Human immunodeficiency virus type 1 and 2 Tat proteins specifically interact with RNA polymerase II.
The Tat-responsive region (TAR) element is a critical RNA regulatory element in the human immunodeficiency virus (HIV) long terminal repeat, which is required for activation of gene expression by the transactivator protein Tat. Recently, we demonstrated by gel-retardation analysis that RNA polymerase II binds to TAR RNA and that Tat prevents this binding even when Tat does not bind to TAR RNA. These results suggested that direct interactions between Tat and RNA polymerase II may prevent RNA polymerase II pausing and lead to Tat-mediated increases in transcriptional 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 that the basic domains of both the HIV-1 and HIV-2 Tat proteins were required for this interaction. Furthermore, "far Western" analysis suggested that the largest subunit of RNA polymerase II was the site for interaction with Tat. The interactions between Tat and RNA polymerase II were of similar magnitude to those detected between RNA polymerase II and the cellular transcription factor RAP30, which stably associates with RNA polymerase II during transcriptional elongation. These studies 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.
Mavankal
G
G
Division of Molecular Virology, University of Texas Southwestern Medical Center, Dallas 75235-8594, USA.
Ignatius Ou
S H
SH
Oliver
H
H
Sigman
D
D
Gaynor
R B
RB
D000818:Animals; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism; D004800:Enzymes, Immobilized; D015967:Gene Expression Regulation, Viral; D015696:Gene Products, tat / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D015498:HIV-2 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008970:Molecular Weight; D011485:Protein Binding; D012319:RNA Polymerase II / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus
D004800:Enzymes, Immobilized; D015696:Gene Products, tat; D054322:tat Gene Products, Human Immunodeficiency Virus; D012319:RNA Polymerase II