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1.
J Mol Biol ; 431(10): 1966-1980, 2019 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-30876916

RESUMO

A comprehensive view of all the structural aspects related to NCp7 is essential to understand how this protein, crucial in many steps of the HIV-1 cycle, binds and anneals nucleic acids (NAs), mainly thanks to two zinc fingers, ZF1 and ZF2. Here, we inspected the structural properties of the available experimental models of NCp7 bound to either DNA or RNA molecules, or free of ligand. Our analyses included the characterization of the relative positioning of ZF1 and ZF2, accessibility measurements and the exhaustive, quantitative mapping of the contacts between amino acids and nucleotides by a recent tessellation method, VLDM. This approach unveiled the intimate connection between NA binding process and the conformations explored by the free protein. It also provided new insights into the functional specializations of ZF1 and ZF2. The larger accessibility of ZF2 in free NCp7 and the consistency of the ZF2/NA interface in different models and conditions give ZF2 the lead of the binding process. ZF1 contributes to stabilize the complexes through various organizations of the ZF1/NA interface. This work outcome is a global binding scheme of NCp7 to DNA and RNA, and an example of how protein-NA complexes are stabilized.

2.
Biochim Open ; 7: 10-25, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30109196

RESUMO

An infectious retroviral particle contains 1000-1500 molecules of the nucleocapsid protein (NC) that cover the diploid RNA genome. NC is a small zinc finger protein that possesses nucleic acid chaperone activity that enables NC to rearrange DNA and RNA molecules into the most thermodynamically stable structures usually those containing the maximum number of base pairs. Thanks to the chaperone activity, NC plays an essential role in reverse transcription of the retroviral genome by facilitating the strand transfer reactions of this process. In addition, these reactions are involved in recombination events that can generate multiple drug resistance mutations in the presence of anti-HIV-1 drugs. The strand transfer reactions rely on base pairing of folded DNA/RNA structures. The molecular mechanisms responsible for NC-mediated strand transfer reactions are presented and discussed in this review. Antiretroviral strategies targeting the NC-mediated strand transfer events are also discussed.

3.
Nucleic Acids Res ; 46(18): 9699-9710, 2018 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-29986076

RESUMO

During HIV-1 assembly and budding, Gag protein, in particular the C-terminal domain containing the nucleocapsid domain (NCd), p1 and p6, is the site of numerous interactions with viral and cellular factors. Most in vitro studies of Gag have used constructs lacking p1 and p6. Here, using NMR spectroscopy, we show that the p1-p6 region of Gag (NCp15) is largely disordered, but interacts transiently with the NCd. These interactions modify the dynamic properties of the NCd. Indeed, using isothermal titration calorimetry (ITC), we have measured a higher entropic penalty to RNA-binding for the NCd precursor, NCp15, than for the mature form, NCp7, which lacks p1 and p6. We propose that during assembly and budding of virions, concomitant with Gag oligomerization, transient interactions between NCd and p1-p6 become salient and responsible for (i) a higher level of structuration of p6, which favours recruitment of budding partners; and (ii) a higher entropic penalty to RNA-binding at specific sites that favours non-specific binding of NCd at multiple sites on the genomic RNA (gRNA). The contributions of p6 and p1 are sequentially removed via proteolysis during Gag maturation such that the RNA-binding specificity of the mature protein is governed by the properties of NCd.

