RESUMEN
How transcription affects genome 3D organization is not well understood. We found that during influenza A (IAV) infection, rampant transcription rapidly reorganizes host cell chromatin interactions. These changes occur at the ends of highly transcribed genes, where global inhibition of transcription termination by IAV NS1 protein causes readthrough transcription for hundreds of kilobases. In these readthrough regions, elongating RNA polymerase II disrupts chromatin interactions by inducing cohesin displacement from CTCF sites, leading to locus decompaction. Readthrough transcription into heterochromatin regions switches them from the inert (B) to the permissive (A) chromatin compartment and enables transcription factor binding. Data from non-viral transcription stimuli show that transcription similarly affects cohesin-mediated chromatin contacts within gene bodies. Conversely, inhibition of transcription elongation allows cohesin to accumulate at previously transcribed intragenic CTCF sites and to mediate chromatin looping and compaction. Our data indicate that transcription elongation by RNA polymerase II remodels genome 3D architecture.
Asunto(s)
Cromatina/metabolismo , Genoma Humano , Subtipo H5N1 del Virus de la Influenza A/metabolismo , Sitios de Unión , Factor de Unión a CCCTC/química , Factor de Unión a CCCTC/metabolismo , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Cromatina/química , Proteínas Cromosómicas no Histona/metabolismo , Flavonoides/farmacología , Humanos , Interferón beta/farmacología , Macrófagos/citología , Macrófagos/metabolismo , Macrófagos/virología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Piperidinas/farmacología , Unión Proteica , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Interferente Pequeño/metabolismo , Transcripción Genética/efectos de los fármacos , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , CohesinasRESUMEN
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/inmunologíaRESUMEN
Paramyxovirus V proteins are known antagonists of the RIG-I-like receptor (RLR)-mediated interferon induction pathway, interacting with and inhibiting the RLR MDA5. We report interactions between the Nipah virus V protein and both RIG-I regulatory protein TRIM25 and RIG-I. We also observed interactions between these host proteins and the V proteins of measles virus, Sendai virus, and parainfluenza virus. These interactions are mediated by the conserved C-terminal domain of the V protein, which binds to the tandem caspase activation and recruitment domains (CARDs) of RIG-I (the region of TRIM25 ubiquitination) and to the SPRY domain of TRIM25, which mediates TRIM25 interaction with the RIG-I CARDs. Furthermore, we show that V interaction with TRIM25 and RIG-I prevents TRIM25-mediated ubiquitination of RIG-I and disrupts downstream RIG-I signaling to the mitochondrial antiviral signaling protein. This is a novel mechanism for innate immune inhibition by paramyxovirus V proteins, distinct from other known V protein functions such as MDA5 and STAT1 antagonism.IMPORTANCE The host RIG-I signaling pathway is a key early obstacle to paramyxovirus infection, as it results in rapid induction of an antiviral response. This study shows that paramyxovirus V proteins interact with and inhibit the activation of RIG-I, thereby interrupting the antiviral signaling pathway and facilitating virus replication.
Asunto(s)
Proteína 58 DEAD Box/metabolismo , Infecciones por Paramyxoviridae/metabolismo , Paramyxoviridae/fisiología , Transducción de Señal , Factores de Transcripción/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Virales/metabolismo , Replicación Viral , Células A549 , Animales , Proteína 58 DEAD Box/genética , Perros , Células HeLa , Humanos , Células de Riñón Canino Madin Darby , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Infecciones por Paramyxoviridae/genética , Receptores Inmunológicos , Factores de Transcripción/genética , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/genética , Proteínas Virales/genéticaRESUMEN
At every step of their replication cycle influenza viruses depend heavily on their host cells. The multifaceted interactions that occur between the virus and its host cell determine the outcome of the infection, including efficiency of progeny virus production, tropism, and pathogenicity. In order to understand viral disease and develop therapies for influenza it is therefore pertinent to study the intricate interplay between influenza viruses and their required host factors. Here, we review the current knowledge on host cell factors required by influenza virus at the different stages of the viral replication cycle. We also discuss the roles of host factors in zoonotic transmission of influenza viruses and their potential for developing novel antivirals.
