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1.
Cell ; 185(12): 2086-2102.e22, 2022 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-35561685

RESUMO

Across biological scales, gene-regulatory networks employ autorepression (negative feedback) to maintain homeostasis and minimize failure from aberrant expression. Here, we present a proof of concept that disrupting transcriptional negative feedback dysregulates viral gene expression to therapeutically inhibit replication and confers a high evolutionary barrier to resistance. We find that nucleic-acid decoys mimicking cis-regulatory sites act as "feedback disruptors," break homeostasis, and increase viral transcription factors to cytotoxic levels (termed "open-loop lethality"). Feedback disruptors against herpesviruses reduced viral replication >2-logs without activating innate immunity, showed sub-nM IC50, synergized with standard-of-care antivirals, and inhibited virus replication in mice. In contrast to approved antivirals where resistance rapidly emerged, no feedback-disruptor escape mutants evolved in long-term cultures. For SARS-CoV-2, disruption of a putative feedback circuit also generated open-loop lethality, reducing viral titers by >1-log. These results demonstrate that generating open-loop lethality, via negative-feedback disruption, may yield a class of antimicrobials with a high genetic barrier to resistance.


Assuntos
Antivirais , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Animais , Antivirais/farmacologia , Farmacorresistência Viral , Redes Reguladoras de Genes/efeitos dos fármacos , Camundongos , SARS-CoV-2/efeitos dos fármacos , Replicação Viral
2.
Cell ; 183(5): 1383-1401.e19, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33159858

RESUMO

Ebola virus (EBOV) causes epidemics with high mortality yet remains understudied due to the challenge of experimentation in high-containment and outbreak settings. Here, we used single-cell transcriptomics and CyTOF-based single-cell protein quantification to characterize peripheral immune cells during EBOV infection in rhesus monkeys. We obtained 100,000 transcriptomes and 15,000,000 protein profiles, finding that immature, proliferative monocyte-lineage cells with reduced antigen-presentation capacity replace conventional monocyte subsets, while lymphocytes upregulate apoptosis genes and decline in abundance. By quantifying intracellular viral RNA, we identify molecular determinants of tropism among circulating immune cells and examine temporal dynamics in viral and host gene expression. Within infected cells, EBOV downregulates STAT1 mRNA and interferon signaling, and it upregulates putative pro-viral genes (e.g., DYNLL1 and HSPA5), nominating pathways the virus manipulates for its replication. This study sheds light on EBOV tropism, replication dynamics, and elicited immune response and provides a framework for characterizing host-virus interactions under maximum containment.


Assuntos
Ebolavirus/fisiologia , Doença pelo Vírus Ebola/genética , Doença pelo Vírus Ebola/virologia , Interações Hospedeiro-Patógeno/genética , Análise de Célula Única , Animais , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Efeito Espectador , Diferenciação Celular , Proliferação de Células , Citocinas/metabolismo , Ebolavirus/genética , Chaperona BiP do Retículo Endoplasmático , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Regulação Viral da Expressão Gênica , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/patologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Interferons/genética , Interferons/metabolismo , Macaca mulatta , Macrófagos/metabolismo , Monócitos/metabolismo , Mielopoese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Transcriptoma/genética
3.
Cell ; 176(3): 625-635.e14, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30682371

RESUMO

Programmed -1 ribosomal frameshifting (-1PRF) is a widely used translation recoding mechanism. HIV-1 expresses Gag-Pol protein from the Gag-coding mRNA through -1PRF, and the ratio of Gag to Gag-Pol is strictly maintained for efficient viral replication. Here, we report that the interferon-stimulated gene product C19orf66 (herein named Shiftless) is a host factor that inhibits the -1PRF of HIV-1. Shiftless (SFL) also inhibited the -1PRF of a variety of mRNAs from both viruses and cellular genes. SFL interacted with the -1PRF signal of target mRNA and translating ribosomes and caused premature translation termination at the frameshifting site. Downregulation of translation release factor eRF3 or eRF1 reduced SFL-mediated premature translation termination. We propose that SFL binding to target mRNA and the translating ribosome interferes with the frameshifting process. These findings identify SFL as a broad-spectrum inhibitor of -1PRF and help to further elucidate the mechanisms of -1PRF.


