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1.
Cell ; 161(6): 1293-1305, 2015 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-26046437

RESUMEN

Dendritic cells (DCs) play a critical role in the immune response to viral infection through the facilitation of cell-intrinsic antiviral activity and the activation of adaptive immunity. HIV-1 infection of DCs triggers an IRF3-dependent innate immune response, which requires the activity of cyclic GAMP synthase (cGAS). We report the results of a targeted RNAi screen utilizing primary human monocyte-derived DCs (MDDCs) to identify immune regulators that directly interface with HIV-1-encoded features to initiate this innate response. Polyglutamine binding protein 1 (PQBP1) emerged as a strong candidate through this analysis. We found that PQBP1 directly binds to reverse-transcribed HIV-1 DNA and interacts with cGAS to initiate an IRF3-dependent innate response. MDDCs derived from Renpenning syndrome patients, who harbor mutations in the PQBP1 locus, possess a severely attenuated innate immune response to HIV-1 challenge, underscoring the role of PQBP1 as a proximal innate sensor of a HIV-1 infection.


Asunto(s)
Proteínas Portadoras/metabolismo , VIH-1/inmunología , Inmunidad Innata , Proteínas Nucleares/metabolismo , Nucleotidiltransferasas/metabolismo , Secuencia de Bases , Línea Celular , Parálisis Cerebral/inmunología , ADN Viral/genética , Proteínas de Unión al ADN , VIH-1/fisiología , Humanos , Discapacidad Intelectual Ligada al Cromosoma X/inmunología , Datos de Secuencia Molecular
2.
Mol Cell ; 81(12): 2656-2668.e8, 2021 06 17.
Artículo en Inglés | MEDLINE | ID: mdl-33930332

RESUMEN

A deficient interferon (IFN) response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been implicated as a determinant of severe coronavirus disease 2019 (COVID-19). To identify the molecular effectors that govern IFN control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human IFN-stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors inhibiting viral entry, RNA binding proteins suppressing viral RNA synthesis, and a highly enriched cluster of endoplasmic reticulum (ER)/Golgi-resident ISGs inhibiting viral assembly/egress. These included broad-acting antiviral ISGs and eight ISGs that specifically inhibited SARS-CoV-2 and SARS-CoV-1 replication. Among the broad-acting ISGs was BST2/tetherin, which impeded viral release and is antagonized by SARS-CoV-2 Orf7a protein. Overall, these data illuminate a set of ISGs that underlie innate immune control of SARS-CoV-2/SARS-CoV-1 infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.


Asunto(s)
Antígenos CD/genética , Interacciones Huésped-Patógeno/genética , Factores Reguladores del Interferón/genética , Interferón Tipo I/genética , SARS-CoV-2/genética , Proteínas Virales/genética , Animales , Antígenos CD/química , Antígenos CD/inmunología , Sitios de Unión , Línea Celular Tumoral , Chlorocebus aethiops , Retículo Endoplásmico/genética , Retículo Endoplásmico/inmunología , Retículo Endoplásmico/virología , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/inmunología , Regulación de la Expresión Génica , Aparato de Golgi/genética , Aparato de Golgi/inmunología , Aparato de Golgi/virología , Células HEK293 , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata , Factores Reguladores del Interferón/clasificación , Factores Reguladores del Interferón/inmunología , Interferón Tipo I/inmunología , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , SARS-CoV-2/inmunología , Transducción de Señal , Células Vero , Proteínas Virales/química , Proteínas Virales/inmunología , Internalización del Virus , Liberación del Virus/genética , Liberación del Virus/inmunología , Replicación Viral/genética , Replicación Viral/inmunología
3.
Nature ; 586(7827): 113-119, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32707573

