The Viral Spike Protein Is Not Involved in the Polarized Sorting of Coronaviruses in Epithelial Cells
By J. Rossen, R. de Beer, G. Godeke, M. Raamsman, Marian C. Horzinek, H. Vennema, P. Rottier. 1998
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 MDCKMHVRcells, 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 MDCKMHVR cells at the nonpermissive temperature, they were again preferentially released from basolateral and apical membranes, respectively. (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 (H. Vennema, G.-J. Godeke, J. W. A. Rossen, W. F. Voorhout, M. C. Horzinek, D.-J. E. Opstelten, and P. J. M. Rottier, EMBO 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.
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Citations in the corpus
- Elicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2 (2020, neutral): “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.”
- Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein (2020, background): “These oligosaccharides participate in S folding (Rossen et al., 1998), affect priming by host proteases (Yang et al.”
- Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus Fusion (2020, unknown): “…glycans contribute to S stability during biogenesis, since inhibition of glycosylation by tunicamycin was reported to yield virions lacking S glycoproteins (Rossen et al., 1998) and likely participate in immune evasion via epitope masking (Du et al., 2016; Walls et al., 2016b; Xiong et al., 2017).”
- Coronavirus Structural Proteins and Virus Assembly (2008, background): “However, one study showed that MHV S protein was not involved in determining the polarized release of virus (148).”