Assuntos
Proteínas de Fusão gag-pol/genética , HIV-1/genética , Sequência de Bases , Mudança da Fase de Leitura do Gene Ribossômico/genética , Regulação Viral da Expressão Gênica/genética , Humanos , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Ribossomos/metabolismo , Replicação Viral/genética
4.
Nature ; 631(8021): 670-677, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38987591

RESUMO

In all organisms, regulation of gene expression must be adjusted to meet cellular requirements and frequently involves helix-turn-helix (HTH) domain proteins1. For instance, in the arms race between bacteria and bacteriophages, rapid expression of phage anti-CRISPR (acr) genes upon infection enables evasion from CRISPR-Cas defence; transcription is then repressed by an HTH-domain-containing anti-CRISPR-associated (Aca) protein, probably to reduce fitness costs from excessive expression2-5. However, how a single HTH regulator adjusts anti-CRISPR production to cope with increasing phage genome copies and accumulating acr mRNA is unknown. Here we show that the HTH domain of the regulator Aca2, in addition to repressing Acr synthesis transcriptionally through DNA binding, inhibits translation of mRNAs by binding conserved RNA stem-loops and blocking ribosome access. The cryo-electron microscopy structure of the approximately 40 kDa Aca2-RNA complex demonstrates how the versatile HTH domain specifically discriminates RNA from DNA binding sites. These combined regulatory modes are widespread in the Aca2 family and facilitate CRISPR-Cas inhibition in the face of rapid phage DNA replication without toxic acr overexpression. Given the ubiquity of HTH-domain-containing proteins, it is anticipated that many more of them elicit regulatory control by dual DNA and RNA binding.


Assuntos
Bacteriófagos , Sistemas CRISPR-Cas , Proteínas de Ligação a DNA , Regulação Viral da Expressão Gênica , Sequências Hélice-Volta-Hélice , Proteínas de Ligação a RNA , Proteínas Virais , Bacteriófagos/química , Bacteriófagos/genética , Bacteriófagos/metabolismo , Bacteriófagos/ultraestrutura , Sítios de Ligação , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Proteínas Associadas a CRISPR/metabolismo , Microscopia Crioeletrônica , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Genes Virais , Modelos Moleculares , Conformação de Ácido Nucleico , Pectobacterium carotovorum/virologia , Biossíntese de Proteínas/genética , Domínios Proteicos , Ribossomos/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/ultraestrutura , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , RNA Viral/ultraestrutura , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/ultraestrutura , Especificidade por Substrato , Transcrição Gênica , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais/ultraestrutura
5.
Nat Immunol ; 18(2): 225-235, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28024153

RESUMO

The mechanisms by which human immunodeficiency virus 1 (HIV-1) avoids immune surveillance by dendritic cells (DCs), and thereby prevents protective adaptive immune responses, remain poorly understood. Here we showed that HIV-1 actively arrested antiviral immune responses by DCs, which contributed to efficient HIV-1 replication in infected individuals. We identified the RNA helicase DDX3 as an HIV-1 sensor that bound abortive HIV-1 RNA after HIV-1 infection and induced DC maturation and type I interferon responses via the signaling adaptor MAVS. Notably, HIV-1 recognition by the C-type lectin receptor DC-SIGN activated the mitotic kinase PLK1, which suppressed signaling downstream of MAVS, thereby interfering with intrinsic host defense during HIV-1 infection. Finally, we showed that PLK1-mediated suppression of DDX3-MAVS signaling was a viral strategy that accelerated HIV-1 replication in infected individuals.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Dendríticas/virologia , Infecções por HIV/imunologia , HIV-1/fisiologia , Evasão da Resposta Imune , Imunidade , Macrófagos/virologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Extratos Celulares , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Estudos de Coortes , RNA Helicases DEAD-box/metabolismo , Células Dendríticas/imunologia , Regulação Viral da Expressão Gênica , Células HEK293 , Infecções por HIV/virologia , Interações Hospedeiro-Patógeno/genética , Humanos , Interferon beta/sangue , Macrófagos/imunologia , Polimorfismo de Nucleotídeo Único , RNA Viral/imunologia , RNA Viral/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais , Carga Viral/genética
6.
Nature ; 623(7987): 608-615, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37938768

