The SARS-CoV-2 envelope and membrane proteins modulate maturation and retention of the spike protein, allowing assembly of virus-like particles

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a β-coronavirus, is the causative agent of the COVID-19 pandemic. Like for other coronaviruses, its particles are composed of four structural proteins: Spike (S), Envelope (E), Membrane (M) and Nucleoprotein (N) proteins. The involvement of each of these proteins and their interactions are critical for assembly and production of β-coronavirus particles. Here, we sought to characterize the interplay of SARS-CoV-2 structural proteins during the viral assembly process. By combining biochemical and imaging assays in infected vs. transfected cells, we show that E and M regulate intracellular trafficking of S as well as its intracellular processing. Indeed, the imaging data reveal that S is re-localized at endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) or Golgi compartments upon co-expression of E or M, as observed in SARS-CoV-2-infected cells, which prevents syncytia formation. We show that a C-terminal retrieval motif in the cytoplasmic tail of S is required for its M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlight that E and M induce a specific maturation of N-glycosylation of S, independently of the regulation of its localization, with a profile that is observed both in infected cells and in purified viral particles. Finally, we show that E, M and N are required for optimal production of virus- like-particles. Altogether, these results highlight how E and M proteins may influence the properties of S proteins and promote the assembly of SARS-CoV-2 viral particles.

Site-specific steric control of SARS-CoV-2 spike glycosylation

“…The SARS-CoV-2 S protein in a viral context is likely under similar constraints; however, the glycan processing is distinct. One factor which could be important is the early budding of the SARS-CoV-2 virion into the ER/Golgi intermediate complex (ERGIC) which may distance the spike protein from glycosyltransferases present in the trans-Golgi compared to Env, which remains attached to the membrane, proximal to glycosyltransferases 69 . The choice of producer cell and culture condition of the virus, in this case Vero cells, which are derived from the kidney cells of Cercopithecus aethiops (African green monkey), may also influence the glycan processing as the expression levels of glycosyltransferase enzymes may account for the diminished attachment of sialic acid and fucose observed on viral S protein compared to recombinant S protein.…”

Linking COVID-19 and heme-driven pathophysiologies: A combined computational-experimental approach

“…Upon binding, the S protein is subjected to proteolytic cleavage by the host cell's transmembrane serine protease subtype 2 (TMPRSS2) [8]. An interplay between the viral proteins membrane protein (M protein), nucleocapsid envelope protein (E protein) and S protein may support the viral budding process [14,15]. Protein 7a act as an accessory protein in virus-host interaction and virus particle formation, which is crucial before release of reproduced virus particles into surrounding areas [16][17][18].…”

Analysis of glycosylation and disulfide bonding of wild-type SARS-CoV-2 spike glycoprotein

“…We evaluated the wild-type SARS-CoV-2 S glycoprotein expressed alone or in combination with the viral membrane (M), envelope (E) and nucleocapsid (N) proteins, which direct the formation of virus-like particles (VLPs) ( 58 , 59 ). In the absence of M, E and N proteins, low levels of the S glycoprotein, presumably in extracellular vesicles, were detected in particles prepared by centrifugation of the supernatants of transiently expressing 293T cells ( Figure 1A – C ).…”

“…Both uncleaved and cleaved SARS-CoV-2 S glycoproteins are incorporated into virus-like particles (VLPs) formed as a result of expression of the SARS-CoV-2 M, E and N proteins ( 59 ) ( Figure 1 ). The S glycoproteins in VLPs are extensively modified by complex carbohydrates, indicating passage through the Golgi compartment.…”