Abstract
By frequently rearranging large regions of the genome, genetic recombination is a major determinant in the plasticity of the human immunodeficiency virus type I (HIV-1) population. In retroviruses, recombination mostly occurs by template switching during reverse transcription. The generation of retroviral vectors provides a means to study this process after a single cycle of infection of cells in culture. Using HIV-1-derived vectors, we present here the first characterization and estimate of the strength of a recombination hot spot in HIV-1 in vivo. In the hot spot region, located within the C2 portion of the gp120 envelope gene, the rate of recombination is up to ten times higher than in the surrounding regions. The hot region corresponds to a previously identified RNA hairpin structure. Although recombination breakpoints in vivo cluster in the top portion of the hairpin, the bias for template switching in this same region appears less marked in a cell-free system. By modulating the stability of this hairpin we were able to affect the local recombination rate both in vitro and in infected cells, indicating that the local folding of the genomic RNA is a major parameter in the recombination process. This characterization of reverse transcription products generated after a single cycle of infection provides insights in the understanding of the mechanism of recombination in vivo and suggests that specific regions of the genome might be prompted to yield different rates of evolution due to the presence of circumscribed recombination hot spots.
Highlights
In their extracellular life, retroviruses store genetic information as two copies of single-stranded RNA molecules
Features of the HIV-1 infectious cycle, such as the poor fidelity of the reverse transcriptase (RT) (4), which boosts the probability of co-packaging non-identical viral RNAs, and the dynamic nature of HIV-1 infection, where de novo infection of CD4ϩ T cells amplifies the possibility of copy choice to occur during the repeated cycles of reverse transcription (5), can explain the extremely high level of genetic recombination documented (6)
Analysis of the reverse transcription products (RTP) found within these cells provided a snapshot of the recombinant population generated in the absence of any type of selection
Summary
DNA Constructs—HIV-1 cis-acting regulatory elements (HXB2 strain) were obtained by PCR amplification from pTRIP-GFP plasmid (22) and cloned in a pKS-derived Knr/origin of replication backbone plasmid (21) following standard cloning techniques. Assuming that only one double-stranded DNA molecule is produced per each vector particle, one-third of the bacterial colonies will be generated by reverse transcription products issued from lacϪ/Ϫ vectors and will have a white phenotype, introducing a bias in the estimation of the frequency of recombination. To accurately estimate the frequency of recombination, another factor to take into account is the background among the white colonies derived from cloning of cellular DNA co-purified with the reverse transcription products. These colonies are distinguished from those issued from the cloning of RTP by the size of the cloned insert and by their restriction pattern. Synthesis of the second DNA strand, BamHI and PstI digestion, ligation, and E. coli transformation were carried out as previously described (21)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