RESUMO

Cell therapies have yielded durable clinical benefits for patients with cancer, but the risks associated with the development of therapies from manipulated human cells are understudied. For example, we lack a comprehensive understanding of the mechanisms of toxicities observed in patients receiving T cell therapies, including recent reports of encephalitis caused by reactivation of human herpesvirus 6 (HHV-6)1. Here, through petabase-scale viral genomics mining, we examine the landscape of human latent viral reactivation and demonstrate that HHV-6B can become reactivated in cultures of human CD4+ T cells. Using single-cell sequencing, we identify a rare population of HHV-6 'super-expressors' (about 1 in 300-10,000 cells) that possess high viral transcriptional activity, among research-grade allogeneic chimeric antigen receptor (CAR) T cells. By analysing single-cell sequencing data from patients receiving cell therapy products that are approved by the US Food and Drug Administration2 or are in clinical studies3-5, we identify the presence of HHV-6-super-expressor CAR T cells in patients in vivo. Together, the findings of our study demonstrate the utility of comprehensive genomics analyses in implicating cell therapy products as a potential source contributing to the lytic HHV-6 infection that has been reported in clinical trials1,6-8 and may influence the design and production of autologous and allogeneic cell therapies.


Assuntos
Linfócitos T CD4-Positivos , Herpesvirus Humano 6 , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos , Ativação Viral , Latência Viral , Humanos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Ensaios Clínicos como Assunto , Regulação Viral da Expressão Gênica , Genômica , Herpesvirus Humano 6/genética , Herpesvirus Humano 6/isolamento & purificação , Herpesvirus Humano 6/fisiologia , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Encefalite Infecciosa/complicações , Encefalite Infecciosa/virologia , Receptores de Antígenos Quiméricos/imunologia , Infecções por Roseolovirus/complicações , Infecções por Roseolovirus/virologia , Análise da Expressão Gênica de Célula Única , Carga Viral
7.
Nature ; 614(7947): 318-325, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36599978

RESUMO

Rare CD4 T cells that contain HIV under antiretroviral therapy represent an important barrier to HIV cure1-3, but the infeasibility of isolating and characterizing these cells in their natural state has led to uncertainty about whether they possess distinctive attributes that HIV cure-directed therapies might exploit. Here we address this challenge using a microfluidic technology that isolates the transcriptomes of HIV-infected cells based solely on the detection of HIV DNA. HIV-DNA+ memory CD4 T cells in the blood from people receiving antiretroviral therapy showed inhibition of six transcriptomic pathways, including death receptor signalling, necroptosis signalling and antiproliferative Gα12/13 signalling. Moreover, two groups of genes identified by network co-expression analysis were significantly associated with HIV-DNA+ cells. These genes (n = 145) accounted for just 0.81% of the measured transcriptome and included negative regulators of HIV transcription that were higher in HIV-DNA+ cells, positive regulators of HIV transcription that were lower in HIV-DNA+ cells, and other genes involved in RNA processing, negative regulation of mRNA translation, and regulation of cell state and fate. These findings reveal that HIV-infected memory CD4 T cells under antiretroviral therapy are a distinctive population with host gene expression patterns that favour HIV silencing, cell survival and cell proliferation, with important implications for the development of HIV cure strategies.


Assuntos
Linfócitos T CD4-Positivos , Regulação Viral da Expressão Gênica , Infecções por HIV , HIV-1 , Latência Viral , Humanos , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA Viral/isolamento & purificação , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/genética , HIV-1/isolamento & purificação , HIV-1/patogenicidade , Memória Imunológica , Microfluídica , Necroptose/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Antirretrovirais/farmacologia , Antirretrovirais/uso terapêutico
8.
Mol Cell ; 78(4): 653-669.e8, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32315601

