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 versus 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 relocalized at endoplasmic reticulum (ER)–Golgi intermediate compartment (ERGIC) or Golgi compartments upon coexpression 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. 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 versus 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 relocalized at endoplasmic reticulum (ER)–Golgi intermediate compartment (ERGIC) or Golgi compartments upon coexpression 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. At the end of 2019, SARS-Cov-2 emerged in China through zoonotic transmission and led to the COVID-19 pandemic, cumulating by end of September 2020 to over 31 million cases and more than 950,000 deaths worldwide (1WHO. Coronavirus Disease (COVID-2019) situation reports.Google Scholar). SARS-CoV-2 belongs to the β-coronavirus genus of the Coronaviridae family that includes SARS-CoV and Middle East Respiratory Virus (MERS-CoV), which are also responsible for severe lower respiratory infections. The main structural components of coronaviruses are the S (Spike) glycoprotein, the M (Membrane) and E (Envelope) transmembrane proteins, and the N nucleoprotein, which form a viral ribonucleoprotein (vRNPs) complex with the 30kb-long viral genomic RNA (vRNA). The S glycoprotein is the major determinant of viral entry in target cells. The M glycoprotein is key for assembly of viral particles by interacting with all other structural proteins (2de Haan C.A. Rottier P.J. Molecular interactions in the assembly of coronaviruses.Adv. Virus Res. 2005; 64: 165-230Crossref PubMed Scopus (250) Google Scholar, 3Masters P.S. The molecular biology of coronaviruses.Adv. Virus Res. 2006; 66: 193-292Crossref PubMed Scopus (1081) Google Scholar), whereas the E protein is a multifunctional protein, supposed to act on viral assembly, release of virions, and pathogenesis (reviewed in (4Schoeman D. Fielding B.C. Coronavirus envelope protein: current knowledge.Virol. J. 2019; 16: 69Crossref PubMed Scopus (1109) Google Scholar)). Specifically, through its oligomerization, E forms an ion-channel termed “viroporin” (5Pervushin K. Tan E. Parthasarathy K. Lin X. Jiang F.L. Yu D. Vararattanavech A. Soong T.W. Liu D.X. Torres J. Structure and inhibition of the SARS coronavirus envelope protein ion channel.PLoS Pathog. 2009; 5: e1000511Crossref PubMed Scopus (159) Google Scholar, 6Parthasarathy K. Ng L. Lin X. Liu D.X. Pervushin K. Gong X. Torres J. Structural flexibility of the pentameric SARS coronavirus envelope protein ion channel.Biophys. J. 2008; 95: L39-L41Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Even though M coordinates virion assembly, an interaction between M and E seems required for the formation of viral particles (7Corse E. Machamer C.E. The cytoplasmic tails of infectious bronchitis virus E and M proteins mediate their interaction.Virology. 2003; 312: 25-34Crossref PubMed Scopus (109) Google Scholar, 8Huang Y. Yang Z.Y. Kong W.P. Nabel G.J. Generation of synthetic severe acute respiratory syndrome coronavirus pseudoparticles: implications for assembly and vaccine production.J. Virol. 2004; 78: 12557-12565Crossref PubMed Scopus (99) Google Scholar, 9Sui J. Aird D.R. Tamin A. Murakami A. Yan M. Yammanuru A. Jing H. Kan B. Liu X. Zhu Q. Yuan Q.A. Adams G.P. Bellini W.J. Xu J. Anderson L.J. et al.Broadening of neutralization activity to directly block a dominant antibody-driven SARS-coronavirus evolution pathway.Plos Pathog. 2008; 4: e1000197Crossref PubMed Scopus (67) Google Scholar). Coronaviruses assembly and budding occur in the lumen of the endoplasmic reticulum (ER)–Golgi intermediate compartment (ERGIC) (10Krijnse-Locker J. Ericsson M. Rottier P.J. Griffiths G. Characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the RER to the Golgi complex requires only one vesicular transport step.J. Cell Biol. 1994; 124: 55-70Crossref PubMed Scopus (263) Google Scholar, 11Stertz S. Reichelt M. Spiegel M. Kuri T. Martinez-Sobrido L. Garcia-Sastre A. Weber F. Kochs G. The intracellular sites of early replication and budding of SARS-coronavirus.Virology. 2007; 361: 304-315Crossref PubMed Scopus (252) Google Scholar). To ensure their accumulation in the ERGIC, M, E, and S proteins contain intracellular trafficking signals that have been identified for some coronavirus species. For example, a dibasic retrieval signal, KxHxx, found at the C-terminus of the cytoplasmic tail of SARS-CoV Spike, allows its recycling via binding to COPI (12McBride C.E. Li J. Machamer C.E. The cytoplasmic tail of the severe acute respiratory syndrome coronavirus spike protein contains a novel endoplasmic reticulum retrieval signal that binds COPI and interaction with membrane Virol. 