RESUMEN

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of the severe pneumonia-like disease coronavirus disease 2019 (COVID-19)1. The development of a vaccine is likely to take at least 12-18 months, and the typical timeline for approval of a new antiviral therapeutic agent can exceed 10 years. Thus, repurposing of known drugs could substantially accelerate the deployment of new therapies for COVID-19. Here we profiled a library of drugs encompassing approximately 12,000 clinical-stage or Food and Drug Administration (FDA)-approved small molecules to identify candidate therapeutic drugs for COVID-19. We report the identification of 100 molecules that inhibit viral replication of SARS-CoV-2, including 21 drugs that exhibit dose-response relationships. Of these, thirteen were found to harbour effective concentrations commensurate with probable achievable therapeutic doses in patients, including the PIKfyve kinase inhibitor apilimod2-4 and the cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825 and ONO 5334. Notably, MDL-28170, ONO 5334 and apilimod were found to antagonize viral replication in human pneumocyte-like cells derived from induced pluripotent stem cells, and apilimod also demonstrated antiviral efficacy in a primary human lung explant model. Since most of the molecules identified in this study have already advanced into the clinic, their known pharmacological and human safety profiles will enable accelerated preclinical and clinical evaluation of these drugs for the treatment of COVID-19.


Asunto(s)
Antivirales/análisis , Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/virología , Evaluación Preclínica de Medicamentos , Reposicionamiento de Medicamentos , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/virología , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Células Epiteliales Alveolares/citología , Células Epiteliales Alveolares/efectos de los fármacos , Betacoronavirus/crecimiento & desarrollo , COVID-19 , Línea Celular , Inhibidores de Cisteína Proteinasa/análisis , Inhibidores de Cisteína Proteinasa/farmacología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hidrazonas , Células Madre Pluripotentes Inducidas/citología , Modelos Biológicos , Morfolinas/análisis , Morfolinas/farmacología , Pandemias , Pirimidinas , Reproducibilidad de los Resultados , SARS-CoV-2 , Bibliotecas de Moléculas Pequeñas/análisis , Bibliotecas de Moléculas Pequeñas/farmacología , Triazinas/análisis , Triazinas/farmacología , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Tratamiento Farmacológico de COVID-19
5.
PLoS Pathog ; 17(11): e1009409, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34843601

RESUMEN

The HIV-1 accessory protein Vpu modulates membrane protein trafficking and degradation to provide evasion of immune surveillance. Targets of Vpu include CD4, HLAs, and BST-2. Several cellular pathways co-opted by Vpu have been identified, but the picture of Vpu's itinerary and activities within membrane systems remains incomplete. Here, we used fusion proteins of Vpu and the enzyme ascorbate peroxidase (APEX2) to compare the ultrastructural locations and the proximal proteomes of wild type Vpu and Vpu-mutants. The proximity-omes of the proteins correlated with their ultrastructural locations and placed wild type Vpu near both retromer and ESCRT-0 complexes. Hierarchical clustering of protein abundances across the mutants was essential to interpreting the data and identified Vpu degradation-targets including CD4, HLA-C, and SEC12 as well as Vpu-cofactors including HGS, STAM, clathrin, and PTPN23, an ALIX-like protein. The Vpu-directed degradation of BST-2 was supported by STAM and PTPN23 and to a much lesser extent by the retromer subunits Vps35 and SNX3. PTPN23 also supported the Vpu-directed decrease in CD4 at the cell surface. These data suggest that Vpu directs targets from sorting endosomes to degradation at multi-vesicular bodies via ESCRT-0 and PTPN23.


Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Infecciones por VIH/virología , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Proteoma/metabolismo , Nexinas de Clasificación/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Proteínas Viroporinas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , VIH-1/fisiología , Células HeLa , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Humanos , Microscopía Electrónica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Transporte de Proteínas , Proteínas Tirosina Fosfatasas no Receptoras/genética , Proteoma/análisis , Nexinas de Clasificación/química , Nexinas de Clasificación/genética , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Viroporinas/genética
6.
Cell ; 135(1): 49-60, 2008 Oct 03.
Artículo en Inglés | MEDLINE | ID: mdl-18854154

RESUMEN

Human Immunodeficiency Viruses (HIV-1 and HIV-2) rely upon host-encoded proteins to facilitate their replication. Here, we combined genome-wide siRNA analyses with interrogation of human interactome databases to assemble a host-pathogen biochemical network containing 213 confirmed host cellular factors and 11 HIV-1-encoded proteins. Protein complexes that regulate ubiquitin conjugation, proteolysis, DNA-damage response, and RNA splicing were identified as important modulators of early-stage HIV-1 infection. Additionally, over 40 new factors were shown to specifically influence the initiation and/or kinetics of HIV-1 DNA synthesis, including cytoskeletal regulatory proteins, modulators of posttranslational modification, and nucleic acid-binding proteins. Finally, 15 proteins with diverse functional roles, including nuclear transport, prostaglandin synthesis, ubiquitination, and transcription, were found to influence nuclear import or viral DNA integration. Taken together, the multiscale approach described here has uncovered multiprotein virus-host interactions that likely act in concert to facilitate the early steps of HIV-1 infection.