RESUMO

Epstein-Barr virus (EBV) is associated with multiple human malignancies. To evade immune detection, EBV switches between latent and lytic programs. How viral latency is maintained in tumors or in memory B cells, the reservoir for lifelong EBV infection, remains incompletely understood. To gain insights, we performed a human genome-wide CRISPR/Cas9 screen in Burkitt lymphoma B cells. Our analyses identified a network of host factors that repress lytic reactivation, centered on the transcription factor MYC, including cohesins, FACT, STAGA, and Mediator. Depletion of MYC or factors important for MYC expression reactivated the lytic cycle, including in Burkitt xenografts. MYC bound the EBV genome origin of lytic replication and suppressed its looping to the lytic cycle initiator BZLF1 promoter. Notably, MYC abundance decreases with plasma cell differentiation, a key lytic reactivation trigger. Our results suggest that EBV senses MYC abundance as a readout of B cell state and highlights Burkitt latency reversal therapeutic targets.


Assuntos
Linfoma de Burkitt/patologia , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ativação Viral , Latência Viral , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Linfócitos B/virologia , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/virologia , Proliferação de Células , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/metabolismo , Feminino , Regulação Viral da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Trends Genet ; 40(9): 772-783, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38821843

RESUMO

To withstand a hostile cellular environment and replicate, viruses must sense, interpret, and respond to many internal and external cues. Retroviruses and DNA viruses can intercept these cues impinging on host transcription factors via cis-regulatory elements (CREs) in viral genomes, allowing them to sense and coordinate context-specific responses to varied signals. Here, we explore the characteristics of viral CREs, the classes of signals and host transcription factors that regulate them, and how this informs outcomes of viral replication, immune evasion, and latency. We propose that viral CREs constitute central hubs for signal integration from multiple pathways and that sequence variation between viral isolates can rapidly rewire sensing mechanisms, contributing to the variability observed in patient outcomes.


Assuntos
Sequências Reguladoras de Ácido Nucleico , Humanos , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/genética , Replicação Viral/genética , Genoma Viral/genética , Interações Hospedeiro-Patógeno/genética , Latência Viral/genética , Regulação Viral da Expressão Gênica/genética
10.
Annu Rev Neurosci ; 42: 249-269, 2019 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-31283901

RESUMO

In 2015, public awareness of Zika virus (ZIKV) rose in response to alarming statistics of infants with microcephaly being born to women who were infected with the virus during pregnancy, triggering global concern over these potentially devastating consequences. Although we have discovered a great deal about the genome and pathogenesis of this reemergent flavivirus since this recent outbreak, we still have much more to learn, including the nature of the virus-host interactions and mechanisms that determine its tropism and pathogenicity in the nervous system, which are in turn shaped by the continual evolution of the virus. Inevitably, we will find out more about the potential long-term effects of ZIKV exposure on the nervous system from ongoing longitudinal studies. Integrating clinical and epidemiological data with a wider range of animal and human cell culture models will be critical to understanding the pathogenetic mechanisms and developing more specific antiviral compounds and vaccines.


Assuntos
Doenças do Sistema Nervoso/virologia , Infecção por Zika virus/fisiopatologia , Adulto , Animais , Encéfalo/embriologia , Encéfalo/patologia , Encéfalo/virologia , Células Cultivadas , Doenças Transmissíveis Emergentes , Surtos de Doenças , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Viral da Expressão Gênica , Vetores Genéticos/genética , Interações entre Hospedeiro e Microrganismos , Humanos , Recém-Nascido , Macaca mulatta , Camundongos , Microbiota , Microcefalia/embriologia , Microcefalia/etiologia , Microcefalia/virologia , Microglia/fisiologia , Modelos Animais , Doenças do Sistema Nervoso/fisiopatologia , Neurogênese , Gravidez , Complicações Infecciosas na Gravidez/fisiopatologia , Receptores Virais/fisiologia , Estudos em Gêmeos como Assunto , Vacinas Virais , Zika virus/imunologia , Zika virus/isolamento & purificação , Zika virus/patogenicidade , Zika virus/fisiologia , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/veterinária
11.
Immunity ; 49(3): 438-448.e5, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30193849