4.
Biochemistry ; 57(30): 4562-4573, 2018 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-30019894

RESUMO

Due to its essential roles in the viral replication cycle and to its highly conserved sequence, the nucleocapsid protein (NCp7) of the human immunodeficiency virus type 1 is a target of choice for inhibiting replication of the virus. Most NCp7 inhibitors identified so far are small molecules. A small number of short peptides also act as NCp7 inhibitors by competing with its nucleic acid (NA) binding and chaperone activities but exhibit antiviral activity only at relatively high concentrations. In this work, in order to obtain more potent NCp7 competitors, we designed a library of longer peptides (10-17 amino acids) whose sequences include most of the NCp7 structural determinants responsible for its specific NA binding and destabilizing activities. Using an in vitro assay, the most active peptide (pE) was found to inhibit the NCp7 destabilizing activity, with a 50% inhibitory concentration in the nanomolar range, by competing with NCp7 for binding to its NA substrates. Formulated with a cell-penetrating peptide (CPP), pE was found to accumulate into HeLa cells, with low cytotoxicity. However, either formulated with a CPP or overexpressed in cells, pE did not show any antiviral activity. In vitro competition experiments revealed that its poor antiviral activity may be partly due to its sequestration by cellular RNAs. The selected peptide pE therefore appears to be a useful tool for investigating NCp7 properties and functions in vitro, but further work will be needed to design pE-derived peptides with antiviral activity.

5.
Biomol NMR Assign ; 12(1): 139-143, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29332151

RESUMO

During HIV-1 assembly, the Pr55Gag polyprotein precursor (Gag) interacts with the genomic RNA, with lipids of the plasma membrane, with host proteins (ALIX, TSG101) through the ESCRT complex, with the viral protein Vpr and are involved in intermolecular interactions with other Pr55Gag proteins. This network of interactions is responsible for the formation of the viral particle, the selection of genomic RNA and the packaging of Vpr. The C-terminal domain of Gag encompassed in NCp15 is involved in the majority of these interactions, either by its nucleocapsid or its p6 domains. We study the NCp15 protein as a model of the C-terminal domain of Gag to better understand the role of this domain in the assembly and budding of HIV-1. Here, we report the 1H, 13C and 15N chemical shift assignments of NCp15 obtained by heteronuclear multidimensional NMR spectroscopy as well as the analysis of its secondary structure in solution. These assignments of NCp15 pave the way for interaction studies with its numerous partners.

6.
J Phys Chem B ; 121(50): 11249-11261, 2017 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-29172512

RESUMO

Recently, a 3-hydroxychromone based nucleoside 3HCnt has been developed as a highly environment-sensitive nucleoside surrogate to investigate protein-DNA interactions. When it is incorporated in DNA, the probe is up to 50-fold brighter than 2-aminopurine, the reference fluorescent nucleoside. Although the insertion of 3HCnt in DNA was previously shown to not alter the overall DNA structure, the possibility of the probe inducing local effects cannot be ruled out. Hence, a systematic structural and dynamic study is required to unveil the 3HCnt's limitations and to properly interpret the data obtained with this universal probe. Here, we investigated by NMR a 12-mer duplex, in which a central adenine was replaced by 3HCnt. The chemical shifts variations and nOe contacts revealed that the 3HCnt is well inserted in the DNA double helix with extensive stacking interactions with the neighbor base pairs. These observations are in excellent agreement with the steady-state and time-resolved fluorescence properties indicating that the 3HCnt fluorophore is protected from the solvent and does not exhibit rotational motion. The 3HCnt insertion in DNA is accompanied by the extrusion of the opposite nucleobase from the double helix. Molecular dynamics simulations using NMR-restraints demonstrated that 3HCnt fluorophore exhibits only translational dynamics. Taken together, our data showed an excellent intercalation of 3HCnt in the DNA double helix, which is accompanied by localized perturbations. This confirms 3HCnt as a highly promising tool for nucleic acid labeling and sensing.


