HIV-1 nucleocapsid protein activates transient melting of least stable parts of the secondary structure of TAR and its complementary sequence

Bernacchi, Serena; Stoylov, Stoyl P.; Piémont, Étienne; Ficheux, Damien; Roques, Bernárd P.; Darlix, Jean Luc; Mély, Yves

doi:10.1006/jmbi.2002.5429

Cited by 129 publications

(233 citation statements)

References 77 publications

Supporting

Mentioning

219

Contrasting

Order By: Relevance

“…This finding indicates that NC exerts its effect upon both DNA and RNA secondary structures. The fact that minus-strand transfer appears to be particularly sensitive to excessively stable structure in the acceptor RNA and to a somewhat smaller extent to the structure in (Ϫ) SSDNA is consistent with the fact that NC induces only very limited melting of the TAR RNA structure compared with melting of the TAR DNA stem-loop (60). It has also been reported that introduction of stabilizing mutations in the poly(A) stem-loop of the acceptor RNA inhibits productive minus-strand transfer (81).…”

Section: Discussionsupporting

confidence: 64%

“…However, when NC and acceptor RNA are both present, there is a dramatic shift in the majority of the TAR DNA molecules to an unfolded state, which blocks self-priming and enables successful strand transfer to occur (25). Interestingly, in vitro studies with mutant HIV-1 NC proteins have demonstrated that the zinc finger motifs are required for NC-dependent inhibition of self-priming and destabilization of highly structured nucleic acids (21, 23, 64 -67), including the TAR stem-loops in (Ϫ) SSDNA (HIV-1 (21,23,60) and feline immunodeficiency virus (68)) and in viral RNA (69).…”

mentioning

confidence: 99%

See 1 more Smart Citation

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: Discussionsupporting

confidence: 64%

mentioning

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 protein noticeably stimulates transfer in reconstituted in vitro systems. The strand annealing and chaperone activities of NC plus its effects on RT-RNase H have been implicated in facilitating the transfer reaction (34,35,39,(43)(44)(45)(46)(47)(48). Analyses in vitro have been used to show that NC inhibits the formation of dead end, self-primed products from the ϪsssDNA, allowing it to be used for transfers (38,49,50).…”

Section: And References Therein and Ref 2)mentioning

confidence: 99%

Mechanism of Minus Strand Strong Stop Transfer in HIV-1 Reverse Transcription

Chen¹,

Balakrishnan²,

Roques

et al. 2003

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Retrovirus minus strand strong stop transfer (minus strand transfer) requires reverse transcriptase-associated RNase H, R sequence homology, and viral nucleocapsid protein. The minus strand transfer mechanism in human immunodeficiency virus-1 was examined in vitro with purified protein and substrates. Blocking donor RNA 5 -end cleavage inhibited transfers when template homology was 19 nucleotides (nt) or less. Cleavage of the donor 5 -end occurred prior to formation of transfer products. This suggests that when template homology is short, transfer occurs through a primer terminus switch-initiated mechanism, which requires cleavage of the donor 5 terminus. On templates with 26-nt and longer homology, transfer occurred before cleavage of the donor 5 terminus. Transfer was unaffected when donor 5 -end cleavages were blocked but was reduced when internal cleavages within the donor were restricted. Based on the overall data, we conclude that in human immunodeficiency virus-1, which contains a 97-nt R sequence, minus strand transfer occurs through an acceptor invasion-initiated mechanism. Transfer is initiated at internal regions of the homologous R sequence without requiring cleavage at the donor 5 -end. The acceptor invades at gaps created by reverse transcriptase-RNase H in the donor-cDNA hybrid. The fragmented donor is eventually strand-displaced by the acceptor, completing the transfer.Reverse transcription, during which the single-stranded viral genomic RNA is converted into the double-stranded DNA, is an essential step in viral replication (see Ref. 1 and reference therein). This process is catalyzed by the virus-encoded enzyme reverse transcriptase (RT). 1 RT has two enzymatic activities, the DNA polymerase and the RNase H activities. Reverse transcription is initiated by the partial annealing between the cellular tRNA Lys3 primer and the viral primer binding site, which is located near the 5Ј-end of the viral RNA. Synthesis proceeds to the 5Ј-end of the genomic RNA, creating the minus strand strong stop DNA (ϪsssDNA), which comprises all of the U5 and R sequences from the 5Ј-untranslated region. During minus strand synthesis, RT simultaneously cleaves the copied RNA using its RNase H activity. This frees the ϪsssDNA, enabling its transfer to the homologous R sequence in the 3Ј-untranslated region and continuation of minus strand synthesis. This step is called minus strand strong stop transfer (minus strand transfer), and it is obligatory for reverse transcription and viral replication. Extensive studies have identified the RT-RNase H activity, R sequence homology, and viral nucleocapsid (NC) protein as important components for minus strand transfer (see Ref. 2 and references therein).Viruses with defective RNase H fail to complete reverse transcription, particularly the minus strand transfer step, indicating that RNase H is required for the transfer event (3-8). HIV-1 RT has been shown to partially cleave the template RNA during polymerization, leaving RNA fragments annealed to the cDNA (9, 10). Since there a...

show abstract

HIV-1 nucleocapsid protein activates transient melting of least stable parts of the secondary structure of TAR and its complementary sequence

Cited by 129 publications

References 77 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

Mechanism of Minus Strand Strong Stop Transfer in HIV-1 Reverse Transcription

Contact Info

Product

Resources

About