VIROLOGY 191, 517-522 (1992) Cell Receptor-Independent Infection by a Neurotropic Murine Coronavirus THOMAS M. GALLAGHER,*, MICHAEL J. BUCHMEIER,* AND STANLEY PERLMANtV2 *Division of Virology, Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California 92037; and Wepartments of Pediatrics and Microbiology, University of Iowa, Iowa City, Iowa 52242 Received June 24, 1992; accepted August 10, 1992 The cellular receptors for a coronavirus, mouse hepatitis virus (MHV), have been recently identified as one or more members of the carcinoembryonic antigen (CEA) family. The neurotropic JHM strain of MHV (MHV-JHM) possesses a highly fusogenic surface (S) glycoprotein. This protein is now shown to promote the spread of MHV into cells lacking the specific CEA-related MHV receptor. Resistant cells are recruited into MHV-induced syncytium with consequent production of progeny virus. Cell-to-cell spread of virus via membrane fusion without the requirement for specific cell surface receptor offers a novel way for virus to spread within infected hosts. o 1992 Academic PWS. IX. Attachment of virus to a host cell is the first step in any viral infection. This process has been shown for many viruses to be mediated by specific cell receptor molecules, although in some cases, receptors for a given virus vary on different cells (3, 4). The cellular receptors for several viruses, including poliovirus, rhi- novirus, echovirus 1, Epstein-Barr virus, human immu- nodeficiency virus (HIV), reovirus, rabies virus, and three coronaviruses, have been identified and partially characterized (1-7). In specific, one or more members of the carcinoembryonic antigen (CEA) family serve as cellular receptors for the A59 and JHM strains of mouse hepatitis virus (MHV) (MHV-A59 and MHV-JHM) and expression of CEA on the surface of some resis- tant cells renders them susceptible to MHV (Z), T.G., S.P., unpublished observations). The viral protein involved in binding to the host cell has been identified for many viruses and in some cases this attachment protein or a second viral protein has been shown to mediate a subsequent plasma mem- brane fusion event (2, 8). This latter function leads to syncytium formation and rapid viral spread in tissue culture cells and, most likely, in animals as well. In the case of MHV, the spike (S) glycoprotein has dual func- tions. S both binds to cellular receptor and induces cell-to-cell fusion (9). Fusion may originate from either external virus (fusion from without) or infected cells (fu- sion from within) and is believed to require the pres- ence of receptor on uninfected cells. Present Address: Department of Microbiology and Immunology, Loyola University Medical Center, Maywood, IL 60153. * To whom correspondence and reprint requests should be ad- dressed at Department of Pediatrics, University of Iowa, Iowa City, IA 52.242. Fax: 319-356-4855. MHV-JHM, like some other coronaviruses, encodes a highly fusogenic S glycoprotein. Fusion is evident following infection with MHV or with either recombi- nant vaccinia virus (VV) or recombinant baculovirus ex- pressing the coronavirus S protein (10, 11). Further- more, the recombinant constructs cause fusion of cells lacking the MHV cellular receptor. These experi- ments indicate that only the S protein is required for cell fusion. The ability of a virus to infect a cell not encoding its cellular receptor would greatly increase its potential host and tissue range. In this report, we show that MHV-JHM is capable of spreading to cells resistant to infection by virions. Spread only occurs when the re- sistant cells are exposed to cell-associated MHV and is a consequence of the strong receptor-independent fu- sion activity of the S protein. Unlike cells of murine origin, hamster and human fibroblasts are resistant to infection with MHV and in some cases, resistance occurs at the level of the virus receptor (2). To demonstrate that the JHM strain was similarly capable of infecting only murine cells, DBT (mouse astrocytoma) (72) BHK (baby hamster kidney), and RKl3 (rabbit kidney clone 13) cells were infected at a multiplicity of 0.2 plaque-forming units/cell (PFU/cell). Virus infection was measured qualitatively by an indi- rect immunofluorescence assay (IFA). At 22 hr postin- oculation (p.i.), only the DBT cell monolayers ex- pressed detectable levels of virus antigen (data not shown) confirming the species specificity of MHV- JHM. The JHM strain used in these experiments en- coded a full-length (4131 nucleotides) S protein (13). In sharp contrast, both BHK and RK13 cells were susceptible to infection with cell-associated MHV-JHM (Fig. 1). In these experiments, DBT cells in suspension 517 0042-6822192 $5.00 CopyrIght 0 1992 by Academic Press. Inc. All rights of reproduction I” any form resewed 518 SHORT COMMUNICATIONS SHORT COMMUNICATIONS 519 were infected with MHV-JHM (0.5 PFU/cell for 1 hr), washed extensively, and seeded onto empty pl