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1.
bioRxiv ; 2024 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-39345625

RESUMO

Crm1 is a highly conserved nuclear exportin that transports >1000 human proteins including ribonucleoprotein (RNP) complexes. The interface between Crm1 and RNP cargos is unknown. The HIV regulatory protein, Rev, was one of the first identified cargos for Crm1 and contains a prototypic nuclear export sequence (NES). We present the cryo-electron microscopy structure of the HIV-1 nuclear export complex (Crm1/Ran-GTP and the Rev/RRE RNP). Rev binds at a previously unseen protein-protein binding site that stabilizes a unique Crm1 dimer and positions two NESs within the Crm1 dimer. The orientation of Rev binding positions the RRE within a charged pocket on the inside of the Crm1 toroid, mediating direct RNA-Ran-GTP contacts, highlighting the significant role of the RRE in the interaction. Structure based mutations, combined with cell-based assays, show that Crm1 has multiple distinct cargo recognition sites and explains how Crm1 can recognize a diverse range of protein and RNP cargos.

2.
Sci Adv ; 10(3): eadi4162, 2024 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-38241362

RESUMO

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.


Assuntos
HIV-1 , NF-kappa B , Animais , NF-kappa B/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , HIV-1/metabolismo , Transdução de Sinais , Ubiquitinação
3.
Elife ; 112022 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-35511220

RESUMO

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.


Assuntos
HIV-1 , HIV-1/química , HIV-1/genética , Fases de Leitura Aberta
4.
Cell Rep ; 39(2): 110690, 2022 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-35417684

RESUMO

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.


Assuntos
Infecções por HIV , Soropositividade para HIV , HIV-1 , HIV-1/genética , Humanos , Processamento de Proteína Pós-Traducional , Proteômica , Ubiquitinação
5.
Mol Cell ; 80(6): 1092-1103.e4, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33248025

RESUMO

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.


Assuntos
COVID-19 , Proteínas do Nucleocapsídeo de Coronavírus/química , Multimerização Proteica , RNA Viral/química , SARS-CoV-2/química , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Humanos , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Domínios Proteicos , RNA Viral/genética , RNA Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo
6.
Mol Cell ; 78(2): 197-209.e7, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32084337

RESUMO

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.


Assuntos
Epistasia Genética , Infecções por HIV/genética , Fator Regulador 7 de Interferon/genética , Fatores de Transcrição/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Infecções por HIV/imunologia , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/patogenicidade , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata/genética , Interferons/genética , Mutação , Transdução de Sinais/genética
7.
Cell Host Microbe ; 26(1): 86-99.e7, 2019 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-31253590

RESUMO

The Cullin-RING E3 ligase (CRL) family is commonly hijacked by pathogens to redirect the host ubiquitin proteasome machinery to specific targets. During HIV infection, CRL5 is hijacked by HIV Vif to target viral restriction factors of the APOBEC3 family for ubiquitination and degradation. Here, using a quantitative proteomics approach, we identify the E3 ligase ARIH2 as a regulator of CRL5-mediated APOBEC3 degradation. The CUL5Vif/CBFß complex recruits ARIH2 where it acts to transfer ubiquitin directly to the APOBEC3 targets. ARIH2 is essential for CRL5-dependent HIV infectivity in primary CD4+ T cells. Furthermore, we show that ARIH2 cooperates with CRL5 to prime other cellular substrates for polyubiquitination, suggesting this may represent a general mechanism beyond HIV infection and APOBEC3 degradation. Taken together, these data identify ARIH2 as a co-factor in the Vif-hijacked CRL5 complex that contributes to HIV infectivity and demonstrate the operation of the E1-E2-E3/E3-substrate ubiquitination mechanism in a viral infection context.


Assuntos
Desaminase APOBEC-3G/metabolismo , Proteínas Culina/metabolismo , Infecções por HIV/patologia , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Ubiquitina-Proteína Ligases/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Humanos , Modelos Teóricos , Proteólise , Proteoma/análise , Replicação Viral
8.
Sci Rep ; 9(1): 5139, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30914719

RESUMO

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.


Assuntos
HIV-1/química , Produtos do Gene rev do Vírus da Imunodeficiência Humana/química , Motivos de Aminoácidos , Células HEK293 , HIV-1/crescimento & desenvolvimento , HIV-1/metabolismo , Humanos , Mutação , Domínios Proteicos , Estabilidade Proteica , Relação Estrutura-Atividade , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo
9.
J Med Chem ; 61(21): 9611-9620, 2018 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-30289719

RESUMO

We synthesized and screened a unique 46 656-member library composed of unnatural amino acids that revealed several hits against RRE IIB RNA. Among the hit peptides identified, peptide 4A5 was found to be selective against competitor RNAs and inhibited HIV-1 Rev-RRE RNA interaction in cell culture in a p24 ELISA assay. Biophysical characterization in a ribonuclease protection assay suggested that 4A5 bound to the stem-loop region in RRE IIB while SHAPE MaP probing with 234 nt RRE RNA indicated additional interaction with secondary Rev binding sites. Taken together, our investigation suggests that HIV replication is inhibited by 4A5 blocking binding of Rev and subsequent multimerization.


