High rate of recombination throughout the human immunodeficiency virus type 1 genome

Abstract

The diploid nature of human immunodeficiency virus type 1 (HIV-1) indicates that recombination serves a central function in virus replication and evolution. Previously, while examining the nature of obligatory primer strand transfers during reverse transcription, a high rate of recombination was observed at the ends of the viral genome within the viral long terminal repeats, prompting the following question: does recombination occur at a high rate throughout the genome? To address this question, two vectors based upon different strains of HIV-1 were utilized. The vectors were comprised predominantly of autologous HIV-1 sequence and were approximately the same size as the parental genome. The proviral progeny of heterodimeric virions were analyzed after a single cycle of replication, and the sequence heterogeneity between the two strains allowed direct examination of recombination crossovers. The results obtained indicate that HIV-1 undergoes approximately two to three recombination events per genome per replication cycle. These results imply that both HIV-1 RNAs are typically utilized during reverse transcription and that recombination is an important aspect of HIV-1 replication.

FIG. 1

HIV-1 vectors and segments of provirus DNA amplified to examine recombination rate. HIV-gpt_HXB2 is based upon HIV-1 strain HXB2 (29). HIV-puro_BCSG3 is based upon HIV-1 strain BCSG3 (14, 49). SV-gpt represents the SV40 early promoter driving expression of the gpt gene. SNV-puro represents the SNV U3 promoter controlling puro gene expression. Segments 1 to 10 are different segments of amplified provirus DNA. The coordinates of the amplified segments are as follows: 1, 611 to 1283; 2, 1261 to 2049; 3, 2028 to 2820; 4, 2801 to 3665; 5, 3547 to 4415; 6, 4271 to 5066; 7, 4932 to 5797; 8, 5778 to 6390; 9, 7635 to 8454; 10, 8336 to 9156 (sequence coordinates according to the HXB2 provirus sequence).

FIG. 2

Protocol for the study of HIV-1 recombination. Individual cell clones containing a single copy of both HIV-gpt_HXB2 and HIV-puro_BCSG3 were established (49). Upon induction, vector virus was harvested and used to inoculate CD4-positive HeLaT4 target cells; this was followed by selection and isolation of target cell clones. Going from a provirus in a producer cell to a provirus in a target cell comprised a single cycle of virus replication (see text). Proviruses in each target cell clone were further analyzed by PCR, the HTA, and DNA sequencing in order to identify recombinants and crossover points.

FIG. 3

Representative example of an HTA. A radioactively labeled single-stranded probe was prepared from HIV-puro_BCSG3 plasmid DNA by asymmetric PCR using the segment 9 primer pair (lane P). Segment 9 is 819 bp long. The sequence difference between the two strains within this segment is 4.4%. The probe was annealed with PCR products amplified from HIV-puro_BCSG3 plasmid DNA (lane S, homoduplex control), HIV-gpt_HXB2 plasmid DNA (lanes H, heteroduplex controls), and genomic DNAs from different target cell clones (lanes 1 to 15). The plus sign indicates a band shifted due to heteroduplex formation. The minus sign indicates a nonshifted homoduplex band. Lanes 3, 7, 9, 16, and 18 are shifted compared to the band for HIV-puro_BCSG3 DNA and were therefore scored as recombinants.

FIG. 4

Comparison of recombination events across the entire HIV-1 genome. The values are percentages of progeny proviruses in which a crossover occurred between the two specified segments. M corresponds to the puromycin resistance gene.