RESUMO
Flaviviruses such as Zika virus and West Nile virus have the potential to cause severe neuropathology if they invade the central nervous system. The type I interferon response is well characterized as contributing to control of flavivirus-induced neuropathogenesis. However, the interferon-stimulated gene (ISG) effectors that confer these neuroprotective effects are less well studied. Here, we used an ISG expression screen to identify Shiftless (SHFL, C19orf66) as a potent inhibitor of diverse positive-stranded RNA viruses, including multiple members of the Flaviviridae (Zika, West Nile, dengue, yellow fever, and hepatitis C viruses). In cultured cells, SHFL functions as a viral RNA-binding protein that inhibits viral replication at a step after primary translation of the incoming genome. The murine ortholog, Shfl, is expressed constitutively in multiple tissues, including the central nervous system. In a mouse model of Zika virus infection, Shfl-/- knockout mice exhibit reduced survival, exacerbated neuropathological outcomes, and increased viral replication in the brain and spinal cord. These studies demonstrate that Shfl is an important antiviral effector that contributes to host protection from Zika virus infection and virus-induced neuropathological disease.
Assuntos
Proteínas de Ligação a RNA/metabolismo , Infecção por Zika virus/patologia , Zika virus/metabolismo , Animais , Linhagem Celular , Efeito Citopatogênico Viral , Modelos Animais de Doenças , Suscetibilidade a Doenças/metabolismo , Suscetibilidade a Doenças/virologia , Flavivirus/genética , Infecções por Flavivirus/genética , Infecções por Flavivirus/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fármacos Neuroprotetores/metabolismo , Proteínas de Ligação a RNA/genética , Replicação Viral/fisiologia , Zika virus/patogenicidade , Infecção por Zika virus/genéticaRESUMO
Bats host a large number of zoonotic viruses, including several viruses that are highly pathogenic to other mammals. The mechanisms underlying this rich viral diversity are unknown, but they may be linked to unique immunological features that allow bats to act as asymptomatic viral reservoirs. Vertebrates respond to viral infection by inducing IFNs, which trigger antiviral defenses through IFN-stimulated gene (ISG) expression. Although the IFN system of several bats is characterized at the genomic level, less is known about bat IFN-mediated transcriptional responses. In this article, we show that IFN signaling in bat cells from the black flying fox (Pteropus alecto) consists of conserved and unique ISG expression profiles. In IFN-stimulated cells, bat ISGs comprise two unique temporal subclusters with similar early induction kinetics but distinct late-phase declines. In contrast, human ISGs lack this decline phase and remained elevated for longer periods. Notably, in unstimulated cells, bat ISGs were expressed more highly than their human counterparts. We also found that the antiviral effector 2-5A-dependent endoribonuclease, which is not an ISG in humans, is highly IFN inducible in black flying fox cells and contributes to cell-intrinsic control of viral infection. These studies reveal distinctive innate immune features that may underlie a unique virus-host relationship in bats.
Assuntos
Antivirais/metabolismo , Quirópteros/imunologia , Endorribonucleases/metabolismo , Fatores Reguladores de Interferon/metabolismo , Viroses/imunologia , Animais , Doenças Assintomáticas , Linhagem Celular , Reservatórios de Doenças , Endorribonucleases/genética , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Fatores Reguladores de Interferon/genética , Interferons/metabolismo , Transdução de SinaisRESUMO
Over the past three decades, the Torpedo californica nicotinic acetylcholine receptor (nAChR) has been one of the most extensively studied membrane protein systems. However, the effects of detergent solubilization on nAChR stability and function are poorly understood. The use of lipid-analog detergents for nAChR solubilization has been shown to preserve receptor stability and functionality. The present study used lipid-analog detergents from phospholipid-analog and cholesterol-analog detergent families for solubilization and affinity purification of the receptor and probed nAChR ion channel function using planar lipid bilayers (PLBs) and stability using analytical size exclusion chromatography (A-SEC) in the detergent-solubilized state. We also examined receptor mobility on the lipidic cubic phase (LCP) by measuring the nAChR mobile fraction and diffusion coefficient through fluorescence recovery after photobleaching (FRAP) experiments using lipid-analog and non-lipid-analog detergents. Our results show that it is possible to isolate stable and functional nAChRs using lipid-analog detergents, with characteristic ion channel currents in PLBs and minimal aggregation as observed in A-SEC. Furthermore, fractional mobility and diffusion coefficient values observed in FRAP experiments were similar to the values observed for these parameters in the recently LCP-crystallized ß(2)-adrenergic receptor. The overall results show that phospholipid-analog detergents with 16 carbon acyl-chains support nAChR stability, functionality and LCP mobility.
