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.

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.

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.

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 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.

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is the pathogen responsible for COVID‐19, a disease which has resulted in the death of millions worldwide. COVID‐19 ranges in severity of symptoms which are related to the inflammatory response. Although the field has progressed significantly, there is still a critical gap in understanding virus‐host lipid interactions that contribute to viral assembly and budding of this lipid‐enveloped virus. There are four structural proteins encoded in the SARS‐CoV‐2 genome: membrane, envelope (E), nucleocapsid, and spike. These proteins give shape to the bilayer lipid coat that encapsulate the genomic core necessary for virus infection and replication. These proteins are essential for viral reproduction but how they interact with each other and how lipid species regulate their assembly is not well understood. It is established that inflammation is common during infection. One enzyme that participates in this process is ceramide kinase (CERK), which synthesizes the pro‐inflammatory lipid, ceramide‐1‐phosphate (C1P). CERK is in fact a therapeutic target for a variety of inflammatory disorders. My primary goal is to define the relationship between CERK activity and SARS‐CoV‐2 assembly. We aim to understand the impact that CERK activity has on the localization and assembly of SARS‐CoV‐2 structural proteins. I am interested in how CERK regulates E protein localization and formation of virus‐like particles and the process by which E‐lipid interactions contributes to membrane curvature changes necessary for formation of new viral particles. Overall, I anticipate that this study will help define the relationships between a host enzyme and host membranes and the assembly of SARS‐CoV‐2.