Author response: Insights into HIV-1 proviral transcription from integrative structure and dynamics of the Tat:AFF4:P-TEFb:TAR complex (2016)

Full text Figures and data Side by side Abstract Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract HIV-1 Tat hijacks the human superelongation complex (SEC) to promote proviral transcription. Here we report the 5.9 Å structure of HIV-1 TAR in complex with HIV-1 Tat and human AFF4, CDK9, and CycT1. The TAR central loop contacts the CycT1 Tat-TAR recognition motif (TRM) and the second Tat Zn2+-binding loop. Hydrogen-deuterium exchange (HDX) shows that AFF4 helix 2 is stabilized in the TAR complex despite not touching the RNA, explaining how it enhances TAR binding to the SEC 50-fold. RNA SHAPE and SAXS data were used to help model the extended (Tat Arginine-Rich Motif) ARM, which enters the TAR major groove between the bulge and the central loop. The structure and functional assays collectively support an integrative structure and a bipartite binding model, wherein the TAR central loop engages the CycT1 TRM and compact core of Tat, while the TAR major groove interacts with the extended Tat ARM. https://doi.org/10.7554/eLife.15910.001 Introduction The human immunodeficiency virus-1 (HIV-1) remains one of the world’s leading health threats. In spite of advances in treatment with antiretrovirals (HAART) (Yeni 2006), it has not been possible to eradicate HIV-1 infection. Even under intensive antiretroviral therapy, resting T cells that harbor latent provirus integrated into their genome survive (Archin et al., 2014). The pool of latent virus is the primary obstacle to eradicating HIV, and thus the mechanisms by which latency is regulated are of paramount interest. The molecular mechanisms regulating HIV-1 transcription have been studied for three decades, yet renewed interest in eradicating HIV-1 infection has lent new urgency to obtaining a more complete understanding. Proviral transcription is silenced by host epigenetic mechanisms and/or deficiency in key cofactors, and is reactivated at the level of both initiation and elongation (Mbonye and Karn 2014; Zhou et al., 2012; Jonkers and Lis 2015). Transcription of the HIV-1 proviral DNA is initiated by RNA polymerase II (Pol II) binding to the HIV promoter, but stalls after a 30–50 nucleotide transcript containing the trans-activation response region (TAR) is formed. Release of the stalled Pol II is dependent on the recruitment of the host super elongation complex (SEC) (He et al., 2010; Sobhian et al., 2010) to the nascent TAR by HIV-1 trans-activator protein (Tat), a central driver of proviral transcriptional activation (Fisher et al., 1986). The SEC is assembled on the AFF1 or AFF4 scaffold protein that interacts through small binding domains with the positive elongation factor b (P-TEFb), composed of CDK9 and Cyclin T1 (CycT1), the transcription factor ELL1/ELL2, and ENL/AF9 (Chou et al., 2013; He et al., 2010; Lin et al., 2010; Sobhian et al., 2010). P-TEFb facilitates promoter escape by phosphorylating two negative elongation factors (DSIF and NELF) as well as the C-terminal domain of Pol II. Overexpression of full-length AFF1, but not a fragment that only binds to P-TEFb, strongly increases Tat transactivation of transcription from the HIV promoter (Lu et al., 2014), indicating that productive HIV transcription requires the whole SEC. Recent crystal structures have revealed how the intrinsically disordered P-TEFb-binding domain of AFF4 folds and binds in a hydrophobic groove on the CycT1 surface (Schulze-Gahmen et al., 2013; Chou et al., 2013). HIV-1 Tat binds to CycT1 in an extended conformation adjacent to AFF4, interacting with residues in the cleft between the two cyclin-fold domains of CycT1, the Tat-TAR recognition motif (TRM), and AFF4 helix α2 (Schulze-Gahmen et al., 2014; Tahirov et al., 2010; Gu et al., 2014). Segments from AFF4, Tat, and CycT1 TRM that are flexible in isolation come together to form a more stable structure with a positively charged surface (Schulze-Gahmen et al., 2014). Biochemical experiments have shown that both Tat and CycT1 make critical and direct contributions to TAR binding (Calnan et al., 1991; Richter et al., 2002b; Garber et al., 1998). In