High Affinity Nucleocapsid Protein Binding to the μΨ RNA Packaging Signal of Rous Sarcoma Virus

Zhou, Jing; McAllen, John K.; Tailor, Yogita; Summers, Michael F.

doi:10.1016/j.jmb.2005.04.046

Cited by 29 publications

(36 citation statements)

References 64 publications

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“…1a) with nucleic acid bases (2)(3)(4)28). RSV (112,113) and MLV (31, 34) NC proteins also engage in similar interactions. While this binding leads to duplex destabilization, it is also expected to result in reduced dissociation kinetics.…”

Section: Discussionmentioning

confidence: 99%

“…These binding measurements were carried out using a fairly random nucleic acid sequence in order to minimize sequence-specific binding effects. Although NCs generally bind nucleic acids nonspecifically, HIV-1 NC (2-4, 28, 40, 41, 105) and MLV NC (31,35,36) have an ϳ10-fold preference for single-stranded TG-or UG-rich sequences, and RSV NC has a preference for G residues (12,31,97,105,112,113).…”

Section: Effects Of Different Retroviral Nc Proteins On Mini-tarmentioning

confidence: 99%

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Retroviral Nucleocapsid Proteins Display Nonequivalent Levels of Nucleic Acid Chaperone Activity

Stewart-Maynard

Cruceanu

Wang

et al. 2008

J Virol

161

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Human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) is a nucleic acid chaperone that facilitates the remodeling of nucleic acids during various steps of the viral life cycle. Two main features of NC's chaperone activity are its abilities to aggregate and to destabilize nucleic acids. These functions are associated with NC's highly basic character and with its zinc finger domains, respectively. While the chaperone activity of HIV-1 NC has been extensively studied, less is known about the chaperone activities of other retroviral NCs. In this work, complementary experimental approaches were used to characterize and compare the chaperone activities of NC proteins from four different retroviruses: HIV-1, Moloney murine leukemia virus (MLV), Rous sarcoma virus (RSV), and human T-cell lymphotropic virus type 1 (HTLV-1). The different NCs exhibited significant differences in their overall chaperone activities, as demonstrated by gel shift annealing assays, decreasing in the order HIV-1 ϳ RSV > MLV Ͼ Ͼ HTLV-1. In addition, whereas HIV-1, RSV, and MLV NCs are effective aggregating agents, HTLV-1 NC, which exhibits poor overall chaperone activity, is unable to aggregate nucleic acids. Measurements of equilibrium binding to single-and double-stranded oligonucleotides suggested that all four NC proteins have moderate duplex destabilization capabilities. Single-molecule DNAstretching studies revealed striking differences in the kinetics of nucleic acid dissociation between the NC proteins, showing excellent correlation between nucleic acid dissociation kinetics and overall chaperone activity.

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Section: Discussionmentioning

confidence: 99%

Section: Effects Of Different Retroviral Nc Proteins On Mini-tarmentioning

confidence: 99%

Retroviral Nucleocapsid Proteins Display Nonequivalent Levels of Nucleic Acid Chaperone Activity

Stewart-Maynard

Cruceanu

Wang

et al. 2008

J Virol

161

View full text Add to dashboard Cite

show abstract

“…Samples were incubated at 35°C for 30 min. Following incubations, samples were supplemented with 6 l of 50% glycerol and then loaded onto 12% native polyacrylamide (37.5:1 acrylamide/bis; polymerized with 0.0375% ammonium persulfate and 0.1125% TEMED) gels in 0.5ϫ Tris borate buffer (44.5 mM Tris base, 44.5 mM boric acid, pH 8.0) (33,63). Gels were electrophoresed at 4°C at 30 -35 mA.…”

Section: Methodsmentioning

confidence: 99%

Analysis of Small Molecule Ligands Targeting the HIV-1 Matrix Protein-RNA Binding Site

Alfadhli

McNett

Eccles

et al. 2013

Journal of Biological Chemistry

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“…31 Consistent with this hypothesis, we recently demonstrated that RSV NC binds μΨ RNA with high affinity, and that mutation of AUG-3 substantially inhibits NC binding. 32 We now report the solution structure of the NC:μΨ complex, determined using a combination of isotope-edited NMR experiments. Our studies show that AUG-3 and a conserved UNCG tetraloop participate directly in binding to the NC protein, and that these binding sites are critical for viral fitness.…”

Section: Introductionmentioning

confidence: 99%

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

Zhou

Bean

Vogt

et al. 2007

Journal of Molecular Biology

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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).

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High Affinity Nucleocapsid Protein Binding to the μΨ RNA Packaging Signal of Rous Sarcoma Virus

Cited by 29 publications

References 64 publications

Retroviral Nucleocapsid Proteins Display Nonequivalent Levels of Nucleic Acid Chaperone Activity

Retroviral Nucleocapsid Proteins Display Nonequivalent Levels of Nucleic Acid Chaperone Activity

Analysis of Small Molecule Ligands Targeting the HIV-1 Matrix Protein-RNA Binding Site

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

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