RESUMEN
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.
Asunto(s)
Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Diseño de Fármacos , VIH-1/efectos de los fármacos , Péptidos/química , Péptidos/farmacología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Secuencia de Aminoácidos , Evaluación Preclínica de Medicamentos , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/química , VIH-1/metabolismo , Células HeLa , Humanos , Modelos Moleculares , Ácidos Nucleicos/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Since currently available therapies against HIV/AIDS still show important drawbacks, the development of novel anti-HIV treatments is a key issue. We recently characterized methylated oligoribonucleotides (mONs) that extensively inhibit HIV-1 replication in primary T cells at nanomolar concentrations. The mONs were shown to target both HIV-1 reverse transcriptase (RT) and the nucleocapsid protein (NC), which is an essential partner of RT during viral DNA synthesis. To further understand the mechanism of such mONs, we studied by isothermal titration calorimetry and fluorescence-based techniques their NC binding properties and ability to inhibit the nucleic acid chaperone properties of NC. Notably, we investigated the ability of mONs to inhibit the NC-induced destabilization of the HIV-1 cTAR (complementary DNA sequence to TAR [transactivation response element]) stem-loop and the NC-promoted cTAR annealing to its complementary sequence, required at the early stage of HIV-1 viral DNA synthesis. Moreover, we compared the activity of the mONs to that of a number of modified and nonmodified oligonucleotides. Results show that the mONs inhibit NC by a competitive mechanism whereby the mONs tightly bind the NC peptide, mainly through nonelectrostatic interactions with the hydrophobic platform at the top of the NC zinc fingers. Taken together, these results favor the notion that the mONs impair the process of the RT-directed viral DNA synthesis by sequestering NC molecules, thus preventing the chaperoning of viral DNA synthesis by NC. These findings contribute to the understanding of the molecular basis for NC inhibition by mONs, which could be used for the rational design of antiretroviral compounds targeting HIV-1 NC protein.
Asunto(s)
VIH-1/metabolismo , Chaperonas Moleculares/antagonistas & inhibidores , Proteínas de la Nucleocápside/antagonistas & inhibidores , Oligorribonucleótidos/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , ADN Viral/biosíntesis , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/metabolismo , Oligorribonucleótidos/química , Oligorribonucleótidos/metabolismoRESUMEN
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.
Asunto(s)
ADN Viral/química , Duplicado del Terminal Largo de VIH , VIH-1/genética , ARN Viral/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Ensayo de Cambio de Movilidad Electroforética , Polarización de Fluorescencia , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido NucleicoRESUMEN
Synthesis of the HIV-1 viral DNA by reverse transcriptase involves two obligatory strand transfer reactions. The second strand transfer corresponds to the annealing of the (-) and (+) DNA copies of the primer binding site (PBS) sequence which is chaperoned by the nucleocapsid protein (NCp7). NCp7 modifies the (+)/(-)PBS annealing mechanism by activating a loop-loop kissing pathway that is negligible without NCp7. To characterize in depth the dynamics of the loop in the NCp7/PBS nucleoprotein complexes, we investigated the time-resolved fluorescence parameters of a (-)PBS derivative containing the fluorescent nucleoside analogue 2-aminopurine at positions 6, 8 or 10. The NCp7-directed switch of (+)/(-)PBS annealing towards the loop pathway was associated to a drastic restriction of the local DNA dynamics, indicating that NCp7 can 'freeze' PBS conformations competent for annealing via the loops. Moreover, the modifications of the PBS loop structure and dynamics that govern the annealing reaction were found strictly dependent on the integrity of the zinc finger hydrophobic platform. Our data suggest that the two NCp7 zinc fingers are required to ensure the specificity and fidelity of the second strand transfer, further underlining the pivotal role played by NCp7 to control the faithful synthesis of viral HIV-1 DNA.
Asunto(s)
Cartilla de ADN/química , VIH-1/genética , Dedos de Zinc , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , 2-Aminopurina/química , Sitios de Unión , ADN Viral/química , Cinética , Mutación , Unión Proteica , Termodinámica , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Reverse transcription of the genomic RNA by reverse transcriptase occurs soon after HIV-1 infection of target cells. The viral nucleocapsid (NC) protein chaperones this process via its nucleic acid annealing activities and its interactions with the reverse transcriptase enzyme. To function, NC needs its two conserved zinc fingers and flanking basic residues. We recently reported a new role for NC, whereby it negatively controls reverse transcription in the course of virus formation. Indeed, deleting its zinc fingers causes reverse transcription activation in virus producer cells. To investigate this new NC function, we used viruses with subtle mutations in the conserved zinc fingers and its flanking domains. We monitored by quantitative PCR the HIV-1 DNA content in producer cells and in produced virions. Results showed that the two intact zinc-finger structures are required for the temporal control of reverse transcription by NC throughout the virus replication cycle. The N-terminal basic residues also contributed to this new role of NC, while Pro-31 residue between the zinc fingers and Lys-59 in the C-terminal region did not. These findings further highlight the importance of NC as a major target for anti-HIV-1 drugs.