Assuntos
Desenho de Fármacos , Genes env , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Peptídeos/farmacologia , Replicação Viral/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Sequência de Bases , Sítios de Ligação , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , HIV-1/genética , Peptídeos/metabolismo , RNA Viral/metabolismo
10.
Elife ; 72018 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-29845934

RESUMO

The HIV-1 Tat protein hijacks P-TEFb kinase to activate paused RNA polymerase II (RNAP II) at the viral promoter. Tat binds additional host factors, but it is unclear how they regulate RNAP II elongation. Here, we identify the cytoplasmic ubiquitin ligase UBE2O as critical for Tat transcriptional activity. Tat hijacks UBE2O to ubiquitinate the P-TEFb kinase inhibitor HEXIM1 of the 7SK snRNP, a fraction of which also resides in the cytoplasm bound to P-TEFb. HEXIM1 ubiquitination sequesters it in the cytoplasm and releases P-TEFb from the inhibitory 7SK complex. Free P-TEFb then becomes enriched in chromatin, a process that is also stimulated by treating cells with a CDK9 inhibitor. Finally, we demonstrate that UBE2O is critical for P-TEFb recruitment to the HIV-1 promoter. Together, the data support a unique model of elongation control where non-degradative ubiquitination of nuclear and cytoplasmic 7SK snRNP pools increases P-TEFb levels for transcriptional activation.


Assuntos
HIV-1/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Ativação Transcricional/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Sequência de Aminoácidos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Cromatina/metabolismo , Diclororribofuranosilbenzimidazol/farmacologia , Células HEK293 , Células HeLa , Humanos , Modelos Biológicos , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição , Ativação Transcricional/efeitos dos fármacos , Enzimas de Conjugação de Ubiquitina/química , Ubiquitinação/efeitos dos fármacos
11.
Annu Rev Virol ; 4(1): 241-260, 2017 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-28961413

RESUMO

Viruses are completely dependent upon cellular machinery to support replication and have therefore developed strategies to co-opt cellular processes to optimize infection and counter host immune defenses. Many viruses, including human immunodeficiency virus type 1 (HIV-1), encode a relatively small number of genes. Viruses with limited genetic content often encode multifunctional proteins that function at multiple stages of the viral replication cycle. In this review, we discuss the functions of HIV-1 regulatory (Tat and Rev) and accessory (Vif, Vpr, Vpu, and Nef) proteins. Each of these proteins has a highly conserved primary activity; however, numerous additional activities have been attributed to these viral proteins. We explore the possibility that HIV-1 proteins leverage their multifunctional nature to alter host transcriptional networks to elicit a diverse set of cellular responses. Although these transcriptional effects appear to benefit the virus, it is not yet clear whether they are strongly selected for during viral evolution or are a ripple effect from the primary function. As our detailed knowledge of these viral proteins improves, we will undoubtedly uncover how the multifunctional nature of these HIV-1 regulatory and accessory proteins, and in particular their transcriptional functions, work to drive viral pathogenesis.


Assuntos
Genes rev , Genes tat , HIV-1/genética , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Transcrição Gênica , Proteínas Virais Reguladoras e Acessórias/metabolismo , HIV-1/química , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Proteínas do Vírus da Imunodeficiência Humana/genética , Humanos , Proteínas Virais Reguladoras e Acessórias/genética , Replicação Viral , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência Humana/genética , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo
12.
Artigo em Inglês | MEDLINE | ID: mdl-28416550

RESUMO

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.