the presence of AFF4, TAR affinity to P-TEFb increases sharply (Schulze-Gahmen et al., 2014). The exact mechanism for the increase in affinity has been unclear, in part due to the lack of a structure of the SEC complex with HIV-1 TAR. To fill the gap in structural understanding, we determined the structure of TAR bound to Tat:AFF4:P-TEFb. We validated our low resolution crystal structure of the TAR complex with data from small angle X-ray scattering (SAXS), hydrogen deuterium exchange (HDX), selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE), binding assays, and previously published biochemical data. The structure of TAR bound to Tat:AFF4:P-TEFb reveals extensive interactions between the TAR loop and a composite protein interaction site, composed of CycT1 TRM and the Tat Zn2+-coordination loop, Tat residues 24–29. Although the Tat ARM is not visible in the electron density maps, its predicted location based on the Tat core position is close to the TAR bulge region in the crystal structure. This constraint, together with biophysical data, were used to develop an integrative model of the ARM, showing it bound through the length of the TAR major groove. AFF4 has no direct contacts with bound TAR in our crystal structure. Instead AFF4 is interacting with the CycT1 TRM and the second Tat Zn2+-coordination loop. Hence, its stimulating activity on TAR binding is likely due to stabilization of the TAR interaction surface of Tat:P-TEFb by AFF4. Our results provide the first structural model, albeit at low resolution, of a critical regulator of HIV-1 latency, the Tat:SEC:TAR complex. Results HDX-MS of Tat:AFF:P-TEFb with and without HIV-1 TAR To localize the regions of the Tat:AFF4:P-TEFb complex that undergo HIV-1 TAR-induced conformational changes, we performed hydrogen-deuterium exchange mass spectrometry (HDX-MS). The HDX rate of a protein region depends on its flexibility, the amount of hydrogen bonding with backbone amides, and solvent accessibility (Hoofnagle et al., 2003; Percy et al., 2012). We compared absolute deuteron incorporation into different segments of Tat:AFF4:P-TEFb in the absence (apo complex) and in the presence of saturating amounts of HIV-1 TAR (TAR complex) (Figure 1A). The observed peptic peptides of the apo complex covered more than 80% of the Tat:AFF4:P-TEFb sequence (Figure 1—figure supplement 1–3). Our HDX data of the apo complex in solution are in good agreement with the crystal structure of Tat:AFF4:P-TEFb (Schulze-Gahmen et al., 2014; Gu et al., 2014) (Figure 1A). The C-terminal segment (residues 59-67) of the synthetic AFF4 peptide (32–67) shows 70% deuteration after 10 s and almost complete deuteration within 3 min (Figure 1B). Although the crystal structure indicates an α-helix in this region, high B-factors indicate high mobility and partial unfolding of this terminal helix in solution, in agreement with the high deuteration levels observed in the HDX experiment. The Tat-TAR recognition motif (TRM; residues 246 to 262 in CycT1, (Das et al., 2004; Garber et al., 1998) also showed almost complete deuteration within 10 s in the apo complex (Figure 1B), which is consistent with multiple conformations for the TRM in the crystal structure (Schulze-Gahmen et al., 2014). Figure 1 with 3 supplements see all Download asset Open asset TAR-dependent changes in the Tat:AFF4:P-TEFb complex. (A) Structure of the Tat:AFF4:P-TEFb complex colored according to the absolute percentage of deuterons incorporated in the presence and absence of TAR after 30 s in D2O. (B) TAR-dependent HDX kinetics of selected segments of Tat:AFF4:P-TEFb complex. Segments with more than 2.5 and 1 amide hydrogens protected in the presence of TAR at time‐point 10 s are colored in dark and light blue, respectively. The HDX kinetics for these segments in the absence of TAR (black) and the presence of TAR (cyan) are shown. https://doi.org/10.7554/eLife.15910.002 Compared with the apo complex, we observed strong protection for the CycT1 TRM and AFF4 helix α2 (residues 59–65) after TAR binding (Figure 1B, dark blue segments) (Figure 1—figure supplement 1–3). These two segments are adjacent to each other in the crystal structure of Tat:AFF4:P-TEFb, consistent with their mutual cooperation in