RESUMEN
Overlapping genes pose an evolutionary dilemma as one DNA sequence evolves under the selection pressures of multiple proteins. Here, we perform systematic statistical and mutational analyses of the overlapping HIV-1 genes tat and rev and engineer exhaustive libraries of non-overlapped viruses to perform deep mutational scanning of each gene independently. We find a "segregated" organization in which overlapped sites encode functional residues of one gene or the other, but never both. Furthermore, this organization eliminates unfit genotypes, providing a fitness advantage to the population. Our comprehensive analysis reveals the extraordinary manner in which HIV minimizes the constraint of overlapping genes and repurposes that constraint to its own advantage. Thus, overlaps are not just consequences of evolutionary constraints, but rather can provide population fitness advantages.
Asunto(s)
Evolución Biológica , VIH-1/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Entropía , Aptitud Genética , Infecciones por VIH/virología , Humanos , Mutación , Sistemas de Lectura Abierta , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The nucleocapsid (N) protein of coronaviruses serves two major functions: compaction of the RNA genome in the virion and regulation of viral gene transcription. It is not clear how the N protein mediates such distinct functions. The N protein contains two RNA-binding domains surrounded by regions of intrinsic disorder. Phosphorylation of the central disordered region promotes the protein's transcriptional function, but the underlying mechanism is not known. Here, we show that the N protein of SARS-CoV-2, together with viral RNA, forms biomolecular condensates. Unmodified N protein forms partially ordered gel-like condensates and discrete 15-nm particles based on multivalent RNA-protein and protein-protein interactions. Phosphorylation reduces these interactions, generating a more liquid-like droplet. We propose that distinct oligomeric states support the two functions of the N protein: unmodified protein forms a structured oligomer that is suited for nucleocapsid assembly, and phosphorylated protein forms a liquid-like compartment for viral genome processing.
Asunto(s)
COVID-19 , Proteínas de la Nucleocápside de Coronavirus/química , Multimerización de Proteína , ARN Viral/química , SARS-CoV-2/química , Proteínas de la Nucleocápside de Coronavirus/genética , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Humanos , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Dominios Proteicos , ARN Viral/genética , ARN Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismoRESUMEN
We have developed a platform for quantitative genetic interaction mapping using viral infectivity as a functional readout and constructed a viral host-dependency epistasis map (vE-MAP) of 356 human genes linked to HIV function, comprising >63,000 pairwise genetic perturbations. The vE-MAP provides an expansive view of the genetic dependencies underlying HIV infection and can be used to identify drug targets and study viral mutations. We found that the RNA deadenylase complex, CNOT, is a central player in the vE-MAP and show that knockout of CNOT1, 10, and 11 suppressed HIV infection in primary T cells by upregulating innate immunity pathways. This phenotype was rescued by deletion of IRF7, a transcription factor regulating interferon-stimulated genes, revealing a previously unrecognized host signaling pathway involved in HIV infection. The vE-MAP represents a generic platform that can be used to study the global effects of how different pathogens hijack and rewire the host during infection.
Asunto(s)
Epistasis Genética , Infecciones por VIH/genética , Factor 7 Regulador del Interferón/genética , Factores de Transcripción/genética , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Infecciones por VIH/inmunología , Infecciones por VIH/patología , Infecciones por VIH/virología , VIH-1/genética , VIH-1/patogenicidad , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata/genética , Interferones/genética , Mutación , Transducción de Señal/genéticaRESUMEN
Viruses are obligate parasites that rely heavily on host cellular processes for replication. The small number of proteins typically encoded by a virus is faced with selection pressures that lead to the evolution of distinctive structural properties, allowing each protein to maintain its function under constraints such as small genome size, high mutation rate, and rapidly changing fitness conditions. One common strategy for this evolution is to utilize small building blocks to generate protein oligomers that assemble in multiple ways, thereby diversifying protein function and regulation. In this review, we discuss specific cases that illustrate how oligomerization is used to generate a single defined functional state, to modulate activity via different oligomeric states, or to generate multiple functional forms via different oligomeric states.