Assuntos
Fármacos Anti-HIV/farmacologia , Antivirais/uso terapêutico , Piridinas/uso terapêutico , Regulação Viral da Expressão Gênica/genética , RNA Viral/genética , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
13.
Sci Rep ; 7: 45394, 2017 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-28345603

RESUMO

Transcription complexes that assemble at the HIV-1 promoter efficiently initiate transcription but generate paused RNA polymerase II downstream from the start site. The virally encoded Tat protein hijacks positive transcription elongation factor b (P-TEFb) to phosphorylate and activate this paused polymerase. In addition, Tat undergoes a series of reversible post-translational modifications that regulate distinct steps of the transcription cycle. To identify additional functionally important Tat cofactors, we performed RNAi knockdowns of sixteen previously identified Tat interactors and found that a novel E3 ligase, PJA2, ubiquitinates Tat in a non-degradative manner and specifically regulates the step of HIV transcription elongation. Interestingly, several different lysine residues in Tat can function as ubiquitin acceptor sites, and variable combinations of these lysines support both full transcriptional activity and viral replication. Further, the polyubiquitin chain conjugated to Tat by PJA2 can itself be assembled through variable ubiquitin lysine linkages. Importantly, proper ubiquitin chain assembly by PJA2 requires that Tat first binds its P-TEFb cofactor. These results highlight that both the Tat substrate and ubiquitin modification have plastic site usage, and this plasticity is likely another way in which the virus exploits the host molecular machinery to expand its limited genetic repertoire.


Assuntos
HIV-1/genética , Transcrição Gênica/genética , Ativação Transcricional/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Regulação Viral da Expressão Gênica/genética , Células HEK293 , Células HeLa , Humanos , Células Jurkat , Fator B de Elongação Transcricional Positiva/genética , Regiões Promotoras Genéticas/genética , Processamento de Proteína Pós-Traducional/genética , RNA Polimerase II/genética , RNA Viral/genética , Replicação Viral/genética
14.
Cell ; 167(7): 1762-1773.e12, 2016 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-27984726

RESUMO

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.


Assuntos
Evolução Biológica , HIV-1/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Entropia , Aptidão Genética , Infecções por HIV/virologia , Humanos , Mutação , Fases de Leitura Aberta , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genética
15.
Crit Rev Biochem Mol Biol ; 51(5): 379-394, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27685368

RESUMO

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.


Assuntos
Multimerização Proteica , Proteínas Virais/química , Viroses/virologia , Vírus/química , Animais , Capsídeo/química , Capsídeo/imunologia , Capsídeo/metabolismo , Ebolavirus/química , Ebolavirus/imunologia , Ebolavirus/metabolismo , Flavivirus/química , Flavivirus/imunologia , Flavivirus/metabolismo , Infecções por Flavivirus/imunologia , Infecções por Flavivirus/metabolismo , Infecções por Flavivirus/virologia , HIV/química , HIV/imunologia , HIV/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/metabolismo , Doença pelo Vírus Ebola/virologia , Humanos , Modelos Moleculares , Conformação Proteica , Proteínas Virais/imunologia , Proteínas Virais/metabolismo , Viroses/imunologia , Viroses/metabolismo , Replicação Viral , Vírus/imunologia , Vírus/metabolismo
16.
Wiley Interdiscip Rev RNA ; 7(4): 470-86, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26929078

RESUMO

HIV replication requires the nuclear export of essential, intron-containing viral RNAs. To facilitate export, HIV encodes the viral accessory protein Rev which binds unspliced and partially spliced viral RNAs and creates a ribonucleoprotein complex that recruits the cellular Chromosome maintenance factor 1 export machinery. Exporting RNAs in this manner bypasses the necessity for complete splicing as a prerequisite for mRNA export, and allows intron-containing RNAs to reach the cytoplasm intact for translation and virus packaging. Recent structural studies have revealed that this entire complex exhibits remarkable plasticity at many levels of organization, including RNA folding, protein-RNA recognition, multimer formation, and host factor recruitment. In this review, we explore each aspect of plasticity from structural, functional, and possible therapeutic viewpoints. WIREs RNA 2016, 7:470-486. doi: 10.1002/wrna.1342 For further resources related to this article, please visit the WIREs website.


Assuntos
Núcleo Celular/metabolismo , HIV-1/fisiologia , RNA Viral/metabolismo , Ribonucleoproteínas/metabolismo , Replicação Viral , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo , Transporte Ativo do Núcleo Celular , Regulação Viral da Expressão Gênica , HIV-1/genética , Humanos , Processamento Pós-Transcricional do RNA
17.
Biochemistry ; 54(42): 6545-54, 2015 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-26422686

RESUMO

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.