Asunto(s)
ADN Viral/biosíntesis , VIH-1/genética , Transcripción Reversa , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Secuencia de Aminoácidos , Línea Celular , Secuencia Conservada , ADN Complementario/análisis , ADN Complementario/biosíntesis , VIH-1/crecimiento & desarrollo , VIH-1/fisiología , Humanos , Datos de Secuencia Molecular , Mutación Puntual , Estructura Terciaria de Proteína , Virión/química , Replicación Viral , Dedos de Zinc , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
The retroviral nucleocapsid proteins (NCs) are small proteins with either one or two conserved zinc fingers flanked by basic domains. NCs play key roles during reverse transcription by chaperoning the obligatory strand transfers. In HIV-1, the first DNA strand transfer relies on the NCp7-promoted destabilization and subsequent annealing of the transactivation response element, TAR with its complementary cTAR sequence. NCp7 chaperone activity relies mainly on its two folded fingers. Since NCs with a unique zinc finger are encoded by gammaretroviruses such as the canonical Moloney murine leukemia virus (MoMuLV), our objective was to characterize, by fluorescence techniques, the binding and chaperone activities of the NCp10 protein of MoMuLV to the TAR sequences of HIV-1. The unique finger and the flanking 12-25 and 40-48 domains of NCp10 were found to bind and destabilize cTAR stem-loop almost as efficiently as the homologous NCp7 protein. The flanking domains were essential for properly positioning the finger and, notably, the Trp35 residue onto cTAR. Thus, the binding and destabilization determinants scattered on the two NCp7 fingers are encoded by the unique finger of NCp10 and its flanking domains. NCp10 also activates the cTAR/TAR annealing reaction, but less efficiently than NCp7, suggesting that the two NCp7 fingers promote in concert the rate-limiting nucleation of the duplex. Due to its ability to mimic NCp7, the simple structure of NCp10 might be useful to design peptidomimetics aimed at inhibiting HIV replication.
Asunto(s)
Duplicado del Terminal Largo de VIH/genética , VIH-1/genética , Virus de la Leucemia Murina de Moloney/metabolismo , Proteínas de la Nucleocápside/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , ADN Viral/química , ADN Viral/genética , ADN Viral/metabolismo , Polarización de Fluorescencia , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Proteínas de la Nucleocápside/química , Unión ProteicaRESUMEN
Conversion of the human immunodeficiency virus type 1 (HIV-1) genomic RNA into the proviral DNA by reverse transcriptase involves two obligatory strand transfers that are chaperoned by the nucleocapsid protein (NC). The second strand transfer relies on the annealing of the (-) and (+) copies of the primer binding site, (-)PBS and (+) PBS, which fold into complementary stem-loops (SLs) with terminal single-stranded overhangs. To understand how NC chaperones their hybridization, we investigated the annealing kinetics of fluorescently labelled (+)PBS with various (-)PBS derivatives. In the absence of NC, the (+)/(-)PBS annealing was governed by a second-order pathway nucleated mainly by the single-stranded overhangs of the two PBS SLs. The annealing reaction appeared to be rate-limited by the melting of the stable G.C-rich stem subsequent to the formation of the partially annealed intermediate. A second pathway nucleated through the loops could be detected, but was very minor. NC(11-55), which consists primarily of the zinc finger domain, increased the (-)/(+) PBS annealing kinetics by about sixfold, by strongly activating the interaction between the PBS loops. NC(11-55) also activated (-)/(+) PBS annealing through the single-strand overhangs, but by a factor of only 2. Full-length NC(1-55) further increased the (-)/(+)PBS annealing kinetics by tenfold. The NC-promoted (-)/(+)PBS mechanism proved to be similar with extended (-)DNA molecules, suggesting that it is relevant in the context of proviral DNA synthesis. These findings favour the notion that the ubiquitous role of NC in the viral life-cycle probably relies on the ability of NC to chaperone nucleic acid hybridization via different mechanisms.