Asunto(s)
Multimerización de Proteína , Proteínas Virales/química , Virosis/virología , Virus/química , Animales , Cápside/química , Cápside/inmunología , Cápside/metabolismo , Ebolavirus/química , Ebolavirus/inmunología , Ebolavirus/metabolismo , Flavivirus/química , Flavivirus/inmunología , Flavivirus/metabolismo , Infecciones por Flavivirus/inmunología , Infecciones por Flavivirus/metabolismo , Infecciones por Flavivirus/virología , VIH/química , VIH/inmunología , VIH/metabolismo , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Fiebre Hemorrágica Ebola/inmunología , Fiebre Hemorrágica Ebola/metabolismo , Fiebre Hemorrágica Ebola/virología , Humanos , Modelos Moleculares , Conformación Proteica , Proteínas Virales/inmunología , Proteínas Virales/metabolismo , Virosis/inmunología , Virosis/metabolismo , Replicación Viral , Virus/inmunología , Virus/metabolismoRESUMEN
Viral regulatory complexes perform critical functions during virus replication and are important targets for therapeutic intervention. In HIV, the Tat and Rev proteins form complexes with multiple viral and cellular factors to direct transcription and export of the viral RNA. These complexes are composed of many proteins and are dynamic, making them difficult to fully recapitulate in vitro Therefore, we developed a cell-based reporter assay to monitor the assembly of viral complexes for inhibitor screening. We screened a small-molecule library and identified multiple hits that inhibit the activity of the viral complexes. A subsequent chemistry effort was focused on a thieno[2,3-b]pyridine scaffold, examples of which inhibited HIV replication and the emergence from viral latency. Notable aspects of the effort to determine the structure-activity relationship (SAR) include migration to the regioisomeric thieno[2,3-c]pyridine ring system and the identification of analogs with single-digit nanomolar activity in both reporter and HIV infectivity assays, an improvement of >100-fold in potency over the original hits. These results validate the screening strategy employed and reveal a promising lead series for the development of a new class of HIV therapeutics.
Asunto(s)
Fármacos Anti-VIH/farmacología , Antivirales/uso terapéutico , Piridinas/uso terapéutico , Regulación Viral de la Expresión Génica/genética , ARN Viral/genética , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
Human immunodeficiency virus (HIV) has a small genome and therefore relies heavily on the host cellular machinery to replicate. Identifying which host proteins and complexes come into physical contact with the viral proteins is crucial for a comprehensive understanding of how HIV rewires the host's cellular machinery during the course of infection. Here we report the use of affinity tagging and purification mass spectrometry to determine systematically the physical interactions of all 18 HIV-1 proteins and polyproteins with host proteins in two different human cell lines (HEK293 and Jurkat). Using a quantitative scoring system that we call MiST, we identified with high confidence 497 HIV-human protein-protein interactions involving 435 individual human proteins, with â¼40% of the interactions being identified in both cell types. We found that the host proteins hijacked by HIV, especially those found interacting in both cell types, are highly conserved across primates. We uncovered a number of host complexes targeted by viral proteins, including the finding that HIV protease cleaves eIF3d, a subunit of eukaryotic translation initiation factor 3. This host protein is one of eleven identified in this analysis that act to inhibit HIV replication. This data set facilitates a more comprehensive and detailed understanding of how the host machinery is manipulated during the course of HIV infection.
