RAPID COMMUNICATION Release of Coronavirus E Protein in Membrane Vesicles from Virus-Infected Cells and E Protein-Expressing Cells Junko Maeda, Akihiko Maeda, and Shinji Makino1 Department of Microbiology, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712; and Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77551-1019 Received May 3, 1999; returned to author for revision May 25, 1999; accepted August 4, 1999 Coronavirus E protein is a small viral envelope protein that plays an essential role in coronavirus assembly; coexpression of coronavirus M and E proteins results in the production of virus-like particles. The present study demonstrated that mouse hepatitis virus (MHV) E protein was released as an integral membrane protein in lipid vesicles from E-protein-expressing mammalian cells, in the absence of other MHV proteins. Furthermore, our data indicated that the E-protein-containing vesicles, which had a slightly lighter buoyant density than that of MHV, were released from MHV-infected cells. These data implied that E protein alone can drive the production and release of coronavirus envelope in the absence of M protein. 1999 Academic Press Coronavirus is an enveloped virus containing a large positive-stranded RNA genome. The coronavirus enve- lope typically contains three virus-specific proteins: S, M, and E. The S protein forms 180- to 90-kDa peplomers, binds receptors on coronavirus-susceptible cells, and induces cell fusion. The M protein, a transmembrane protein, is the most abundant glycoprotein in infected cells as well as in the virus particle. The M protein has three domains; these include a short N-terminal ectodo- main, a triple-spanning transmembrane domain, and a C-terminal endodomain. The E protein, previously re- ferred to as the small membrane protein or sM protein, is an acylated protein and is present only in minute amounts in infected cells as well as in the virus enve- lope. The structure inside the viral envelope is a helical nucleocapsid that consists of viral RNA and N protein. Each coronavirus-specific protein is translated from each of six to eight species of virus-specific mRNAs that have a 39-coterminal nested set structure (1). E protein is crucial to coronavirus assembly. Coex- pression of coronavirus M protein and E protein results in the production of virus-like particles (VLPs), while expression of M protein alone does not produce VLPs (24). Complementation studies using two defective in- terfering (DI) RNAs of a prototypic coronavirus, mouse hepatitis virus (MHV), showed that E and M proteins are both required for the production of MHV particles con- taining the viral nucleocapsid (5). E protein also plays a role in coronavirus morphogenesis, because MHV mu- tants encoding mutated E protein are morphologically aberrant compared to wild-type MHV (6). Even though the importance of E protein in coronavirus assembly is known, more detailed characterization of its function has been limited because of its very low abundance in the virus and in infected cells. In infected cells E protein has been shown to be an integral membrane protein (3), yet its presence as an integral membrane protein in virus particles has not been demonstrated. The orientation of E protein in the membrane is also unknown. In the present study we demonstrated that E-protein- containing membrane vesicles were released from E- protein-expressing cells. Furthermore, our data indicated that the E-protein-containing membrane vesicles were released from MHV-infected cells. These data were un- expected, because M protein is also considered essen- tial for coronavirus envelope formation. The data pre- sented here suggest the possibility that E protein alone can drive the production and release of the coronavirus envelope in the absence of M protein. We used a Sindbis virus expression vector, pSinRep5 (7) (Invitrogen, San Diego, CA), to express MHV E protein and M protein at high levels in mammalian cells. This vector contains a promoter for subgenomic transcription and the Sindbis virus nonstructural protein genes 14 that are required for replication of vector RNAs. We constructed one Sindbis vector that expressed MHV M 1To whom correspondence and reprint requests should be ad- dressed at Department of Microbiology and Immunology, The Univer- sity of Texas Medical Branch at Galveston, Galveston, TX 77551-1019. Fax: (409) 772-5065. E-mail: shmakinoutmb.edu. Virology 263, 265272 (1999) Article ID viro.1999.9955, available online at on 0042-6822/99 $30.00 Copyright 1999 by Academic Press All rights of reproduction in any form reserved. 265 protein (pSinM) and another that expressed MHV E pro- tein (pSinE) by respectively inserting gene 6 (encoding the M protein) from the JHM strain of MHV (MHV-JHM) and gene 5b (encoding the E protein) from the A59 strain of MHV (MHV-A59) downstream of the promoter for