Asunto(s)
VIH-1/genética , Proteínas de la Nucleocápside/fisiología , Transcripción Reversa , Humanos , Cinética , Chaperonas Moleculares , Conformación de Ácido Nucleico , Fenómenos Fisiológicos de los VirusAsunto(s)
VIH/efectos de los fármacos , VIH/fisiología , Proteínas de la Nucleocápside/antagonistas & inhibidores , Proteínas de la Nucleocápside/metabolismo , Secuencia de Aminoácidos , Animales , Fármacos Anti-VIH/farmacología , Evaluación Preclínica de Medicamentos , VIH/química , Humanos , Datos de Secuencia Molecular , Proteínas de la Nucleocápside/química , Ensamble de Virus/efectos de los fármacosRESUMEN
The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) is formed of two highly conserved CCHC zinc fingers flanked by small basic domains. NC is required for the two obligatory strand transfers in viral DNA synthesis through its nucleic acid chaperoning properties. The first DNA strand transfer relies on NC's ability to bind and destabilize the secondary structure of complementary transactivation response region (cTAR) DNA, to inhibit self-priming, and to promote the annealing of cTAR to TAR RNA. To further investigate NC chaperone properties, our aim was to identify by fluorescence spectroscopy and gel electrophoresis, the NC structural determinants for cTAR binding and destabilization, and for the inhibition of self-primed DNA synthesis on a model system using a series of NC mutants and HIV-1 reverse transcriptase. NC destabilization and self-priming inhibition properties were found to be supported by the two fingers in their proper context and the basic (29)RAPRKKG(35) linker. The strict requirement of the native proximal finger suggests that its hydrophobic platform (Val13, Phe16, Thr24 and Ala25) is crucial for binding, destabilization and inhibition of self-priming. In contrast, only partial folding of the distal finger is required, probably for presenting the Trp37 residue in an appropriate orientation. Also, Trp37 and the hydrophobic residues of the proximal finger appear to be essential for the propagation of the melting from the cTAR ends up to the middle of the stem. Finally, both N-terminal and C-terminal basic domains contribute to cTAR binding but not to its destabilization.
Asunto(s)
Proteínas de la Cápside/química , Proteínas de Unión al ADN/química , Productos del Gen gag/química , Duplicado del Terminal Largo de VIH/fisiología , VIH-1/genética , Transcripción Reversa/fisiología , Proteínas Virales/química , Secuencia de Aminoácidos , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Replicación del ADN/genética , Replicación del ADN/fisiología , ADN Viral/genética , ADN Viral/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Productos del Gen gag/genética , Productos del Gen gag/metabolismo , Duplicado del Terminal Largo de VIH/genética , VIH-1/fisiología , Humanos , Datos de Secuencia Molecular , Mutación/genética , Conformación de Ácido Nucleico , Pliegue de Proteína , Transcripción Reversa/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Dedos de Zinc/genética , Dedos de Zinc/fisiología , Productos del Gen gag del Virus de la Inmunodeficiencia HumanaRESUMEN
The nucleocapsid protein NCp7 of HIV-1 possesses nucleic acid chaperone properties that are critical for the two obligatory strand transfer reactions required for the synthesis of a complete proviral DNA by reverse transcriptase. The first DNA strand transfer relies on the destabilization by NCp7 of double-stranded segments of the transactivation response region (TAR) sequence at the 3' end of the genomic RNA and the complementary sequence cTAR at the 3' terminus of minus strong-stop DNA, the early product of reverse transcription. In order to determine the dynamics of NCp7-mediated nucleic acid destabilization, we investigated by time-resolved fluorescence spectroscopy and two photon fluorescence correlation spectroscopy, the interaction of a doubly labeled cTAR sequence with NC(12-55) containing NCp7 CCHC zinc fingers and flanking basic amino acid residues. From the chemical rates and the activation energy associated with the conformational fluctuations observed in the absence of NC, it is concluded that such fluctuations are associated with the opening and closing of the double-stranded terminal segments of cTAR. The destabilizing activity of NC(12-55) occurs mainly through a major increase of the opening rate constant of cTAR. Moreover, NC appears to augment the number of pathways between the open and closed states of cTAR, suggesting that it initiates melting of base-pairs at different locations within the terminal segments of cTAR. This activity of NC on the dynamics of cTAR secondary structure is thought to be critical for the formation of the cTAR-TAR complex, which is essential for the specificity and extent of proviral DNA synthesis by reverse transcriptase.
