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1.
EMBO Rep ; 23(11): e51709, 2022 11 07.
Artículo en Inglés | MEDLINE | ID: mdl-36094794

RESUMEN

Phosphatidylserine (PS) is a critical lipid factor in the assembly and spread of numerous lipid-enveloped viruses. Here, we describe the ability of the Ebola virus (EBOV) matrix protein eVP40 to induce clustering of PS and promote viral budding in vitro, as well as the ability of an FDA-approved drug, fendiline, to reduce PS clustering and subsequent virus budding and entry. To gain mechanistic insight into fendiline inhibition of EBOV replication, multiple in vitro assays were run including imaging, viral budding and viral entry assays. Fendiline lowers PS content in mammalian cells and PS in the plasma membrane, where the ability of VP40 to form new virus particles is greatly lower. Further, particles that form from fendiline-treated cells have altered particle morphology and cannot significantly infect/enter cells. These complementary studies reveal the mechanism by which EBOV matrix protein clusters PS to enhance viral assembly, budding, and spread from the host cell while also laying the groundwork for fundamental drug targeting strategies.


Asunto(s)
Ebolavirus , Fiebre Hemorrágica Ebola , Animales , Fiebre Hemorrágica Ebola/metabolismo , Ebolavirus/fisiología , Fosfatidilserinas/metabolismo , Fendilina/metabolismo , Proteínas de la Matriz Viral/metabolismo , Ensamble de Virus , Análisis por Conglomerados , Mamíferos/metabolismo
2.
J Biol Chem ; 296: 100103, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33214224

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first discovered in December 2019 in Wuhan, China, and expeditiously spread across the globe causing a global pandemic. Research on SARS-CoV-2, as well as the closely related SARS-CoV-1 and MERS coronaviruses, is restricted to BSL-3 facilities. Such BSL-3 classification makes SARS-CoV-2 research inaccessible to the majority of functioning research laboratories in the United States; this becomes problematic when the collective scientific effort needs to be focused on such in the face of a pandemic. However, a minimal system capable of recapitulating different steps of the viral life cycle without using the virus' genetic material could increase accessibility. In this work, we assessed the four structural proteins from SARS-CoV-2 for their ability to form virus-like particles (VLPs) from human cells to form a competent system for BSL-2 studies of SARS-CoV-2. Herein, we provide methods and resources of producing, purifying, fluorescently and APEX2-labeling of SARS-CoV-2 VLPs for the evaluation of mechanisms of viral budding and entry as well as assessment of drug inhibitors under BSL-2 conditions. These systems should be useful to those looking to circumvent BSL-3 work with SARS-CoV-2 yet study the mechanisms by which SARS-CoV-2 enters and exits human cells.


Asunto(s)
Proteínas de la Envoltura de Coronavirus/genética , Proteínas de la Nucleocápside/genética , SARS-CoV-2/crecimiento & desarrollo , Glicoproteína de la Espiga del Coronavirus/genética , Proteínas de la Matriz Viral/genética , Virión/crecimiento & desarrollo , Materiales Biomiméticos/química , Materiales Biomiméticos/metabolismo , Contención de Riesgos Biológicos/clasificación , Proteínas de la Envoltura de Coronavirus/metabolismo , Expresión Génica , Genes Reporteros , Regulación Gubernamental , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Electrónica , Proteínas de la Nucleocápside/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/ultraestructura , Glicoproteína de la Espiga del Coronavirus/metabolismo , Proteínas de la Matriz Viral/metabolismo , Virión/genética , Virión/metabolismo , Virión/ultraestructura , Ensamble de Virus/fisiología , Internalización del Virus , Liberación del Virus/fisiología , Proteína Fluorescente Roja
3.
bioRxiv ; 2020 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-33024964

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first discovered in December 2019 in Wuhan, China and expeditiously spread across the globe causing a global pandemic. While a select agent designation has not been made for SARS-CoV-2, closely related SARS-CoV-1 and MERS coronaviruses are classified as Risk Group 3 select agents, which restricts use of the live viruses to BSL-3 facilities. Such BSL-3 classification make SARS-CoV-2 research inaccessible to the majority of functioning research laboratories in the US; this becomes problematic when the collective scientific effort needs to be focused on such in the face of a pandemic. In this work, we assessed the four structural proteins from SARS-CoV-2 for their ability to form viruslike particles (VLPs) from human cells to form a competent system for BSL-2 studies of SARS-CoV-2. Herein, we provide methods and resources of producing, purifying, fluorescently and APEX2-labeling of SARS-CoV-2 VLPs for the evaluation of mechanisms of viral budding and entry as well as assessment of drug inhibitors under BSL-2 conditions.

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