Asunto(s)
Infecciones por VIH/virología , VIH-1/fisiología , Interacciones Huésped-Patógeno , Proteínas/metabolismo , Replicación Viral , Línea Celular , Humanos , Interferencia de ARN , Técnicas del Sistema de Dos Híbridos
7.
J Virol ; 95(22): e0099621, 2021 10 27.
Artículo en Inglés | MEDLINE | ID: mdl-34468177

RESUMEN

Zika virus (ZIKV) is a mosquito-borne pathogen classified by the World Health Organization (WHO) as a public health emergency of international concern in 2016, and it is still identified as a priority disease. Although most infected individuals are asymptomatic or show mild symptoms, a risk of neurologic complications is associated with infection in adults. Additionally, infection during pregnancy is directly linked to microcephaly and other congenital malformations. Since there are no currently available vaccines or approved therapeutics for this virus, there is a critical unmet need in developing treatments to prevent future ZIKV outbreaks. Toward this end, we performed a large-scale cell-based high-content screen of 51,520 chemical compounds to identify potential antiviral drug candidates. The compound (2E)-N-benzyl-3-(4-butoxyphenyl)prop-2-enamide (SBI-0090799) was found to inhibit replication of multiple ZIKV strains and in different cell systems. SBI-0090799 did not affect viral entry or RNA translation but suppressed RNA replication by preventing the formation of the membranous replication compartment. Selection of drug-resistant viruses identified single-amino-acid substitutions in the N-terminal region of nonstructural protein NS4A, arguing this is the likely drug target. These resistance mutations rescued viral RNA replication and restored the formation of the membranous replication compartment. This mechanism of action is similar to clinically approved NS5A inhibitors for hepatitis C virus (HCV). Taken together, SBI-0090799 represents a promising lead candidate for the development of an antiviral treatment against ZIKV infection for the mitigation of severe complications and potential resurgent outbreaks of the virus. IMPORTANCE This study describes the elucidation of (2E)-N-benzyl-3-(4-butoxyphenyl)prop-2-enamide (SBI-0090799) as a selective and potent inhibitor of Zika virus (ZIKV) replication using a high-throughput screening approach. Mapping and resistance studies, supported by electron microscopy observations, indicate that the small molecule is functioning through inhibition of NS4A-mediated formation of ZIKV replication compartments in the endoplasmic reticulum (ER). Intriguingly, this defines a novel nonenzymatic target and chemical matter for the development of a new class of ZIKV antivirals. Moreover, chemical modulation affecting this nonstructural protein mirrors the identification and development of hepatitis C virus (HCV) NS5A inhibitor daclatasvir and its derivatives, similarly interfering with the formation of the viral replication compartment and also targeting a protein with no enzymatic activity, which have been part of a curative strategy for HCV.


Asunto(s)
Antivirales/farmacología , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Replicación Viral/efectos de los fármacos , Infección por el Virus Zika/tratamiento farmacológico , Virus Zika/efectos de los fármacos , Animales , Astrocitos , Chlorocebus aethiops , Células Dendríticas , Células HEK293 , Humanos , Cultivo Primario de Células , Células Vero , Compartimentos de Replicación Viral/efectos de los fármacos
8.
J Virol ; 94(4)2020 01 31.
Artículo en Inglés | MEDLINE | ID: mdl-31776276