forming the TAR binding site. In contrast to CycT1 and AFF4, Tat exhibited a lesser degree of protection, localized to the N-terminal segment (residues 1-33) (Figure 1B, light blue segments). Surprisingly, the Tat ARM did not show any significant protection in HDX after TAR binding despite its well-established role in TAR binding (Weeks and Crothers, 1991; Calnan et al., 1991) (Figure 1B). This observation indicates that Tat ARM–TAR interactions are mediated primarily by amino acid side chains, without extensive hydrogen bonding and solvent exclusion of main-chain amide protons. SHAPE mapping of the Tat:AFF4:P-TEFb complex on the HIV 5’UTR We performed SHAPE analysis (Merino et al., 2005) to map the interactions of the Tat:AFF4:P-TEFb complex on the HIV 5’UTR, which includes the TAR sequence at its 5’ end. Experiments were done using the 344 nt-long 5’ end of the HIV genome (Heng et al., 2012) in the presence and absence of the Tat-AFF4-P-TEFb complex. Upon protein complex binding, the major SHAPE changes cluster in the 5’ TAR region of the 5’UTR (Figure 2A,B) with significant SHAPE changes observed around the bulge region at A17, the bulge region A22-U25, as well as the loop region C30-A35. As expected, no significant reactivity changes were observed in the regions 3’ to TAR. The SHAPE reactivity for some nucleotides in bulge A22-U25 and loop C30-A35 is extremely high (above 3) in the absence of Tat:AFF4:P-TEFb and remains high even after Tat:AFF4:P-TEFb binding. TAR binding reduces SHAPE reactivity by 0.3-0.4 and 0.2-1.2 for nucleotides in the bulge and loop region, respectively. The pattern of changes in SHAPE reactivity for TAR after binding the SEC is significantly different from changes observed after TAR binding to a Tat peptide covering the ARM region (Kenyon et al., 2015). This difference is expected because of additional contacts between CycT1 and TAR, which lead to higher affinity and more specific interactions between TAR and the SEC (Richter et al., 2002b; Zhang et al., 2000) compared to interactions between TAR and a small arginine-rich peptide from Tat. Because reduction in SHAPE activity correlates with reduced flexibility of the respective RNA regions (McGinnis et al., 2012), the results for the TAR-SEC complex indicate a stabilization of the bulge and loop structure through direct contacts between TAR and the SEC (Figure 2). Figure 2 with 1 supplement see all Download asset Open asset SHAPE profiles of the HIV-1 5’UTR in the presence and absence of the SEC. (A) SHAPE reactivity of the entire HIV-1 5’UTR either alone or in complex with the SEC are shown in red and blue, respectively. SHAPE reactivity profiles are normalized using the 2/8 rule, where the top 2% of reactive nucleotides are excluded and the average of the next 8% of reactive nucleotides is used as the normalization factor. The left insert is a zoomed-in view of the TAR region. The right insert shows the secondary structure of TAR color-coded by the SHAPE profile of the TAR region alone and the SHAPE changes in the TAR region upon SEC binding. (B) SHAPE changes of the HIV-1 5’UTR upon SEC binding. https://doi.org/10.7554/eLife.15910.006 SAXS of the SEC complex with TAR SAXS data were collected for Tat:AFF4:P-TEFb complex in the presence and absence of TAR (Figure 3A). A comparison of SAXS data reveals significant differences in the P(r) function (Figure 3B), radius of gyration (RG), and the Porod exponent (Table 1). The RG of the TAR complex increases by 2.8 Å upon TAR binding. The Porod exponent, in contrast, is smaller for the TAR complex than the apo complex. The Porod exponent is a measure of the compactness of a molecule, with a larger exponent indicating a more compact structure. The TAR complex appears slightly less compact than the apo structure, which agrees with the differences in the shape of the P(r) function. The P(r) curve for the TAR complex is shifted to larger radii, with especially large differences in the distance distributions at r values between 70 and 130 Å. Figure 3 Download asset Open asset SAXS analysis of the apo and TAR-bound Tat:AFF4:P-TEFb complexes. (A) Experimental intensities for the apo complex (left) and the TAR complex (right). Inserts in the top right corner