In addition to encoding the proteins that coat the viral membrane, env also encodes a well-known functional RNA structure, the Rev responsive element (RRE). For the recombinants containing breakpoints in the RRE region the functionality of this RNA module was therefore also tested. Being involved in the regulation of the timing and the balance among the various forms of unspliced and partially or completely spliced RNAs, RRE is essential for viral replication [30] . Failure to properly regulate this process results in either a decrease or complete halt in viral production [30] . The functionality of chimaeric RREs was tested by measuring viral titres obtained upon transfection of cells with a plasmid containing the proviral sequence of the molecular clone NL4.3 of HIV-1, in which we had replaced the native NL4.3 RRE with that of the various chimaeric RREs. To uncouple the effects on RNA-folding caused by the introduced RRE sequences from those altering the amino acid sequence of expressed proteins, we used a variant of NL4.3 that does not express Env (NL4.3-Env 2 ) [31] , and a plasmid encoding the wild-type Env was co-transfected to complement the production of gp120 and gp41 proteins. In order to increase the statistical power of the analysis, additional chimaeric RREs were constructed using parental sequences other than those employed in our cell culture recombinant generation experiments (following a PCR procedure described in Materials and Methods) and tested for their functionality. As can be seen in Table 1 , the viral titres obtained with every chimaeric RRE sequence we tested were both similar to those obtained with nonrecombinant parental RRE sequences and markedly higher than that observed when the RRE was replaced with a non-viral sequence (see Materials and Methods). This result therefore clearly indicated that recombinants generated by breakpoints within the RRE generally retain the functionality of this element.