To determine the functionality of individual recombinant envelopes at the protein level, full-length recombinant envelope genes containing breakpoints of interest were constructed by successive PCR, as described in Material and Methods. Each fulllength recombinant gene was then cloned in the pcDNA3.1 expression vector, and used to transfect HEK 293T cells together with the pNL4.3-Env 2 -Luc plasmid, to generate viral particles pseudotyped with the recombinant envelope of interest. The functionality of the recombinant envelopes was then tested after transduction of HEK 293T-CD4 + -CCR5 + cells at a multiplicity of infection of 0.1, by measuring luciferase expression in these cells 48 hours after transduction. Since target cells cannot synthesize new viral envelope proteins, infection was limited to reverse transcription and, potentially, integration. The luciferase values observed therefore reflected the relative success of viral entry into the target cells. For this analysis recombinants derived from parental env sequences that yielded the strongest positive signals in this single cycle test were chosen (parental sequences A-Q461, C-CAP210, G-1033 and O-32, see Table 2 for their relative genetic distance) due to the higher reliability of the luciferase signal. The parental env sequences were used as controls. As for the functional analysis of the RRE, additional recombinants involving combinations of parental sequences -other than those involved in the experiments of recombination in cell culture, but carrying breakpoints in the same regions -were also tested. These additional recombinant env sequences were generated by PCR, as described for the reconstitution of the full-length env gene.