Contributions of viral splice sites and cis-regulatory elements to lentivirus vector function

The mobile transgene constructs of most human immunodeficiency virus (HIV)- based lentivirus vectors currently in use contain viral long terminal repea ts, a 5' untranslated region, gag sequences, and env sequences that include the Rev-responsive element (RRE). In this study, we examined the possibili ty of deleting HIV splice sites and gag and env sequences from an HN type 1 recombinant vector established in our laboratory as part of our ongoing ef forts to improve this vector system. Mutations in the major splice donor si te (SD) markedly reduced viral RNA expression but had little effect on vect or titer. Deletion of gag or env sequences, excluding RRE, led to a moderat e reduction in vector titer. Interestingly, deletion of RRE slightly reduce d viral RNA expression but markedly impaired vector function. Combined dele tions of RRE, gag (except for the first 40 nucleotides), env, and the SD mu tation resulted in a twofold increase in cytoplasmic viral RNA expression a nd a recovery of vector efficiency to similar to 50% of the wild-type level . This increase in cytoplasmic RNA levels is likely to be due, at least in part, to effects of the TE671 host cells, a human rhabdomyosarcoma cell lin e used for vector production in our system, on the cytoplasmic distribution of spliced and unspliced viral RNA. These results show that optimal lentiv irus vector function can be maintained in the absence of multiple essential viral elements.