Tat functions to stimulate the elongation properties of transcription complexes paused by the duplicated TAR RNA element of human immunodeficiency virus 2

Abstract

In this study we have defined the in vitro requirements for transcriptional regulation of the HIV-2 LTR in response to the HIV-1 and HIV-2 Tat proteins and addressed potential mechanisms of Tat function. HIV-2 contains a duplicated TAR RNA stem-loop structure in contrast to the single stem-loop structure found in HIV-1 TAR RNA. We demonstrated that the HIV-2 proximal TAR RNA stem-loop structure was more important for in vitro transcriptional activation by the HIV-1 and HIV-2 Tat proteins than the distal TAR RNA stem-loop though this downstream TAR element itself was able to confer Tat-responsiveness. The role of the two HIV-2 TAR RNA stem-loop bulge sequences was less critical than the loop sequences for in vitro transcriptional activation by Tat. In addition, we demonstrated that replacing the HIV-2 TATA element with that of HIV-1 markedly reduced the overall level of Tat activation. The role of the Tat-1 and Tat-2 proteins on the synthesis of HIV-1 and HIV-2 promoter proximal and promoter distal transcripts was then investigated. In contrast to the HIV-1 promoter, the HIV-2 promoter generated abundant levels of short transcripts in vitro transcription assays likely due to the structure of its duplicated TAR element. Both Tat-1 and Tat-2 increased the level of transcripts extending to the end of the HIV-1 and HIV-2 TAR elements as well as the level of transcripts extending more than 500 nucleotides from the transcription initiation site. However, the synthesis of transcripts within 30 nucleotides of the HIV-2 LTR transcription initiation site was unchanged in either the presence or absence of Tat while the level of transcripts extending increasing distances from the HIV-2 LTR transcription initiation site were progressively stimulated in the presence of Tat. Though the HIV-1 Tat protein was a stronger inducer of HIV-1 LTR transcription than the HIV-2 Tat protein, we did not detect differences in the binding of these proteins to the HIV-1 and HIV-2 TAR RNAs. This suggested that differences in their transactivation properties may be due to alterations in their association with RNA polymerase II or associated elongation factors. (ABSTRACT TRUNCATED AT 250 WORDS)