RESUMO

Recognition of viral RNA by the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) initiates innate antiviral immune response. How the binding of viral RNA to and activation of the RLRs are regulated remains enigmatic. In this study, we identified ZCCHC3 as a positive regulator of the RLRs including RIG-I and MDA5. ZCCHC3 deficiency markedly inhibited RNA virus-triggered induction of downstream antiviral genes, and ZCCHC3-deficient mice were more susceptible to RNA virus infection. ZCCHC3 was associated with RIG-I and MDA5 and functions in two distinct processes for regulation of RIG-I and MDA5 activities. ZCCHC3 bound to dsRNA and enhanced the binding of RIG-I and MDA5 to dsRNA. ZCCHC3 also recruited the E3 ubiquitin ligase TRIM25 to the RIG-I and MDA5 complexes to facilitate its K63-linked polyubiquitination and activation. Thus, ZCCHC3 is a co-receptor for RIG-I and MDA5, which is critical for RLR-mediated innate immune response to RNA virus.


Assuntos
Proteína DEAD-box 58/metabolismo , Infecções por Vírus de RNA/imunologia , Vírus de RNA/fisiologia , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica , Células HEK293 , Humanos , Imunidade Inata , Helicase IFIH1 Induzida por Interferon/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , RNA Viral/imunologia , Proteínas de Ligação a RNA/genética , Células THP-1 , Fatores de Transcrição/metabolismo , Ubiquitinação
12.
Cell ; 150(6): 1147-57, 2012 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-22980978

RESUMO

Transcription elongation is increasingly recognized as an important mechanism of gene regulation. Here, we show that microprocessor controls gene expression in an RNAi-independent manner. Microprocessor orchestrates the recruitment of termination factors Setx and Xrn2, and the 3'-5' exoribonuclease, Rrp6, to initiate RNAPII pausing and premature termination at the HIV-1 promoter through cleavage of the stem-loop RNA, TAR. Rrp6 further processes the cleavage product, which generates a small RNA that is required to mediate potent transcriptional repression and chromatin remodeling at the HIV-1 promoter. Using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-seq), we identified cellular gene targets whose transcription is modulated by microprocessor. Our study reveals RNAPII pausing and premature termination mediated by the co-operative activity of ribonucleases, Drosha/Dgcr8, Xrn2, and Rrp6, as a regulatory mechanism of RNAPII-dependent transcription elongation.


Assuntos
Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Regulação Viral da Expressão Gênica , HIV-1/genética , RNA Helicases/metabolismo , RNA Polimerase II/metabolismo , Transcrição Gênica , Sequência de Bases , Montagem e Desmontagem da Cromatina , Imunoprecipitação da Cromatina , DNA Helicases , Repetição Terminal Longa de HIV , Humanos , Dados de Sequência Molecular , Enzimas Multifuncionais , Regiões Promotoras Genéticas , Interferência de RNA , RNA Viral/química , RNA Viral/genética , Fatores de Transcrição/metabolismo
13.
Cell ; 151(7): 1569-80, 2012 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-23260143

RESUMO

Many signaling circuits face a fundamental tradeoff between accelerating their response speed while maintaining final levels below a cytotoxic threshold. Here, we describe a transcriptional circuitry that dynamically converts signaling inputs into faster rates without amplifying final equilibrium levels. Using time-lapse microscopy, we find that transcriptional activators accelerate human cytomegalovirus (CMV) gene expression in single cells without amplifying steady-state expression levels, and this acceleration generates a significant replication advantage. We map the accelerator to a highly self-cooperative transcriptional negative-feedback loop (Hill coefficient ∼7) generated by homomultimerization of the virus's essential transactivator protein IE2 at nuclear PML bodies. Eliminating the IE2-accelerator circuit reduces transcriptional strength through mislocalization of incoming viral genomes away from PML bodies and carries a heavy fitness cost. In general, accelerators may provide a mechanism for signal-transduction circuits to respond quickly to external signals without increasing steady-state levels of potentially cytotoxic molecules.