Assuntos
Cromonas/química , DNA/química , Fluorescência , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico
7.
Bioorg Med Chem Lett ; 27(11): 2506-2509, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28400233

RESUMO

SHAPE chemistry (selective 2'-hydroxyl acylation analyzed by primer extension) has been developed to specifically target flexible nucleotides (often unpaired nucleotides) independently to their purine or pyrimidine nature for RNA secondary structure determination. However, to the best of our knowledge, the structure of 2'-O-acylation products has never been confirmed by NMR or X-ray data. We have realized the acylation reactions between cNMP and NMIA under SHAPE chemistry conditions and identified the acylation products using standard NMR spectroscopy and LC-MS/MS experiments. For cAMP and cGMP, the major acylation product is the 2'-O-acylated compound (>99%). A trace amount of N-acylated cAMP has also been identified by LC-UV-MS2. While for cCMP, the isolated acylation products are composed of 96% of 2'-O-acylated, 4% of N,O-diacylated, and trace amount of N-acylated compounds. In addition, the characterization of the major 2'-O-acylated compound by NMR showed slight differences in the conformation of the acylated sugar between the three cyclic nucleotides. This interesting result should be useful to explain some unexpected reactivity of the SHAPE chemistry.


Assuntos
Nucleotídeos/química , Acilação , Espectroscopia de Ressonância Magnética , Nitrosaminas/química , Conformação de Ácido Nucleico , RNA/química , Espectrometria de Massas em Tandem
8.
Sci Rep ; 7: 43954, 2017 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-28266653

RESUMO

Werner syndrome is caused by mutations in the WRN gene encoding WRN helicase. A knowledge of WRN helicase's DNA unwinding mechanism in vitro is helpful for predicting its behaviors in vivo, and then understanding their biological functions. In the present study, for deeply understanding the DNA unwinding mechanism of WRN, we comprehensively characterized the DNA unwinding properties of chicken WRN helicase core in details, by taking advantages of single-molecule fluorescence resonance energy transfer (smFRET) method. We showed that WRN exhibits repetitive DNA unwinding and translocation behaviors on different DNA structures, including forked, overhanging and G-quadruplex-containing DNAs with an apparently limited unwinding processivity. It was further revealed that the repetitive behaviors were caused by reciprocating of WRN along the same ssDNA, rather than by complete dissociation from and rebinding to substrates or by strand switching. The present study sheds new light on the mechanism for WRN functioning.


Assuntos
Galinhas , DNA Helicases/metabolismo , DNA/metabolismo , Helicase da Síndrome de Werner/metabolismo , Animais , Transferência Ressonante de Energia de Fluorescência , Imagem Individual de Molécula
9.
Sci Rep ; 7: 42865, 2017 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-28216645

RESUMO

3'-5' exonucleases are frequently found to be associated to polymerases or helicases domains in the same enzyme or could function as autonomous entities. Here we uncovered that Candida albicans Pif1 (CaPif1) displays a 3'-5' exonuclease activity besides its main helicase activity. These two latter activities appear to reside on the same polypeptide and the new exonuclease activity could be mapped to the helicase core domain. We clearly show that CaPif1 displays exclusively exonuclease activity and unambiguously establish the directionality of the exonuclease activity as the 3'-to-5' polarity. The enzyme appears to follow the two-metal-ion driven hydrolyzing activity exhibited by most of the nucleases, as shown by its dependence of magnesium and also by the identification of aspartic residues. Interestingly, an excellent correlation could be found between the presence of the conserved residues and the exonuclease activity when testing activities on Pif1 enzymes from eight fungal organisms. In contrast to others proteins endowed with the double helicase/exonuclease functionality, CaPif1 differs in the fact that the two activities are embedded in the same helicase domain and not located on separated domains. Our findings may suggest a biochemical basis for mechanistic studies of Pif1 family helicases.


