Complex interactions of HIV-1 nucleocapsid protein with oligonucleotides

Fisher, Robert J.; Fivash, Matthew J.; Stephen, Andrew; Hagan, Nathan; Shenoy, Shilpa R.; Medaglia, Maxine V.; Smith, Lindsey; Worthy, Karen M.; Simpson, John T.; Shoemaker, Robert H.; McNitt, Karen L.; Johnson, Daniel K. N.; Hixson, Catherine V.; Gorelick, Robert J.; Fabris, Daniele; Henderson, Louis E.; Rein, Alan

doi:10.1093/nar/gkj442

Cited by 86 publications

(94 citation statements)

References 42 publications

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“…Binding of Gag to RNA is presumably mediated by the nucleocapsid (NC) domain, with a major contribution from the two zinc fingers within this domain (9,14). Therefore, we assessed the role of RNA-binding in assembly in vivo using mutants in which zinccoordinating cysteines were replaced with serines or in which the entire NC domain was deleted.…”

Section: Resultsmentioning

confidence: 99%

Functional Redundancy in HIV-1 Viral Particle Assembly

O’Carroll

Crist

Mirro

et al. 2012

J Virol

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Expression of a retroviral Gag protein in mammalian cells leads to the assembly of virus particles. In vitro, recombinant Gag proteins are soluble but assemble into virus-like particles (VLPs) upon addition of nucleic acid. We have proposed that Gag undergoes a conformational change when it is at a high local concentration and that this change is an essential prerequisite for particle assembly; perhaps one way that this condition can be fulfilled is by the cooperative binding of Gag molecules to nucleic acid. We have now characterized the assembly in human cells of HIV-1 Gag molecules with a variety of defects, including (i) inability to bind to the plasma membrane, (ii) near-total inability of their capsid domains to engage in dimeric interaction, and (iii) drastically compromised ability to bind RNA. We find that Gag molecules with any one of these defects still retain some ability to assemble into roughly spherical objects with roughly correct radius of curvature. However, combination of any two of the defects completely destroys this capability. The results suggest that these three functions are somewhat redundant with respect to their contribution to particle assembly. We suggest that they are alternative mechanisms for the initial concentration of Gag molecules; under our experimental conditions, any two of the three is sufficient to lead to some semblance of correct assembly.

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Section: Resultsmentioning

confidence: 99%

Functional Redundancy in HIV-1 Viral Particle Assembly

O’Carroll

Crist

Mirro

et al. 2012

J Virol

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“…1) that are critical for RNA-binding specificity, nucleic acid chaperone activity Darlix et al 2011), and gRNA packaging (Gorelick et al 1999b;Kafaie et al 2008). Investigations into the RNAbinding specificity of Gag have previously studied the binding of the NC protein to RNA and DNA (Dannull et al 1994;Fisher et al 1998Fisher et al , 2006Vuilleumier et al 1999;Avilov et al 2009;Athavale et al 2010), and more recently the binding of Gag to short oligonucleotides has also been investigated (Stephen et al 2007;Wu et al 2010;Jones et al 2011). In this study, we investigate the binding of Gag and NC to longer ∼100-nt RNAs derived from the gRNA 5 ′ UTR.…”

Section: Thementioning

confidence: 99%

“…Previous studies have shown that NC binds RNAs in a largely nonspecific manner via electrostatic interactions between the nucleic acid backbone and the basic residues in the protein (Levin et al 2005;Fisher et al 2006;Vo et al 2009a;Darlix et al 2011;Wu et al 2012). However, binding to certain RNA motifs, especially single-stranded UG sequences (Fisher et al 1998), or exposed G residues in loop regions such as SL3 (De Guzman et al 1998), involves high-affinity binding in which the zinc finger (ZF) aromatic residues stack with the G base in a hydrophobic pocket formed between the two ZFs (De Guzman et al 1998;Darlix et al 2011).…”

