S-acylation controls SARS-CoV-2 membrane lipid organization and enhances infectivity

SARS-CoV-2 virions are surrounded by a lipid bilayer that contains membrane proteins such as spike, responsible for target-cell binding and virus fusion. We found that during SARS-CoV-2 infection, spike becomes lipid modified, through the sequential action of the S-acyltransferases ZDHHC20 and 9. Particularly striking is the rapid acylation of spike on 10 cytosolic cysteines within the ER and Golgi. Using a combination of computational, lipidomics, and biochemical approaches, we show that this massive lipidation controls spike biogenesis and degradation, and drives the formation of localized ordered cholesterol and sphingolipid-rich lipid nanodomains in the early Golgi, where viral budding occurs. Finally, S-acylation of spike allows the formation of viruses with enhanced fusion capacity. Our study points toward S-acylating enzymes an

Distinct Roles for Carbohydrate and Protein Receptors in Coronavirus Infection

Coronaviruses (CoVs) are common human and animal pathogens. In humans, four endemic CoV species together account for one third of mild respiratory infections worldwide. More severe and frequently fatal respiratory pathologies are caused by recent CoV outbreaks that resulted from occasional zoonotic spillover from animal CoV reservoirs, namely, SARS-CoV in 2002, MERS-CoV in 2012, and SARS-CoV-2 in 2019. Because CoVs threaten global health, any chance of relieving CoV’s threat on human populations would rely heavily on our understanding of the mechanistic requirements for CoV tropism, whose major determinant is at the level of viral entry. CoVs have evolved to use a single viral protein for entry: spike (S). The S protein binds viruses to host cell receptors and catalyzes virus-cell membrane fusion for entry. Uniquely, CoV S proteins contain at least two receptor binding domains, a domain A that generally engages host sialic acids, and a domain B that recognizes host transmembrane proteins. A putative advantage of this bivalent binding is elevated CoV zoonotic potential, for each binding domain can, theoretically, independently evolve affinity to distinct host factors. To test this hypothesis, we aimed to identify roles for each receptor for the S proteins of two beta-coronaviruses, the prototypic MHV-CoV strain JHM and human MERS-CoV. We focused on three distinct stages of the CoV life cycle: (1) CoV particle-cell binding; (2) CoV particle-cell entry; (3) CoV cell-to-cell spread via cell-cell fusion. For MHV-JHM S protein, we identified its interaction with host sialic acids. This interaction is responsible for the majority of the particle binding mediated by the S protein, which assists in JHM-CoV entry into various target cells, and is sufficient for cell-to-cell spread. For the S protein of MERS-CoV, we found its interaction with host sialic acids is similarly sufficient for cell-cell fusion. Furthermore, we identified single-nucleotide changes in both S protein-sialic acid binding S domain As that conferred elevated cell-binding and cell-cell fusion capabilities. This study highlights distinctive CoV receptor binding domain activities in the infection process and raise the possibility that CoVs utilizes them for facile zoonotic transmission and intercellular spread within infected organism

Assembly and Entry of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2): Evaluation Using Virus-Like Particles

Research on infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is currently restricted to BSL-3 laboratories. SARS-CoV2 virus-like particles (VLPs) offer a BSL-1, replication-incompetent system that can be used to evaluate virus assembly and virus-cell entry processes in tractable cell culture conditions. Here, we describe a SARS-CoV2 VLP system that utilizes nanoluciferase (Nluc) fragment complementation to track assembly and entry. We utilized the system in two ways. Firstly, we investigated the requirements for VLP assembly. VLPs were produced by concomitant synthesis of three viral membrane proteins, spike (S), envelope (E), and matrix (M), along with the cytoplasmic nucleocapsid (N). We discovered that VLP production and secretion were highly dependent on N proteins. N proteins from related betacoronaviruses variably substituted for the homologous SARS-CoV2 N, and chimeric betacoronavirus N proteins effectively supported VLP production if they contained SARS-CoV2 N carboxy-terminal domains (CTD). This established the CTDs as critical features of virus particle assembly. Secondly, we utilized the system by investigating virus-cell entry. VLPs were produced with Nluc peptide fragments appended to E, M, or N proteins, with each subsequently inoculated into target cells expressing complementary Nluc fragments. Complementation into functional Nluc was used to assess virus-cell entry. We discovered that each of the VLPs were effective at monitoring virus-cell entry, to various extents, in ways that depended on host cell susceptibility factors. Overall, we have developed and utilized a VLP system that has proven useful in identifying SARS-CoV2 assembly and entry features

A single C-terminal residue controls SARS-CoV-2 spike trafficking and incorporation into VLPs.

The spike (S) protein of SARS-CoV-2 is delivered to the virion assembly site in the ER-Golgi Intermediate Compartment (ERGIC) from both the ER and cis-Golgi in infected cells. However, the relevance and modulatory mechanism of this bidirectional trafficking are unclear. Here, using structure-function analyses, we show that S incorporation into virus-like particles (VLP) and VLP fusogenicity are determined by coatomer-dependent S delivery from the cis-Golgi and restricted by S-coatomer dissociation. Although S mimicry of the host coatomer-binding dibasic motif ensures retrograde trafficking to the ERGIC, avoidance of the host-like C-terminal acidic residue is critical for S-coatomer dissociation and therefore incorporation into virions or export for cell-cell fusion. Because this C-terminal residue is the key determinant of SARS-CoV-2 assembly and fusogenicity, our work provides a framework for the export of S protein encoded in genetic vaccines for surface display and immune activation

Limited Variation between SARS-CoV-2-Infected Individuals in Domain Specificity and Relative Potency of the Antibody Response against the Spike Glycoprotein

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is arranged as a trimer on the virus surface, composed of three S1 and three S2 subunits. Infected and vaccinated individuals generate antibodies against spike, which can neutralize the virus. Most antibodies target the receptor-binding domain (RBD) and N-terminal domain (NTD) of S1; however, antibodies against other regions of spike have also been isolated. The interhost variability in domain specificity and relative neutralization efficacy of the antibodies is still poorly characterized. To this end, we tested serum and plasma samples collected from 85 coronavirus disease 2019 (COVID-19) convalescent subjects. Samples were analyzed using seven immunoassays that employ different domains, subunits, and oligomeric forms of spike to capture the antibodies. Samples were also tested for their neutralization of pseudovirus containing SARS-CoV-2 spike and of replication-competent SARS-CoV-2. While the total amount of anti-spike antibodies produced varied among convalescent subjects, we observed an unexpectedly fixed ratio of RBD- to NTD-targeting antibodies. The relative potency of the response (defined as the measured neutralization efficacy relative to the total level of spike-targeting antibodies) also exhibited limited variation between subjects and was not associated with the overall amount of antispike antibodies produced. These studies suggest that host-to-host variation in the polyclonal response elicited against SARS-CoV-2 spike in early pandemic subjects is primarily limited to the quantity of antibodies generated rather than their domain specificity or relative neutralization potency.This article is published as Van Ert, Hanora A., Dana W. Bohan, Kai Rogers, Mohammad Fili, Roberth A. Rojas Chávez, Enya Qing, Changze Han et al. "Limited variation between SARS-CoV-2-infected individuals in domain specificity and relative potency of the antibody response against the spike glycoprotein." Microbiology Spectrum 10, no. 1 (2022): e02676-21. DOI: 10.1128/spectrum.02676-21. Copyright 2022 Van Ert et al. Attribution 4.0 International (CC BY 4.0). Posted with permission