Assuntos
Candida albicans/enzimologia , DNA Helicases/química , DNA Helicases/metabolismo , Exonucleases/metabolismo , Sequência de Aminoácidos , Candida albicans/química , Sequência Conservada , Exonucleases/química , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Hidrólise , Magnésio/metabolismo , Domínios Proteicos
10.
J Am Chem Soc ; 139(6): 2520-2528, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28112929

RESUMO

DNA methylation patterns, which are critical for gene expression, are replicated by DNA methyltransferase 1 (DNMT1) and ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) proteins. This replication is initiated by the recognition of hemimethylated CpG sites and further flipping of methylated cytosines (mC) by the Set and Ring Associated (SRA) domain of UHRF1. Although crystallography has shed light on the mechanism of mC flipping by SRA, tools are required to monitor in real time how SRA reads DNA and flips the modified nucleobase. To accomplish this aim, we have utilized two distinct fluorescent nucleobase surrogates, 2-thienyl-3-hydroxychromone nucleoside (3HCnt) and thienoguanosine (thG), incorporated at different positions into hemimethylated (HM) and nonmethylated (NM) DNA duplexes. Large fluorescence changes were associated with mC flipping in HM duplexes, showing the outstanding sensitivity of both nucleobase surrogates to the small structural changes accompanying base flipping. Importantly, the nucleobase surrogates marginally affected the structure of the duplex and its affinity for SRA at positions where they were responsive to base flipping, illustrating their promise as nonperturbing probes for monitoring such events. Stopped-flow studies using these two distinct tools revealed the fast kinetics of SRA binding and sliding to NM duplexes, consistent with its reader role. In contrast, the kinetics of mC flipping was found to be much slower in HM duplexes, substantially increasing the lifetime of CpG-bound UHRF1, and thus the probability of recruiting DNMT1 to faithfully duplicate the DNA methylation profile. The fluorescence-based approach using these two different fluorescent nucleoside surrogates advances the mechanistic understanding of the UHRF1/DNMT1 tandem and the development of assays for the identification of base flipping inhibitors.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Citosina/metabolismo , DNA/metabolismo , Termodinâmica , Proteínas Estimuladoras de Ligação a CCAAT/química , Citosina/química , DNA/química , Metilação de DNA , Replicação do DNA , Fluorescência , Humanos , Cinética , Estrutura Molecular
11.
Nucleic Acids Res ; 44(7): 3432-47, 2016 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-26883628

RESUMO

Experimental characterization of the structural couplings in free B-DNA in solution has been elusive, because of subtle effects that are challenging to tackle. Here, the exploitation of the NMR measurements collected on four dodecamers containing a substantial set of dinucleotide sequences provides new, consistent correlations revealing the DNA intrinsic mechanics. The difference between two successive residual dipolar couplings (ΔRDCs) involving C6/8-H6/8, C3'-H3' and C4'-H4' vectors are correlated to the(31)P chemical shifts (δP), which reflect the populations of the BI and BII backbone states. The δPs are also correlated to the internucleotide distances (Dinter) involving H6/8, H2' and H2″ protons. Calculations of NMR quantities on high resolution X-ray structures and controlled models of DNA enable to interpret these couplings: the studied ΔRDCs depend mostly on roll, while Dinterare mainly sensitive to twist or slide. Overall, these relations demonstrate how δP measurements inform on key inter base parameters, in addition to probe the BI↔BII backbone equilibrium, and shed new light into coordinated motions of phosphate groups and bases in free B-DNA in solution. Inspection of the 5' and 3' ends of the dodecamers also supplies new information on the fraying events, otherwise neglected.


Assuntos
DNA de Forma B/química , Desoxirribonucleotídeos/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico
12.
RNA ; 22(4): 506-17, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26826129

RESUMO

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.


Assuntos
HIV-1/genética , RNA Viral/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Sequência de Bases , Sequências Repetidas Invertidas , Cinética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Estabilidade de RNA , Elementos de Resposta
13.
J Biol Chem ; 291(7): 3468-82, 2016 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-26668324

