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1.
Nature ; 630(8017): 712-719, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38839957

RESUMEN

Genetic screens have transformed our ability to interrogate cellular factor requirements for viral infections1,2, but most current approaches are limited in their sensitivity, biased towards early stages of infection and provide only simplistic phenotypic information that is often based on survival of infected cells2-4. Here, by engineering human cytomegalovirus to express single guide RNA libraries directly from the viral genome, we developed virus-encoded CRISPR-based direct readout screening (VECOS), a sensitive, versatile, viral-centric approach that enables profiling of different stages of viral infection in a pooled format. Using this approach, we identified hundreds of host dependency and restriction factors and quantified their direct effects on viral genome replication, viral particle secretion and infectiousness of secreted particles, providing a multi-dimensional perspective on virus-host interactions. These high-resolution measurements reveal that perturbations altering late stages in the life cycle of human cytomegalovirus (HCMV) mostly regulate viral particle quality rather than quantity, establishing correct virion assembly as a critical stage that is heavily reliant on virus-host interactions. Overall, VECOS facilitates systematic high-resolution dissection of the role of human proteins during the infection cycle, providing a roadmap for in-depth study of host-herpesvirus interactions.


Asunto(s)
Citomegalovirus , Genoma Viral , Virión , Replicación Viral , Citomegalovirus/genética , Citomegalovirus/fisiología , Humanos , Genoma Viral/genética , Replicación Viral/genética , Virión/genética , Virión/metabolismo , Infecciones por Citomegalovirus/virología , Sistemas CRISPR-Cas/genética , Interacciones Huésped-Patógeno/genética , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Ensamble de Virus/genética , Liberación del Virus , Línea Celular
2.
Mol Cell ; 83(3): 481-495, 2023 02 02.
Artículo en Inglés | MEDLINE | ID: mdl-36334591

RESUMEN

Viral reproduction is contingent on viral protein synthesis that relies on the host ribosomes. As such, viruses have evolved remarkable strategies to hijack the host translational apparatus in order to favor viral protein production and to interfere with cellular innate defenses. Here, we describe the approaches viruses use to exploit the translation machinery, focusing on commonalities across diverse viral families, and discuss the functional relevance of this process. We illustrate the complementary strategies host cells utilize to block viral protein production and consider how cells ensure an efficient antiviral response that relies on translation during this tug of war over the ribosome. Finally, we highlight potential roles mRNA modifications and ribosome quality control play in translational regulation and innate immunity. We address these topics in the context of the COVID-19 pandemic and focus on the gaps in our current knowledge of these mechanisms, specifically in viruses with pandemic potential.


Asunto(s)
COVID-19 , Biosíntesis de Proteínas , Virosis , Virus , Humanos , COVID-19/genética , COVID-19/inmunología , Pandemias , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/inmunología , ARN Viral/genética , ARN Viral/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunología , Virosis/genética , Virosis/inmunología , Virus/genética , Virus/inmunología , Ribosomas/genética , Ribosomas/inmunología , Ribosomas/virología
3.
Cell Rep ; 39(11): 110954, 2022 06 14.
Artículo en Inglés | MEDLINE | ID: mdl-35671758

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to shutoff of protein synthesis, and nsp1, a central shutoff factor in coronaviruses, inhibits cellular mRNA translation. However, the diverse molecular mechanisms employed by nsp1 as well as its functional importance are unresolved. By overexpressing various nsp1 mutants and generating a SARS-CoV-2 mutant, we show that nsp1, through inhibition of translation and induction of mRNA degradation, targets translated cellular mRNA and is the main driver of host shutoff during infection. The propagation of nsp1 mutant virus is inhibited exclusively in cells with intact interferon (IFN) pathway as well as in vivo, in hamsters, and this attenuation is associated with stronger induction of type I IFN response. Therefore, although nsp1's shutoff activity is broad, it plays an essential role, specifically in counteracting the IFN response. Overall, our results reveal the multifaceted approach nsp1 uses to shut off cellular protein synthesis and uncover nsp1's explicit role in blocking the IFN response.


Asunto(s)
COVID-19 , Proteínas no Estructurales Virales , Línea Celular , Humanos , Estabilidad del ARN , SARS-CoV-2 , Proteínas no Estructurales Virales/metabolismo
4.
bioRxiv ; 2022 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-35313595

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 19 (COVID-19) pandemic. Despite its urgency, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis and its ability to antagonize innate immune responses. SARS-CoV-2 leads to shutoff of cellular protein synthesis and over-expression of nsp1, a central shutoff factor in coronaviruses, inhibits cellular gene translation. However, the diverse molecular mechanisms nsp1 employs as well as its functional importance in infection are still unresolved. By overexpressing various nsp1 mutants and generating a SARS-CoV-2 mutant in which nsp1 does not bind ribosomes, we untangle the effects of nsp1. We uncover that nsp1, through inhibition of translation and induction of mRNA degradation, is the main driver of host shutoff during SARS-CoV-2 infection. Furthermore, we find the propagation of nsp1 mutant virus is inhibited specifically in cells with intact interferon (IFN) response as well as in-vivo , in infected hamsters, and this attenuation is associated with stronger induction of type I IFN response. This illustrates that nsp1 shutoff activity has an essential role mainly in counteracting the IFN response. Overall, our results reveal the multifaceted approach nsp1 uses to shut off cellular protein synthesis and uncover the central role it plays in SARS-CoV-2 pathogenesis, explicitly through blockage of the IFN response.

5.
Nature ; 594(7862): 240-245, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33979833

RESUMEN

The coronavirus SARS-CoV-2 is the cause of the ongoing pandemic of COVID-191. Coronaviruses have developed a variety of mechanisms to repress host mRNA translation to allow the translation of viral mRNA, and concomitantly block the cellular innate immune response2,3. Although several different proteins of SARS-CoV-2 have previously been implicated in shutting off host expression4-7, a comprehensive picture of the effects of SARS-CoV-2 infection on cellular gene expression is lacking. Here we combine RNA sequencing, ribosome profiling and metabolic labelling of newly synthesized RNA to comprehensively define the mechanisms that are used by SARS-CoV-2 to shut off cellular protein synthesis. We show that infection leads to a global reduction in translation, but that viral transcripts are not preferentially translated. Instead, we find that infection leads to the accelerated degradation of cytosolic cellular mRNAs, which facilitates viral takeover of the mRNA pool in infected cells. We reveal that the translation of transcripts that are induced in response to infection (including innate immune genes) is impaired. We demonstrate this impairment is probably mediated by inhibition of nuclear mRNA export, which prevents newly transcribed cellular mRNA from accessing ribosomes. Overall, our results uncover a multipronged strategy that is used by SARS-CoV-2 to take over the translation machinery and to suppress host defences.


Asunto(s)
COVID-19/metabolismo , COVID-19/virología , Interacciones Huésped-Patógeno , Biosíntesis de Proteínas , SARS-CoV-2/patogenicidad , Regiones no Traducidas 5'/genética , COVID-19/genética , COVID-19/inmunología , Línea Celular , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata/genética , Biosíntesis de Proteínas/genética , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Ribosomas/metabolismo , Proteínas no Estructurales Virales/metabolismo
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