Asunto(s)
Interacciones Huésped-Patógeno/genética , Gripe Humana/genética , Gripe Humana/virología , Orthomyxoviridae/crecimiento & desarrollo , Replicación Viral , Humanos , Gripe Humana/tratamiento farmacológico , Orthomyxoviridae/patogenicidad , VirulenciaAsunto(s)
Vacuna nCoV-2019 mRNA-1273/administración & dosificación , Vacuna BNT162/administración & dosificación , COVID-19/epidemiología , ChAdOx1 nCoV-19/administración & dosificación , SARS-CoV-2/patogenicidad , Vacuna nCoV-2019 mRNA-1273/inmunología , Adulto , Vacuna BNT162/inmunología , COVID-19/inmunología , COVID-19/prevención & control , COVID-19/virología , Vacunas contra la COVID-19/inmunología , ChAdOx1 nCoV-19/inmunología , Femenino , Humanos , Inmunización Secundaria , Inmunogenicidad Vacunal , Masculino , SARS-CoV-2/inmunología , SARS-CoV-2/aislamiento & purificación , Carga ViralRESUMEN
UNLABELLED: Bats are important reservoirs for several viruses, many of which cause lethal infections in humans but have reduced pathogenicity in bats. As the innate immune response is critical for controlling viruses, the nature of this response in bats and how it may differ from that in other mammals are of great interest. Using next-generation transcriptome sequencing (mRNA-seq), we profiled the transcriptional response of Pteropus vampyrus bat kidney (PVK) cells to Newcastle disease virus (NDV), an avian paramyxovirus known to elicit a strong innate immune response in mammalian cells. The Pteropus genus is a known reservoir of Nipah virus (NiV) and Hendra virus (HeV). Analysis of the 200 to 300 regulated genes showed that genes for interferon (IFN) and antiviral pathways are highly upregulated in NDV-infected PVK cells, including genes for beta IFN, RIG-I, MDA5, ISG15, and IRF1. NDV-infected cells also upregulated several genes not previously characterized to be antiviral, such as RND1, SERTAD1, CHAC1, and MORC3. In fact, we show that MORC3 is induced by both IFN and NDV infection in PVK cells but is not induced by either stimulus in human A549 cells. In contrast to NDV infection, HeV and NiV infection of PVK cells failed to induce these innate immune response genes. Likewise, an attenuated response was observed in PVK cells infected with recombinant NDVs expressing the NiV IFN antagonist proteins V and W. This study provides the first global profile of a robust virus-induced innate immune response in bats and indicates that henipavirus IFN antagonist mechanisms are likely active in bat cells. IMPORTANCE: Bats are the reservoir host for many highly pathogenic human viruses, including henipaviruses, lyssaviruses, severe acute respiratory syndrome coronavirus, and filoviruses, and many other viruses have also been isolated from bats. Viral infections are reportedly asymptomatic or heavily attenuated in bat populations. Despite their ecological importance to viral maintenance, research into their immune system and mechanisms for viral control has only recently begun. Nipah virus and Hendra virus are two paramyxoviruses associated with high mortality rates in humans and whose reservoir is the Pteropus genus of bats. Greater knowledge of the innate immune response of P. vampyrus bats to viral infection may elucidate how bats serve as a reservoir for so many viruses.
