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. 2000 Mar;74(5):2333-42.
doi: 10.1128/jvi.74.5.2333-2342.2000.

Characterization of the coronavirus mouse hepatitis virus strain A59 small membrane protein E

M J Raamsman  1 , J K Locker, A de Hooge, A A de Vries, G Griffiths, H Vennema, P J Rottier
Affiliations

Characterization of the coronavirus mouse hepatitis virus strain A59 small membrane protein E

M J Raamsman et al. J Virol. 2000 Mar.

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.

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Figures

FIG. 1
FIG. 1
(A) Amino acid sequence of the MHV-A59 E protein and its hydropathy profile as determined by Kyte and Doolittle (23) with a 8-residue moving window. Peaks extending upwards indicate hydrophobic domains; those pointing downwards represent hydrophilic regions. (B) Structure of the fusion protein for antibody preparation against E protein. A construct was prepared consisting of the ectodomain of the EAV GS protein (residues 23 to 184) and the MHV-A59 E protein (residues 49 to 83, underlined in panel A) preceded by a 6-histidine stretch.
FIG. 2
FIG. 2
Kinetics of appearance of the E protein in MHV-A59-infected cells. MHV-infected and mock-infected OST7-1 cells were labeled with 35S-labeled amino acids (80 μCi/10-cm2 dish) for different 1-h periods starting at the indicated times after infection. Combined lysates of cells and culture media were then prepared, and immunoprecipitations were carried out with different antisera. (A) Proteins precipitated with the polyclonal anti-MHV serum were analyzed in 15% PAG. (B) Proteins precipitated by the anti-E (αE) or the preE serum were analyzed in 15% PAG. Radioactivities in the bands representing the different viral proteins were quantitated, taking for M all the different forms, including the lower band indicated by an asterisk. The results are compiled in panel C. They were normalized by placing the added total of all measurements for each protein at 100 and expressing each measurement as the fraction of this total.
FIG. 3
FIG. 3
Stability of the E protein in MHV-A59 infection. MHV-infected OST7-1 cells were labeled with 35S-labeled amino acids (80 μCi/10-cm2 dish) for 30 min starting at 6 hpi. Combined lysates of cells and culture media were prepared either immediately or after different chase periods as indicated. Viral proteins were immunoprecipitated with polyclonal anti-MHV serum (αMHV) or anti-E (αE) serum and analyzed in 15% (A) and 20% (B) PAG, respectively. The radioactivities in the viral proteins were quantitated, including for S protein also the cleaved form of the protein and for M the faster-migrating band indicated by an asterisk (C). The results are compiled in panel C. For each protein the radioactivity in the chase samples was related to that observed after the pulse labeling, which was set at 100.
FIG. 4
FIG. 4
Stability of the expressed E protein. vTF7-3-infected OST7-1 cells transfected with pT7Ts-E, pTUM-M, or pAVI02 plasmids containing the E, the M, and the EAV GS genes, respectively, were labeled with 35S-labeled amino acids (80 μCi/10-cm2 dish) for 1 h starting at 6 hpi. Cell lysates were prepared immediately after the labeling or after various chase times as indicated, and immunoprecipitations were carried out with the antisera anti-E (αE), pre-anti-E (preαE), and anti-MHV (αMHV). Proteins were analyzed in 20% PAG.
FIG. 5
FIG. 5
E protein membrane integration without cleavage of a signal sequence. (A) RNA transcribed in vitro from plasmid pT7Ts-E was translated in a rabbit reticulocyte lysate in the presence or absence of canine microsomal membranes (MM). In parallel, vTF7-3-infected OST7-1 cells transfected with pT7Ts-E were labeled (80 μCi/10-cm2 dish) from 6 to 7 hpi and then lysed. Immunoprecipitations were carried out with the anti-E (αE) serum and the preE serum, and the proteins were analyzed in 20% PAG. (B) A similar OST7-1-derived immunoprecipitate of the E protein was split up into two parts, which were both incubated in 1 M Tris-Cl (pH 8.0), one of which also contained 1 M hydroxylamine. Samples were then diluted with Laemmli sample buffer and analyzed in 20% PAG. Untreated control immunoprecipitates prepared with anti-E and preE serum were included in the analysis for comparison. Note that the presence of hydroxylamine caused a broadening of the protein band in the right lane.
FIG. 6
FIG. 6
Location of the carboxy terminus of the membrane-integrated E protein. BHK-21 cells expressing the M protein or the E protein were permeabilized by using either SLO or Triton X-100 and stained for immunofluorescence with the indicated antibodies. In some cases, the SLO-permeabilized cells were first treated with proteinase K (Prot.K) before the staining, while in other cases the SLO-permeabilized and proteinase K-treated cells were additionally permeabilized by using Triton X-100 and then incubated with the antibodies. αMC, peptide antiserum to the M protein's C-terminal tail; αMN, monoclonal antibody J1.3 specific for the M protein's N terminus.
FIG. 7
FIG. 7
Topology of the E protein in MHV-A59 virions: the carboxy-terminal domain is not exposed on the outside. (A) Immunoaffinity isolation of virions. OST7-1 cells grown in a 10-cm2 dish and infected with MHV-A59 were labeled with 35S-labeled amino acids at 6 to 9 hpi, after which the cells were lysed and processed for immunoprecipitation with anti-MHV (αMHV) and anti-E (αE) (right panel). The culture medium containing released virus particles was clarified, and aliquots were first incubated with the antibodies as indicated (for explanation, see previous figures; αS, monoclonal antibody J7.6 recognizing an epitope in the S ectodomain; C, control monoclonal antibody) and subsequently with Staphylococcus aureus bacteria to adsorb the antibodies and associated viral particles, which were analyzed in 20% PAG. (B) Protease protection analysis. The culture medium of a similarly labeled 10-cm2 dish of infected cells was split and either treated with proteinase K (Prot. K) or incubated without enzyme. Concentrated lysis buffer was then added, and standard immunoprecipitations were carried out with the antibodies indicated (for explanations, see the legends to previous figures). Proteins were analyzed in 20% PAG. From the upper panel, which was exposed for 4 h, the region containing the E protein is shown after a longer exposure (4 days) in the lower panel.
FIG. 8
FIG. 8
Electron microscopic analysis. (A) Epon section of BHK-21 cell expressing the E protein and fixed at 4 h posttransfection. The E protein induces the formation of electron-dense membrane structures that are often continuous with the rough ER (arrowheads). (B to D) The same structures in thawed cryosections double labeled for E (5-nm gold; arrows) and Rab-1 (10-nm gold; arrowheads). Panels: B, an MHV-infected cell fixed at 5.30 hpi, C and D, BHK-21 cells expressing the E protein. Bars, 100 nm.
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