Asunto(s)
Duplicado del Terminal Largo de VIH , VIH-1/genética , Proteínas de la Nucleocápside/química , Secuencia de Bases , ADN de Cadena Simple/química , Magnesio/química , Conformación de Ácido Nucleico , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/metabolismo , Conformación Proteica , Espectrometría de Fluorescencia , TemperaturaRESUMEN
The nucleocapsid protein NCp7 of HIV-1 possesses a nucleic acid chaperone activity that is critical in minus and plus strand transfer during reverse transcription. The minus strand transfer notably relies on the ability of NCp7 to destabilize the stable stem with five contiguous, double-stranded segments of both the TAR sequence at the 3' end of the viral genome and the complementary sequence, cTAR, in minus strong-stop DNA. In order to examine the nature and the extent of NCp7 destabilizing activity, we investigated, by absorbance and fluorescence spectroscopy, the interaction of TAR and cTAR with a (12-55)NCp7 peptide containing the zinc-finger motifs but lacking the ability to aggregate the oligonucleotides. The absorbance changes in the UV band of cTAR show that seven to eight base-pairs, on average, are melted per oligonucleotide at a ratio of one peptide to 7.5 nucleotides. In contrast, the melting of TAR does not exceed an average of one base-pair per oligonucleotide. This may be linked to the greater stability of TAR, since a strong correlation between NCp7 destabilizing effect and oligonucleotide stability was observed. The effect of (12-55)NCp7 on the stem terminus was investigated by using a cTAR molecule doubly labeled at the 3' and 5' ends by a donor/acceptor couple. In the absence of the peptide, about 80 % of the oligonucleotides are in a dark non-fluorescent state, having a close proximity of the two dyes. The remaining 20 % are distributed between three fluorescent species, having either the terminal segment, the two terminal segments or all segments of the stem melted. This is in line with a fraying mechanism wherein the stem terminus fluctuates rapidly between open and closed states. Addition of (12-55)NCp7 shifts the equilibrium toward the open species, suggesting that NC enhances fraying of the stem terminus. Taken together, our data suggest that NCp7 activates the transient opening of base-pairs in the least stable parts of the stem. Also, this activity of NCp7 was found to be dependent on the zinc-finger motifs, since no melting was observed with a fingerless NCp7 peptide.
Asunto(s)
Proteínas de la Cápside , Cápside/metabolismo , Productos del Gen gag/metabolismo , Duplicado del Terminal Largo de VIH/genética , VIH-1/genética , Conformación de Ácido Nucleico , ARN Viral/química , ARN Viral/metabolismo , Proteínas Virales , Emparejamiento Base , Secuencia de Bases , Cápside/química , Cristalografía por Rayos X , Productos del Gen gag/química , Modelos Moleculares , Datos de Secuencia Molecular , ARN Viral/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Alineación de Secuencia , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Relación Estructura-Actividad , Dedos de Zinc , Productos del Gen gag del Virus de la Inmunodeficiencia HumanaRESUMEN
The integrase (IN) encoded by the Saccharomyces cerevisiae retrovirus-like element Ty3 has features found in retrovirus IN proteins including the catalytic triad, an amino-terminal zinc-binding motif, and a nuclear localization sequence. Mutations in the amino- and carboxyl-terminal domains of Ty3 IN cause reduced accumulation of full-length cDNA in the viruslike particles. We show that the reduction in cDNA is accompanied by reduced amounts of early intermediates such as minus-strand, strong-stop DNA. Expression of a capsid (CA)-IN fusion protein (CA-IN) complemented catalytic site and nuclear localization mutants, but not DNA mutants. However, expression of a fusion of CA, reverse transcriptase (RT), and IN (CA-RT-IN) complemented transposition of catalytic site and nuclear localization signal mutants, increased the amount of cDNA in some of the mutants, and complemented transposition of several mutants to low frequencies. Expression of a CA-RT-IN protein with a Ty3 IN catalytic site mutation did not complement transposition of either a Ty3 catalytic site mutant or a nuclear localization mutant but did increase the amount of cDNA in several mutants and complement at least one of the cDNA mutants for transposition. These in vivo data support a model in which independent IN domains can contribute to reverse transcription and integration. We conclude that during reverse transcription, the Ty3 IN domain interacts closely with the polymerase domain and may even constitute a domain within a heterodimeric RT. These studies also suggest that during integration the IN catalytic site and at least portions of the IN carboxyl-terminal domain act in cis.