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HIV Rev Response Element (RRE) Directs Assembly of the Rev Homooligomer Into Discrete Asymmetric Complexes

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HIV Rev Response Element (RRE) Directs Assembly of the Rev Homooligomer Into Discrete Asymmetric Complexes HIV Rev response element (RRE) directs assembly of the Rev homooligomer into discrete asymmetric complexes Matthew D. Daughertya,c, David S. Boothb,c, Bhargavi Jayaramanc, Yifan Chengc, and Alan D. Frankelc,1 aChemistry and Chemical Biology Graduate Program, bGraduate Group in Biophysics, and cDepartment of Biochemistry and Biophysics, University of California, San Francisco, CA 94158 Communicated by Keith R. Yamamoto, University of California, San Francisco, CA, June 2, 2010 (received for review February 25, 2010) RNA is a crucial structural component of many ribonucleoprotein (RNP) complexes, including the ribosome, spliceosome, and signal A recognition particle, but the role of RNA in guiding complex forma- tion is only beginning to be explored. In the case of HIV, viral re- plication requires assembly of an RNP composed of the Rev protein B homooligomer and the Rev response element (RRE) RNA to mediate nuclear export of unspliced viral mRNAs. Assembly of the functional Rev-RRE complex proceeds by cooperative oligo- merization of Rev on the RRE scaffold and utilizes both protein-pro- tein and protein-RNA interactions to organize complexes with high specificity. The structures of the Rev protein and a peptide-RNA complex are known, but the complete RNP is not, making it unclear to what extent RNA defines the composition and architecture of Rev-RNA complexes. Here we show that the RRE controls the oli- gomeric state and solubility of Rev and guides its assembly into discrete Rev-RNA complexes. SAXS and EM data were used to de- rive a structural model of a Rev dimer bound to an essential RRE hairpin and to visualize the complete Rev-RRE RNP, demonstrating Fig. 1. Rev and the RRE. (A) Domain structure of Rev used in this study. (B) that RRE binding drives assembly of Rev homooligomers into Sequence and predicted secondary structure of the 242-nt RRE and stem IIB asymmetric particles, reminiscent of the role of RNA in organizing fragments used. more complex RNP machines, such as the ribosome, composed of additional sites by protein-protein interactions (10). It is not many different protein subunits. Thus, the RRE is not simply a BIOCHEMISTRY passive scaffold onto which proteins bind but instead actively de- known if an oligomeric assembly also is important for other steps fines the protein composition and organization of the RNP. in the export pathway. The structure of the Rev ARM bound to stem IIB (PDB code: nuclear export ∣ ribonucleoprotein assembly ∣ RNA-protein recognition 1ETF) (7) and recent crystal structures of Rev dimers (PDB codes: 3LPH and 2X7L) (11) illuminate essential protein-RNA omplex retroviruses encode essential regulatory proteins that and protein-protein interactions that mediate Rev-RRE assem- Cdirect nuclear export of the viral RNA genome at late stages bly. These results, and models generated from genetic and in the viral life cycle (1). In the case of HIV, Rev binds to the Rev biochemical mapping (12), provide an initial representation of response element (RRE), a ∼350-nt structured RNA found in the Rev-RRE structure, but a complete understanding of how the introns of unspliced viral mRNAs, and interacts with the Crm1 RRE organizes the functional RNP has been hampered by low nuclear export receptor to facilitate transport to the cytoplasm protein solubility, typically limited to 1–5 μM (13, 14) and an before splicing is completed (1, 2). In this way, assembly of inability to define the Rev oligomerization state (14, 15). These the Rev-RRE ribonucleoprotein (RNP) complex is coupled to limitations, and the observation that Rev-RNA complexes form expression of the virion structural proteins and packaging of long filaments when prepared by denaturation-renaturation in the genomic RNA. vitro (13, 16), have called into question the existence of discrete In addition to RRE binding, oligomerization of Rev along the Rev-RRE complexes. However, recent biochemical analyses with RNA is required for export (3). Early studies defined one specific Rev purified under native conditions indicate that RNA plays a site in the RRE, known as stem IIB, as necessary, but not suffi- crucial role in cooperatively forming high-affinity, functional cient, for in vivo function (2, 4–6). Rev binds to stem IIB using a RNPs (9) and suggest that the method of Rev preparation has 17-amino acid α-helical arginine-rich motif (ARM), whose inter- action is well understood at the biochemical and structural levels important consequences for assembly. We surmised that natively (7, 8). However, full RNA export activity requires more than prepared Rev, combined with