Section: The Zinc Fingers In Gag Are Required For High-affinity Psi Rmentioning

confidence: 99%

Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging

Webb

Jones

Parent

et al. 2013

RNA

144

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Despite the vast excess of cellular RNAs, precisely two copies of viral genomic RNA (gRNA) are selectively packaged into new human immunodeficiency type 1 (HIV-1) particles via specific interactions between the HIV-1 Gag and the gRNA psi (ψ) packaging signal. Gag consists of the matrix (MA), capsid, nucleocapsid (NC), and p6 domains. Binding of the Gag NC domain to ψ is necessary for gRNA packaging, but the mechanism by which Gag selectively interacts with ψ is unclear. Here, we investigate the binding of NC and Gag variants to an RNA derived from ψ (Psi RNA), as well as to a non-ψ region (TARPolyA). Binding was measured as a function of salt to obtain the effective charge (Z eff ) and nonelectrostatic (i.e., specific) component of binding, K d(1M) . Gag binds to Psi RNA with a dramatically reduced K d(1M) and lower Z eff relative to TARPolyA. NC, GagΔMA, and a dimerization mutant of Gag bind TARPolyA with reduced Z eff relative to WT Gag. Mutations involving the NC zinc finger motifs of Gag or changes to the G-rich NC-binding regions of Psi RNA significantly reduce the nonelectrostatic component of binding, leading to an increase in Z eff . These results show that Gag interacts with gRNA using different binding modes; both the NC and MA domains are bound to RNA in the case of TARPolyA, whereas binding to Psi RNA involves only the NC domain. Taken together, these results suggest a novel mechanism for selective gRNA encapsidation.

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“…70 RNA constructs lacking salient structural motifs were used to unambiguously identify binding sites located in single-stranded regions of the two conformers (Scheme 1), in agreement with the preference of NC for unpaired nucleotides. [71][72][73][74] This work demonstrated that specific interactions with the apical loop or the stem-bulge motif can have contrasting effects on RNA dimerization. In fact, loop binding was shown to hamper the palindrome's participation in intermolecular basepairing, in direct competition with the initial step of RNA dimerization.…”

Section: Introductionmentioning

confidence: 97%

Understanding the Isomerization of the HIV-1 Dimerization Initiation Domain by the Nucleocapsid Protein

Turner

Hagan

Fabris

2007

Journal of Molecular Biology

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The specific binding of HIV-1 nucleocapsid (NC) to the hinge region of the kissing-loop (KL) dimer formed by stemloop 1 (SL1) can have significant consequences on its ability to isomerize into the corresponding extended duplex (ED) form. The binding determinants and the effects on the isomerization process were investigated in vitro by a concerted strategy involving ad hoc RNA mutants and electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry, which enabled us to characterize the stoichiometry and conformational state of all possible protein-RNA and RNA-RNA assemblies present simultaneously in solution. For the first time, NC-hinge interactions were observed in constructs including at least one unpaired guanine at the 5′-end of the loop-loop duplex, whereas no interactions were detected when the unpaired guanine was placed at its 3′-end. This binding mode is supported by the presence of a grip-like motif described by recent crystal structures, which is formed by the 5′-purines of both hairpins held together by mutual stacking interactions. Using tandem mass spectrometry, hinge interactions were clearly shown to reduce the efficiency of KL/ED isomerization without inducing its complete block. This outcome is consistent with the partial stabilization of the extra-helical grip by the bound protein, which can hamper the purine components from parting ways and initiate the strand exchange process. These findings confirm that the broad binding and chaperone activities of NC induce unique effects that are clearly dependent on the structural context of the cognate nucleic acid substrate. For this reason, the presence of multiple binding sites on the different forms assumed by SL1 can produce seemingly contrasting effects that contribute to a fine modulation of the two-step process of RNA dimerization and isomerization.

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Complex interactions of HIV-1 nucleocapsid protein with oligonucleotides

Cited by 86 publications

References 42 publications

Functional Redundancy in HIV-1 Viral Particle Assembly

Functional Redundancy in HIV-1 Viral Particle Assembly

Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging

Understanding the Isomerization of the HIV-1 Dimerization Initiation Domain by the Nucleocapsid Protein

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