RESUMO

An essential step of human immunodeficiency virus type 1 (HIV-1) reverse transcription is the first strand transfer that requires base pairing of the R region at the 3'-end of the genomic RNA with the complementary r region at the 3'-end of minus-strand strong-stop DNA (ssDNA). HIV-1 nucleocapsid protein (NC) facilitates this annealing process. Determination of the ssDNA structure is needed to understand the molecular basis of NC-mediated genomic RNA-ssDNA annealing. For this purpose, we investigated ssDNA using structural probes (nucleases and potassium permanganate). This study is the first to determine the secondary structure of the full-length HIV-1 ssDNA in the absence or presence of NC. The probing data and phylogenetic analysis support the folding of ssDNA into three stem-loop structures and the presence of four high-affinity binding sites for NC. Our results support a model for the NC-mediated annealing process in which the preferential binding of NC to four sites triggers unfolding of the three-dimensional structure of ssDNA, thus facilitating interaction of the r sequence of ssDNA with the R sequence of the genomic RNA. In addition, using gel retardation assays and ssDNA mutants, we show that the NC-mediated annealing process does not rely on a single pathway (zipper intermediate or kissing complex).


Assuntos
Códon de Terminação , DNA de Cadeia Simples/química , DNA Viral/química , HIV-1/metabolismo , Modelos Moleculares , Proteínas do Nucleocapsídeo/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Sítios de Ligação , DNA Recombinante/química , DNA Recombinante/isolamento & purificação , DNA Recombinante/metabolismo , DNA de Cadeia Simples/isolamento & purificação , DNA de Cadeia Simples/metabolismo , DNA Viral/isolamento & purificação , DNA Viral/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Cinética , Peso Molecular , Mutação , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Proteínas do Nucleocapsídeo/metabolismo , Filogenia , Conformação Proteica , RNA Viral/química , RNA Viral/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo
14.
Biochemistry ; 53(35): 5601-12, 2014 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-25102280

RESUMO

We investigated how the intrinsic sequence-dependent properties probed via the phosphate linkages (BI ↔ BII equilibrium) influence the preferred shape of free DNA, and how this affects the nucleosome formation. First, this exploits NMR solution studies of four B-DNA dodecamers that together cover 39 base pairs of the 5' half of the sequence 601, of special interest for nucleosome formation. The results validate our previous prediction of a systematic, general sequence effect on the intrinsic backbone BII propensities. NMR provides new evidence that the backbone behavior is intimately coupled to the minor groove width. Second, application of the backbone behavior predictions to the full sequence 601 and other relevant sequences demonstrates that alternation of intrinsic low and high BII propensities, coupled to intrinsic narrow and wide minor grooves, largely coincides with the sinusoidal variations of the DNA minor groove width observed in crystallographic structures of the nucleosome. This correspondence is much poorer with low affinity sequences. Overall, the results indicate that nucleosome formation involves an indirect readout process implicating pre-existing DNA minor groove conformations. It also illustrates how the prediction of the intrinsic structural DNA behavior offers a powerful framework to gain explanatory insight on how proteins read DNA.


Assuntos
DNA de Forma B/química , Nucleossomos/química , Sequência de Bases , DNA de Forma B/genética , Humanos , Substâncias Macromoleculares/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/química , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética
15.
PLoS One ; 9(7): e102150, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25029439

RESUMO

The HIV-1 nucleocapsid protein (NC) is a small basic protein containing two zinc fingers (ZF) separated by a short linker. It is involved in several steps of the replication cycle and acts as a nucleic acid chaperone protein in facilitating nucleic acid strand transfers occurring during reverse transcription. Recent analysis of three-dimensional structures of NC-nucleic acids complexes established a new property: the unpaired guanines targeted by NC are more often inserted in the C-terminal zinc finger (ZF2) than in the N-terminal zinc finger (ZF1). Although previous NMR dynamic studies were performed with NC, the dynamic behavior of the linker residues connecting the two ZF domains remains unclear. This prompted us to investigate the dynamic behavior of the linker residues. Here, we collected 15N NMR relaxation data and used for the first time data at several fields to probe the protein dynamics. The analysis at two fields allows us to detect a slow motion occurring between the two domains around a hinge located in the linker at the G35 position. However, the amplitude of motion appears limited in our conditions. In addition, we showed that the neighboring linker residues R29, A30, P31, R32, K33 displayed restricted motion and numerous contacts with residues of ZF1. Our results are fully consistent with a model in which the ZF1-linker contacts prevent the ZF1 domain to interact with unpaired guanines, whereas the ZF2 domain is more accessible and competent to interact with unpaired guanines. In contrast, ZF1 with its large hydrophobic plateau is able to destabilize the double-stranded regions adjacent to the guanines bound by ZF2. The linker residues and the internal dynamics of NC regulate therefore the different functions of the two zinc fingers that are required for an optimal chaperone activity.