Asunto(s)
Quirópteros/inmunología , Reservorios de Enfermedades/virología , Perfilación de la Expresión Génica , Infecciones por Henipavirus/inmunología , Inmunidad Innata , Interferones/inmunología , Virus Nipah/inmunología , Animales , Quirópteros/genética , Quirópteros/virología , Virus Hendra/genética , Virus Hendra/inmunología , Virus Hendra/fisiología , Infecciones por Henipavirus/genética , Infecciones por Henipavirus/virología , Humanos , Evasión Inmune , Interferones/genética , Virus de la Enfermedad de Newcastle/genética , Virus de la Enfermedad de Newcastle/inmunología , Virus de la Enfermedad de Newcastle/fisiología , Virus Nipah/genética , Virus Nipah/fisiologíaRESUMEN
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 VirusRESUMEN
Arenaviruses merit significant interest as important human pathogens, since several of them cause severe hemorrhagic fever disease that is associated with high morbidity and significant mortality. Currently, there are no FDA-licensed arenavirus vaccines available, and current antiarenaviral therapy is limited to an off-labeled use of the nucleoside analog ribavirin, which has limited prophylactic efficacy. The pyrimidine biosynthesis inhibitor A3, which was identified in a high-throughput screen for compounds that blocked influenza virus replication, exhibits a broad-spectrum antiviral activity against negative- and positive-sense RNA viruses, retroviruses, and DNA viruses. In this study, we evaluated the antiviral activity of A3 against representative Old World (lymphocytic choriomeningitis virus) and New World (Junin virus) arenaviruses in rodent, monkey, and human cell lines. We show that A3 is significantly more efficient than ribavirin in controlling arenavirus multiplication and that the A3 inhibitory effect is in part due to its ability to interfere with viral RNA replication and transcription. We document an additive antiarenavirus effect of A3 and ribavirin, supporting the potential combination therapy of ribavirin and pyrimidine biosynthesis inhibitors for the treatment of arenavirus infections.
Asunto(s)
Antivirales/farmacología , Infecciones por Arenaviridae/virología , Arenavirus/efectos de los fármacos , Pirimidinas/antagonistas & inhibidores , Animales , Infecciones por Arenaviridae/metabolismo , Arenavirus/genética , Arenavirus/fisiología , Línea Celular , Evaluación Preclínica de Medicamentos , Humanos , Pirimidinas/biosíntesis , Replicación Viral/efectos de los fármacosRESUMEN
We previously performed a small interfering RNA (siRNA) screen and identified serum- and glucocorticoid-regulated kinase 1 (SGK1) as a host factor required for influenza A virus replication. However, the role of SGK1 in the influenza viral life cycle has never been examined. In this study, we demonstrate that SGK1 is required for optimal replication of influenza virus, using the SGK1 inhibitor GSK 650394 and SGK1-specific siRNAs. We also demonstrate that SGK1 is required for viral ribonucleoprotein nuclear export.
Asunto(s)
Transporte Activo de Núcleo Celular , Interacciones Huésped-Patógeno , Proteínas Inmediatas-Precoces/metabolismo , Virus de la Influenza A/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Ribonucleoproteínas/metabolismo , Replicación Viral , Benzoatos/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/metabolismo , Línea Celular , Técnicas de Silenciamiento del Gen , Humanos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
Influenza viruses continue to pose a major public health threat worldwide and options for antiviral therapy are limited by the emergence of drug-resistant virus strains. The antiviral cytokine, interferon (IFN) is an essential mediator of the innate immune response and influenza viruses, like many viruses, have evolved strategies to evade this response, resulting in increased replication and enhanced pathogenicity. A cell-based assay that monitors IFN production was developed and applied in a high-throughput compound screen to identify molecules that restore the IFN response to influenza virus infected cells. We report the identification of compound ASN2, which induces IFN only in the presence of influenza virus infection. ASN2 preferentially inhibits the growth of influenza A viruses, including the 1918 H1N1, 1968 H3N2 and 2009 H1N1 pandemic strains and avian H5N1 virus. In vivo, ASN2 partially protects mice challenged with a lethal dose of influenza A virus. Surprisingly, we found that the antiviral activity of ASN2 is not dependent on IFN production and signaling. Rather, its IFN-inducing property appears to be an indirect effect resulting from ASN2-mediated inhibition of viral polymerase function, and subsequent loss of the expression of the viral IFN antagonist, NS1. Moreover, we identified a single amino acid mutation at position 499 of the influenza virus PB1 protein that confers resistance to ASN2, suggesting that PB1 is the direct target. This two-pronged antiviral mechanism, consisting of direct inhibition of virus replication and simultaneous activation of the host innate immune response, is a unique property not previously described for any single antiviral molecule.
