructs EF-H0 and FJ-T6. Asterisks indicate inactivating mutations in the gfp gene; a functional gfp gene can be reconstituted by template switching events during reverse transcription. B. Experimental procedure used to study recombination events.

Fig. 2

Representative results of flow cytometry analyses. A. Analyses of the producer cell line. Left panel shows that >97% of cells express HSA and Thy markers, encoded by the two parent proviruses. Right panel shows that the producer cells lack GFP expression. B. Analyses of target Hut/R5 cells after infection. Left panel shows cells expressing HSA or Thy, whereas the right panel shows cells expressing GFP.

Fig. 3

Distribution of distance between polymorphic sites in the target pol region and between sites in which crossover were observed.

Fig. 4

Patterns of identified recombinant pol gene sequences. The region encoding PR, RT, and IN are shown in white boxes on top whereas identified recombinants are shown below. Sequences derived from subtype A HIV-1 EF (GenBank EF545108) and FJ (Genbank FJ864679) are shown as black and gray boxes, respectively. Nucleotide 0 in the scale corresponds to the nucleotide number 2318 in both EF and FJ sequences. Locations of cPPT and the G-rich region hypothesized to be form the G-quartet are indicated. Open diamonds indicate positions of mutations that confer resistance to anti-viral drugs. Three black squares located in PR, RT, and IN denote amino acid I84, Q151, and Q148, respectively, which when mutated can confer resistance to antiviral inhibitors.

Fig. 5

One-round recombination rates measured across the target pol region. The recombination rate of each 25-nt region is shown. Location of cPPT and G-rich region are indicated. Nucleotide numbering is the same as in Fig. 4.