show the Guinier plots for each data set. (B) The distance distribution plots were computed from the experimental data and normalized to the maximum value of unity. https://doi.org/10.7554/eLife.15910.008 Table 1 Overall parameters of Tat:AFF4:P-TEFb complexes determined by SAXS https://doi.org/10.7554/eLife.15910.009 ComplexRG, Å*Dmax, Å*Porod exponent*Tat:AFF4:P-TEFb31.21263.6TAR:Tat:AFF4:P-TEFb33.91343.4 * Parameters were determined using the program SCÅTTER (Förster et al., 2010). Crystal structure of the SEC complex with TAR To clearly define the interactions between TAR and Tat:P-TEFb, and to determine if direct interactions existed between TAR and AFF4, we solved the crystal structure of the TAR:Tat:SEC complex at 5.9 Å resolution (Table 2). The structure was solved by molecular replacement with one copy of the Tat:AFF4:P-TEFb complex (PDB ID 4OGR) in the asymmetric unit. After rigid-body and torsion angle molecular dynamics refinement with deformable elastic network (DEN) restraints (Table 2) (Schröder et al., 2010; Brunger et al., 2012) strong density for the TAR RNA became visible in the Fo-Fc electron density map (Figure 4—figure supplement 1) which allowed placement of one TAR molecule (PDB ID 1ARJ) into the asymmetric unit. The complex was further refined to R/Rfree = 0.211/0.311 with good geometry (Table 2) and an excellent fit to the density (Figure 4—figure supplement 2). Table 2 X-ray data collection and refinement statistics for Tat:AFF4:P-TEFb with TAR21. https://doi.org/10.7554/eLife.15910.010 Data collectionSpace groupP3221Cell dimensions: a, b, c146.87, 146.87, 103.75Resolution (Å)*50.0-5.9 (6.1–5.9)Unique reflections*3607 (339)I/σ(I)*10.22 (0.35)Rsym (%)*17.8 (>100)CC1/2 high resolution shell0.24Completeness (%)*100.0 (100.0)Redundancy*9.68 (10.25)Twin fraction†0.03Temperature (K)100Mosaicity (°)0.175 RefinementResolution (Å)50.0-5.9No. reflections3595DEN refinement parameter§γ=0, wDEN=300Rwork/Rfree0.223/0.315Average B-factor (Å2)400R.m.s. deviationsBond lengths (Å)0.0027Bond angles (º)0.81Ramachandran plot#Favored (%)92.01%Allowed (%)6.43%Disallowed (%)1.57% *Values in parentheses are for the highest resolution shell. †Value from Ctruncate (see Table 2—source data 1) §parameter for DEN refinement in CNS (see Table 2—source data 1) #Values from COOT. Table 2—source data 1 Diffraction data and refinement analysis. Twinning analysis by L-test in Ctruncate (A) and Xtriage indicate no twinning. Blue curve is acentric untwinned data, green curve acentric twinned data, and red curve observed data from Tat:AFF4:P-TEFb-TAR co-crystal. (B, C) Optimization of DEN refinement parameters using the web service for low resolution crystal structure refinement (https://portal.sbgrid.org/d/apps/den/). (D) Log-file of twinning analysis in CNS. https://doi.org/10.7554/eLife.15910.011 Download elife-15910-table2-data1-v3.pdf Table 2—source data 2 RSCC and RSR=sum|r-r_calc|/sum[r+r_calc| using 2fofc map and D*Fcalc. https://doi.org/10.7554/eLife.15910.012 Download elife-15910-table2-data2-v3.txt Figure 4 with 2 supplements see all Download asset Open asset Ribbon diagram of the crystal structure of HIV-1 TAR in complex with Tat:AFF4:P-TEFb. The top panel shows an overview of the complex with the TAR loop next to CycT1 TRM (yellow) and Tat (red), and the TAR bulge close to Tat R49, the start of the Tat ARM. Zn2+ ions are shown as grey spheres. The close-up view below highlights the interactions of the Zn2+ binding loop and CycT1 TRM with the TAR-loop and the role of AFF4 (blue) in indirectly stabilizing the Tat-CycT1 surface involved in TAR interactions. TAR nucleotides with reduced SHAPE activity (< −0.2) in the presence of SEC are colored blue, those with increased SHAPE activity (> 0.2) are colored red. https://doi.org/10.7554/eLife.15910.013 The extended TAR loop region 29-33 contributes the main interactions with the SEC. It interacts with the protein complex surface composed of CycT1 TRM and the Tat cysteine-rich region, especially the Zn2+-coordinating loop (Tat 24-29). This structure rules out direct interactions between TAR and AFF4 α2. TAR loop nucleotides G32 and G33 abut the Tat Zn2+ -coordination loop (24-29) and CycT TRM, residues 259-261. The backbone phosphates