Asunto(s)
Antivirales/farmacología , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Virus de la Influenza A/efectos de los fármacos , Interferones/biosíntesis , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Animales , Antivirales/química , Células Cultivadas , Perros , Inhibidores Enzimáticos/química , Haplorrinos , Humanos , Indoles/química , Virus de la Influenza A/fisiología , Ratones , Ratones Endogámicos BALB C , Replicación Viral/efectos de los fármacosRESUMEN
Compound A3 was identified in a high-throughput screen for inhibitors of influenza virus replication. It displays broad-spectrum antiviral activity, and at noncytotoxic concentrations it is shown to inhibit the replication of negative-sense RNA viruses (influenza viruses A and B, Newcastle disease virus, and vesicular stomatitis virus), positive-sense RNA viruses (Sindbis virus, hepatitis C virus, West Nile virus, and dengue virus), DNA viruses (vaccinia virus and human adenovirus), and retroviruses (HIV). In contrast to mammalian cells, inhibition of viral replication by A3 is absent in chicken cells, which suggests species-specific activity of A3. Correspondingly, the antiviral activity of A3 can be linked to a cellular protein, dihydroorotate dehydrogenase (DHODH), which is an enzyme in the de novo pyrimidine biosynthesis pathway. Viral replication of both RNA and DNA viruses can be restored in the presence of excess uracil, which promotes pyrimidine salvage, or excess orotic acid, which is the product of DHODH in the de novo pyrimidine biosynthesis pathway. Based on these findings, it is proposed that A3 acts by depleting pyrimidine pools, which are crucial for efficient virus replication.
Asunto(s)
Antivirales/farmacología , Indoles/farmacología , Oxadiazoles/farmacología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Pirimidinas/biosíntesis , Replicación Viral/efectos de los fármacos , Virus/efectos de los fármacos , Animales , Autorradiografía , Carbamatos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Pollos , Cartilla de ADN/genética , Dihidroorotato Deshidrogenasa , Furanos , Humanos , Lamivudine , Mamíferos , Nevirapina , Pirimidinas/metabolismo , Pirrolidinonas , Raltegravir Potásico , Especificidad de la Especie , Sulfonamidas , Factores de Tiempo , Virus/crecimiento & desarrollo , beta-Galactosidasa/metabolismoRESUMEN
In the quest for heightened protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, we engineered a prototype vaccine utilizing the plant expression system of Nicotiana benthamiana, to produce a recombinant SARS-CoV-2 virus-like particle (VLP) vaccine presenting the S-protein from the Beta (B.1.351) variant of concern (VOC). This innovative vaccine, formulated with either a squalene oil-in-water emulsion or a synthetic CpG oligodeoxynucleotide adjuvant, demonstrated efficacy in a golden Syrian Hamster challenge model. The Beta VLP vaccine induced a robust humoral immune response, with serum exhibiting neutralization not only against SARS-CoV-2 Beta but also cross-neutralizing Delta and Omicron pseudoviruses. Protective efficacy was demonstrated, evidenced by reduced viral RNA copies and mitigated weight loss and lung damage compared to controls. This compelling data instills confidence in the creation of a versatile platform for the local manufacturing of potential pan-sarbecovirus vaccines, against evolving viral threats.