Assuntos
Infecções por Citomegalovirus/virologia , Citomegalovirus/genética , Redes Reguladoras de Genes , Aptidão Genética , Citomegalovirus/fisiologia , Retroalimentação Fisiológica , Fibroblastos/virologia , Regulação Viral da Expressão Gênica , Humanos , Proteínas Imediatamente Precoces/metabolismo , Corpos de Inclusão Viral/metabolismo , Imagem com Lapso de Tempo , Transativadores/metabolismo , Ativação Transcricional , Replicação Viral
14.
Cell ; 149(2): 358-70, 2012 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-22500802

RESUMO

The function of the Vibrio 7(th) pandemic island-1 (VSP-1) in cholera pathogenesis has remained obscure. Utilizing chromatin immunoprecipitation sequencing and RNA sequencing to map the regulon of the master virulence regulator ToxT, we identify a TCP island-encoded small RNA that reduces the expression of a previously unrecognized VSP-1-encoded transcription factor termed VspR. VspR modulates the expression of several VSP-1 genes including one that encodes a novel class of di-nucleotide cyclase (DncV), which preferentially synthesizes a previously undescribed hybrid cyclic AMP-GMP molecule. We show that DncV is required for efficient intestinal colonization and downregulates V. cholerae chemotaxis, a phenotype previously associated with hyperinfectivity. This pathway couples the actions of previously disparate genomic islands, defines VSP-1 as a pathogenicity island in V. cholerae, and implicates its occurrence in 7(th) pandemic strains as a benefit for host adaptation through the production of a regulatory cyclic di-nucleotide.


Assuntos
AMP Cíclico/biossíntese , Nucleotídeos Cíclicos/metabolismo , Vibrio cholerae/metabolismo , Vibrio cholerae/patogenicidade , Animais , Proteínas de Bactérias , Sequência de Bases , Regulação Viral da Expressão Gênica , Ilhas Genômicas , Humanos , Intestinos/microbiologia , Redes e Vias Metabólicas , Camundongos , Dados de Sequência Molecular , Fósforo-Oxigênio Liases , RNA não Traduzido/metabolismo , RNA Viral/metabolismo , Alinhamento de Sequência , Fatores de Transcrição , Vibrio cholerae/genética , Virulência
15.
Nature ; 589(7841): 306-309, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33208949

RESUMO

CrAss-like phages are a recently described expansive group of viruses that includes the most abundant virus in the human gut1-3. The genomes of all crAss-like phages encode a large virion-packaged protein2,4 that contains a DFDxD sequence motif, which forms the catalytic site in cellular multisubunit RNA polymerases (RNAPs)5. Here, using Cellulophaga baltica crAss-like phage phi14:2 as a model system, we show that this protein is a DNA-dependent RNAP that is translocated into the host cell along with the phage DNA and transcribes early phage genes. We determined the crystal structure of this 2,180-residue enzyme in a self-inhibited state, which probably occurs before virion packaging. This conformation is attained with the help of a cleft-blocking domain that interacts with the active site and occupies the cavity in which the RNA-DNA hybrid binds. Structurally, phi14:2 RNAP is most similar to eukaryotic RNAPs that are involved in RNA interference6,7, although most of the phi14:2 RNAP structure (nearly 1,600 residues) maps to a new region of the protein fold space. Considering this structural similarity, we propose that eukaryal RNA interference polymerases have their origins in phage, which parallels the emergence of the mitochondrial transcription apparatus8.


Assuntos
Bacteriófagos/classificação , Bacteriófagos/enzimologia , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Flavobacteriaceae/virologia , Bacteriófagos/genética , Domínio Catalítico , Sistema Livre de Células , Cristalografia por Raios X , DNA de Cadeia Simples/biossíntese , DNA de Cadeia Simples/genética , RNA Polimerases Dirigidas por DNA/genética , Evolução Molecular , Regulação Viral da Expressão Gênica , Genes Virais/genética , Modelos Biológicos , Modelos Moleculares , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Interferência de RNA , Transcrição Gênica
16.
Nature ; 593(7859): 362-371, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34012080

RESUMO

Unlike the human genome that comprises mostly noncoding and regulatory sequences, viruses have evolved under the constraints of maintaining a small genome size while expanding the efficiency of their coding and regulatory sequences. As a result, viruses use strategies of transcription and translation in which one or more of the steps in the conventional gene-protein production line are altered. These alternative strategies of viral gene expression (also known as gene recoding) can be uniquely brought about by dedicated viral enzymes or by co-opting host factors (known as host dependencies). Targeting these unique enzymatic activities and host factors exposes vulnerabilities of a virus and provides a paradigm for the design of novel antiviral therapies. In this Review, we describe the types and mechanisms of unconventional gene and protein expression in viruses, and provide a perspective on how future basic mechanistic work could inform translational efforts that are aimed at viral eradication.


