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1、JOURNAL OFVIROLOGY, 0022-538X/00/$04.0010 Mar. 2000, p. 23332342Vol. 74, No. 5 Copyright 2000, American Society for Microbiology. All Rights Reserved. Characterization of the Coronavirus Mouse Hepatitis Virus Strain A59 Small Membrane Protein E MARTIN J. B. RAAMSMAN,1JACOMINE KRIJNSE LOCKER,2ALPHONS
2、DEHOOGE,1 ANTOINE A. F.DEVRIES,1GARETH GRIFFITHS,2HARRY VENNEMA,1ANDPETER J. M. ROTTIER1* Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Institute of Virology, and Institute of Biomembranes, Utrecht University, 3584 CL Utrecht, The Netherlands,1 and European Molecu
3、lar Biology Laboratory, Heidelberg, Germany2 Received 23 June 1999/Accepted 2 December 1999 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 pro
4、tein 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 othe
5、rwise 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 wi
6、th 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 modifi cations of the polypeptide observed. Immunofl uorescence analysis of cells expressing the E protein demonstrated that the hydro
7、philic 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 th
8、e 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
9、, apparently con- sisting 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
10、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 n
11、ucleocapsid (N) protein into a helical nucleocapsid. The sur- rounding envelope contains three, and sometimes four, mem- brane proteins. The spike (S) protein, a type I glycoprotein, occurs as trimers that constitute the characteristic surface pro- jections. These function primarily in virus entry,
12、being respon- sible for binding to the receptor on the target cell and for mediating fusion of viral and cellular membranes. The mem- brane (M) protein is a triple-spanning glycoprotein. It is the most abundant envelope protein component having essential functions in virus assembly. The hemagglutini
13、n-esterase pro- tein is present in only a subset of coronaviruses. The type I glycoprotein occurs in virions in disulfi de-linked homodimeric form. Its biological role in the virus life cycle has not been well established. The small envelope (E) protein was only recently recognized as a structural c
14、omponent of the coronavirion (12, 26, 48, 49). Although very little is still known about its features, the E protein appears to be surprisingly important for assembly of the viral envelope. By coexpression of the genes encoding the mouse hepatitis virus strain A59 (MHV-A59) membrane pro- teins we sh
15、owed that virus-like particles (VLPs) morphologi- cally mimicking normal virions were produced only when the E protein was present, while the S protein was dispensable (48). Similar particles were observed after coexpression of transmis- sible gastroenteritis virus (TGEV) membrane proteins (1a). Cor
16、onavirus E proteins vary in size from about 76 to 109 amino acids (for a review, see reference 38). Consistent with their membrane association (12, 40, 48, 49), the proteins are generally quite hydrophobic in nature, particularly in their N-terminal half (see Fig. 1). The MHV-A59 E protein was reported to be acylated on the basis of a biochemical assay (49), but attempts to directly label the TGEV protein with palmitic acid failed (12). By immunofl uorescence, the E pro- tein was observed in in
