Structure, sequence, and position of the stem-loop in tar determine transcriptional elongation by tat through the HIV-1 long terminal repeat.

The human immunodeficiency virus (HIV-1)-encoded trans-activator (tat) increases HIV gene expression and replication. Previously, we demonstrated that tat facilitates elongation of transcription through the HIV-1 long terminal repeat (LTR) and that short transcripts corresponding to prematurely terminated RNA are released and accumulate in the absence of tat. Here, using a transient expression assay, we tested clustered and compensatory mutations, as well as 3' deletions, in the trans-acting responsive region (tar) and observed that the primary sequence in the loop and secondary structure in the stem of the stem-loop in tar are required for trans-activation by tat. Insertions in the 5' region of tar revealed that tar must be near the site of HIV-1 initiation of transcription for trans-activation by tat. Deletions (3') and an insertion in tar demonstrated that an intact stem-loop is required for the recovery of prematurely terminated transcripts. Short and full-length transcripts were observed also with HIV type 2 (HIV-2) in the absence and presence of tat, respectively. We conclude that an intact stem-loop in tar is essential for trans-activation by tat and that initiation of transcription by HIV-1 promoter factors and elongation of transcription by tat are coupled.