SARS-CoV-2 viral budding and entry can be modeled using BSL-2 level virus-like particles

Dhabaleswar Patra,Caroline B. Plescia,Ranjan Sengupta,Robert V. Stahelin,Souad Amiar,Yuan Su,Emily A. David

doi:10.1074/jbc.ra120.016148

Dhabaleswar Patra, Caroline B. Plescia + Show 5 more

Open Access

https://doi.org/10.1074/jbc.ra120.016148

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Abstract

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.

Highlights

Severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) was first discovered in December 2019 in Wuhan, China, and expeditiously spread across the globe causing a global pandemic
We examined the ability of viral structural proteins to produce virus-like particles (VLPs) and found that M alone was not sufficient to support VLP formation, but coexpression of M with N or S was the minimal requirement for VLP formation
E protein was found to enrich VLP production, highlighting the important role E must play in viral assembly and release

Summary

Introduction

Severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) was first discovered in December 2019 in Wuhan, China, and expeditiously spread across the globe causing a global pandemic. 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. 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. The role of E in assembly and budding is enigmatic, though it has been shown to be crucial for proper assembly of SARSCoV-1 viral particles and serves as a viroporin altering ion transport [2, 8, 9]

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