Characterization of the coronavirus mouse hepatitis virus strain A59 smallmembrane protein E

The small envelope (E) protein has recently been shown to play an essential role in the assembly of coronaviruses. Expression studies revealed that fo r formation of the viral envelope, actually only the E protein and the memb rane (M) protein are required. Since little is known about this generally l ow-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 wit h similar kinetics as the other viral structural proteins, The protein appe ared to be quite stable both during infection and when expressed individual ly using a vaccinia virus expression system. Consistent with the lack of a predicted cleavage site, the protein was found to become integrated in memb ranes without involvement of a cleaved signal peptide, nor were any other m odifications of the polypeptide observed. Immunofluorescence analysis of ce lls expressing the E protein demonstrated that the hydrophilic tail is expo sed 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 topolo gical model in which the polypeptide is buried within the membrane, spannin g the lipid bilayer once, possibly twice, and exposing only its carboxy-ter minal domain. Finally, electron microscopic studies demonstrated that expre ssion of the E protein in cells induced the formation of characteristic mem brane structures also observed in MHV-AJ9-infected cells, apparently consis ting of masses of tubular, smooth, convoluted membranes. As judged by their colabeling with antibodies to E and to Rab-1, a marker for the intermediat e compartment and endoplasmic reticulum, the E protein accumulates in and i nduces curvature into these pre-Golgi membranes where coronaviruses have be en shown earlier to assemble by budding.