Luciferase values determined for each recombinant were plotted as a function of the corresponding breakpoint position ( Figure 3 ). Recombinants with breakpoints falling within the six hot regions indicated in Figure 2B were preferentially characterized. It was apparent that most of the severely defective recombinants contained breakpoints in hot regions 2 and 3 of the recombination rate distribution (Figure 3 ). Given this data, we approximated a probability of Env functionality being disrupted by breakpoints falling within each of the six high recombination-rate regions. Since the parental sequences themselves were not uniformly functional ( Figure 3 ), a situation that is probably common in nature, for each recombinant an estimate of loss of functionality was calculated by dividing the luciferase value obtained with that recombinant by the one of the least functional parental sequence involved in its generation. Recombinants displaying values between those of the two parental sequences were considered to white-grey hatched box: partially deleted and therefore non-functional U3 sequence; white boxes: HIV envelope sequences studied, env X and env Y stand for sequences of two different isolates (isolate X on the donor RNA and isolate Y on the acceptor, respectively); black box: marker lacZ' bacterial gene; grey box: partial sequence of the bacterial gene malT, inserted in the reverse orientation. The approximate location of the BamHI site (present only on the donor RNA) and of the PstI site present on both RNAs is also indicated. The path followed by reverse transcription for generating the BamHI + /LacZ' + recombinants studied in the present work is indicated schematically. doi:10.1371/journal.ppat.1000418.g001 retain functionality (and assigned a functionality value of 1). Of note, none of the recombinants yielded functionality values higher than that of the most functional parent from which it was generated. Values from recombinants containing breakpoints within the same region of the six hot regions were pooled, and a functionality loss value for each region was averaged ( Figure 3 ). The most significant loss of functionality was observed in regions 2, 3, and 6.