HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 AND TYPE-2 TAT PROTEINS SPECIFICALLY INTERACT WITH RNA-POLYMERASE-II

The Tat-responsive region (TAR) element is a critical RNA regulatory e lement in the human immunodeficiency virus (HIV) long terminal repeat, which is required for activation of gene expression by the transactiv ator protein Tat, Recently, we demonstrated by gel-retardation analysi s that RNA polymerase II binds to TAR RNA and that Tat prevents this b inding even when Tat does not bind to TAR RNA, These results suggested that direct interactions between Tat and RNA polymerase II may preven t RNA polymerase II pausing and lead to Tat-mediated increases in tran scriptional elongation. To test this possibility, we performed protein interaction studies with RNA polymerase II and both the HIV-1 and the closely related HIV-2 Tat protein, These studies indicated that both the HIV-1 and HIV-2 Tat proteins could specifically interact with RNA polymerase II, Mutagenesis of both HIV-1 and HIV-2 Tat demonstrated th at the basic domains of both the HIV-1 and HIV-2 Tat proteins were req uired for this interaction. Furthermore, ''far Western'' analysis sugg ested that the largest subunit of RNA polymerase II was the site for i nteraction with Tat. The interactions between Tat and RNA polymerase I I were of similar magnitude to those detected between RNA polymerase I I and the cellular transcription factor RAP30, which stably associates with RNA polymerase II during transcriptional elongation, These studi es are consistent with the model that RNA polymerase II is a cellular target for Tat resulting in Tat-mediated increases in transcriptional elongation from the HIV long terminal repeat.