Envelope glycoprotein interactions in coronavirus assembly.

175

204

The small envelope (E) protein has recently been shown to play an essential role in the assembly of coronaviruses. Expression studies revealed that for formation of the viral envelope, actually only the E protein and the membrane (M) protein are required. Since little is known about this generally low-abundance virion component, we have characterized the E protein of mouse hepatitis virus strain A59 (MHV-A59), an 83-residue polypeptide. Using an antiserum to the hydrophilic carboxy terminus of this otherwise hydrophobic protein, we found that the E protein was synthesized in infected cells with similar kinetics as the other viral structural proteins. The protein appeared to be quite stable both during infection and when expressed individually using a vaccinia virus expression system. Consistent with the lack of a predicted cleavage site, the protein was found to become integrated in membranes without involvement of a cleaved signal peptide, nor were any other modifications of the polypeptide observed. Immunofluorescence analysis of cells expressing the E protein demonstrated that the hydrophilic tail is exposed on the cytoplasmic side. Accordingly, this domain of the protein could not be detected on the outside of virions but appeared to be inside, where it was protected from proteolytic degradation. The results lead to a topological model in which the polypeptide is buried within the membrane, spanning the lipid bilayer once, possibly twice, and exposing only its carboxy-terminal domain. Finally, electron microscopic studies demonstrated that expression of the E protein in cells induced the formation of characteristic membrane structures also observed in MHV-A59-infected cells, apparently consisting of masses of tubular, smooth, convoluted membranes. As judged by their colabeling with antibodies to E and to Rab-1, a marker for the intermediate compartment and endoplasmic reticulum, the E protein accumulates in and induces curvature into these pre-Golgi membranes where coronaviruses have been shown earlier to assemble by budding.Coronaviruses, a family of viruses belonging to the newly established order of the Nidovirales (for reviews, see references 8 and 37) have enveloped virions containing a nonsegmented, plus-stranded RNA genome. The RNA is packaged by the nucleocapsid (N) protein into a helical nucleocapsid. The surrounding envelope contains three, and sometimes four, membrane proteins. The spike (S) protein, a type I glycoprotein, occurs as trimers that constitute the characteristic surface projections. These function primarily in virus entry, being responsible for binding to the receptor on the target cell and for mediating fusion of viral and cellular membranes. The membrane (M) protein is a triple-spanning glycoprotein. It is the most abundant envelope protein component having essential functions in virus assembly. The hemagglutinin-esterase protein is present in only a subset of coronaviruses. The type I glycoprotein occurs in virions in disulfide-linked homodimeric form. Its biological role in the vi...

Section: Discussionmentioning

confidence: 93%

Characterization of the Coronavirus Mouse Hepatitis Virus Strain A59 Small Membrane Protein E

Raamsman

Locker

Hooge

et al. 2000

175

204

“…It will thus be important to understand the basis of its selective inclusion into particles, and, conversely, the reason why other proteins are excluded. It has been previously shown that there are specific associations between the M and S proteins while M is coalescing into higher-order arrays, and these may be key to recruiting S into budding viral particles (36,38,39). However, much remains to be learned of the molecular details of this process, and an active role for E protein cannot be ruled out.…”

Section: Discussionmentioning

confidence: 97%

“…in the endoplasmic reticulum (36,38,39). S multimers must somehow fit specifically into the interstices of the arrays of M (or M and E) monomers without contributing much to their overall stability.…”

mentioning

confidence: 99%

Retargeting of Coronavirus by Substitution of the Spike Glycoprotein Ectodomain: Crossing the Host Cell Species Barrier