RESUMEN

Influenza A virus (IAV) is a human respiratory pathogen that causes yearly global epidemics, as well as sporadic pandemics due to human adaptation of pathogenic strains. Efficient replication of IAV in different species is, in part, dictated by its ability to exploit the genetic environment of the host cell. To investigate IAV tropism in human cells, we evaluated the replication of IAV strains in a diverse subset of epithelial cell lines. HeLa cells were refractory to the growth of human H1N1 and H3N2 viruses and low-pathogenic avian influenza (LPAI) viruses. Interestingly, a human isolate of the highly pathogenic avian influenza (HPAI) H5N1 virus successfully propagated in HeLa cells to levels comparable to those in a human lung cell line. Heterokaryon cells generated by fusion of HeLa and permissive cells supported H1N1 virus growth, suggesting the absence of a host factor(s) required for the replication of H1N1, but not H5N1, viruses in HeLa cells. The absence of this factor(s) was mapped to reduced nuclear import, replication, and translation, as well as deficient viral budding. Using reassortant H1N1:H5N1 viruses, we found that the combined introduction of nucleoprotein (NP) and hemagglutinin (HA) from an H5N1 virus was necessary and sufficient to enable H1N1 virus growth. Overall, this study suggests that the absence of one or more cellular factors in HeLa cells results in abortive replication of H1N1, H3N2, and LPAI viruses, which can be circumvented upon the introduction of H5N1 virus NP and HA. Further understanding of the molecular basis of this restriction will provide important insights into the virus-host interactions that underlie IAV pathogenesis and tropism.IMPORTANCE Many zoonotic avian influenza A viruses have successfully crossed the species barrier and caused mild to life-threatening disease in humans. While human-to-human transmission is limited, there is a risk that these zoonotic viruses may acquire adaptive mutations enabling them to propagate efficiently and cause devastating human pandemics. Therefore, it is important to identify viral determinants that provide these viruses with a replicative advantage in human cells. Here, we tested the growth of influenza A virus in a subset of human cell lines and found that abortive replication of H1N1 viruses in HeLa cells can be circumvented upon the introduction of H5N1 virus HA and NP. Overall, this work leverages the genetic diversity of multiple human cell lines to highlight viral determinants that could contribute to H5N1 virus pathogenesis and tropism.


Asunto(s)
Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/metabolismo , Tropismo Viral/genética , Células A549 , Animales , Aves , Línea Celular , Perros , Células HEK293 , Células HeLa , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/metabolismo , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Virus de la Influenza A/patogenicidad , Gripe Aviar/genética , Gripe Aviar/metabolismo , Gripe Humana/genética , Gripe Humana/virología , Células de Riñón Canino Madin Darby , Tropismo Viral/inmunología , Replicación Viral/genética
9.
Nature ; 463(7282): 813-7, 2010 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-20027183

RESUMEN

Influenza A virus is an RNA virus that encodes up to 11 proteins and this small coding capacity demands that the virus use the host cellular machinery for many aspects of its life cycle. Knowledge of these host cell requirements not only informs us of the molecular pathways exploited by the virus but also provides further targets that could be pursued for antiviral drug development. Here we use an integrative systems approach, based on genome-wide RNA interference screening, to identify 295 cellular cofactors required for early-stage influenza virus replication. Within this group, those involved in kinase-regulated signalling, ubiquitination and phosphatase activity are the most highly enriched, and 181 factors assemble into a highly significant host-pathogen interaction network. Moreover, 219 of the 295 factors were confirmed to be required for efficient wild-type influenza virus growth, and further analysis of a subset of genes showed 23 factors necessary for viral entry, including members of the vacuolar ATPase (vATPase) and COPI-protein families, fibroblast growth factor receptor (FGFR) proteins, and glycogen synthase kinase 3 (GSK3)-beta. Furthermore, 10 proteins were confirmed to be involved in post-entry steps of influenza virus replication. These include nuclear import components, proteases, and the calcium/calmodulin-dependent protein kinase (CaM kinase) IIbeta (CAMK2B). Notably, growth of swine-origin H1N1 influenza virus is also dependent on the identified host factors, and we show that small molecule inhibitors of several factors, including vATPase and CAMK2B, antagonize influenza virus replication.