Assuntos
DNA/metabolismo , HIV-1 , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/metabolismo , RNA/metabolismo , Dedos de Zinco , Modelos Moleculares , Rotação
16.
Virus Res ; 193: 2-15, 2014 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-24907482

RESUMO

This review aims at briefly presenting a retrospect on the retroviral nucleocapsid protein (NC), from an unspecific nucleic acid binding protein (NABP) to an all-in-one viral protein with multiple key functions in the early and late phases of the retrovirus replication cycle, notably reverse transcription of the genomic RNA and viral DNA integration into the host genome, and selection of the genomic RNA together with the initial steps of virus morphogenesis. In this context we will discuss the notion that NC protein has a flexible conformation and is thus a member of the growing family of intrinsically disordered proteins (IDPs) where disorder may account, at least in part, for its function as a nucleic acid (NA) chaperone and possibly as a protein chaperone vis-à-vis the viral DNA polymerase during reverse transcription. Lastly, we will briefly review the development of new anti-retroviral/AIDS compounds targeting HIV-1 NC because it represents an ideal target due to its multiple roles in the early and late phases of virus replication and its high degree of conservation.


Assuntos
Proteínas do Nucleocapsídeo/metabolismo , Retroviridae/fisiologia , Animais , Antivirais/farmacologia , Proteínas de Transporte/metabolismo , Humanos , Complexos Multiproteicos/metabolismo , Nucleocapsídeo/antagonistas & inibidores , Nucleocapsídeo/metabolismo , Proteínas do Nucleocapsídeo/genética , Ligação Proteica , RNA Viral/genética , RNA Viral/metabolismo , Retroviridae/efeitos dos fármacos , Transcrição Reversa , Ribonucleoproteínas/metabolismo , Montagem de Vírus
17.
Nucleic Acids Res ; 42(2): 1065-78, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24153111

RESUMO

The HIV-1 transactivator of transcription (Tat) protein is thought to stimulate reverse transcription (RTion). The Tat protein and, more specifically, its (44-61) domain were recently shown to promote the annealing of complementary DNA sequences representing the HIV-1 transactivation response element TAR, named dTAR and cTAR, that plays a key role in RTion. Moreover, the kinetic mechanism of the basic Tat(44-61) peptide in this annealing further revealed that this peptide constitutes a representative nucleic acid annealer. To further understand the structure-activity relationships of this highly conserved domain, we investigated by electrophoresis and fluorescence approaches the binding and annealing properties of various Tat(44-61) mutants. Our data showed that the Tyr47 and basic residues of the Tat(44-61) domain were instrumental for binding to cTAR through stacking and electrostatic interactions, respectively, and promoting its annealing with dTAR. Furthermore, the annealing efficiency of the mutants clearly correlates with their ability to rapidly associate and dissociate the complementary oligonucleotides and to promote RTion. Thus, transient and dynamic nucleic acid interactions likely constitute a key mechanistic component of annealers and the role of Tat in the late steps of RTion. Finally, our data suggest that Lys50 and Lys51 acetylation regulates Tat activity in RTion.


