During the Early Phase of HIV-1 DNA Synthesis, Nucleocapsid Protein Directs Hybridization of the TAR Complementary Sequences via the Ends of their Double-stranded Stem

Godet, Julien; Rocquigny, Hugues de; Chinnappan, Raja; Glasser, Nicole; Ficheux, Damien; Darlix, Jean‐Luc; Mély, Yves

doi:10.1016/j.jmb.2005.12.038

Cited by 68 publications

(140 citation statements)

References 55 publications

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“…Peptide concentration was determined using an extinction coefficient of 5,700 M Ϫ1 cm Ϫ1 at 280 nm for nonlabeled peptides, 33,000 M Ϫ1 cm Ϫ1 at 400 nm for the MFLlabeled peptides, and 80,000 M Ϫ1 cm Ϫ1 at 555 nm for LRh-labeled NC. Note that the nucleic acid chaperone properties (75,76) of the labeled NC peptide were tested as described previously (72) and found to be close to those of the unlabeled NC, indicating that the MFL label marginally perturbs the peptide structure and activity (data not shown).…”

Section: Methodsmentioning

confidence: 52%

The HIV-1 Nucleocapsid Protein Recruits Negatively Charged Lipids To Ensure Its Optimal Binding to Lipid Membranes

Kempf

Postupalenko

Bora

et al. 2015

J Virol

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The HIV-1 Gag polyprotein precursor composed of the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains orchestrates virus assembly via interactions between MA and the cell plasma membrane (PM) on one hand and NC and the genomic RNA on the other hand. As the Gag precursor can adopt a bent conformation, a potential interaction of the NC domain with the PM cannot be excluded during Gag assembly at the PM. To investigate the possible interaction of NC with lipid membranes in the absence of any interference from the other domains of Gag, we quantitatively characterized by fluorescence spectroscopy the binding of the mature NC protein to large unilamellar vesicles (LUVs) used as membrane models. We found that NC, either in its free form or bound to an oligonucleotide, was binding with high affinity (ϳ10 7 M ؊1 ) to negatively charged LUVs. The number of NC binding sites, but not the binding constant, was observed to decrease with the percentage of negatively charged lipids in the LUV composition, suggesting that NC and NC/oligonucleotide complexes were able to recruit negatively charged lipids to ensure optimal binding. However, in contrast to MA, NC did not exhibit a preference for phosphatidylinositol-(4,5)-bisphosphate. These results lead us to propose a modified Gag assembly model where the NC domain contributes to the initial binding of the bent form of Gag to the PM. IMPORTANCEThe NC protein is a highly conserved nucleic acid binding protein that plays numerous key roles in HIV-1 replication. While accumulating evidence shows that NC either as a mature protein or as a domain of the Gag precursor also interacts with host proteins, only a few data are available on the possible interaction of NC with lipid membranes. Interestingly, during HIV-1 assembly, the Gag precursor is thought to adopt a bent conformation where the NC domain may interact with the plasma membrane. In this context, we quantitatively characterized the binding of NC, as a free protein or as a complex with nucleic acids, to lipid membranes and showed that the latter constitute a binding platform for NC. Taken together, our data suggest that the NC domain may play a role in the initial binding events of Gag to the plasma membrane during HIV-1 assembly.T he nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) is a small basic nucleic acid binding protein with two strictly conserved CCHC motifs that strongly bind zinc (1). Mature NC results from the protease-mediated cleavage of the Gag polyprotein precursor and plays key roles in HIV-1 replication (2-5). During the early steps, NC acts as a cofactor of the reverse transcriptase (RT), to promote the initiation of reverse transcription (6-8), as well as the two obligatory strand transfers (9, 10). Moreover, NC also reduces RT pauses at the initiation step (11-13), increases the overall RT processivity (14, 15), promotes synthesis through pause sites (16-19), helps to remove the RNA fragments resulting from the RT RNase H activity (20), and may contribute to the pro...

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Section: Methodsmentioning

confidence: 52%

The HIV-1 Nucleocapsid Protein Recruits Negatively Charged Lipids To Ensure Its Optimal Binding to Lipid Membranes

Kempf

Postupalenko

Bora

et al. 2015

J Virol

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“…In conclusion, our results are consistent with the formation of an extended kissing loop complex TAR-cTAR as suggested, in previous studies performed at low NC:nt ratios. However, they do not exclude the possibility that the "zipper" pathway is preferred at high NC:nt ratios (Godet et al 2006;Liu et al 2007;Vo et al 2009b).…”

Section: Discussionmentioning

confidence: 96%

“…NC promotes the annealing of the TAR RNA hairpin with the cTAR DNA hairpin. This is an essential step in the first strand transfer process and has thus been the subject of many studies (Godet et al 2006;Liu et al 2007;Vo et al 2009a,b;Godet and Mély 2010). The NC-mediated destabilization of the secondary structures of TAR and cTAR stem-loops is one of the initial steps in the annealing process.…”

Section: Introductionmentioning

confidence: 99%

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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“…[51][52][53] However, NC-mediated fraying of the NA structure depends on the overall stability of the NA and requires natural mismatches as for wild-type TAR in order to take place. 46,54,57 This NC-mediated fraying of the NA structure largely relies on the hydrophobic plateau of the ZnF domain. 58 The second NC chaperoning component consists in NC-directed annealing of two NAs with complementary sequences when present in the same complex, namely TAR and cTAR sequences.…”

Section: O N O T D I S T R I B U T Ementioning

confidence: 99%

Properties and functions of the nucleocapsid protein in virus assembly

2010

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During the Early Phase of HIV-1 DNA Synthesis, Nucleocapsid Protein Directs Hybridization of the TAR Complementary Sequences via the Ends of their Double-stranded Stem

Cited by 68 publications

References 55 publications

The HIV-1 Nucleocapsid Protein Recruits Negatively Charged Lipids To Ensure Its Optimal Binding to Lipid Membranes

The HIV-1 Nucleocapsid Protein Recruits Negatively Charged Lipids To Ensure Its Optimal Binding to Lipid Membranes

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

Properties and functions of the nucleocapsid protein in virus assembly

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