Asunto(s)
Factores Biológicos/genética , Factores Biológicos/fisiología , Interacciones Huésped-Patógeno/fisiología , Virus de la Influenza A/crecimiento & desarrollo , Gripe Humana/genética , Gripe Humana/virología , Replicación Viral/fisiología , Animales , Línea Celular , Chlorocebus aethiops , Biblioteca de Genes , Genoma Humano/genética , Interacciones Huésped-Patógeno/genética , Humanos , Subtipo H1N1 del Virus de la Influenza A/clasificación , Subtipo H1N1 del Virus de la Influenza A/crecimiento & desarrollo , Virus de la Influenza A/clasificación , Interferencia de ARN , Células Vero , Internalización del Virus
10.
J Virol ; 88(14): 7987-97, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24807708

RESUMEN

Detection of cytosolic nucleic acids by pattern recognition receptors leads to the induction of type I interferons (IFNs) and elicits the innate immune response. We report here the identification of RIOK3 as a novel adaptor protein that is essential for the cytosolic nucleic acid-induced type I IFN production and for the antiviral response to gammaherpesvirus through two independent kinome-wide RNA interference screens. RIOK3 knockdown blocks both cytosolic double-stranded B-form DNA and double-stranded RNA-induced IRF3 activation and IFN-ß production. In contrast, the overexpression of RIOK3 activates IRF3 and induces IFN-ß. RIOK3 functions downstream of TBK1 and upstream of IRF3 activation. Furthermore, RIOK3 physically interacts with both IRF3 and TBK1 and is necessary for the interaction between TBK1 and IRF3. In addition, global transcriptome analysis shows that the expression of many gene involved antiviral responses is dependent on RIOK3. Thus, knockdown of RIOK3 inhibits cellular antiviral responses against both DNA and RNA viruses (herpesvirus and influenza A virus). Our data suggest that RIOK3 plays a critical role in the antiviral type I IFN pathway by bridging TBK1 and IRF3. Importance: The innate immune response, such as the production of type I interferons, acts as the first line of defense, limiting infectious pathogens directly and shaping the adaptive immune response. In this study, we identified RIOK3 as a novel regulator of the antiviral type I interferon pathway. Specifically, we found that RIOK3 physically interacts with TBK1 and IRF3 and bridges the functions between TBK1 and IRF3 in the activation of type I interferon pathway. The identification of a cellular kinase that plays a role the type I interferon pathway adds another level of complexity in the regulation of innate immunity and will have implications for developing novel strategies to combat viral infection.


Asunto(s)
Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/inmunología , Interferón Tipo I/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Línea Celular , Gammaherpesvirinae/inmunología , Humanos , Virus de la Influenza A/inmunología , Unión Proteica , Transcriptoma
11.
Proc Natl Acad Sci U S A ; 108(41): 17117-22, 2011 Oct 11.
Artículo en Inglés | MEDLINE | ID: mdl-21949408

RESUMEN

Glucocorticoids can inhibit inflammation by abrogating the activity of NF-κB, a family of transcription factors that regulates the production of proinflammatory cytokines. To understand the molecular mechanism of repression of NF-κB activity by glucocorticoids, we performed a high-throughput siRNA oligo screen to identify novel genes involved in this process. Here, we report that loss of p53, a tumor suppressor protein, impaired repression of NF-κB target gene transcription by glucocorticoids. Additionally, loss of p53 also impaired transcription of glucocorticoid receptor (GR) target genes, whereas upstream NF-κB and glucocorticoid receptor signaling cascades remained intact. We further demonstrate that p53 loss severely impaired glucocorticoid rescue of death in a mouse model of LPS shock. Our findings unveil a new role for p53 in the repression of NF-κB by glucocorticoids and suggest important implications for treatment of the proinflammatory microenvironments found in tumors with aberrant p53 activity.


Asunto(s)
FN-kappa B/metabolismo , Receptores de Glucocorticoides/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Dexametasona/farmacología , Ensayos Analíticos de Alto Rendimiento , Lipopolisacáridos/toxicidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mapas de Interacción de Proteínas , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Choque Séptico/inducido químicamente , Choque Séptico/genética , Choque Séptico/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética
12.
bioRxiv ; 2024 Jul 10.
Artículo en Inglés | MEDLINE | ID: mdl-39026801

