AFF4 binding to Tat-P-TEFb indirectly stimulates TAR recognition of super elongation complexes at the HIV promoter.

Ursula Schulze-Gahmen,Qiang Zhou,Huasong Lu,Tom Alber

doi:10.7554/elife.02375

Abstract

Superelongation complexes (SECs) are essential for transcription elongation of many human genes, including the integrated HIV-1 genome. At the HIV-1 promoter, the viral Tat protein binds simultaneously to the nascent TAR RNA and the CycT1 subunit of the P-TEFb kinase in a SEC. To understand the preferential recruitment of SECs by Tat and TAR, we determined the crystal structure of a quaternary complex containing Tat, P-TEFb, and the SEC scaffold, AFF4. Tat and AFF4 fold on the surface of CycT1 and interact directly. Interface mutations in the AFF4 homolog AFF1 reduced Tat-AFF1 affinity in vivo and Tat-dependent transcription from the HIV promoter. AFF4 binding in the presence of Tat partially orders the CycT1 Tat-TAR recognition motif and increases the affinity of Tat-P-TEFb for TAR 30-fold. These studies indicate that AFF4 acts as a two-step filter to increase the selectivity of Tat and TAR for SECs over P-TEFb alone.DOI: http://dx.doi.org/10.7554/eLife.02375.001.

Highlights

Transcription of the human immunodeficiency virus (HIV) genome by RNA polymerase II (Pol II), like the expression of many cellular genes, is largely regulated at the step of transcript elongation. (Levine, 2011; Lin et al, 2011; Luo et al, 2012; Zhou et al, 2012)
Pol II is recruited to the HIV promoter and initiates transcription, which stalls after a 30–50 nucleotide transcript containing the trans-activating response region (TAR) is formed
Since AFF4 binds close to the TAR recognition motif (TRM) in the Tat-AFF4-positive elongation factor b (P-TEFb) structure, we investigated the effect of AFF4 on TAR binding

Summary

Introduction

Transcription of the HIV genome by RNA polymerase II (Pol II), like the expression of many cellular genes, is largely regulated at the step of transcript elongation. (Levine, 2011; Lin et al, 2011; Luo et al, 2012; Zhou et al, 2012). The HIV Tat protein bound to a host super elongation complex (SEC) recognizes TAR and releases the paused polymerase (He et al, 2010; Sobhian et al, 2010). It is yet unclear how TAR and Tat recruit SECs in preference to other complexes in the cell that contain SEC subunits. P-TEFb and the transcriptional elongation factors ELL2 and ENL/AF9 associate with hydrophobic segments in the approximately 1200-amino-acid AFF1 or AFF4 scaffold, together forming the SEC (He et al, 2010; Lin et al, 2010; Sobhian et al, 2010). Overexpression of an AFF1 fragment that binds to P-TEFb, but not the other components of SECs, has a strong inhibitory effect on HIV transcription, indicating that productive HIV transcription requires a complete SEC (Lu et al, 2013a)

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Results

Conclusion