Asunto(s)
COVID-19 , Animales , Cricetinae , Humanos , COVID-19/prevención & control , Mesocricetus , SARS-CoV-2 , Vacunas contra la COVID-19/genética , Glicoproteína de la Espiga del Coronavirus , Anticuerpos Antivirales , Anticuerpos NeutralizantesRESUMEN
Molecular farming of vaccines has been heralded as a cheap, safe and scalable production platform. In reality, however, differences in the plant biosynthetic machinery, compared to mammalian cells, can complicate the production of viral glycoproteins. Remodelling the secretory pathway presents an opportunity to support key post-translational modifications, and to tailor aspects of glycosylation and glycosylation-directed folding. In this study, we applied an integrated host and glyco-engineering approach, NXS/T Generation™, to produce a SARS-CoV-2 prefusion spike trimer in Nicotiana benthamiana as a model antigen from an emerging virus. The size exclusion-purified protein exhibited a characteristic prefusion structure when viewed by transmission electron microscopy, and this was indistinguishable from the equivalent mammalian cell-produced antigen. The plant-produced protein was decorated with under-processed oligomannose N-glycans and exhibited a site occupancy that was comparable to the equivalent protein produced in mammalian cell culture. Complex-type glycans were almost entirely absent from the plant-derived material, which contrasted against the predominantly mature, complex glycans that were observed on the mammalian cell culture-derived protein. The plant-derived antigen elicited neutralizing antibodies against both the matched Wuhan and heterologous Delta SARS-CoV-2 variants in immunized hamsters, although titres were lower than those induced by the comparator mammalian antigen. Animals vaccinated with the plant-derived antigen exhibited reduced viral loads following challenge, as well as significant protection from SARS-CoV-2 disease as evidenced by reduced lung pathology, lower viral loads and protection from weight loss. Nonetheless, animals immunized with the mammalian cell-culture-derived protein were better protected in this challenge model suggesting that more faithfully reproducing the native glycoprotein structure and associated glycosylation of the antigen may be desirable.
RESUMEN
The SARS-CoV-2 pandemic demonstrated the need for potent and broad-spectrum vaccines. This study reports the development and testing of a lumpy skin disease virus (LSDV)-vectored vaccine against SARS-CoV-2, utilizing stabilized spike and conserved nucleocapsid proteins as antigens to develop robust immunogenicity. Construction of the vaccine (LSDV-SARS2-S,N) was confirmed by polymerase chain reaction (PCR) amplification and sequencing. In vitro characterization confirmed that cells infected with LSDV-SARS2-S,N expressed SARS-CoV-2 spike and nucleocapsid protein. In BALB/c mice, the vaccine elicited high magnitude IFN-γ ELISpot responses (spike: 2808 SFU/106 splenocytes) and neutralizing antibodies (ID50 = 6552). Testing in hamsters, which emulate human COVID-19 disease progression, showed the development of high titers of neutralizing antibodies against the Wuhan and Delta SARS-CoV-2 variants (Wuhan ID50 = 2905; Delta ID50 = 4648). Additionally, hamsters vaccinated with LSDV-SARS2-S,N displayed significantly less weight loss, lung damage, and reduced viral RNA copies following SARS-CoV-2 infection with the Delta variant as compared to controls, demonstrating protection against disease. These data demonstrate that LSDV-vectored vaccines display promise as an effective SARS-CoV-2 vaccine and as a potential vaccine platform for communicable diseases in humans and animals. Further efficacy testing and immune response analysis, particularly in non-human primates, are warranted.
Asunto(s)
COVID-19 , Virus de la Dermatosis Nodular Contagiosa , Vacunas , Animales , Cricetinae , Bovinos , Ratones , Humanos , SARS-CoV-2/genética , Vacunas contra la COVID-19 , COVID-19/prevención & control , Anticuerpos Neutralizantes , Ratones Endogámicos BALB C , Proteínas de la Nucleocápside , Anticuerpos Antivirales , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Influenza A Virus (IAV) is a recurring respiratory virus with limited availability of antiviral therapies. Understanding host proteins essential for IAV infection can identify targets for alternative host-directed therapies (HDTs). Using affinity purification-mass spectrometry and global phosphoproteomic and protein abundance analyses using three IAV strains (pH1N1, H3N2, H5N1) in three human cell types (A549, NHBE, THP-1), we map 332 IAV-human protein-protein interactions and identify 13 IAV-modulated kinases. Whole exome sequencing of patients who experienced severe influenza reveals several genes, including scaffold protein AHNAK, with predicted loss-of-function variants that are also identified in our proteomic analyses. Of our identified host factors, 54 significantly alter IAV infection upon siRNA knockdown, and two factors, AHNAK and coatomer subunit COPB1, are also essential for productive infection by SARS-CoV-2. Finally, 16 compounds targeting our identified host factors suppress IAV replication, with two targeting CDK2 and FLT3 showing pan-antiviral activity across influenza and coronavirus families. This study provides a comprehensive network model of IAV infection in human cells, identifying functional host targets for pan-viral HDT.
