The replication of human immunodeficiency virus (HIV) is dependent on the function of two small virus coded regulatory proteins, TAT and REV. REV protein acts post-transcriptionally to modulate the splicing, extra- nuclear transport and/or translational utilization of unspliced or partially spliced viral RNAs. REV mediates its function by interacting with a highly structured viral mRNA sequence, RRE (REV responsive element) of about 236 nt. located in the ENV ORF. A) Studies using 80 or so RRE mutants have demonstrated that i) REV binding to RRE RNA is necessary but not sufficient for REV function in vivo; ii) REV binding to RRE is dependent on the presence of a 5'(50)CACUAUGGG(58)3' in the context of an unique secondary structure; iii) of the 56..GGG..58 sequence the G-56 is critical and a minimum of 2 Gs are required for RRE function; iv) other homopolymers cannot substitute, and v) mutations distal to the 3 Gs that result in base-pairing of the Gs eliminate binding. B) We have identified a putative nuclear factor that binds to a sub-domain of RRE in vitro distinct from the REV recognition region. C) We have devised a chimeric RRE construct replacing the REV responsive domain with an unrelated RNA, namely the MS2 phage translational operator sequence. This chimera was activated by a REV-MS2 coat protein fusion that tethered REV to the heterologous RNA via the MS2 protein. These fusion proteins have allowed us to dissect the function of distinct domains within REV and RRE. The above molecular studies have enabled us to devise genetic therapeutic approaches using RNA decoys to impede REV function in natural HIV infection. We have extended and refined our earlier studies on the effect of HIV-1 NEF protein on LTR transcription. Stable Hela cell and Jurkat lymphoid cell lines expressing NEF from HIV-1 LTR, CMV early promoter or MMTV or RSV LTR have been developed. NEF expression in these cell lines was consistently associated with repression of HIV-1 LTR transcription. Although the mechanism(s) of NEF induced repression were not deduced, they are unlikely to be mediated by GTP binding or GTPase activities. NEF from four different HIV-1 isolates were expressed and purified. All the NEF proteins were devoid of GTP binding or GTPase activities although they possessed measurable auto-kinase activity. To examine pathogenic potential of NEF, several transgenic mouse lines expressing NEF either from the HIV-1 LTR or MMTV LTR were constructed. HIV-1 LTR linked NEF expression was confined to skin, particularly in the basal cells of the epithelium and the Langhans cells. Interestingly, NEF expression was associated with papillomatous skin lesions that varied in severity depending on the level of NEF expression. MMTV LTR driven NEF expression was observed in the mammary and salivary glands, seminal vesicles and the testes. In all these organs, no obvious pathology was associated with NEF expression. A small number of MMTV NEF transgenic mice also expressed NEF in the skin where it was associated with papillomas.