Kuo

Godeke

Raamsman

et al. 2000

333

525

Coronaviruses generally have a narrow host range, infecting one or just a few species. Using targeted RNA recombination, we constructed a mutant of the coronavirus mouse hepatitis virus (MHV) in which the ectodomain of the spike glycoprotein (S) was replaced with the highly divergent ectodomain of the S protein of feline infectious peritonitis virus. The resulting chimeric virus, designated fMHV, acquired the ability to infect feline cells and simultaneously lost the ability to infect murine cells in tissue culture. This reciprocal switch of species specificity strongly supports the notion that coronavirus host cell range is determined primarily at the level of interactions between the S protein and the virus receptor. The isolation of fMHV allowed the localization of the region responsible for S protein incorporation into virions to the carboxyterminal 64 of the 1,324 residues of this protein. This establishes a basis for further definition of elements involved in virion assembly. In addition, fMHV is potentially the ideal recipient virus for carrying out reverse genetics of MHV by targeted RNA recombination, since it presents the possibility of selecting recombinants, no matter how defective, that have regained the ability to replicate in murine cells.The family Coronaviridae contains the causative agents of a number of significant respiratory and enteric diseases affecting humans, other mammals, and birds (55). One of the hallmarks of this family is that most of its members exhibit a very strong degree of host species specificity, the molecular basis of which is thought to reside in the particularity of the interactions of individual viruses with their corresponding host cell receptors.Coronaviruses have positive-stranded RNA genomes, on the order of 30 kb in length, that are packaged by a nucleocapsid protein (N) into helical ribonucleoprotein structures (31). The nucleocapsid is incorporated into viral particles by budding through the membrane of the intermediate compartment between the endoplasmic reticulum and the Golgi complex (26, 57). Subsequent to budding, it may acquire a spherical, possibly icosahedral superstructure (43,44). The virion envelope surrounding the nucleocapsid contains a minimal set of three structural proteins: the membrane glycoprotein (M), the small envelope protein (E), and the spike glycoprotein (S). In some coronaviruses, other proteins may also be present; these include a hemagglutinin-esterase (HE) (34, 54) and the product of the internal open reading frame of the N gene (I protein) (12, 53), neither of which is essential for virus infectivity.M is the most abundant of the virion structural proteins. It spans the membrane bilayer three times, having a short aminoterminal domain on the exterior of the virus and a large carboxy terminus, containing more than half the mass of the molecule, in the virion interior (48). By contrast, E is a minor structural protein, in both size and stoichiometry, and was only relatively recently identified as a constituent of viral particles (17,33,...

“…They are trimeric type I integral membrane proteins, organized into subdomains necessary for receptor engagement (S1) and membrane fusion (S2) (20). S has been shown to be incorporated into virions by interactions with M (45,51,52). In infected cells and in S-transfected cells, S can be transported to the cell surface, where it mediates fusion with adjoining cells, resulting in the formation of syncytia (2,11,39).…”

mentioning

confidence: 99%

S Protein of Severe Acute Respiratory Syndrome-Associated Coronavirus Mediates Entry into Hepatoma Cell Lines and Is Targeted by Neutralizing Antibodies in Infected Patients

Hofmann

Hattermann

Marzi

et al. 2004

184

204

The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) causes severe pneumonia with a fatal outcome in approximately 10% of patients. SARS-CoV is not closely related to other coronaviruses but shares a similar genome organization. Entry of coronaviruses into target cells is mediated by the viral S protein. We functionally analyzed SARS-CoV S using pseudotyped lentiviral particles (pseudotypes). The SARSCoV S protein was found to be expressed at the cell surface upon transient transfection. Coexpression of SARS-CoV S with human immunodeficiency virus-based reporter constructs yielded viruses that were infectious for a range of cell lines. Most notably, viral pseudotypes harboring SARS-CoV S infected hepatoma cell lines but not T-and B-cell lines. Infection of the hepatoma cell line Huh-7 was also observed with replicationcompetent SARS-CoV, indicating that hepatocytes might be targeted by SARS-CoV in vivo. Inhibition of vacuolar acidification impaired infection by SARS-CoV S-bearing pseudotypes, indicating that S-mediated entry requires low pH. Finally, infection by SARS-CoV S pseudotypes but not by vesicular stomatitis virus G pseudotypes was efficiently inhibited by a rabbit serum raised against SARS-CoV particles and by sera fromSARS patients, demonstrating that SARS-CoV S is a target for neutralizing antibodies and that such antibodies are generated in SARS-CoV-infected patients. Our results show that viral pseudotyping can be employed for the analysis of SARS-CoV S function. Moreover, we provide evidence that SARS-CoV infection might not be limited to lung tissue and can be inhibited by the humoral immune response in infected patients.