RESUMEN

Defining the subset of cellular factors governing SARS-CoV-2 replication can provide critical insights into viral pathogenesis and identify targets for host-directed antiviral therapies. While a number of genetic screens have previously reported SARS-CoV-2 host dependency factors, these approaches relied on utilizing pooled genome-scale CRISPR libraries, which are biased towards the discovery of host proteins impacting early stages of viral replication. To identify host factors involved throughout the SARS-CoV-2 infectious cycle, we conducted an arrayed genome-scale siRNA screen. Resulting data were integrated with published datasets to reveal pathways supported by orthogonal datasets, including transcriptional regulation, epigenetic modifications, and MAPK signalling. The identified proviral host factors were mapped into the SARS-CoV-2 infectious cycle, including 27 proteins that were determined to impact assembly and release. Additionally, a subset of proteins were tested across other coronaviruses revealing 17 potential pan-coronavirus targets. Further studies illuminated a role for the heparan sulfate proteoglycan perlecan in SARS-CoV-2 viral entry, and found that inhibition of the non-canonical NF-kB pathway through targeting of BIRC2 restricts SARS-CoV-2 replication both in vitro and in vivo. These studies provide critical insight into the landscape of virus-host interactions driving SARS-CoV-2 replication as well as valuable targets for host-directed antivirals.

13.
Front Pharmacol ; 13: 1029093, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36532738

RESUMEN

The translation initiation complex 4F (eIF4F) is a rate-limiting factor in protein synthesis. Alterations in eIF4F activity are linked to several diseases, including cancer and infectious diseases. To this end, coronaviruses require eIF4F complex activity to produce proteins essential for their life cycle. Efforts to target coronaviruses by abrogating translation have been largely limited to repurposing existing eIF4F complex inhibitors. Here, we report the results of a high throughput screen to identify small molecules that disrupt eIF4F complex formation and inhibit coronavirus RNA and protein levels. Of 338,000 small molecules screened for inhibition of the eIF4F-driven, CAP-dependent translation, we identified SBI-1232 and two structurally related analogs, SBI-5844 and SBI-0498, that inhibit human coronavirus OC43 (HCoV-OC43; OC43) with minimal cell toxicity. Notably, gene expression changes after OC43 infection of Vero E6 or A549 cells were effectively reverted upon treatment with SBI-5844 or SBI-0498. Moreover, SBI-5844 or SBI-0498 treatment effectively impeded the eIF4F complex assembly, with concomitant inhibition of newly synthesized OC43 nucleocapsid protein and OC43 RNA and protein levels. Overall, we identify SBI-5844 and SBI-0498 as small molecules targeting the eIF4F complex that may limit coronavirus transcripts and proteins, thereby representing a basis for developing novel therapeutic modalities against coronaviruses.

14.
ACS Infect Dis ; 8(7): 1265-1279, 2022 07 08.
Artículo en Inglés | MEDLINE | ID: mdl-35766385

RESUMEN

There is a pressing need for host-directed therapeutics that elicit broad-spectrum antiviral activities to potentially address current and future viral pandemics. Apratoxin S4 (Apra S4) is a potent Sec61 inhibitor that prevents cotranslational translocation of secretory proteins into the endoplasmic reticulum (ER), leading to anticancer and antiangiogenic activity both in vitro and in vivo. Since Sec61 has been shown to be an essential host factor for viral proteostasis, we tested Apra S4 in cellular models of viral infection, including SARS-CoV-2, influenza A virus, and flaviviruses (Zika, West Nile, and Dengue virus). Apra S4 inhibited viral replication in a concentration-dependent manner and had high potency particularly against SARS-CoV-2 and influenza A virus, with subnanomolar activity in human cells. Characterization studies focused on SARS-CoV-2 revealed that Apra S4 impacted a post-entry stage of the viral life-cycle. Transmission electron microscopy revealed that Apra S4 blocked formation of stacked double-membrane vesicles, the sites of viral replication. Apra S4 reduced dsRNA formation and prevented viral protein production and trafficking of secretory proteins, especially the spike protein. Given the potent and broad-spectrum activity of Apra S4, further preclinical evaluation of Apra S4 and other Sec61 inhibitors as antivirals is warranted.


