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 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. After establishing the minimal system requirements for VLP production, we examined their morphological relevance with transmission electron microscopy (TEM). VLPs produced with all four viral structural proteins were approximately 100 nm in diameter and bared the characteristic coronavirus crown or ‘corona’. We next sought to evaluate the entry competency of our VLPs. GFP‐tagged VLPs were generated by fluorescently tagging one of the four structural proteins used to produce VLPs. Once incorporation of the GFP‐tagged protein into VLPs was confirmed, we used these GFP‐VLPs to infect HEK293 cells. GFP‐VLPs indeed did enter HEK293 cells and properly colocalized with endocytic markers Rab5 and LAMP1 in accordance with live virus data. To further evaluate viral entry, we made the VLP entry assay accessible to TEM analysis by replacing the GFP tag with an ascorbate peroxidase (APEX2) tag, which when oxidized produces a dark brown precipitate visible on micrographs. APEX2‐VLPs were found to be entry‐competent as well. In addition to entry, APEX2‐VLPs yield the ability to visualize VLP assembly at the ER‐Golgi intermediary complex (ERGIC) and for the first time we show localization of the structural proteins during SARS‐CoV‐2 VLP assembly, budding, and egress. In total, this research provides ample resources for other BSL‐2 laboratories interested in joining the growing field to try and understand SARS‐CoV‐2 assembly, budding, and entry dynamics, biochemical and biophysical questions on the four structural proteins, and drug screening of viral assembly, budding, and/or entry inhibitors.

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