Single cycle recombination assays were performed using a system previously developed by our laboratory [29] . HIV-1 env fragments from group M subtypes A, C, D and G, and from group O viral DNA were amplified by PCR from infected PBMCs obtained from patients and cloned in plasmids (called genomic plasmids), which differ for the genetic marker present downstream (in the sense of reverse transcription) of the sequence in which recombination is studied (Figure 1 ). All constructs were verified by sequencing. The trans-complementation plasmids, pCMV R8.2 [40] encoding HIV-1 Gag, Pol, and accessory proteins, and pHCMV-G [41] encoding the Vesicular Stomatitis Virus envelope protein were co-transfected into 293T cells with the two genomic plasmids to produce defective retrovirus particles which were then used to transduce MT4 cells as previously described [29] . The reverse transcription products were purified from the cytoplasmic fraction of transduced cells using the method described by Hirt [42] . The purified double stranded DNA was digested with DpnI for 2 h at 37uC (in order to eliminate possible contaminating DNA of bacterial origin) prior to PCR amplification as previously described [29] . The amplified product was purified after electrophoresis on agarose gel, digested with PstI and BamHI, ligated to an appropriate plasmid vector and used to transform E. coli. Plating on IPTG/X-Gal containing agar plates allowed blue/ white screening of recombinant and parental colonies, respectively [29] . The frequency of recombination was determined by computing the number of blue colonies over the total number of colonies as described in reference [29] . Recombination breakpoints were identified by full-length sequencing of the env portion of the recombinant clones.