specific RNAs derived from the 230-nt of the ∼350-nt structured RRE, as well as two Rev oligo- RRE, might form stable, defined RNPs amenable to biophysical merization domains (Fig. 1) (2, 3, 5, 6). The RRE drives assembly study. of a highly cooperative complex with the Rev homooligomer, with ∼500 an affinity -fold higher than Rev binding to stem IIB alone Author contributions: M.D.D. and A.D.F. designed research; M.D.D., D.S.B., and B.J. (9). The oligomerization domains and large RRE structure both performed research; M.D.D., D.S.B., B.J., Y.C., and A.D.F. analyzed data; and M.D.D., are required for tight complex assembly in vitro, correlating with D.S.B., and A.D.F. wrote the paper. their requirement for RNA export activity in vivo and demon- The authors declare no conflict of interest. strating that proper RNP formation is essential for its biological 1To whom correspondence should be addressed. E-mail: [email protected]. function (9). Kinetic analyses suggest an ordered assembly of Rev This article contains supporting information online at www.pnas.org/lookup/suppl/ monomers on the RRE, initiated at stem IIB and propagated at doi:10.1073/pnas.1007022107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1007022107 PNAS ∣ July 13, 2010 ∣ vol. 107 ∣ no. 28 ∣ 12481–12486 Downloaded by guest on September 26, 2021 A IIB:Rev Here we show that RNA binding indeed drives assembly of AB500 discrete Rev-RRE complexes and provides initial structural 6 B -5 1:4 characterizations of two complexes. We find that RNA, or 500µM GB1-Rev mAU RNA surrogates such as a negatively charged protein fusion or 0 oxyanionic counterions, greatly enhance Rev solubility, and that 100 78910 1500 B binding to specific RRE RNAs prevents uncontrolled growth of Rev filaments and determines its precise oligomerization state. 1000 mAU A 1:2 Small-angle X-ray scattering (SAXS) of a dimeric assembly inter- 200µM GB1-Rev 500 mediate and electron microscopy of the full Rev-RRE complex 10 Molar mass (kDa) 0 show that each forms defined RNP particles with distinct lobed Rayleigh ratio (1/cm) (10 ) 78910 0 B features that lack the obvious symmetry observed in Rev dimer 7.5 8 8.5 2000 C structures and expected for complexes assembled with a homo- mL 1:1 oligomeric protein. These asymmetric structures are reminiscent mAU 1000 IIB of RNPs composed of heterooligomeric subunits, such as the C A alone 2.5 0 ribosome, and indicate that the RRE plays an active role in de- 1:4 78910 fining the overall architecture of the RNP. -5 mL D 1:4 1:2 1:1 Results 1:1 100 1:2 Fraction IIB A B A B A B C RNA Controls the Solubility and Oligomerization State of Rev. The importance of Rev oligomerization and RNA binding for HIV RNA export is well known, but attempts to understand the IIB molecular basis of Rev-RRE assembly have been hampered by 10 Molar mass (kDa) low Rev solubility and uncontrolled oligomerization (14, 15). Rayleigh ratio (1/cm) (10 ) 0 78910 We recently described how Rev purified under nondenaturing mL conditions assembles into a cooperative, high-affinity oligomeric complex with the RRE that correlates with its activity in vivo (9). Given that these binding properties had not been described pre- Fig. 2. RNA controls the oligomerization state of Rev. (A) MALS measure- viously, we reasoned that preparing Rev-RNA complexes under ment from SEC of 200 μM (red) and 500 μM (black) GB1-Rev in the absence similar native conditions might generate samples suitable for of RNA. Light scattering is shown as a function of elution volume (dashed structural characterization. lines, left axis). Calculated molar masses are shown for each peak (solid lines, right axis). (B) SEC of 200 μM GB1-Rev in the presence of 50 μM(Top), 100 μM We considered two important differences between our proto- Middle μ Bottom – ( ), or 200 M( ) IIB34 RNA, with absorbance monitored at col (9) and others (3, 4, 12 15). First, we expressed Rev as a 260 nm. The profile of 10 μM IIB34 alone is shown in the bottom panel. fusion to GB1, a commonly used expression tag derived from Arrows indicate fractions analyzed by native PAGE (D). (C) MALS (dashed the well-folded B1 domain of streptococcal protein G (17). Sec- lines, left axis) and calculated molar masses (solid lines, right axis) determined ond, we did not remove RNA during the purification, even after from the SEC peaks in B, with 50 μM unbound IIB34 shown for reference. cleaving Rev from the GB1 fusion, and thus the preparation con- (D) Native gel analyses of SEC fractions (B), with unbound IIB34 RNA in tained endogenous Escherichia coli RNAs. To assess the influence the left lane. of each factor, we purified GB1-fused Rev (termed GB1-Rev) and removed RNA with RNases and high salt washes, typically coupled multiangle light scattering (MALS) and refractive index obtaining GB1-Rev at substantially higher concentrations measurements (Fig. 2A and Table S1).
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