The focus of this protocol is mouse hepatitis virus (MHV), with occasional references to other coronaviruses. Many of these protocols can be easily adapted to other coronaviruses. Protocols for propagating MHV in DBT and 17CL‐1 cells; the storage and titration of viral stocks; purification of MHV on sucrose gradients; and the generation of recombinant viruses by a cDNA assembly method and by targeted recombination will be presented. Protocols are also included for the propagation of DBT, 17CL‐1, and L2 cells used for growing and titrating MHV, and for the growth of BHK‐R cells and FCWF cells. The latter two cell lines are used for regenerating infectious MHV by an in vitro cDNA assembly protocol and by a targeted recombination protocol, respectively, allowing reverse genetic manipulation of these viruses. An additional protocol for the maintenance of the large plasmids used for generating recombinant MHVs will also be presented. Curr. Protoc. Microbiol . 21:15E.1.1‐15E.1.46. © 2011 by John Wiley & Sons, Inc.

Mouse Hepatitis Virus (MHV) is a single-stranded positive sense RNA virus with the ability to promote acute and chronic diseases in mice. The MHV spike protein (S) is a major virulence determinant which in addition to binding to cellular receptors to mediate cell entry and facilitate virus spread to adjacent cells by cell-cell fusion, also is a molecular mimic of the FcγRII receptor. This molecular mimicry of FcγRII by the MHV S protein is also exhibited by other lineage 2a betacoronaviruses, with the exception of the human coronavirus HCoV-OC43. In this work we undertook a mutational analysis to attempt to identify specific amino acid sequences within the spike glycoprotein crucial for molecular mimicry of FcγRII. Although we were unsuccessful in isolating mutant viruses which were specifically defective in that property, we identified several mutations with interesting phenotypes. Mutation of the cysteine in position 547 to alanine and alanine replacements at residues 581–586 was lethal. Replacing proline 939 with the corresponding HCoV-OC43 residue, leucine, decreased the ability MHV to induce cell-cell fusion, providing experimental support for an earlier proposal that residues 929–944 make up the fusion peptide of the MHV S protein.