Asunto(s)
VIH-1/química , VIH-1/metabolismo , Interacciones Huésped-Patógeno , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas/fisiología , Marcadores de Afinidad , Secuencia de Aminoácidos , Secuencia Conservada , Factor 3 de Iniciación Eucariótica/química , Factor 3 de Iniciación Eucariótica/metabolismo , Células HEK293 , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Proteasa del VIH/metabolismo , VIH-1/fisiología , Proteínas del Virus de la Inmunodeficiencia Humana/análisis , Proteínas del Virus de la Inmunodeficiencia Humana/química , Proteínas del Virus de la Inmunodeficiencia Humana/aislamiento & purificación , Humanos , Inmunoprecipitación , Células Jurkat , Espectrometría de Masas , Unión Proteica , Reproducibilidad de los Resultados , Replicación ViralRESUMEN
Many ribonucleoprotein (RNP) complexes assemble into large, organized structures in which protein subunits are positioned by interactions with RNA and other proteins. Here we demonstrate that HIV Rev, constrained in size by a limited viral genome, also forms an organized RNP by assembling a homo-oligomer on the Rev response element (RRE) RNA. Rev subunits bind cooperatively to discrete RNA sites using an oligomerization domain and an adaptable protein-RNA interface, forming a complex with 500-fold higher affinity than the tightest single interaction. High-affinity binding correlates strongly with RNA export activity. Rev utilizes different surfaces of its alpha-helical RNA-binding domain to recognize several low-affinity binding sites, including the well-characterized stem IIB site and an additional site in stem IA. We propose that adaptable RNA-binding surfaces allow the Rev oligomer to assemble economically into a discrete, stable RNP and provide a mechanistic role for Rev oligomerization during the HIV life cycle.
Asunto(s)
Genoma Viral/genética , VIH/genética , ARN Viral/metabolismo , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/química , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Bases , Sitios de Unión , Modelos Biológicos , Datos de Secuencia Molecular , Mutación/genética , Conformación de Ácido Nucleico , Péptidos/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Transporte de ARN , ARN Viral/química , ARN Viral/genética , Elementos de Respuesta/genéticaRESUMEN
The HIV-1 protein Rev facilitates the nuclear export of intron-containing viral mRNAs by recognizing a structured RNA site, the Rev-response-element (RRE), contained in an intron. Rev assembles as a homo-oligomer on the RRE using its α-helical arginine-rich-motif (ARM) for RNA recognition. One unique feature of this assembly is the repeated use of the ARM from individual Rev subunits to contact distinct parts of the RRE in different binding modes. How the individual interactions differ and how they contribute toward forming a functional complex is poorly understood. Here we examine the thermodynamics of Rev-ARM peptide binding to two sites, RRE stem IIB, the high-affinity site that nucleates Rev assembly, and stem IA, a potential intermediate site during assembly, using NMR spectroscopy and isothermal titration calorimetry (ITC). NMR data indicate that the Rev-IIB complex forms a stable interface, whereas the Rev-IA interface is highly dynamic. ITC studies show that both interactions are enthalpy-driven, with binding to IIB being 20-30 fold tighter than to IA. Salt-dependent decreases in affinity were similar at both sites and predominantly enthalpic in nature, reflecting the roles of electrostatic interactions with arginines. However, the two interactions display strikingly different partitioning between enthalpy and entropy components, correlating well with the NMR observations. Our results illustrate how the variation in binding modes to different RRE target sites may influence the stability or order of Rev-RRE assembly and disassembly, and consequently its function.
Asunto(s)
VIH-1/fisiología , ARN Viral/química , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/química , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Calorimetría , Genes env/genética , VIH-1/química , VIH-1/genética , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , Dominios y Motivos de Interacción de Proteínas , ARN Viral/genética , Termodinámica , Ensamble de Virus/genética , Ensamble de Virus/fisiología , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The Tat proteins of HIV-1 and simian immunodeficiency virus (SIV) are essential for activating viral transcription. In addition, Tat stimulates nuclear factor κB (NF-κB) signaling pathways to regulate viral gene expression although its molecular mechanism is unclear. Here, we report that Tat directly activates NF-κB through the interaction with TRAF6, which is an essential upstream signaling molecule of the canonical NF-κB pathway. This interaction increases TRAF6 oligomerization and auto-ubiquitination, as well as the synthesis of K63-linked polyubiquitin chains to further activate the NF-κB pathway and HIV-1 transcription. Moreover, ectopic expression of TRAF6 significantly activates HIV-1 transcription, whereas TRAF6 knockdown inhibits transcription. Furthermore, Tat-mediated activation of NF-κB through TRAF6 is conserved among HIV-1, HIV-2, and SIV isolates. Our study uncovers yet another mechanism by which HIV-1 subverts host transcriptional pathways to enhance its own transcription.