Assuntos
Antivirais/farmacologia , Antivirais/uso terapêutico , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Interações entre Hospedeiro e Microrganismos/genética , Viroses/tratamento farmacológico , Viroses/virologia , Animais , Mudança da Fase de Leitura do Gene Ribossômico/efeitos dos fármacos , Mudança da Fase de Leitura do Gene Ribossômico/genética , Regulação Viral da Expressão Gênica/genética , Genoma Viral/efeitos dos fármacos , Genoma Viral/genética , Humanos , Splicing de RNA/efeitos dos fármacos , Splicing de RNA/genética
17.
Nature ; 596(7873): 558-564, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34408324

RESUMO

Viral pathogens are an ongoing threat to public health worldwide. Analysing their dependence on host biosynthetic pathways could lead to effective antiviral therapies1. Here we integrate proteomic analyses of polysomes with functional genomics and pharmacological interventions to define how enteroviruses and flaviviruses remodel host polysomes to synthesize viral proteins and disable host protein production. We find that infection with polio, dengue or Zika virus markedly modifies polysome composition, without major changes to core ribosome stoichiometry. These viruses use different strategies to evict a common set of translation initiation and RNA surveillance factors from polysomes while recruiting host machineries that are specifically required for viral biogenesis. Targeting these specialized viral polysomes could provide a new approach for antiviral interventions. For example, we find that both Zika and dengue use the collagen proline hydroxylation machinery to mediate cotranslational modification of conserved proline residues in the viral polyprotein. Genetic or pharmacological inhibition of proline hydroxylation impairs nascent viral polyprotein folding and induces its aggregation and degradation. Notably, such interventions prevent viral polysome remodelling and lower virus production. Our findings delineate the modular nature of polysome specialization at the virus-host interface and establish a powerful strategy to identify targets for selective antiviral interventions.


Assuntos
Flavivirus/crescimento & desenvolvimento , Flavivirus/metabolismo , Interações Hospedeiro-Patógeno , Hidroxilação , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Prolina/metabolismo , Biossíntese de Proteínas , Linhagem Celular , Colágeno/química , Colágeno/metabolismo , Vírus da Dengue/genética , Vírus da Dengue/crescimento & desenvolvimento , Flavivirus/química , Regulação Viral da Expressão Gênica , Genômica , Fatores Celulares Derivados do Hospedeiro/antagonistas & inibidores , Fatores Celulares Derivados do Hospedeiro/metabolismo , Interações Hospedeiro-Patógeno/genética , Humanos , Sítios Internos de Entrada Ribossomal , Chaperonas Moleculares/metabolismo , Iniciação Traducional da Cadeia Peptídica , Poliovirus/genética , Poliovirus/crescimento & desenvolvimento , Polirribossomos/química , Polirribossomos/metabolismo , Agregados Proteicos , Dobramento de Proteína , Mapas de Interação de Proteínas , Proteólise , Proteômica , Zika virus/genética , Zika virus/crescimento & desenvolvimento
18.
Mol Cell ; 74(1): 59-72.e3, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30745087

RESUMO

Bacillus phages use a communication system, termed "arbitrium," to coordinate lysis-lysogeny decisions. Arbitrium communication is mediated by the production and secretion of a hexapeptide (AimP) during lytic cycle. Once internalized, AimP reduces the expression of the negative regulator of lysogeny, AimX, by binding to the transcription factor, AimR, promoting lysogeny. We have elucidated the crystal structures of AimR from the Bacillus subtilis SPbeta phage in its apo form, bound to its DNA operator and in complex with AimP. AimR presents intrinsic plasticity, sharing structural features with the RRNPP quorum-sensing family. Remarkably, AimR binds to an unusual operator with a long spacer that interacts nonspecifically with the receptor TPR domain, while the HTH domain canonically recognizes two inverted repeats. AimP stabilizes a compact conformation of AimR that approximates the DNA-recognition helices, preventing AimR binding to the aimX promoter region. Our results establish the molecular basis of the arbitrium communication system.


