HIV Rev response element (RRE) directs assembly of the Rev homooligomer into discrete asymmetric complexes

Abstract

RNA is a crucial structural component of many ribonucleoprotein (RNP) complexes, including the ribosome, spliceosome, and signal recognition particle, but the role of RNA in guiding complex formation is only beginning to be explored. In the case of HIV, viral replication requires assembly of an RNP composed of the Rev protein 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 oligomerization of Rev on the RRE scaffold and utilizes both protein-protein 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 oligomeric state and solubility of Rev and guides its assembly into discrete Rev-RNA complexes. SAXS and EM data were used to derive a structural model of a Rev dimer bound to an essential RRE hairpin and to visualize the complete Rev-RRE RNP, demonstrating that RRE binding drives assembly of Rev homooligomers into asymmetric particles, reminiscent of the role of RNA in organizing more complex RNP machines, such as the ribosome, composed of many different protein subunits. Thus, the RRE is not simply a passive scaffold onto which proteins bind but instead actively defines the protein composition and organization of the RNP.nuclear export | ribonucleoprotein assembly | RNA-protein recognition C omplex retroviruses encode essential regulatory proteins that direct nuclear export of the viral RNA genome at late stages in the viral life cycle (1). In the case of HIV, Rev binds to the Rev response element (RRE), a ∼350-nt structured RNA found in the introns of unspliced viral mRNAs, and interacts with the Crm1 nuclear export receptor to facilitate transport to the cytoplasm before splicing is completed (1, 2). In this way, assembly of the Rev-RRE ribonucleoprotein (RNP) complex is coupled to expression of the virion structural proteins and packaging of the genomic RNA.In addition to RRE binding, oligomerization of Rev along the RNA is required for export (3). Early studies defined one specific site in the RRE, known as stem IIB, as necessary, but not sufficient, for in vivo function (2, 4-6). Rev binds to stem IIB using a 17-amino acid α-helical arginine-rich motif (ARM), whose interaction is well understood at the biochemical and structural levels (7, 8). However, full RNA export activity requires more than 230-nt of the ∼350-nt structured RRE, as well as two Rev oligomerization domains (Fig. 1) (2, 3, 5, 6). The RRE drives assembly of a highly cooperative complex with the Rev homooligomer, with an affinity ∼500-fold higher than Rev binding to stem IIB alone (9). The oligomerization domains and large RRE structure both are required for tight complex assembly in vitro, correlating with their requirement for RNA export activity in vivo and demonst...

Nuclear Magnetic Resonance Structure Revealed that the Human Polyomavirus JC Virus Agnoprotein Contains an α-Helix Encompassing the Leu/Ile/Phe-Rich Domain

“…Like agnoprotein, a variety of viral proteins reported in the literature form dimers and oligomers, including hepatitis C virus nonstructural protein 4B (65), Ebola virus VP40 (66), polyomavirus LT-Ag (67,68), and HIV-1 proteins (Rev, Vpr, Vif, and Vpu) (44,(69)(70)(71)(72). For instance, Rev binds to HIV RNA and regulates the transport of unspliced or partially spliced RNA molecules from the nucleus to the cytoplasm.…”

Biochemical Analysis of the Complex between the Tetrameric Export Adapter Protein Rec of HERV-K/HML-2 and the Responsive RNA Element RcRE pck30

“…Therefore, we propose that the binding of RcRE pck30 might induce a conformational change in Rec that tightens the interaction between the subunits. This is again comparable to Rev binding because the RRE seems to control the oligomeric state of Rev as well (12).…”

“…A sequential binding of Rev to one RRE was proposed with the multimeric assembly nucleated by the high-affinity binding of Rev (12,13,40). The binding of additional Rev dimers occur to already bound Rev proteins and adjacent sites on the RNA with less affinity (13,40).…”

Analysis of the Relationship between the Structure and Function of the HIV-1 Rev Response Element (RRE)

“…This export is initiated by the binding of HIV Rev protein to the RRE, a cis-acting RNA element found in a highly conserved region of env in these mRNAs (67,75,151,251). Functionally, the RRE has been modeled as a scaffold for binding Rev molecules (48). The primary Rev binding site on the RRE has been mapped to stem IIB (41,56,125), and stem I/I' has been shown to provide secondary Rev binding sites (51,155).…”

“…A series of genetic, biochemical, and biophysical studies have contributed to our understanding of Rev structure and some molecular details of the assembly of NL4-3 Rev and the RRE (12,13,49,50,53,116,118,154,185,228). For a long time, the only structural information of Rev came from an NMR study (13) and circular dichroism experiments (225) using a 23 amino acid synthetic polypeptide corresponding to the ARM domain of Rev (residue 34 to 50) bound to the stem IIB of RRE containing the high affinity Rev binding site (13).…”