Asunto(s)
VIH-1 , FN-kappa B , Animales , FN-kappa B/metabolismo , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/metabolismo , VIH-1/metabolismo , Transducción de Señal , UbiquitinaciónRESUMEN
Splicing factor 1 (SF1) binds to the branch point sequence (BPS) of mammalian introns and is believed to be important for the splicing of some, but not all, introns. To help identify BPSs, particularly those that depend on SF1, we generated a BPS profile model in which SF1 binding affinity data, validated by branch point mapping, were iteratively incorporated into computational models. We searched a data set of 117,499 human introns for best matches to the SF1 Affinity Model above a threshold, and counted the number of matches at each intronic position. After subtracting a background value, we found that 87.9% of remaining high-scoring matches identified were located in a region upstream of 3'-splice sites where BPSs are typically found. Since U2AF65 recognizes the polypyrimidine tract (PPT) and forms a cooperative RNA complex with SF1, we combined the SF1 model with a PPT model computed from high affinity binding sequences for U2AF65. The combined model, together with binding site location constraints, accurately identified introns bound by SF1 that are candidates for SF1-dependent splicing.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Intrones , Modelos Genéticos , Factores de Transcripción/metabolismo , Secuencia de Bases , Sitios de Unión , Humanos , Factores de Empalme de ARN , ARN Mensajero/química , Análisis de Secuencia de ARNRESUMEN
RNA is a crucial structural component of many ribonucleoprotein (RNP) complexes, including the ribosome, spliceosome, and signal recognition particle, but the role of RNA in guiding complex formation is only beginning to be explored. In the case of HIV, viral replication requires assembly of an RNP composed of the Rev protein homooligomer and the Rev response element (RRE) RNA to mediate nuclear export of unspliced viral mRNAs. Assembly of the functional Rev-RRE complex proceeds by cooperative oligomerization of Rev on the RRE scaffold and utilizes both protein-protein and protein-RNA interactions to organize complexes with high specificity. The structures of the Rev protein and a peptide-RNA complex are known, but the complete RNP is not, making it unclear to what extent RNA defines the composition and architecture of Rev-RNA complexes. Here we show that the RRE controls the oligomeric state and solubility of Rev and guides its assembly into discrete Rev-RNA complexes. SAXS and EM data were used to derive a structural model of a Rev dimer bound to an essential RRE hairpin and to visualize the complete Rev-RRE RNP, demonstrating that RRE binding drives assembly of Rev homooligomers into asymmetric particles, reminiscent of the role of RNA in organizing more complex RNP machines, such as the ribosome, composed of many different protein subunits. Thus, the RRE is not simply a passive scaffold onto which proteins bind but instead actively defines the protein composition and organization of the RNP.