2007; PubMed Scopus Google Scholar). a recycling of S may its to with M, which at the ERGIC, S accumulation at the virion budding Here, we at the interplay between S and the other structural proteins, E, M, and N of to its with we that some assembly between SARS-CoV-2 and other Specifically, we at how E, M, and N regulate S intracellular trafficking and as its by which is for SARS-CoV B. X. B. E. The spike glycoprotein of the coronavirus contains a in of the Res. PubMed Scopus Google Scholar). SARS-CoV been to induce the release of particles M. F. of spike and membrane coronavirus PubMed Scopus Google Scholar), we also at the of SARS-CoV-2 proteins required for production of We the and of the S glycoprotein in cells upon with SARS-CoV-2 versus of an at or We in SARS-CoV-2-infected cells and their both a S as and a form of S, as which is from by B. X. B. E. The spike glycoprotein of the coronavirus contains a in of the Res. PubMed Scopus Google Scholar), an protein the cell secretory G. at the from protein to and Cell Biol. PubMed Scopus Google Scholar). in cells, we observed a lower form of as a as which as with in SARS-CoV-2-infected cells, which that some other viral proteins influence the maturation of We also the form that in cells transfected with S both and the forms in purified SARS-CoV-2 viral we of particles S in the of cells S that some other viral proteins are required for of particles. Finally, through to the of S we of in infected cells versus to in cells which that some other SARS-CoV-2 proteins regulate S as well as We also that S of to in transfected cells that both viral and processing. To which SARS-CoV-2 proteins influence S and we S with E, M, or N structural proteins in transfected cells. we the of we found a of upon S coexpression with E or M coexpression with N influence the of S, as with S and that the form that in SARS-CoV-2-infected cells at upon coexpression of S with E or with M and as with S or with N that the Altogether, these results that both E and M influence and maturation of SARS-CoV-2 S. the S protein is Y. D. M. of the SARS-CoV-2 PubMed Scopus Google Scholar), we that the of versus forms in their maturation To we of S transfected cells versus S and E, M, or N cells with which the from the profile of these S versus with E, M, or N that is a of and that the of E or M the maturation of S. that we also forms of between and which may a form of or a form of as X. Liu Y. X. Li D. L. L. T. J. X. J. K. et of spike glycoprotein of SARS-CoV-2 on virus entry and its with PubMed Scopus Google Scholar). the E and M proteins of some other coronaviruses are in the regulation of S M. F. of spike and membrane coronavirus PubMed Scopus Google and is found in the compartments of the cell secretory G. at the from protein to and Cell Biol. PubMed Scopus Google Scholar), we that the in SARS-CoV-2 S between infected versus transfected cells to a in S intracellular We the of SARS-CoV-2 S versus with other structural viral proteins, as with we found that the intracellular S in SARS-CoV-2-infected cells in that contain a of the also of compartments to the T. T. Y. A. G. A. The and Golgi proteins through and a of the intermediate Cell Biol. PubMed Scopus Google Scholar), whereas S in cells the cell in cells, we found that SARS-CoV-2 E or M with S its in or as by its with that both E and M the of S. through of transfected cells S at the cell with E or M lower Altogether, that E and M induce the retention of S in We found that of SARS-CoV-2 S the formation of syncytia in transfected cells, in the formation in cells the of S at the cell and that all required to mediate at the of these cells. we and syncytia S with E or M, whereas activity by S upon N Altogether, these results that E and M regulate the of S, by its intracellular retention assembly sites in the or which prevents the formation of syncytia by the and are compartments of the secretory that are of in which is G. at the from protein to and Cell Biol. PubMed Scopus Google Scholar), with the and maturation of SARS-CoV-2 S upon its coexpression with E and M in coexpression of SARS-CoV-2 S with E S intracellular E of some other coronaviruses is supposed to act as a (4Schoeman D. Fielding B.C. Coronavirus envelope protein: current knowledge.Virol. J. 2019; 16: 69Crossref PubMed Scopus (1109) Google and as of some other coronaviruses Machamer C.E. The of infectious bronchitis virus E protein the secretory and is for release of infectious Virol. PubMed Scopus (67) Google or S. T. F. F. B. B. F.L. The of the hepatitis virus and its from glycoprotein specific and of Pathog. PubMed Scopus Google Scholar, T. G.P. The ion activity of the virus protein transport through the Golgi Cell Biol. PubMed Scopus Google Scholar, virus protein the secretory pathway. of compartments early Golgi transport Biol. 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K. protein from hepatitis virus and of secretory Virol. 