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

Abstract

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...

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