The Viral Spike Protein Is Not Involved in the Polarized Sorting of Coronaviruses in Epithelial Cells

Abstract

Coronaviruses are assembled by budding into a pre-Golgi compartment from which they are transported along the secretory pathway to leave the cell. In cultured epithelial cells, they are released in a polarized fashion; depending on the virus and cell type, they are sorted preferentially either to the apical domain or to the basolateral plasma membrane domain. In this study, we investigated the role of the coronavirus spike protein, because of its prominent position in the virion the prime sorting candidate, in the directionality of virus release. Three independent approaches were taken. (i) The inhibition of N glycosylation by tunicamycin resulted in the synthesis of spikeless virions. The absence of spikes, however, did not influence the polarity in the release of virions. Thus, murine hepatitis virus strain A59 (MHV-A59) was still secreted from the basolateral membranes of mTAL and LMR cells and from the apical sides of MDCK MHVR cells, whereas transmissible gastroenteritis virus (TGEV) was still released from the apical surfaces of LMR cells. (ii) Spikeless virions were also studied by using the MHV-A59 temperature-sensitive mutant Albany 18. When these virions were produced in infected LMR and MDCK MHVR cells at the nonpermissive temperature, they were again preferentially released from basolateral and apical membranes, respectively. J. 15:2020-2028, 1996). The spikeless particles produced in mTAL cells by using recombinant Semliki Forest viruses to express these two genes of MHV-A59 were specifically released from basolateral membranes, i.e., with the same polarity as that of wild-type MHV-A59. Our results thus consistently demonstrate that the spike protein is not involved in the directional sorting of coronaviruses in epithelial cells. In addition, our observations with tunicamycin show that contrary to the results with some secretory proteins, the N-linked oligosaccharides present on the viral M proteins of coronaviruses such as TGEV also play no role in viral sorting. The implications of these conclusions are discussed. (iii) We recently demonstrated that coronavirus-like particles resembling normal virions were assembled and released when the envelope proteins M and E were coexpressed in cells (, EMBO

The glycosylation status of the murine hepatitis coronavirus M protein affects the interferogenic capacity of the virus in vitro and its ability to replicate in the liver but not the brain

“…Furthermore, M protein glycosylation was also shown not to be critical for interaction with the S protein (de Haan et al, 1999). These observations are consistent with studies that used tunicamycin (Rossen et al, 1998;Stern and Sefton, 1982) and monensin (Niemann et al, 1982) to inhibit glycosylation in infected cells.…”

“…In contrast, the M protein released from cells infected with Alb244 or Alb246 migrated in the gel as a single band with approximately the same electrophoretic mobility as the unglycosylated M protein species released from Alb138-or Alb139-infected cells. The M proteins released from cells infected with Alb248 or Alb250 appeared as a smear in the gel, quite similar to the M proteins from group I coronaviruses, which carry Nlinked sugars (Rossen et al, 1998;Vennema et al, 1990). The appearance of the M proteins as a smear in the gel is probably the result of extensive, heterogeneous modifications of the sugar side chains.…”

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

“…We previously showed that coronavirus S glycoproteins are densely decorated by heterogeneous N-linked glycans protruding from the trimer surface (Walls et al, 2016b;Walls et al, 2019;Xiong et al, 2018). These oligosaccharides participate in S folding (Rossen et al, 1998), affect priming by host proteases (Yang et al, 2015b), and might modulate antibody recognition (Pallesen et al, 2017;Walls et al, 2019). SARS-CoV-2 S comprise 22 N-linked glycosylation sequons per protomer and oligosaccharides are resolved in the cryo-EM map for 16 of these sites (Figure 4).…”

“…As the coronavirus S glycoprotein is surface-exposed and mediates entry into host cells, it is the main target of neutralizing antibodies (Abs) upon infection and the focus of therapeutic and vaccine design. S trimers are extensively decorated with N-linked glycans that are important for proper folding (Rossen et al, 1998) and for modulating accessibility to host proteases and neutralizing Abs (Walls et al, 2016b;Walls et al, 2019;Xiong et al, 2018;Yang et al, 2015b). We previously characterized potent human-neutralizing Abs from rare memory B cells of individuals infected with SARS-CoV (Traggiai et al, 2004) or MERS-CoV (Corti et al, 2015) in complex with SARS-CoV S and MERS-CoV S to provide molecular-level information of the mechanism of competitive inhibition of S B attachment to the host receptor .…”

Influenza virus morphogenesis and budding

“…For example, although measles virus glycoproteins H and F are transported in a random fashion or to basolateral membrane, respectively, virus budding occurred predominantly from the apical surface of polarized MDCK cells (Maisner et al, 1998). Similarly, the spike protein of coronavirus is not involved in the polarized budding of this virus (Rossen et al, 1998). Moreover, Marburg virus buds predominantly from the basolateral surface, while its glycoprotein is transported to the apical surface (Sanger et al, 2001).…”