2003; PubMed Scopus Google Scholar). at all retention at to upon at for to the Golgi We that E a of the of We these results in transfected cells these cells have a lower trafficking with cells in E to the cell secretory in both cell to the influence of E on trafficking to the we the accumulation of at the cell of transfected cells at for by E the and of cell Altogether, these results that SARS-CoV-2 E protein the cell secretory the retention of which also includes S. that for SARS-CoV S protein, a dibasic retrieval signal at the C-terminus of its cytoplasmic tail allows S recycling via binding to COPI (12McBride C.E. Li J. Machamer C.E. The cytoplasmic tail of the severe acute respiratory syndrome coronavirus spike protein contains a novel endoplasmic reticulum retrieval signal that binds COPI and interaction with membrane Virol. 2007; PubMed Scopus Google Scholar). a recycling of S its to with M, which at the virion assembly to the of motif in the cytoplasmic tail of SARS-CoV-2 S we sought to the is we the of M on retention of a of SARS-CoV-2 S, from which the the dibasic retrieval signal, with S, we found that with M in cells, intracellular retention the in with of which that retrieval signal allows S recycling M-mediated retention of SARS-CoV-2 S. in to the of M to induce E with induce its intracellular retention which with results that E induce the retention of S by modulating the cell secretory and than by directly interacting with S. we observed that the of E, with to the than for S in versus that E induces the retention of the cells in to the of the retrieval To these we the of cells or in with the other viral structural proteins. we found that and syncytia than S of its accumulation at the cell to of the recycling with that M, E, intracellular trafficking the coexpression of and M in formation of whereas coexpression of and E to the between the intracellular retention of SARS-CoV-2 S and its as we the of in cells with E or M. with with E whereas coexpression of M its that the M-mediated retention of S and its are on the C-terminal retention whereas retention of S and its are to of the cell secretory pathway. we of to with E or M and M is to induce retention of for a of the N by E and M independently of their to induce S Altogether, these results that E and M induce the retention of SARS-CoV-2 S via Indeed, M induces intracellular retention of S through interaction with S, upon its retrieval by its cytoplasmic tail of S, whereas E may induce the retention of S by intracellular with coronaviruses that the intracellular retention of S by M is for assembly of infectious particles and that the of E is for budding of particles M. F. of spike and membrane coronavirus PubMed Scopus Google Scholar). is in with results that M and E induce and intracellular retention of S, and that SARS-CoV-2 assembly and budding may a to we sought to the required to induce the formation of SARS-CoV-2 in we transfected cells with of S versus S in with E, M, N or with all structural proteins. At we the cell and we purified particles by through a in we found that S in the of of E, M, or N with S the of S. of S with both E and N or with both M and N the of S in the though to an in cell we found that coexpression of the of E, M, and N with S a production of with a of S in the in the cell that all structural proteins are required for an optimal of which induces their from cells. We also found that N with S its upon coexpression with S, E, and M a of E, M, and N for budding and of we the of the versus forms that in of cells We found that the form and in the of purified particles upon S coexpression with E or M, as with with S or with S and N Altogether, these results that E, M, and N are required for optimal production of S in its N-glycosylation Here, we sought to SARS-CoV-2 with other coronaviruses of assembly and production of its results also that are to SARS-CoV-2 or that been Specifically, we found by the retention of SARS-CoV-2 S the cells, E and M proteins a that only allows its to the virion assembly also its to a and its cell which prevents syncytia formation. we also show that independently of their on S E or M coexpression with S the maturation of the N of S We show that E induces the retention of S by the cell secretory independently of the retrieval motif by S cytoplasmic E from and been to form ion L. G. 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The M, E, and N structural proteins of the severe acute respiratory syndrome coronavirus are required for assembly, and release of Virol. 2008; PubMed Scopus Google proteins in the production of that for their to S. results that for coronaviruses, E and M are for the formation of viral that a for E and M are to with each other Liu D.X. The in coronavirus of the coronavirus infectious bronchitis virus envelope protein in the compartments and interaction between the envelope and membrane Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). E in membrane or of A. Griffiths G. H. Rottier P.J. Characterization of the coronavirus mouse hepatitis virus membrane protein Virol. PubMed Scopus Google Scholar, F. P.S. of E of mouse hepatitis virus a for E protein in coronavirus Virol. PubMed Google Scholar). The of N is more complex and N is to form proteins of coronavirus of and N protein with an RNA PubMed Scopus Google Scholar, M. Liu B. S. 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