NCp7 Activates HIV-1Lai RNA Dimerization by Converting a Transient Loop-Loop Complex into a Stable Dimer

Muriaux, Delphine; Rocquigny, Hugues de; Roques, B. P.; Paoletti, Jacques

doi:10.1074/jbc.271.52.33686

Cited by 175 publications

(150 citation statements)

References 51 publications

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“…Tubes containing 125 pmol of NCp7 in monomer buffer were incubated at 37°C for 30 minutes. The concentration of NCp7 (around one protein per 20 nucleotides) allows complete tight dimerization of the homologous HIV-1 RNA (25). Since NCp7 binding to the RNA changes the electrophoretic properties of the RNA and prevents the analysis of monomer and dimer levels, an SDS/phenol extraction step is necessary.…”

Section: Ncp7-mediated Tight Dimerization Assaymentioning

confidence: 99%

“…This protein was expressed in Escherichia coli and purified by chromatography (for further details, see (24)). We based our NCp7-mediated dimerization assay on the protocol described by Muriaux et al for an HIV-1 Lai isolate RNA fragment and nucleocapsid protein (25). Five picomoles of RNA were denatured in water for 2 minutes at 90°C and quench cooled on ice for 2 minutes.…”

Section: Ncp7-mediated Tight Dimerization Assaymentioning

confidence: 99%

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Elements Located Upstream and Downstream of the Major Splice Donor Site Influence the Ability of HIV-2 Leader RNA To Dimerize in Vitro

et al. 2003

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An essential step in the replication cycle of all retroviruses is the dimerization of genomic RNA prior to or during budding and maturation of the viral particle. In HIV-1 a 5′ leader region site referred to as stem-loop1 (SL1) promotes RNA dimerization in vitro and influences dimerization in vivo. In HIV-2, two sequences promote dimerization of RNA fragments in vitro: the 5′-end of the primerbinding site (PBS) and a stem loop homologous to the HIV-1 SL1 sequence. Because HIV-2 RNA constructs of different lengths use these two dimerization signals disproportionately, we hypothesized that other sequences could modulate their relative utilization. Here, we characterized the influence of sequences upstream and downstream of the major splice donor site on the formation of HIV-2 RNA dimers in vitro using a variety of RNA constructs and dimerization/electrophoresis protocols. We first assayed the formation of loose or tight dimers for 1-444 and 1-561 model RNAs. Although both RNAs could form PBS-dependent loose dimers, the 1-561 RNA was unable to make SL1-dependent tight dimers. Using RNAs truncated at their 5′ and/or 3′ ends and by making compensatory base substitutions we found that two elements interfere with the formation of SL1-dependent tight dimers. The cores of these elements are located at nucleotides 189-196 and 543-550. Our results suggest that base pairing between these sequences prevents the formation of SL1- † This research was supported by the National Institutes of Health grant number AI45388 to J.S.L.

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Section: Ncp7-mediated Tight Dimerization Assaymentioning

confidence: 99%

Section: Ncp7-mediated Tight Dimerization Assaymentioning

confidence: 99%

Elements Located Upstream and Downstream of the Major Splice Donor Site Influence the Ability of HIV-2 Leader RNA To Dimerize in Vitro

et al. 2003

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“…1b). This extended duplex is associated with the stabilization of the dimer observed in vitro upon heat treatment at 55°C or by the addition of the viral nucleocapsid protein NCp7 (17).…”

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confidence: 99%

Mechanism of Hairpin-Duplex Conversion for the HIV-1 Dimerization Initiation Site