Asunto(s)
Productos del Gen rev/química , Productos del Gen rev/metabolismo , VIH/genética , Citoplasma/genética , Citoplasma/metabolismo , Productos del Gen rev/genética , VIH/metabolismo , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Péptidos/genética , Péptidos/metabolismo , Proteínas/genética , Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Elementos de Respuesta , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Dispersión del Ángulo Pequeño , Replicación Viral/genéticaRESUMEN
To fully understand how pathogens infect their host and hijack key biological processes, systematic mapping of intra-pathogenic and pathogen-host protein-protein interactions (PPIs) is crucial. Due to the relatively small size of viral genomes (usually around 10-100 proteins), generation of comprehensive host-virus PPI maps using different experimental platforms, including affinity tag purification-mass spectrometry (AP-MS) and yeast two-hybrid (Y2H) approaches, can be achieved. Global maps such as these provide unbiased insight into the molecular mechanisms of viral entry, replication and assembly. However, to date, only two-hybrid methodology has been used in a systematic fashion to characterize viral-host protein-protein interactions, although a deluge of data exists in databases that manually curate from the literature individual host-pathogen PPIs. We will summarize this work and also describe an AP-MS platform that can be used to characterize viral-human protein complexes and discuss its application for the HIV genome.
Asunto(s)
Infecciones por VIH/metabolismo , VIH-1/metabolismo , Factores Celulares Derivados del Huésped/metabolismo , Interacciones Huésped-Patógeno , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Mapeo de Interacción de Proteínas/métodos , Cromatografía de Afinidad , Clonación Molecular , Genoma Viral , Infecciones por VIH/virología , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Proteínas del Virus de la Inmunodeficiencia Humana/aislamiento & purificación , Humanos , Células Jurkat , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Fracciones Subcelulares/metabolismo , TransfecciónRESUMEN
HIV-1 Tat enhances viral transcription elongation by forming a ribonucleoprotein complex with transactivating responsive (TAR) RNA and P-TEFb, an elongation factor composed of cyclin T1 (CycT1) and Cdk9 that phosphorylates the C-terminal domain of RNA polymerase II. Previous studies have shown that Lys-28 in the activation domain (AD) of Tat is essential for HIV-1 transcription and replication and is acetylated by p300/CBP-associated factor (PCAF), but the mechanistic basis of the Lys-28 requirement is unknown. Here, we show that Lys-28 acetylation modulates the affinity and stability of HIV-1 Tat-CycT1-TAR complexes by enhancing an interaction with the CycT1 Tat-TAR recognition motif. High-affinity assembly correlates strongly with stimulation of transcription elongation in vitro and Tat activation in vivo. In marked contrast, bovine lentiviral Tat proteins have evolved a high-affinity TAR interaction that does not require PCAF-mediated acetylation of the Tat AD or CycT1 for RNA binding, whereas HIV-2 Tat has evolved an intermediate mechanism that uses a duplicated TAR element and CycT1 to enhance RNA affinity and consequently transcription activation. The coevolution of Tat acetylation, CycT1 dependence, and TAR binding affinity is seen in viral replication assays using Tat proteins that rely on CycT1 for TAR binding but are acetylation deficient, where compensatory mutations rapidly accrue in TAR to generate high-affinity, CycT1-independent complexes reminiscent of the bovine viruses. Thus, lysine acetylation can be used to modulate and evolve the strength of a viral-host RNA-protein complex, thereby tuning the levels of transcription elongation.
Asunto(s)
Productos del Gen tat/metabolismo , ARN/genética , Transcripción Genética/genética , Acetilación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Ciclinas/química , Ciclinas/genética , Ciclinas/metabolismo , Productos del Gen tat/genética , VIH-1/genética , VIH-1/metabolismo , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Lisina/genética , Lisina/metabolismo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Filogenia , Unión Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Overlapping coding regions balance selective forces between multiple genes. One possible division of nucleotide sequence is that the predominant selective force on a particular nucleotide can be attributed to just one gene. While this arrangement has been observed in regions in which one gene is structured and the other is disordered, we sought to explore how overlapping genes balance constraints when both protein products are structured over the same sequence. We use a combination of sequence analysis, functional assays, and selection experiments to examine an overlapped region in HIV-1 that encodes helical regions in both Env and Rev. We find that functional segregation occurs even in this overlap, with each protein spacing its functional residues in a manner that allows a mutable non-binding face of one helix to encode important functional residues on a charged face in the other helix. Additionally, our experiments reveal novel and critical functional residues in Env and have implications for the therapeutic targeting of HIV-1.