Asunto(s)
Tratamiento Farmacológico de COVID-19 , Virus de la Influenza A , Infección por el Virus Zika , Virus Zika , Antivirales/farmacología , Antivirales/uso terapéutico , Depsipéptidos , Humanos , Pandemias , SARS-CoV-2 , Infección por el Virus Zika/tratamiento farmacológico
15.
Cell Rep ; 34(2): 108628, 2021 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-33440148

RESUMEN

Recent studies have profiled the innate immune signatures in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and suggest that cellular responses to viral challenge may affect disease severity. Yet the molecular events that underlie cellular recognition and response to SARS-CoV-2 infection remain to be elucidated. Here, we find that SARS-CoV-2 replication induces a delayed interferon (IFN) response in lung epithelial cells. By screening 16 putative sensors involved in sensing of RNA virus infection, we found that MDA5 and LGP2 primarily regulate IFN induction in response to SARS-CoV-2 infection. Further analyses revealed that viral intermediates specifically activate the IFN response through MDA5-mediated sensing. Additionally, we find that IRF3, IRF5, and NF-κB/p65 are the key transcription factors regulating the IFN response during SARS-CoV-2 infection. In summary, these findings provide critical insights into the molecular basis of the innate immune recognition and signaling response to SARS-CoV-2.


Asunto(s)
Inmunidad Innata , Helicasa Inducida por Interferón IFIH1/metabolismo , SARS-CoV-2/fisiología , COVID-19/patología , COVID-19/virología , Línea Celular , Células Epiteliales/citología , Células Epiteliales/inmunología , Células Epiteliales/virología , Humanos , Células Madre Pluripotentes Inducidas/citología , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Interferones/genética , Interferones/metabolismo , ARN Helicasas/metabolismo , Interferencia de ARN , ARN Bicatenario/metabolismo , ARN Interferente Pequeño/metabolismo , SARS-CoV-2/inmunología , SARS-CoV-2/aislamiento & purificación , Transducción de Señal , Factor de Transcripción ReIA/metabolismo , Replicación Viral
16.
Nat Microbiol ; 6(10): 1319-1333, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34556855

RESUMEN

The fate of influenza A virus (IAV) infection in the host cell depends on the balance between cellular defence mechanisms and viral evasion strategies. To illuminate the landscape of IAV cellular restriction, we generated and integrated global genetic loss-of-function screens with transcriptomics and proteomics data. Our multi-omics analysis revealed a subset of both IFN-dependent and independent cellular defence mechanisms that inhibit IAV replication. Amongst these, the autophagy regulator TBC1 domain family member 5 (TBC1D5), which binds Rab7 to enable fusion of autophagosomes and lysosomes, was found to control IAV replication in vitro and in vivo and to promote lysosomal targeting of IAV M2 protein. Notably, IAV M2 was observed to abrogate TBC1D5-Rab7 binding through a physical interaction with TBC1D5 via its cytoplasmic tail. Our results provide evidence for the molecular mechanism utilised by IAV M2 protein to escape lysosomal degradation and traffic to the cell membrane, where it supports IAV budding and growth.


Asunto(s)
Autofagia , Evasión Inmune , Virus de la Influenza A/fisiología , Antivirales/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Interacciones Huésped-Patógeno , Humanos , Virus de la Influenza A/patogenicidad , Lisosomas/metabolismo , Unión Proteica , Proteínas de la Matriz Viral/metabolismo , Replicación Viral , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7
17.
bioRxiv ; 2020 Sep 30.
Artículo en Inglés | MEDLINE | ID: mdl-33024967

RESUMEN

A deficient interferon response to SARS-CoV-2 infection has been implicated as a determinant of severe COVID-19. To identify the molecular effectors that govern interferon control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human interferon stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors that inhibited viral entry, nucleic acid binding proteins that suppressed viral RNA synthesis, and a highly enriched cluster of ER and Golgi-resident ISGs that inhibited viral translation and egress. These included the type II integral membrane protein BST2/tetherin, which was found to impede viral release, and is targeted for immune evasion by SARS-CoV-2 Orf7a protein. Overall, these data define the molecular basis of early innate immune control of viral infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.