Bernacchi

Ennifar

Tóth

et al. 2005

Journal of Biological Chemistry

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We have used the dimerization initiation site of HIV-1 genomic RNA as a model to investigate hairpin-duplex interconversion with a combination of fluorescence, UV melting, gel electrophoresis, and x-ray crystallographic techniques. Fluorescence studies with molecular beacons and crystallization experiments with 23-nucleotide dimerization initiation site fragments showed that the ratio of hairpin to duplex formed after annealing in water essentially depends on RNA concentration and not on cooling kinetics. With natural sequences allowing to form the most stable duplex, and thus also the loop-loop complex (or "kissing complex"), concentrations as low as 3 M in strands are necessary to obtain a majority of the hairpin form. With a mutated sequence preventing kissing complex formation, a majority of hairpins was even obtained at 80 M in strands. However, this did not prevent an efficient conversion from hairpin to duplex in the presence of salts. Kinetic considerations are in favor of duplex formation from intermediates involving hairpins engaged in cruciform dimers rather than from free strands. The very first step of formation of such a cruciform intermediate could be trapped in a crystal structure. This mechanism might be significant for the dynamics of small RNAs beyond the strict field of HIV-1.Stem-loops and bulged loops are common motifs found in RNA secondary structure and are often involved in RNA-protein or RNA-RNA recognition. Because of the self-complementarity of stems, a stem-loop structure can also adopt an alternative duplex structure containing unpaired nucleotides. This hairpin-duplex equilibrium also represents a major problem in structural studies of nucleic acids by NMR and x-ray crystallography, both requiring concentrated samples. Strategies using a mixture of unlabeled and 15 N-labeled oligonucleotides were developed to discriminate between the two species by NMR (1, 2). However, many attempts at crystallizing RNA or DNA stem-loop structures led unexpectedly to duplex crystal structures (3-6). This was even the case for the unusually stable RNA tetraloops belonging to the GNRA, UNCG, and CUUG families, which are supposed to be nucleation points for RNA folding because of their stability and quick folding kinetics (see "Discussion" in Ref. 7). Significantly, this problem does not seem to spread to NMR studies because several RNA stem-loop structures were successfully solved by using this technique, whereas most of the hairpin structures described by x-ray crystallography were obtained from complexes with proteins (8 -10) or were embedded in a larger RNA context, as for tRNAs, hammerhead ribozyme, and ribosome structures. The only notable exception is the GAGA tetraloop found in the sarcin-ricin loop, successfully crystallized as a hairpin (11,12).The present study focuses on the dimerization initiation site (DIS) 4 of HIV-1 genomic RNA that has been extensively studied by several biochemical and biophysical methods. This strongly conserved stem-loop sequence (13) (Fig. 1a) was shown to be cruc...

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“…Heat treatment or incubation with the viral nucleic acid-binding nucleocapsid protein, which acts as a chaperone, melts the DIS stem and triggers the formation of a dimer with extended interstrand base pairing. This transition is usually referred to as the conversion of the loose dimer (kissing-loop complex) into the tight dimer form (extended duplex) (23,24).…”

mentioning

confidence: 99%

The Dimer Initiation Site Hairpin Mediates Dimerization of the Human Immunodeficiency Virus, Type 2 RNA Genome

Dirac

Huthoff

Kjems

et al. 2001

Journal of Biological Chemistry

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The untranslated leader of retroviral RNA genomes encodes multiple structural signals that are critical for virus replication. In the human immunodeficiency virus, type 1 (HIV-1) leader, a hairpin structure with a palindrome-containing loop is termed the dimer initiation site (DIS), because it triggers in vitro RNA dimerization through base pairing of the loop-exposed palindromes (kissing loops). Controversy remains regarding the region responsible for HIV-2 RNA dimerization. Different studies have suggested the involvement of the transactivation region, the primer binding site, and a hairpin structure that is the equivalent of the HIV-1 DIS hairpin. We have performed a detailed mutational analysis of the HIV-2 leader RNA, and we also used antisense oligonucleotides to probe the regions involved in dimerization. Our results unequivocally demonstrate that the DIS hairpin is the main determinant for HIV-2 RNA dimerization. The 6-mer palindrome sequence in the DIS loop is essential for dimer formation. Although the sequence can be replaced by other 6-mer palindromes, motifs that form more than two A/U base pairs do not dimerize efficiently. The inability to form stable kissingloop complexes precludes formation of dimers with more extended base pairing. Structure probing of the DIS hairpin in the context of the complete HIV-2 leader RNA suggests a 5-base pair elongation of the DIS stem as it is proposed in current RNA secondary structure models. This structure is supported by phylogenetic analysis of leader RNA sequences from different viral isolates, indicating that RNA genome dimerization occurs by a similar mechanism for all members of the human and simian immunodeficiency viruses.

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NCp7 Activates HIV-1Lai RNA Dimerization by Converting a Transient Loop-Loop Complex into a Stable Dimer

Cited by 175 publications

References 51 publications

Elements Located Upstream and Downstream of the Major Splice Donor Site Influence the Ability of HIV-2 Leader RNA To Dimerize in Vitro

Elements Located Upstream and Downstream of the Major Splice Donor Site Influence the Ability of HIV-2 Leader RNA To Dimerize in Vitro

Mechanism of Hairpin-Duplex Conversion for the HIV-1 Dimerization Initiation Site

The Dimer Initiation Site Hairpin Mediates Dimerization of the Human Immunodeficiency Virus, Type 2 RNA Genome

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