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

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 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. 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.

SARS CoV-2 Nucleoprotein Enhances the Infectivity of Lentiviral Spike Particles

“…Significant advances have been achieved in recent times, ranging from optimization of pseudotyping protocols to the application of these for screening entry inhibitors (Hu et al, 2020). The roles of structural proteins such as M, E, and N in assisting viral assembly and particle formation have also been explored (Plescia et al, 2020). Along similar lines, leading studies have deciphered the manifold cellular associations and interactions of various SARS CoV-2 genes, in processes as diverse as host translation inhibition, disruption of splicing, immune evasion, and protein trafficking, among others (Banerjee et al, 2020;Schubert et al, 2020;Xia et al, 2020;Flower et al, 2021).…”

“…Numerous studies have demonstrated that the spike protein may be employed to generate stably pseudotyped lentiviral, retroviral, and vesicular stomatitis virus particles (Crawford et al, 2020;Johnson et al, 2020;Plescia et al, 2020;Shang et al, 2020;Neerukonda et al, 2021). Given that live SARS CoV-2 is a biosafety level-3 agent, these pseudotyping approaches have greatly facilitated investigations undertaken within lower containment facilities.…”

A Summary of the SARS-CoV-2 Vaccines and Technologies Available or under Development

“…The VLPs are composed of several structural viral proteins (co-expressed or admixed) [ 145 , 146 ]. These VLPs are manufactured viral proteins—S, M, and E with or without N—co-expressed and budding from eukaryotic producer cells [ 147 , 148 ]. These particles are similar to the virus but lack the viral genome.…”

SARS CoV-2 nucleoprotein enhances the infectivity of lentiviral spike particles

“…Significant advances have been achieved in recent times, ranging from optimization of pseudotyping protocols to the application of these for screening entry inhibitors 9 . The roles of structural proteins such as M, E, and N in assisting viral assembly and particle formation have also been explored 6 . Along similar lines, leading studies have deciphered the manifold cellular associations and interactions of various SARS CoV-2 genes, in processes as diverse as host translation inhibition, disruption of splicing, immune evasion, and protein trafficking, among others [15][16][17][18] .…”

“…Numerous studies have demonstrated that the spike protein may be employed to generate stably pseudotyped lentiviral, retroviral, and vesicular stomatitis virus particles [3][4][5][6][7] . Given that live SARS CoV-2 is a biosafety level-3 agent, these pseudotyping approaches have greatly facilitated investigations undertaken within lower containment facilities.…”