Human Immunodeficiency Virus Type 1 Recombination: Rate, Fidelity, and Putative Hot Spots

Abstract

Previously, we reported that human immunodeficiency virus type 1 (HIV-1) recombines approximately two to three times per genome per replication cycle, an extremely high rate of recombination given the relatively small genome size of HIV-1. However, a recombination hot spot involving sequence of nonretroviral origin was identified in the vector system utilized, raising the possibility that this hot spot skewed the rate of recombination, and the rate of recombination observed was an overestimation. To address this issue, an HIV-1-derived vector system was used to examine the rate of recombination between autologous HIV-1 sequences after restricting replication to a single cycle in the absence of this hot spot. Viral DNA and RNA were analyzed by a combination of the heteroduplex tracking assay, restriction enzyme analysis, DNA sequencing, and reverse transcription-PCR. The results indicate that HIV-1 undergoes recombination at a minimum rate of 2.8 crossovers per genome per cycle. Again, this is a very high rate given the small size of the HIV-1 genome. The results also suggested that there might be local hot spots of recombination at different locations throughout the genome since 13 of the 33 strand transfers identified by DNA sequencing shared the same site of recombination with one or two other clones. Furthermore, identification of crossover segments also allowed examination of mutations at the point of recombination, since it has been predicted from some studies of cell-free systems that mutations may occur with a frequency of 30 to 50% at crossover junctions. However, DNA sequence analysis of crossover junctions indicated that homologous recombination during viral replication was not particularly mutagenic, indicating that there are other factors or conditions not yet reproduced in cell-free systems which contribute to fidelity during retroviral recombination.

Dynamics of HIV-1 recombination in its natural target cells

“…We could identify 3-13 RSTE in each provirus, yielding an average of seven and a half RSTE per wild-type genome (Fig. 3B), or approximately twice the rate reported during infection of HeLa-CD4 cells (19)(20)(21).…”

“…Prior studies quantifying HIV-1 RT-strand transfer frequencies across the HIV-1 genome have been conducted only during infection of HeLa-CD4 fibroblastic cells and have demonstrated a minimal average of three to four RSTE per virus per round of replication (19)(20)(21). Because the intracellular environments of diverse cell types vary, we sought to determine the frequency of RT-strand transfer directly during HIV-1 infection of a natural target of HIV-1 infection.…”

Coestimation of recombination, substitution and molecular adaptation rates by approximate Bayesian computation

“…Nevertheless, there is space for improvement. First, we have assumed a uniform recombination rate along sequences, although some studies revealed the presence of hotspots in HIV-1 (for example, Zhuang et al, 2002). Second, we have estimated molecular adaptation for the whole sequence, although estimating adaptation at local (codon) level is possible (for example, by using the Bayesian hierarchical model developed by Bazin et al, 2010).…”

The Structure of HIV-1 Genomic RNA in the gp120 Gene Determines a Recombination Hot Spot in Vivo

“…At present, indications on this issue are extremely scarce. An analysis of ten intra-subtype B recombinant clones generated in cell culture highlighted the presence of a few putative hot spots along the genome (18), but no molecular characterization of these hot spots was carried out. On the other hand, in vitro studies have highlighted the existence of local fluctuations in the frequency of strand transfer on different sequences leading to the proposal of various mechanistic sketches (10 -12, 19, 20).…”