Asunto(s)
VIH-1 , VIH-1/química , VIH-1/genética , Sistemas de Lectura AbiertaRESUMEN
Viruses must effectively remodel host cellular pathways to replicate and evade immune defenses, and they must do so with limited genomic coding capacity. Targeting post-translational modification (PTM) pathways provides a mechanism by which viruses can broadly and rapidly transform a hostile host environment into a hospitable one. We use mass spectrometry-based proteomics to quantify changes in protein abundance and two PTM types-phosphorylation and ubiquitination-in response to HIV-1 infection with viruses harboring targeted deletions of a subset of HIV-1 genes. PTM analysis reveals a requirement for Aurora kinase activity in HIV-1 infection and identified putative substrates of a phosphatase that is degraded during infection. Finally, we demonstrate that the HIV-1 Vpr protein inhibits histone H1 ubiquitination, leading to defects in DNA repair.
Asunto(s)
Infecciones por VIH , Seropositividad para VIH , VIH-1 , VIH-1/genética , Humanos , Procesamiento Proteico-Postraduccional , Proteómica , UbiquitinaciónRESUMEN
BACKGROUND: The increasing availability of HIV-host interaction datasets, including both physical and genetic interactions, has created a need for software tools to integrate and visualize the data. Because these host-pathogen interactions are extensive and interactions between human proteins are found within many different databases, it is difficult to generate integrated HIV-human interaction networks. RESULTS: We have developed a web-based platform, termed GPS-Prot http://www.gpsprot.org, that allows for facile integration of different HIV interaction data types as well as inclusion of interactions between human proteins derived from publicly-available databases, including MINT, BioGRID and HPRD. The software has the ability to group proteins into functional modules or protein complexes, generating more intuitive network representations and also allows for the uploading of user-generated data. CONCLUSIONS: GPS-Prot is a software tool that allows users to easily create comprehensive and integrated HIV-host networks. A major advantage of this platform compared to other visualization tools is its web-based format, which requires no software installation or data downloads. GPS-Prot allows novice users to quickly generate networks that combine both genetic and protein-protein interactions between HIV and its human host into a single representation. Ultimately, the platform is extendable to other host-pathogen systems.
Asunto(s)
Infecciones por VIH/metabolismo , VIH-1 , Interacciones Huésped-Patógeno , Programas Informáticos , Biología de Sistemas/métodos , Humanos , Internet , Mapeo de Interacción de Proteínas , Interfaz Usuario-ComputadorRESUMEN
We have analyzed host cell genes linked to HIV replication that were identified in nine genome-wide studies, including three independent siRNA screens. Overlaps among the siRNA screens were very modest (<7% for any pairwise combination), and similarly, only modest overlaps were seen in pairwise comparisons with other types of genome-wide studies. Combining all genes from the genome-wide studies together with genes reported in the literature to affect HIV yields 2,410 protein-coding genes, or fully 9.5% of all human genes (though of course some of these are false positive calls). Here we report an "encyclopedia" of all overlaps between studies (available at http://www.hostpathogen.org), which yielded a more extensively corroborated set of host factors assisting HIV replication. We used these genes to calculate refined networks that specify cellular subsystems recruited by HIV to assist in replication, and present additional analysis specifying host cell genes that are attractive as potential therapeutic targets.
