Impact of the Terminal Bulges of HIV-1 cTAR DNA on its Stability and the Destabilizing Activity of the Nucleocapsid Protein NCp7

Beltz, Hervé; Azoulay, Joel; Bernacchi, Serena; Clamme, Jean‐Pierre; Ficheux, Damien; Roques, Bernárd P.; Darlix, Jean‐Luc; Mély, Yves

doi:10.1016/s0022-2836(03)00244-4

Cited by 79 publications

(147 citation statements)

References 71 publications

Supporting

Mentioning

137

Contrasting

Order By: Relevance

“…Mutations that convert bulges in the lower portion of TAR DNA to paired bases (56,62) reduce the ability of NC to destabilize the structure (62) and lead to an increase in self-priming (56). Recent studies also show that NC destabilizes the bottom half of TAR DNA more efficiently than the upper half (62,63). It is of interest that in the absence of acceptor, NC has little or no effect on the amount of SP products that are synthesized (Fig.…”

Section: Discussionmentioning

confidence: 99%

Alteration of Nucleic Acid Structure and Stability Modulates the Efficiency of Minus-Strand Transfer Mediated by the HIV-1 Nucleocapsid Protein

Heilman-Miller

Levin

2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

During human immunodeficiency virus type 1 minusstrand transfer, the nucleocapsid protein (NC) facilitates annealing of the complementary repeat regions at the 3-ends of acceptor RNA and minus-strand strong-stop DNA ((؊) SSDNA). In addition, NC destabilizes the highly structured complementary trans-activation response element (TAR) stem-loop (TAR DNA) at the 3-end of (؊) SSDNA and inhibits TAR-induced self-priming, a deadend reaction that competes with minus-strand transfer. To investigate the relationship between nucleic acid secondary structure and NC function, a series of truncated (؊) SSDNA and acceptor RNA constructs were used to assay minus-strand transfer and self-priming in vitro. The results were correlated with extensive enzymatic probing and mFold analysis. As the length of (؊) SSDNA was decreased, self-priming increased and was highest when the DNA contained little more than TAR DNA, even if NC and acceptor were both present; in contrast, truncations within TAR DNA led to a striking reduction or elimination of self-priming. However, destabilization of TAR DNA was not sufficient for successful strand transfer: the stability of acceptor RNA was also crucial, and little or no strand transfer occurred if the RNA was highly stable. Significantly, NC may not be required for in vitro strand transfer if (؊) SSDNA and acceptor RNA are small, relatively unstructured molecules with low thermodynamic stabilities. Collectively, these findings demonstrate that for efficient NC-mediated minus-strand transfer, a delicate thermodynamic balance between the RNA and DNA reactants must be maintained.Reverse transcription consists of a complex series of reactions catalyzed by the virion-associated enzyme reverse transcriptase (RT) 1 that lead to conversion of the single-stranded RNA genome into an integration-competent linear doublestranded DNA (1). This process is facilitated by host and viral accessory proteins, one of which is the viral nucleocapsid protein (NC). Human immunodeficiency virus type 1 (HIV-1) NC is a small, highly basic, nucleic acid-binding protein with two zinc fingers, each containing the invariant CCHC metal ion-binding motif (2-5). NC functions as a nucleic acid chaperone in an ATP-independent manner (6) and catalyzes nucleic acid conformational rearrangements that lead to the formation of the most thermodynamically stable structure ). This activity is required for efficient reverse transcription and allows NC to promote intermolecular annealing of nucleic acids with significant stretches of base complementarity (7, 12-26), destabilization of secondary structures in RNA and DNA templates that are responsible for RT pausing (27-30), unwinding of primer tRNA (19, 31-34), primer placement (12,15,19,32,33,(35)(36)(37)(38), and the initiation step (39 -41).NC nucleic acid chaperone activity is also critical for the minus-strand (reviewed in Refs. 8, 10, and 11) and plus-strand (18,20,22,42) transfer events that are required to complete elongation of minus-and plus-strand DNAs and to generate the long te...

show abstract

Section: Discussionmentioning

confidence: 99%

Alteration of Nucleic Acid Structure and Stability Modulates the Efficiency of Minus-Strand Transfer Mediated by the HIV-1 Nucleocapsid Protein

Heilman-Miller

Levin

2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…[124][125][126][127][128][129][130][131][132][133][134][135][136] This chaperone activity, facilitating restructuring of the nucleic acid complex, has been attributed to both an aggregating ability of NC and a facility for duplex destabilization. 125,131,[137][138][139][140][141][142][143][144][145] This destabilization, however, is weak, 143,144,146,147 and will not completely melt DNA without a complementary strand. 6,138,148,149 Stretching and relaxation cycles can also reveal information on the kinetics of protein association and dissociation.…”

Section: Figure 11mentioning

confidence: 99%

Mechanisms of DNA binding determined in optical tweezers experiments

McCauley¹,

Williams²

2006

Biopolymers

View full text Add to dashboard Cite

The last decade has seen rapid development in single molecule manipulation of RNA and DNA. Measuring the response force for a particular manipulation has allowed the free energies of various nucleic acid structures and configurations to be determined. Optical tweezers represent a class of single molecule experiments that allows the energies and structural dynamics of DNA to be probed up to and beyond the transition from the double helix to its melted single strands. These experiments are capable of high force resolution over a wide dynamic range. Additionally, these investigations may be compared with results obtained when the nucleic acids are in the presence of proteins or other binding ligands. These ligands may bind into the major or minor groove of the double helix, intercalate between bases or associate with an already melted single strand of DNA. By varying solution conditions and the pulling dynamics, energetic and dynamic information may be deduced about the mechanisms of binding to nucleic acids, providing insight into the function of proteins and the utility of drug treatments. © 2006 Wiley Periodicals, Inc. Biopolymers 85:154–168, 2007.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

show abstract

“…NC destabilizes the cTAR DNA to a much greater extent than the more stable TAR RNA (Bernacchi et al 2002). More generally, the destabilizing activity of NC has been shown to be inversely correlated with oligonucleotide stability (Bernacchi et al 2002;Beltz et al 2003Beltz et al , 2005. It has been shown, by timeresolved fluorescence spectroscopy, that there are preferentially destabilized segments in the lower part of the cTAR DNA molecule (Godet et al 2013).…”

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

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Impact of the Terminal Bulges of HIV-1 cTAR DNA on its Stability and the Destabilizing Activity of the Nucleocapsid Protein NCp7

Cited by 79 publications

References 71 publications

Alteration of Nucleic Acid Structure and Stability Modulates the Efficiency of Minus-Strand Transfer Mediated by the HIV-1 Nucleocapsid Protein

Alteration of Nucleic Acid Structure and Stability Modulates the Efficiency of Minus-Strand Transfer Mediated by the HIV-1 Nucleocapsid Protein

Mechanisms of DNA binding determined in optical tweezers experiments

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

Contact Info

Product

Resources

About