Assuntos
Fagos Bacilares/metabolismo , Lisogenia , Proteínas Virais/metabolismo , Fagos Bacilares/genética , Bacillus subtilis/virologia , DNA/metabolismo , Regulação Viral da Expressão Gênica , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Transdução de Sinais , Relação Estrutura-Atividade , Proteínas Virais/química , Proteínas Virais/genética
19.
Mol Cell ; 74(1): 196-211.e11, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30799147

RESUMO

The compendium of RNA-binding proteins (RBPs) has been greatly expanded by the development of RNA-interactome capture (RIC). However, it remained unknown if the complement of RBPs changes in response to environmental perturbations and whether these rearrangements are important. To answer these questions, we developed "comparative RIC" and applied it to cells challenged with an RNA virus called sindbis (SINV). Over 200 RBPs display differential interaction with RNA upon SINV infection. These alterations are mainly driven by the loss of cellular mRNAs and the emergence of viral RNA. RBPs stimulated by the infection redistribute to viral replication factories and regulate the capacity of the virus to infect. For example, ablation of XRN1 causes cells to be refractory to SINV, while GEMIN5 moonlights as a regulator of SINV gene expression. In summary, RNA availability controls RBP localization and function in SINV-infected cells.


Assuntos
Células Epiteliais/virologia , Perfilação da Expressão Gênica/métodos , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Sindbis virus/genética , Transcriptoma , Neoplasias do Colo do Útero/virologia , Regiões 5' não Traduzidas , Sítios de Ligação , Células Epiteliais/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Feminino , Regulação Viral da Expressão Gênica , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Ligação Proteica , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas do Complexo SMN , Sindbis virus/crescimento & desenvolvimento , Sindbis virus/metabolismo , Sindbis virus/patogenicidade , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Replicação Viral
20.
Proc Natl Acad Sci U S A ; 121(10): e2315860121, 2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38408244

RESUMO

Human cytomegalovirus (HCMV) is a prevalent pathogen that establishes life-long latent infection in hematopoietic cells. While this infection is usually asymptomatic, immune dysregulation leads to viral reactivation, which can cause significant morbidity and mortality. However, the mechanisms underpinning reactivation remain incompletely understood. The HCMV major immediate early promoter (MIEP)/enhancer is a key factor in this process, as its transactivation from a repressed to active state helps drive viral gene transcription necessary for reactivation from latency. Numerous host transcription factors bind the MIE locus and recruit repressive chromatin modifiers, thus impeding virus reactivation. One such factor is CCCTC-binding protein (CTCF), a highly conserved host zinc finger protein that mediates chromatin conformation and nuclear architecture. However, the mechanisms by which CTCF contributes to HCMV latency were previously unexplored. Here, we confirm that CTCF binds two convergent sites within the MIE locus during latency in primary CD14+ monocytes, and following cellular differentiation, CTCF association is lost as the virus reactivates. While mutation of the MIE enhancer CTCF binding site does not impact viral lytic growth in fibroblasts, this mutant virus fails to maintain latency in myeloid cells. Furthermore, we show the two convergent CTCF binding sites allow looping to occur across the MIEP, supporting transcriptional repression during latency. Indeed, looping between the two sites diminishes during virus reactivation, concurrent with activation of MIE transcription. Taken together, our data reveal that three-dimensional chromatin looping aids in the regulation of HCMV latency and provides insight into promoter/enhancer regulation that may prove broadly applicable across biological systems.


Assuntos
Infecções por Citomegalovirus , Citomegalovirus , Humanos , Cromatina/genética , Citomegalovirus/genética , Infecções por Citomegalovirus/genética , Regulação Viral da Expressão Gênica , Regiões Promotoras Genéticas , Ativação Viral/genética , Latência Viral/genética
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