18.
bioRxiv ; 2020 Apr 17.
Artículo en Inglés | MEDLINE | ID: mdl-32511357

RESUMEN

The emergence of novel SARS coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of severe pneumonia-like disease designated as coronavirus disease 2019 (COVID-19). To date, more than 2.1 million confirmed cases and 139,500 deaths have been reported worldwide, and there are currently no medical countermeasures available to prevent or treat the disease. As the development of a vaccine could require at least 12-18 months, and the typical timeline from hit finding to drug registration of an antiviral is >10 years, repositioning of known drugs can significantly accelerate the development and deployment of therapies for COVID-19. To identify therapeutics that can be repurposed as SARS-CoV-2 antivirals, we profiled a library of known drugs encompassing approximately 12,000 clinical-stage or FDA-approved small molecules. Here, we report the identification of 30 known drugs that inhibit viral replication. Of these, six were characterized for cellular dose-activity relationships, and showed effective concentrations likely to be commensurate with therapeutic doses in patients. These include the PIKfyve kinase inhibitor Apilimod, cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825, and ONO 5334, and the CCR1 antagonist MLN-3897. Since many of these molecules have advanced into the clinic, the known pharmacological and human safety profiles of these compounds will accelerate their preclinical and clinical evaluation for COVID-19 treatment.

19.
Mol Cell Biol ; 39(3)2019 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-30397075

RESUMEN

The nuclear factor kappa B (NF-κB) family of transcription factors plays a central role in coordinating the expression of genes that control inflammation, immune responses, cell proliferation, and a variety of other biological processes. In an attempt to identify novel regulators of this pathway, we performed whole-genome RNA interference (RNAi) screens in physiologically relevant human macrophages in response to lipopolysaccharide and tumor necrosis factor alpha (TNF-α). The top hit was SNW1, a splicing factor and transcriptional coactivator. SNW1 does not regulate the cytoplasmic components of the NF-κB pathway but complexes with the NF-κB heterodimer in the nucleus for transcriptional activation. We show that SNW1 detaches from its splicing complex (formed with SNRNP200 and SNRNP220) upon NF-κB activation and binds to NF-κB's transcriptional elongation partner p-TEFb. We also show that SNW1 is indispensable for the transcriptional elongation of NF-κB target genes such as the interleukin 8 (IL-8) and TNF genes. SNW1 is a unique protein previously shown to be involved in both splicing and transcription, and in this case, its role involves binding to the NF-κB-p-TEFb complex to facilitate transcriptional elongation of some NF-κB target genes.


Asunto(s)
FN-kappa B/metabolismo , Coactivadores de Receptor Nuclear/genética , Coactivadores de Receptor Nuclear/metabolismo , Regulación de la Expresión Génica , Humanos , Macrófagos/metabolismo , Factor B de Elongación Transcripcional Positiva/metabolismo , Regiones Promotoras Genéticas , Transducción de Señal , Células THP-1 , Factor de Transcripción ReIA/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Activación Transcripcional , Factor de Necrosis Tumoral alfa/metabolismo
20.
Elife ; 82019 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-30717826

RESUMEN

Many viral pathogens target innate sensing cascades and/or cellular transcription factors to suppress antiviral immune responses. Here, we show that the accessory viral protein U (Vpu) of HIV-1 exerts broad immunosuppressive effects by inhibiting activation of the transcription factor NF-κB. Global transcriptional profiling of infected CD4 +T cells revealed that vpu-deficient HIV-1 strains induce substantially stronger immune responses than the respective wild type viruses. Gene set enrichment analyses and cytokine arrays showed that Vpu suppresses the expression of NF-κB targets including interferons and restriction factors. Mutational analyses demonstrated that this immunosuppressive activity of Vpu is independent of its ability to counteract the restriction factor and innate sensor tetherin. However, Vpu-mediated inhibition of immune activation required an arginine residue in the cytoplasmic domain that is critical for blocking NF-κB signaling downstream of tetherin. In summary, our findings demonstrate that HIV-1 Vpu potently suppresses NF-κB-elicited antiviral immune responses at the transcriptional level.


Asunto(s)
VIH-1/crecimiento & desarrollo , VIH-1/inmunología , Interacciones Huésped-Patógeno , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Evasión Inmune , Inmunidad Innata , FN-kappa B/antagonistas & inhibidores , Proteínas Reguladoras y Accesorias Virales/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Células Cultivadas , Regulación hacia Abajo , Humanos , Transcripción Genética
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