Assuntos
Detergentes/química , Fosfolipídeos/química , Receptores Nicotínicos/metabolismo , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Colesterol/química , Colesterol/metabolismo , Cromatografia em Gel , Detergentes/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Potenciais da Membrana/fisiologia , Fosfolipídeos/metabolismo , Estabilidade Proteica , Receptores Nicotínicos/isolamento & purificação , Reprodutibilidade dos Testes , Solubilidade , Torpedo/metabolismoRESUMO
Among mammals, bats are particularly rich in zoonotic viruses, including flaviviruses. Certain bat species can be productively yet asymptomatically infected with viruses that cause overt disease in other species. However, little is known about the antiviral effector repertoire in bats relative to other mammals. Here, we report the black flying fox receptor transporter protein 4 (RTP4) as a potent interferon (IFN)-inducible inhibitor of human pathogens in the Flaviviridae family, including Zika, West Nile, and hepatitis C viruses. Mechanistically, RTP4 associates with the flavivirus replicase, binds viral RNA, and suppresses viral genome amplification. Comparative approaches revealed that RTP4 undergoes positive selection, that a flavivirus can mutate to escape RTP4-imposed restriction, and that diverse mammalian RTP4 orthologs exhibit striking patterns of specificity against distinct Flaviviridae members. Our findings reveal an antiviral mechanism that has likely adapted over 100 million years of mammalian evolution to accommodate unique host-virus genetic conflicts.
Assuntos
Antivirais/imunologia , Flavivirus/efeitos dos fármacos , Interações Hospedeiro-Patógeno , Interferons/metabolismo , Interferons/farmacologia , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/farmacologia , Animais , Antivirais/farmacologia , Linhagem Celular , Quirópteros/genética , Quirópteros/virologia , Feminino , Flavivirus/genética , Genoma Viral , Interações Hospedeiro-Patógeno/genética , Humanos , Interferons/genética , Masculino , Mamíferos/genética , Especificidade da Espécie , Replicação Viral , Vírus/efeitos dos fármacos , Vírus/genéticaRESUMO
The endoplasmic reticulum (ER) is an architecturally diverse organelle that serves as a membrane source for the replication of multiple viruses. Flaviviruses, including yellow fever virus, West Nile virus, dengue virus and Zika virus, induce unique single-membrane ER invaginations that house the viral replication machinery1. Whether this virus-induced ER remodelling is vulnerable to antiviral pathways is unknown. Here, we show that flavivirus replication at the ER is targeted by the interferon (IFN) response. Through genome-scale CRISPR screening, we uncovered an antiviral mechanism mediated by a functional gene pairing between IFI6 (encoding IFN-α-inducible protein 6), an IFN-stimulated gene cloned over 30 years ago2, and HSPA5, which encodes the ER-resident heat shock protein 70 chaperone BiP. We reveal that IFI6 is an ER-localized integral membrane effector that is stabilized through interactions with BiP. Mechanistically, IFI6 prophylactically protects uninfected cells by preventing the formation of virus-induced ER membrane invaginations. Notably, IFI6 has little effect on other mammalian RNA viruses, including the related Flaviviridae family member hepatitis C virus, which replicates in double-membrane vesicles that protrude outwards from the ER. These findings support a model in which the IFN response is armed with a membrane-targeted effector that discriminately blocks the establishment of virus-specific ER microenvironments that are required for replication.