Assuntos
Repetição Terminal Longa de HIV , HIV-1 , Transcrição Reversa , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química
18.
PLoS One ; 7(6): e38905, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22745685

RESUMO

HIV-1 nucleocapsid protein (NC) is involved in the rearrangement of nucleic acids occurring in key steps of reverse transcription. The protein, through its two zinc fingers, interacts preferentially with unpaired guanines in single-stranded sequences. In mini-cTAR stem-loop, which corresponds to the top half of the cDNA copy of the transactivation response element of the HIV-1 genome, NC was found to exhibit a clear preference for the TGG sequence at the bottom of mini-cTAR stem. To further understand how this site was selected among several potential binding sites containing unpaired guanines, we probed the intrinsic dynamics of mini-cTAR using (13)C relaxation measurements. Results of spin relaxation time measurements have been analyzed using the model-free formalism and completed by dispersion relaxation measurements. Our data indicate that the preferentially recognized guanine in the lower part of the stem is exempt of conformational exchange and highly mobile. In contrast, the unrecognized unpaired guanines of mini-cTAR are involved in conformational exchange, probably related to transient base-pairs. These findings support the notion that NC preferentially recognizes unpaired guanines exhibiting a high degree of mobility. The ability of NC to discriminate between close sequences through their dynamic properties contributes to understanding how NC recognizes specific sites within the HIV genome.


Assuntos
DNA Viral/metabolismo , HIV-1/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , DNA Viral/química , Ensaio de Desvio de Mobilidade Eletroforética , HIV-1/genética , Espectroscopia de Ressonância Magnética , Conformação de Ácido Nucleico , Proteínas do Nucleocapsídeo/química , Ligação Proteica
19.
J Mol Biol ; 410(4): 565-81, 2011 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-21762801

RESUMO

One salient feature of reverse transcription in retroviruses, notably in the human immunodeficiency virus type 1, is that it requires the homologous nucleocapsid (NC) protein acting as a chaperoning partner of the genomic RNA template and the reverse transcriptase, from the initiation to the completion of viral DNA synthesis. This short review on the NC protein of human immunodeficiency virus type 1 aims at briefly presenting the flexible nature of NC protein, how it interacts with nucleic acids via its invariant zinc fingers and flanking basic residues, and the possible mechanisms that account for its multiple functions in the early steps of virus replication, notably in the obligatory strand transfer reactions during viral DNA synthesis by the reverse transcriptase enzyme.


Assuntos
HIV-1/metabolismo , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/metabolismo , Sequência de Aminoácidos , DNA Viral/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ácidos Nucleicos/metabolismo , Ligação Proteica
20.
Nucleic Acids Res ; 39(18): 8148-62, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21724607

RESUMO

Annealing of the TAR RNA hairpin to the cTAR DNA hairpin is required for the minus-strand transfer step of HIV-1 reverse transcription. HIV-1 nucleocapsid protein (NC) plays a crucial role by facilitating annealing of the complementary hairpins. To gain insight into the mechanism of NC-mediated TAR RNA-DNA annealing, we used structural probes (nucleases and potassium permanganate), gel retardation assays, fluorescence anisotropy and cTAR mutants under conditions allowing strand transfer. In the absence of NC, cTAR DNA-TAR RNA annealing depends on nucleation through the apical loops. We show that the annealing intermediate of the kissing pathway is a loop-loop kissing complex involving six base-pairs and that the apical stems are not destabilized by this loop-loop interaction. Our data support a dynamic structure of the cTAR hairpin in the absence of NC, involving equilibrium between both the closed conformation and the partially open 'Y' conformation. This study is the first to show that the apical and internal loops of cTAR are weak and strong binding sites for NC, respectively. NC slightly destabilizes the lower stem that is adjacent to the internal loop and shifts the equilibrium toward the 'Y' conformation exhibiting at least 12 unpaired nucleotides in its lower part.


Assuntos
DNA Viral/química , Repetição Terminal Longa de HIV , HIV-1/genética , RNA Viral/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Ensaio de Desvio de Mobilidade Eletroforética , Polarização de Fluorescência , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico
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