Asunto(s)
COVID-19 , Subtipo H5N1 del Virus de la Influenza A , Virus de la Influenza A , Gripe Humana , Humanos , Virus de la Influenza A/genética , Gripe Humana/genética , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/metabolismo , Proteómica , Replicación Viral/genética , SARS-CoV-2 , Antivirales/metabolismo , Interacciones Huésped-Patógeno/genéticaRESUMEN
Increasing antiviral drug resistance is a major concern for treating influenza, especially in a pandemic setting when the availability of a protective vaccine is uncertain. Resistance is often an issue with drugs directed at viral proteins and for small RNA viruses; there are also a limited number of viral proteins that are amenable to inhibition by a small molecule. A new approach that is gaining support is that cellular proteins, which facilitate virus replication, may be used as alternative targets. Whereas drugs directed at viral proteins tend to be virus-specific, drugs directed at host targets have the potential to have broad-spectrum antiviral activity as many viruses may share a dependency on that host function. For influenza virus, we have very limited knowledge of which cellular factors are involved in virus replication, let alone which of these have suitable properties to serve as drug targets. Through the use of high-throughput RNA interference screens, several studies have addressed this gap in our knowledge. The resulting datasets provide new insight into host pathways that are involved in the influenza virus replication cycle and identify specific host factors in these pathways that may serve as potential targets for future antiviral drug development.
Asunto(s)
Antivirales/farmacología , Interacciones Huésped-Patógeno/efectos de los fármacos , Orthomyxoviridae/patogenicidad , Humanos , Gripe Humana/tratamiento farmacológicoRESUMEN
Molecular responses to influenza A virus (IAV) infections vary between mammalian species. To identify conserved and species-specific molecular responses, we perform a comparative study of transcriptomic data derived from blood cells, primary epithelial cells, and lung tissues collected from IAV-infected humans, ferrets, and mice. The molecular responses in the human host have unique functions such as antigen processing that are not observed in mice or ferrets. Highly conserved gene coexpression modules across the three species are enriched for IAV infection-induced pathways including cell cycle and interferon (IFN) signaling. TDRD7 is predicted as an IFN-inducible host factor that is up-regulated upon IAV infection in the three species. TDRD7 is required for antiviral IFN response, potentially modulating IFN signaling via the JAK/STAT/IRF9 pathway. Identification of the common and species-specific molecular signatures, networks, and regulators of IAV infection provides insights into host-defense mechanisms and will facilitate the development of novel therapeutic interventions against IAV infection.
