In vitro formation of short RNA polymerase II transcripts that terminate within the HIV-1 and HIV-2 promoter-proximal downstream regions.

Trans-activation of HIV-1 transcription by the viral regulatory protein Tat has been proposed recently to overcome a block to RNA polymerase II elongation in vivo imposed by 5'-untranslated leader sequences. Interestingly, however, only full-length transcripts, rather than prematurely terminated HIV RNAs, are synthesized in most cell-free transcription extracts. Here, we describe an in vitro system in which induction of a highly efficient RNA polymerase II termination or cotranscriptional RNA processing event creates short HIV RNAs with 3' ends that map to a region immediately downstream of the HIV-1 or HIV-2 promoters. Termination in vitro is sequence dependent, generating short HIV-1 RNAs of 58-61 nucleotides that resemble in vivo transcripts observed in the absence of Tat, and a distinct, longer transcript of approximately 125-130 nucleotides from the HIV-2 promoter. Deletion of promoter-proximal HIV-2 downstream sequences results in the loss of a discrete RNA but also fails to restore wild-type transcription, indicating that termination actually is specified at the promoter and occurs at a site positioned by one or more elements located immediately upstream of the 3' end of the short RNAs. Experiments with recombinant HIV-2 promoters and nucleoside analogs indicate that this event involves a concerted interaction between the promoter and orientation-dependent leader sequences and that RNA secondary structure formation may also be required. These data provide direct evidence for abbreviated HIV transcripts and an in vitro approach to understanding the roles of cellular and viral regulatory proteins that mediate this process at the HIV promoters.