of nucleotides 30–32 contact basic residues in the CycT TRM region (Figure 4). This orientation places the N-terminus of the Tat ARM (Arg49) at the widest point in the TAR major groove, which is 13 Å wide between nucleobases C24 and G34. However, there is not interpretable electron density present for residues in the Tat ARM region at the limited resolution of this crystal structure. This seems consistent with the lack of protection of this region seen in the HDX experiment (Figure 1B), but it is important not to overinterpret this observation given the limitations of the 5.9 Å map. The structure of the TAR complex with Tat:AFF4:P-TEFb shows no direct contacts between AFF4 and TAR. Instead, AFF4 helix α2 and its N-terminal loop connection to helix α1 are interacting with CycT1 TRM and Tat helix α1. The interaction network between these three protein regions most likely stabilizes the protein interaction surface for TAR and indirectly increases Tat:SEC affinity for TAR in the presence of AFF4 (Schulze-Gahmen et al., 2014). Modeling of Tat ARM and CycT1 TRM The Tat ARM and CycT TRM are key components for TAR binding by the SEC. Since the crystal structure shows no electron density for Tat ARM and the molecular replacement model for CycT1-TRM segment adopts multiple conformations we modeled the Tat ARM and refined the structure of the CycT1-TRM segment using the MODELLER 9.15 loop modeling protocol (Šali and Blundell, 1993; Fiser and Šali, 2003). Using hierarchical clustering and DOPE scoring (Shen and Sali, 2006) we obtained five best scoring clusters with precisions between 2.5 and 6.3 Å (Figure 5). The computed small angle scattering fits well with SAXS data obtained from the TAR complex in solution with χ=0.86 (Figure 5—figure supplement 1). The CycT1 TRM segment is anchored by CycT1 Cys261, which coordinates one of the Tat Zn2+ ions and displays structural variability in the center part of the TRM (Figure 5B). The Tat ARM region is N-terminally anchored by Tat Tyr47, which binds to a pocket on the CycT1 surface and interacts with CycT1 residues Leu44, Asp47, and Cys111 (Figure 5C). From there, the ARM (residues 49-57) extends into the major groove of TAR from the TAR bulge the backbone the side in the Tat ARM are in where form previously interactions with the TAR and phosphates and in the TAR backbone et al., 1993; et al., Figure with 1 supplement see all Download asset Open asset Ribbon diagram of the crystal structure of the TAR-SEC complex modeled Tat ARM and CycT1 TRM (A) of the whole complex with multiple conformations for ARM and TRM, using the as in Figure (B) of the interactions between and CycT1 The complex is degree around to Tat ARM is N-terminally anchored by Tat to CycT1. The extended ARM is in the TAR major groove, and close to the TAR bulge and TAR of AFF4 on TAR binding and transcriptional activation The strong protection of AFF4 helix α2 (residues observed in HDX experiments due to direct interactions between the AFF4 helix 2 and TAR or to an stabilization of the structure of AFF4 after TAR binding. To between these two we the of in the side of AFF4 helix α2 on TAR binding. We performed mobility assays with TAR and Tat:P-TEFb complex in the presence of or AFF4 fragment (residues (Figure The AFF4 and AFF4 show a and reduction in TAR binding, while the of the other is The in the absence of AFF4 shows a increased for TAR binding by Tat:P-TEFb = compared to the in the presence of AFF4 = The small of surface in AFF4 helix α2 on TAR binding indicates that of direct interactions with TAR structural stabilization of the collectively by (Schulze-Gahmen et al., 2014) is the major role of AFF4 α2. Figure Download asset Open asset AFF4 in helix α2 TAR binding. (A) mobility assays with TAR and of Tat:P-TEFb in the absence or presence of AFF4 fragment or AFF4 In the absence of AFF4, affinity for TAR is reduced 50-fold. and TAR affinity 3 and respectively. (B) cells containing the integrated HIV-1 were with the and/or as were in with the level of activity in cells with an to The one from three To the to HIV-1 we the of on HIV-1 transcription in HIV-1 These assays were performed with the AFF1 scaffold because Tat has a on HIV transcription with AFF1 than with AFF4 (Lu et