Asunto(s)
Enfermedades Transmisibles , Virus de la Influenza A , Gripe Humana , Infecciones por Orthomyxoviridae , Animales , Antivirales , Hurones/metabolismo , Humanos , Virus de la Influenza A/fisiología , Gripe Humana/genética , Interferones/metabolismo , Ratones , Infecciones por Orthomyxoviridae/genética , RibonucleoproteínasRESUMEN
Nipah virus is an emerging pathogen that causes severe disease in humans. It expresses several antagonist proteins that subvert the immune response and that may contribute to its pathogenicity. Studies of its biology are difficult due to its high pathogenicity and requirement for biosafety level 4 containment. We integrated experimental and computational methods to elucidate the effects of Nipah virus immune antagonists. Individual Nipah virus immune antagonists (phosphoprotein and V and W proteins) were expressed from recombinant Newcastle disease viruses, and the responses of infected human monocyte-derived dendritic cells were determined. We developed an ordinary differential equation model of the infectious process that that produced results with a high degree of correlation with these experimental results. In order to simulate the effects of wild-type virus, the model was extended to incorporate published experimental data on the time trajectories of immune-antagonist production. These data showed that the RNA-editing mechanism utilized by the wild-type Nipah virus to produce immune antagonists leads to a delay in the production of the most effective immune antagonists, V and W. Model simulations indicated that this delay caused a disconnection between attenuation of the antiviral response and suppression of inflammation. While the antiviral cytokines were efficiently suppressed at early time points, some early inflammatory cytokine production occurred, which would be expected to increase vascular permeability and promote virus spread and pathogenesis. These results suggest that Nipah virus has evolved a unique immune-antagonist strategy that benefits from controlled expression of multiple antagonist proteins with various potencies.
Asunto(s)
Células Dendríticas/virología , Sistema Inmunológico/virología , Modelos Teóricos , Virus Nipah/inmunología , Células Dendríticas/inmunología , Humanos , Virus Nipah/patogenicidad , Fosfoproteínas/inmunología , Edición de ARN , Factores de Tiempo , Proteínas Virales/inmunología , Proteínas Estructurales Virales/inmunologíaRESUMEN
Nipah virus (NiV) is predicted to encode four proteins from its P gene (P, V, W, and C) via mRNA editing and an alternate open reading frame. By use of specific antibodies, the expression of the V, W, and C proteins in NiV-infected cells has now been confirmed. Analysis of the P-gene transcripts shows a ratio of P:V:W mRNA of 1:1:1, but this differs over time, with greater proportions of V and W transcripts observed as the infection progresses. Eighty-two percent of transcripts are edited, with up to 11 G insertions observed. This exceptionally high editing frequency ensures expression of the V and W proteins.
Asunto(s)
Regulación Viral de la Expresión Génica , Virus Nipah/fisiología , Fosfoproteínas/biosíntesis , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Proteínas Virales/biosíntesis , Proteínas Estructurales Virales/biosíntesis , Línea Celular , Humanos , Mutagénesis InsercionalRESUMEN
The Nipah virus (NiV) phosphoprotein (P) gene encodes the C, P, V, and W proteins. P, V, and W, have in common an amino-terminal domain sufficient to bind STAT1, inhibiting its interferon (IFN)-induced tyrosine phosphorylation. P is also essential for RNA-dependent RNA polymerase function. C is encoded by an alternate open reading frame (ORF) within the common amino-terminal domain. Mutations within residues 81 to 113 of P impaired its polymerase cofactor function, as assessed by a minireplicon assay, but these mutants retained STAT1 inhibitory function. Mutations within the residue 114 to 140 region were identified that abrogated interaction with and inhibition of STAT1 by P, V, and W without disrupting P polymerase cofactor function. Recombinant NiVs were then generated. A G121E mutation, which abrogated inhibition of STAT1, was introduced into a C protein knockout background (C(ko)) because the mutation would otherwise also alter the overlapping C ORF. In cell culture, relative to the wild-type virus, the C(ko) mutation proved attenuating but the G121E mutant virus replicated identically to the C(ko) virus. In cells infected with the wild-type and C(ko) viruses, STAT1 was nuclear despite the absence of tyrosine phosphorylation. This latter observation mirrors what has been seen in cells expressing NiV W. In the G121E mutant virus-infected cells, STAT1 was not phosphorylated and was cytoplasmic in the absence of IFN stimulation but became tyrosine phosphorylated and nuclear following IFN addition. These data demonstrate that the gene for NiV P encodes functions that sequester inactive STAT1 in the nucleus, preventing its activation and suggest that the W protein is the dominant inhibitor of STAT1 in NiV-infected cells.