Specific Interactions Between HIV-1 Nucleocapsid Protein and the TAR Element

Kanevsky, Igor; Chaminade, Françoise; Ficheux, Damien; Moumen, Abdeladim; Gorelick, Robert J.; Negroni, Matteo; Darlix, Jean‐Luc; Fossé, Philippe

doi:10.1016/j.jmb.2005.03.046

Cited by 41 publications

(72 citation statements)

References 69 publications

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“…Two of these A-U base pairs are located in the U4-U6 region, resulting in an unpaired U4C5U6 motif similar to the U23C24U25 bulge. These findings are consistent with chemical probing data showing some reactivity for the residues of the U4C5U6 motif, but less than for the U23C24U25 bulge and the apical loop (Kanevsky et al 2005). Significant motions, like those already described for the U23C24U25 bulge (Zhang et al 2006 probably occur for the U4C5U6 motif, increasing thus the potential complexity and flexibility of the TAR hairpin.…”

Section: Discussionsupporting

confidence: 91%

“…Isothermal calorimetry titrations showed that TAR contained one high affinity site (110 nM), possibly with a second site of intermediate affinity (Heng et al 2012). Kanevsky et al (2005) showed that the apical loop G32 and G33 residue constitute a binding site for NC, this is probably not the case for G34, which has been shown to be involved in a transient cross-loop base pair (Huthoff et al 2004;Lee et al 2014). This finding was supported by the results of biophysical studies on the dynamic properties of the TAR apical loop demonstrating that G32 was highly dynamic (Dethoff et al 2008), constituting a favorable binding site for NC.…”

Section: Discussionmentioning

confidence: 99%

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Insights into the mechanisms of RNA secondary structure destabilization by the HIV-1 nucleocapsid protein

Belfetmi¹,

Zargarian²,

Tisné³

et al. 2016

RNA

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The mature HIV-1 nucleocapsid protein NCp7 (NC) plays a key role in reverse transcription facilitating the two obligatory strand transfers. Several properties contribute to its efficient chaperon activity: preferential binding to single-stranded regions, nucleic acid aggregation, helix destabilization, and rapid dissociation from nucleic acids. However, little is known about the relationships between these different properties, which are complicated by the ability of the protein to recognize particular HIV-1 stem-loops, such as SL1, SL2, and SL3, with high affinity and without destabilizing them. These latter properties are important in the context of genome packaging, during which NC is part of the Gag precursor. We used NMR to investigate destabilization of the full-length TAR (trans activating response element) RNA by NC, which is involved in the first strand transfer step of reverse transcription. NC was used at a low protein:nucleotide (nt) ratio of 1:59 in these experiments. NMR data for the imino protons of TAR identified most of the base pairs destabilized by NC. These base pairs were adjacent to the loops in the upper part of the TAR hairpin rather than randomly distributed. Gel retardation assays showed that conversion from the initial TAR-cTAR complex to the fully annealed form occurred much more slowly at the 1:59 ratio than at the higher ratios classically used. Nevertheless, NC significantly accelerated the formation of the initial complex at a ratio of 1:59.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Insights into the mechanisms of RNA secondary structure destabilization by the HIV-1 nucleocapsid protein

Belfetmi¹,

Zargarian²,

Tisné³

et al. 2016

RNA

Self Cite

View full text Add to dashboard Cite

show abstract

“…In addition, NC binding to TAR RNA and its role in destabilizing the hairpin during the minus-strand transfer step of reverse transcription is well established (Kanevsky et al 2005(Kanevsky et al , 2011Vo et al 2009b;Levin et al 2010;Heng et al 2012). In an HIV-1 variant that does not depend on Tat-TAR interaction for transcription activation, TAR is not required for gRNA packaging (Das et al 2007), but TAR destabilization leads to aberrant RNA dimerization and packaging (Das et al 2012).…”

Section: Discussionmentioning

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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“…This view is supported by the observation that purified p53 protein could not discriminate between RNAs in vitro even when the RNAs displayed dramatically different apparent affinities for p53 in yeast. Three sequence-nonspecific RNA-binding proteins were compared with p53: the E. coli S12 ribosomal protein (Coetzee et al 1994), the double-stranded RNA-binding domain of the double-stranded RNA-dependent protein kinase (Carlson et al 2003), and the zinc finger domain of the HIV-1 nucleocapsid protein (Kanevsky et al 2005). Both S12 and HIV-1 NC have been discussed as ''RNA chaperones,'' an activity that has also been mentioned for p53.…”

Section: Discussion P53 Binds Rna In Yeastmentioning

confidence: 99%

Recognition of RNA by the p53 tumor suppressor protein in the yeast three-hybrid system

Riley

Cassiday

Kumar

et al. 2006

RNA

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The p53 tumor suppressor protein is a homotetrameric transcription factor whose gene is mutated in nearly half of all human cancers. In an unrelated screen of RNA/protein interactions using the yeast three-hybrid system, we inadvertently detected p53 interactions with several different RNAs. A literature review revealed previous reports of both sequence-specific and -nonspecific interactions between p53 and RNA. Using yeast three-hybrid selections to identify preferred RNA partners for p53, we failed to identify primary RNA sequences or obvious secondary structures required for p53 binding. The cationic p53 C-terminus was shown to be required for RNA binding in yeast. We show that while p53 strongly discriminates between certain RNAs in the yeast three-hybrid assay, the same RNAs are bound equally by p53 in vitro. We further show that the p53 RNA-binding preferences in yeast are mirrored almost exactly by a recombinant tetrameric form of the HIV-1 nucleocapsid (NC) protein thought to be a sequence-nonspecific RNA-binding protein. However, the possibility of specific RNA binding by p53 could not be ruled out because p53 and HIV-1 NC displayed certain differences in RNA-binding preference. We conclude that (1) p53 binds RNA in vivo, (2) RNA binding by p53 is largely sequence-nonspecific in the yeast nucleus, (3) some structure-specific RNA binding by p53 cannot be ruled out, and (4) caution is required when interpreting results of RNA screens in the yeast threehybrid system because sequence-dependent differences in RNA folding and display can masquerade as sequence-dependent differences in protein recognition.

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Specific Interactions Between HIV-1 Nucleocapsid Protein and the TAR Element

Cited by 41 publications

References 69 publications

Insights into the mechanisms of RNA secondary structure destabilization by the HIV-1 nucleocapsid protein

Insights into the mechanisms of RNA secondary structure destabilization by the HIV-1 nucleocapsid protein

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

Recognition of RNA by the p53 tumor suppressor protein in the yeast three-hybrid system

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