Asunto(s)
Bases de Datos de Proteínas , Genoma Humano/genética , VIH/fisiología , Interacciones Huésped-Patógeno/genética , Replicación Viral , Análisis por Conglomerados , Humanos , Internet , Unión ProteicaRESUMEN
We report a comparative study in which a single-molecule fluorescence resonance energy transfer approach was used to examine how the binding of two families of HIV-1 viral proteins to viral RNA hairpins locally changes the RNA secondary structures. The single-molecule fluorescence resonance energy transfer results indicate that the zinc finger protein (nucleocapsid) locally melts the TAR RNA and RRE-IIB RNA hairpins, whereas arginine-rich motif proteins (Tat and Rev) may strengthen the hairpin structures through specific binding interactions. Competition experiments show that Tat and Rev can effectively inhibit the nucleocapsid-chaperoned annealing of complementary DNA oligonucleotides to the TAR and RRE-IIB RNA hairpins, respectively. The competition binding data presented here suggest that the specific nucleic acid binding interactions of Tat and Rev can effectively compete with the general nucleic acid binding/chaperone functions of the nucleocapsid protein, and thus may in principle help regulate critical events during the HIV life cycle.
Asunto(s)
Arginina/metabolismo , VIH-1/metabolismo , Proteínas de la Nucleocápside/metabolismo , ARN Viral/metabolismo , Dedos de Zinc , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Ensayo de Cambio de Movilidad Electroforética , Transferencia Resonante de Energía de Fluorescencia , Duplicado del Terminal Largo de VIH/genética , Cinética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Unión Proteica , ARN Viral/química , ARN Viral/genética , Elementos de Respuesta/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/química , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
Human immunodeficiency virus type 1 (HIV-1) transcription is regulated by the viral Tat protein, which relieves a block to elongation by recruiting an elongation factor, P-TEFb, to the viral promoter. Here, we report the discovery of potent Tat inhibitors that utilize a localization signal to target a dominant negative protein to its site of action. Fusing the Tat activation domain to some splicing factors, particularly to the Arg-Ser (RS) domain of U2AF65, creates Tat inhibitors that localize to subnuclear speckles, sites where pre-mRNA processing factors are stored for assembly into transcription complexes. A U2AF65 fusion named T-RS interacts with the nonphosphorylated C-terminal domain of RNA polymerase II (RNAP II) via its RS domain and is loaded into RNAP II holoenzyme complexes. T-RS is recruited efficiently to the HIV-1 promoter in a TAR-independent manner before RNAP II hyperphosphorylation but not to cellular promoters. The "preloading" of T-RS into HIV-1 preinitiation complexes prevents the entry of active Tat molecules, leaving the complexes in an elongation-incompetent state and effectively suppressing HIV-1 replication. The ability to deliver inhibitors to transcription complexes through the use of targeting/localization signals may provide new avenues for designing viral and transcription inhibitors.
Asunto(s)
Regulación Viral de la Expresión Génica , Productos del Gen tat/antagonistas & inhibidores , VIH-1/metabolismo , Regiones Promotoras Genéticas , Transcripción Genética , Replicación Viral , Secuencia de Aminoácidos , Productos del Gen tat/metabolismo , Células HeLa , Humanos , Cinética , Modelos Biológicos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Linfocitos T/metabolismo , Linfocitos T/virologíaRESUMEN
HIV-1 Rev is an essential viral regulatory protein that facilitates the nuclear export of intron-containing viral mRNAs. It is organized into structured, functionally well-characterized motifs joined by less understood linker regions. Our recent competitive deep mutational scanning study confirmed many known constraints in Rev's established motifs, but also identified positions of mutational plasticity, most notably in surrounding linker regions. Here, we probe the mutational limits of these linkers by testing the activities of multiple truncation and mass substitution mutations. We find that these regions possess previously unknown structural, functional or regulatory roles, not apparent from systematic point mutational approaches. Specifically, the N- and C-termini of Rev contribute to protein stability; mutations in a turn that connects the two main helices of Rev have different effects in different contexts; and a linker region which connects the second helix of Rev to its nuclear export sequence has structural requirements for function. Thus, Rev function extends beyond its characterized motifs, and is tuned by determinants within seemingly plastic portions of its sequence. Additionally, Rev's ability to tolerate many of these massive truncations and substitutions illustrates the overall mutational and functional robustness inherent in this viral protein.