Full-length sequences of recombinant env genes were reconstituted, using an overlapping PCR procedure. We separately amplified the region from the 59 end of the acceptor gene (using primer Topo59 annealing to positions 5966-5990 of the reference strain HXB2) to the breakpoint position (using a specific primer encompassing the region of the breakpoint) and from the 39 end of the donor gene (primer Donor39, HXB2 positions 8785-8819) to the breakpoint position (also in this case with a specific primer). These PCR products, overlapping by approximately 30 nucleotides around the breakpoint site, were mixed at equal ratios and used as templates to generate the full-length recombinant env gene using primer Topo59 and Donor39. All PCR reactions were run with Phusion DNA polymerase (Finnzymes, Finland) for 30 cycles. PCR products were gel purified and ligated to pCDNA3.1 Topo (Invitrogen, CA, USA). For RRE functionality assays, a portion of the envelope gene containing the RRE of pNL4.3-Env 2 -Luc (nucleotides 7646 to 8046) was replaced with the corresponding sequence of parental or recombinant envelope genes or, as a negative control, a 400 nt sequence from the Drosophila melanogaster desoxynucleoside kinase gene (DdNK). All constructs were verified by sequencing.