al., 2015). In the used a Tat that an important for Tat activity et al., 1998). It is thus to the increase in affinity for P-TEFb (Lu et al., 2014). AFF1 strongly the activation of HIV-1 by all with observed and were either or (Figure The results from and transcription assays are consistent with the structural that helix α2 a significant role in TAR binding and the crystal structure shows no direct contact between of TAR on SEC binding have TAR that transactivation activity of TAR and TAR binding to a the ARM region of Tat et al., with strongly reduced transactivation activity and which are below and in the TAR To further AFF4 function in TAR binding we determined binding for and to Tat:P-TEFb with or without AFF4 (Figure in mobility assays In the absence of AFF4, all three showed strongly reduced binding affinity compared to the TAR. However, in the presence of AFF4, the affinity of TAR was increased to and binding of the was to a increased compared to TAR (Figure The of bulge by of AFF4 is consistent with the placement of the bulge to the Tat The that is of a bipartite of TAR binding to the SEC. with the of the Tat and the adjacent N-terminal of the CycT1 This region is the part that is by AFF4. The nucleobases of the binding on TAR, the with extended regions of the C-terminal part of the CycT1 TRM and with the Tat ARM. Figure Download asset Open asset of TAR on Tat:P-TEFb binding in the absence and presence of AFF4. mobility assays with TAR were performed as in Figure TAR and et al., show large in binding to Tat:P-TEFb, but in the presence of AFF4. Experiments were performed in The the from three Discussion of we how HIV-1 TAR binds to HIV-1 Tat from some of structural of TAR in complex with or peptide of Tat et al., et al., et al., 1993; and et al., Tat not function alone in human it hijacks the host of CDK9 and Cyclin T1 (P-TEFb), ELL1/ELL2, and ENL/AF9 et al., 2010; He et al., 2010; et al., 2012; et al., 2013). It has been for that the interaction of Tat with P-TEFb the of its interaction with TAR. a key in the has been to the structural for TAR binding to Tat in the of the SEC. it has to structural data on HIV TAR in the of the functional human complex. We have determined the first crystal structure of a TAR:Tat:SEC complex to 5.9 Å resolution (Figure 4). the resolution, the are to out of TAR. We a of biophysical to support and the crystal structure. These data important changes, especially in AFF4, that are not from the crystal structure The Tat ARM region was in the crystal structure. The Tat ARM, as well as the disordered CycT1 TRM were modeled as loop into the crystal structure (Figure 5). In agreement with biochemical data et al., Garber et al., Richter et al., HIV-1 TAR binds with the loop region interacting with CycT1-TRM and the Zn2+-coordinating core region of Tat, while the TAR bulge is close to the expected location of Tat ARM (Figure 5). Results from HDX experiments for the ARM (Figure 1) indicate a high flexibility for the Tat ARM even after binding to TAR. the Tat peptide of its within 10 TAR was bound or This region of Tat is in the crystal structure through and the are protected by hydrogen through of the peptide is protected from the that is involved in hydrogen bonding in the crystal structure. This that the ARM main-chain is extended and bound to TAR. The five Tat ARM structures in integrative modeling are all in an extended conformation in the TAR major groove (Figure which the basic residues in the ARM to make contacts with in the TAR bulge and with the In contrast, the TAR central loop binds to a more protein composed of the CycT1 TRM and the second Tat Zn2+ loop, Tat residues Although AFF4 has no direct interactions with TAR, residues in helix α2 Tat helix α2 and the Zn2+ loop, and the CycT1 This network of AFF4 which stabilizes CycT1 and Tat conformations in the TAR interacting region, is the likely for the increased binding affinity of Tat:P-TEFb for TAR in the presence of AFF4 (Figure with the bipartite of binding by the crystal structure and dynamics data, AFF4 is to binding of TAR in the bulge and the region below the bulge (Figure This is to the and stabilizing role of AFF4 on the TAR interacting surface composed of CycT1 TRM and Tat loop, Our