OPTIMAL TAT-MEDIATED ACTIVATION OF THE HIV-1 LTR PROMOTER REQUIRES A FULL-LENGTH TAR RNA HAIRPIN

HIV-1 transcription from the LTR promoter is activated by the viral Ta t protein through interaction with the nascent TAR RNA hairpin structu re. The mechanism of Tat-mediated transcriptional activation has been extensively investigated with LTR-CAT reporter genes in transient tran sfections and, more recently, in infection experiments with mutant HIV -1 variants, Several discrepancies between these two assay systems hav e been reported. For instance, whereas opening of the lower part of th e TAR RNA stem does not affect the promoter activity of an LTR-CAT pla smid in transient assays, the corresponding virus mutant is fully repl ication-impaired, With the aim to resolve this controversy, we have ex amined the activity of a set of TAR RNA mutants in transient transfect ion experiments with a variety of cell types, We now demonstrate that truncated TAR motifs exhibit a severe, but cell-type dependent transcr iption defect. Whereas full LTR activity is measured in COS cells that have been used regularly in previous transfection assays, a severe de fect is apparent in a variety of human cell lines, including T cell li nes that are typically used in HIV-I replication studies, These result s suggest the presence of a human protein that participates in Tat-med iated transcriptional activation through binding to the lower part of the TAR stem. Several candidate co-factors have been reported in liter ature. This study resolves the discrepancy between transfection and in fection studies on the requirements of the fewer TAR stem structure. T he evidence also implies that LTR transcription studies should be perf ormed preferentially in human cell types.