Solution Structure of the Rous Sarcoma Virus Nucleocapsid Protein: μΨ RNA Packaging Signal Complex

Abstract

The 5′-untranslated region (5′-UTR) of retroviral genomes contains elements required for genome packaging during virus assembly. For many retroviruses, the packaging elements reside in noncontiguous segments that span most or all of the 5′-UTR. The Rous sarcoma virus (RSV) is an exception in that its genome can be efficiently packaged by a relatively short, 82-nucleotide segment of the 5′-UTR called μΨ. The RSV 5′-UTR also contains three translational start codons (AUG-1, -2 and -3) that have been controvertibly implicated in translation initiation and genome packaging, one of which (AUG-3) resides within the μΨ sequence. We recently demonstrated that μΨ is capable of binding to the cognate RSV nucleocapsid protein (NC) with high affinity (dissociation constant K d ~2 nM), and that residues of AUG-3 are essential for tight binding. We now report the solution structure of the NC:μΨ complex, determined using NMR data obtained for samples containing 13 C, 15 N-labeled NC and 2 H-enriched, nucleotide-specifically-protonated RNAs. Upon NC binding, μΨ adopts a stable secondary structure that consists of three stem loops (SL-A, SL-B and SL-C) and an 8-base pair stem (O3). Binding is mediated by NC's two zinc knuckle domains. The N-terminal knuckle interacts with a conserved U(217)GCG tetraloop (a member of the UNCG family; N = A,U,G or C), and the C-terminal zinc knuckle binds to residues that flank SL-A, including residues of AUG-3. Mutations of critical nucleotides in these sequences compromise or abolish viral infectivity. Our studies reveal novel structural features important for NC:RNA binding, and support the hypothesis that AUG-3 is conserved for genome packaging rather than translational control. KeywordsRous sarcoma virus; ribonucleic acid (RNA); psi-site (μΨ); nucleocapsid (NC) protein; UNCG tetraloop; nuclear magnetic resonance (NMR) Abbreviations usedA, adenosine; C, cytidine; G, guanosine; GST, glutathione-S-transferase; HIV-1, human immunodeficiency virus type-1; HMQC, heteronuclear multiple quantum coherence; HSQC, heteronuclear single quantumn coherence; ITC, isothermal titration calorimetry; MLV, Moloney Murine Leukaemia Virus; NC, nucleocapsid protein; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; ORF, open reading frame; PBS, primer binding site; RMSD, root-mean-square deviation; ROESY, rotating frame Overhauser effect spectroscopy; RSV, Rous sarcoma virus; SD, splice-donor site; U, uridine; UTR, unstranslated region *Corresponding author E-mail address of the corresponding author: summers@hhmi.umbc.edu, Phone: (410)-455-2527 FAX: (410)-455-1174 Depositions: The atomic coordinates are available from the RCSB Protein Data Bank with accession codes rcsb039591 (RCSB) and 2IHX (PDB).

The structural biology of HIV assembly

“…The NC/RNA interactions visualized in these structures undoubtedly contribute to the specificity of genomic RNA packaging, although the full mechanism by which HIV-1 Gag packages dimeric RNA genomes has yet to be delineated. Even greater progress on the mechanism of genomic RNA packaging has been made for RSV and MLV, where complexes of NC with minimal Ψ RNA elements are now available [87,88]. In the MLV case, the high-affinity NC binding site is initially sequestered by base pairing in the monomeric form of the viral RNA, and then becomes exposed in the dimeric RNA, providing an elegant mechanism for coupling genome dimerization and encapsidation [87].…”

Structure determination and dynamics of protein–RNA complexes by NMR spectroscopy

“…This second approach is used most frequently and often leads to a significant improvement of the protein expression yield. Many RNA binding proteins have been expressed in such bacterial strains [17,29,34,39,45].…”

“…In this case, the DNA coding for the protein of interest was cloned into a plasmid containing an N-terminal or a C-terminal purification tag. The most commonly used tags have been the poly-histidine tag [1, 5-7, 9, 15-17, 19, 25, 26, 28, 29, 33-35, 37, 42, 44, 47] and the glutathione S-transferase (GST) tag [10,13,21,24,32,39,40,45]. The fusion protein overexpressed in E.…”

“…Glu-Asn-Leu-Tyr-Phe-Gln-Gly and cleaves between the glutamine and the glycine [21,25,26,37], and the PreScission protease that specifically recognize Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro segment and cleaves between the glutamine and the glycine [10,13,45]. In these cases, during cloning, the DNA sequence encoding for a thrombin, a Tev or a PreScission cleavage site was inserted between the fused tag and the peptide of interest.…”

“…Because of precipitation in presence of Pf1 phage and severe line-broadening using 5% polyacrylamide gel, high-quality 1 H-13 C RDCs could be obtained only for the three short stem-loops in isolation but not for the protein-RNA complex between the nucleocapsid domain of the retroviral Gag polyprotein and the 101 nucleotide core encapsidation segment of the MoMuLV ψ site (ψ RNA) [10]. The same problem occurred in the structure determination of the 82-nucleotide segment of the 5'UTR μψ RNA packaging signal and the NC protein [45]. However, orienting different helical domains in nucleic acids has already been shown several times for medium size to large RNA molecules.…”