Assuntos
HIV-1/fisiologia , RNA Viral/química , Produtos do Gene rev do Vírus da Imunodeficiência Humana/química , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Calorimetria , Genes env/genética , HIV-1/química , HIV-1/genética , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Domínios e Motivos de Interação entre Proteínas , RNA Viral/genética , Termodinâmica , Montagem de Vírus/genética , Montagem de Vírus/fisiologia , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genética
18.
ACS Chem Biol ; 10(11): 2441-7, 2015 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-26322864

RESUMO

In drug discovery, small molecules must often discriminate between healthy and diseased cells. This feat is usually accomplished by binding to a protein that is preferentially expressed in the target cell or on its surface. However, in many cases, the expression of an individual protein may not generate sufficient cyto-selectivity. Here, we demonstrate that bispecific molecules can better discriminate between similar cell types by exploiting their simultaneous affinity for two proteins. Inspired by the natural product FK506, we designed molecules that have affinity for both FKBP12 and HIV protease. Using cell-based reporters and live virus assays, we observed that these compounds preferentially accumulated in cells that express both targets, mimicking an infected lymphocyte. Treatment with FKBP12 inhibitors reversed this partitioning, while overexpression of FKBP12 protein further promoted it. The partitioning into the target cell type could be tuned by controlling the properties of the linker and the affinities for the two proteins. These results show that bispecific molecules create significantly better potential for cyto-selectivity, which might be especially important in the development of safe and effective antivirals and anticancer compounds.


Assuntos
Anticorpos Biespecíficos/química , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Regulação da Expressão Gênica , Protease de HIV/genética , Proteína 1A de Ligação a Tacrolimo/genética , Citometria de Fluxo , Protease de HIV/metabolismo , Humanos , Estrutura Molecular , Proteína 1A de Ligação a Tacrolimo/antagonistas & inibidores , Proteína 1A de Ligação a Tacrolimo/metabolismo
19.
Elife ; 3: e04120, 2014 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-25486594

RESUMO

The HIV-1 protein Rev controls a critical step in viral replication by mediating the nuclear export of unspliced and singly-spliced viral mRNAs. Multiple Rev subunits assemble on the Rev Response Element (RRE), a structured region present in these RNAs, and direct their export through the Crm1 pathway. Rev-RRE assembly occurs via several Rev oligomerization and RNA-binding steps, but how these steps are coordinated to form an export-competent complex is unclear. Here, we report the first crystal structure of a Rev dimer-RRE complex, revealing a dramatic rearrangement of the Rev-dimer upon RRE binding through re-packing of its hydrophobic protein-protein interface. Rev-RNA recognition relies on sequence-specific contacts at the well-characterized IIB site and local RNA architecture at the second site. The structure supports a model in which the RRE utilizes the inherent plasticity of Rev subunit interfaces to guide the formation of a functional complex.


Assuntos
HIV-1/genética , RNA Viral/genética , Elementos de Resposta , Replicação Viral/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genética , Transporte Ativo do Núcleo Celular , Sítios de Ligação , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Cristalografia por Raios X , Citosol/metabolismo , Citosol/virologia , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Células HEK293 , HIV-1/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Carioferinas/genética , Carioferinas/metabolismo , Modelos Moleculares , Ligação Proteica , Splicing de RNA , RNA Viral/química , RNA Viral/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Linfócitos T/virologia , Produtos do Gene rev do Vírus da Imunodeficiência Humana/química , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo , Proteína Exportina 1
20.
Elife ; 3: e04121, 2014 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-25486595

RESUMO

The HIV Rev protein routes viral RNAs containing the Rev Response Element (RRE) through the Crm1 nuclear export pathway to the cytoplasm where viral proteins are expressed and genomic RNA is delivered to assembling virions. The RRE assembles a Rev oligomer that displays nuclear export sequences (NESs) for recognition by the Crm1-Ran(GTP) nuclear receptor complex. Here we provide the first view of an assembled HIV-host nuclear export complex using single-particle electron microscopy. Unexpectedly, Crm1 forms a dimer with an extensive interface that enhances association with Rev-RRE and poises NES binding sites to interact with a Rev oligomer. The interface between Crm1 monomers explains differences between Crm1 orthologs that alter nuclear export and determine cellular tropism for viral replication. The arrangement of the export complex identifies a novel binding surface to possibly target an HIV inhibitor and may point to a broader role for Crm1 dimerization in regulating host gene expression.


Assuntos
HIV-1/genética , Carioferinas/genética , RNA Viral/genética , Receptores Citoplasmáticos e Nucleares/genética , Elementos de Resposta , Proteína ran de Ligação ao GTP/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genética , Transporte Ativo do Núcleo Celular , Sítios de Ligação , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Cristalografia por Raios X , Citosol/metabolismo , Citosol/virologia , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Células HEK293 , HIV-1/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Carioferinas/química , Carioferinas/metabolismo , Modelos Moleculares , Ligação Proteica , Multimerização Proteica , Splicing de RNA , RNA Viral/química , RNA Viral/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Linfócitos T/virologia , Replicação Viral/genética , Proteína ran de Ligação ao GTP/química , Proteína ran de Ligação ao GTP/metabolismo , Produtos do Gene rev do Vírus da Imunodeficiência Humana/química , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo , Proteína Exportina 1
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