Within genomic regions where sequence identity is high, a trigger for recombination could be the presence of secondary The luciferase values that were determined for the four parental strains used to generate the recombinants are represented, as a frame of reference, by lilac bars on the right. The six recombination-prone regions defined in Figure 2B are shaded in pale blue and annotated accordingly above the graph. The value of loss of functionality approximated for each region is given in bold in the top part of the graph. Three M/O inter-type recombinants were also tested (AO456, AO7810, and AO8090, breakpoint positions 6508, 7810, and 8090, respectively, of the HXB2 reference strain), resulting in a complete loss of functionality, probably due to the higher sequence divergence between the parental isolates. In order to preserve the homogeneity of the dataset to intersubtype recombinants, these recombinants are neither presented in the figure, nor were considered for the calculation of the average functionality of the recombinants, described in the main text. doi:10.1371/journal.ppat.1000418.g003 structures [34] . The highest recombination peak within the second region in Figure 2B (corresponding with the C2 portion of gp120) coincides with a recombination hot-spot that is determined by the presence of a stable RNA hairpin structure [29, 35, 36] , while the fourth hot region ( Figure 2B ) corresponds to the RRE RNA structure that is highly conserved amongst all HIV isolates [37] . It is therefore possible that RNA secondary structures also contribute to the high rates of recombination observed at some of the other recombination hot regions. Noteworthy, the functionality of the RRE was retained even when crossing genetically distant isolates as for inter-group M/O recombinants (Figure 2A) , supporting the possibility that regions of the genome harbouring functional RNA structures, which are generally more conserved within the population, provide a mechanism for crossing distantly related retroviruses and are possibly important for recombination of RNA viruses in general. With respect to selection of recombinant genes at the protein level, experiments involving lattice proteins have shown that genes encoding proteins that tolerate mutations also tend to be recombination tolerant [2] . Since the env gene displays a degree of diversity between isolates from different HIV-1 group M subtypes ( [38] and references herein) that is two to three times higher than the genome average, we anticipated that the manifest mutation tolerance of env might predispose it to high recombination tolerance. However, we show that this is not the case with certain regions within the gp120 encoding portions of env (particularly region 2 described in the present work in Figure 3 ) tending not to tolerate recombination well.