Host lipids required for Membrane protein matrix formation in new severe acute respiratory syndrome‐related coronavirus (SARS‐CoV‐2), ribonucleocapsid packaging and viral assembly

The current model of SARS‐CoV‐2 assembly proposes a multistage process: (i) the viral RNA coated with the nucleoprotein (N) forms the ribonucleoprotein (RNP), (ii) traffics to the endoplasmic reticulum‐Golgi apparatus intermediate compartment (ERGIC)‐derived vesicles to (iii) interact with the membrane (M) assembled along with spike (S) and envelope (E) proteins. Coronaviruses’ M has been described as required for new virus particle assembly. To date, biochemical and structural information on M are unavailable and key host factors required for M lattice formation, trafficking and interaction with the RNP are poorly characterized. The objective of this research project is to progress our understanding of SARS‐CoV‐2 assembly. Our central hypothesis is that M through its interaction with host lipids coordinates viral protein interactions and RNP packaging as key processes for SARS‐CoV‐2 assembly and spread. The role of lipids as molecules regulating the viral life cycle is poorly investigated. Our current data demonstrates a clear binding of cell expressed M to a Golgi resident sphingolipid. Furthermore, mutations at the sphingolipid binding motif resulted in deficient pull down of M protein by sphingolipid coated beads. In cells, the M sphingolipid‐binding mutant failed to retain S protein at viral assembly membranes resulting in a reduced entry capacity of newly formed virus‐like particles. Our current study is providing a first insight on the direct role of host lipids during SARS‐CoV‐2 assembly, orchestrated by the M protein.

SARS-CoV-2 Viral Budding and Entry can be Modeled Using BSL-2 Level Virus-Like Particles

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.

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

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.

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

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.