The green fluorescent protein (GFP)-expressing MHV68 bacterial artificial chromosome (BAC) infectious clone has been described elsewhere (RcHV68A98.01 [28] ), and mutants were generated by allelic exchange as previously described [67] . To generate the MHV68.DHS BAC, a targeting region consisting of 566 nt upstream and 568 nt downstream of the mutation site was ligated into pGS284 between BglII and NotI restriction sites and electroporated into the S17lpir strain of E. coli. The MHV68-YFP BAC infectious clone was a generous gift from Dr. Samuel Speck (Emory University) [29] , and MHV68-YFP.DHS was generated using the same methodology as MHV68.DHS. The targeting vector for the mutant rescue BAC (MHV68.MR) was generated by ligating the region around wild-type muSOX into pGS284. Targeting vector-containing cells were cross-streaked with BACcontaining GS500 cells and successful recombinants were identified by colony PCR and subsequent digest with PsiI. BAC DNA was isolated from positive clones using the Qiagen Large-Construct kit (Qiagen). BAC variants were verified by restriction digests with EcoRI and PsiI and sequencing of the region surrounding the recombination site. BAC-derived MHV68 virus was produced by transfecting 2 mg of BAC DNA into NIH 3T3 cells using SuperFect (Qiagen). Virus was then amplified in NIH 3T12 cells and titered by plaque assays on NIH 3T3 cells. Before infecting mice, the loxP-flanked BAC vector sequence was removed from the recombinant viruses by passaging the virus over Vero cells expressing Cre recombinase (kindly provided by Dr. Samuel Speck, Emory University) [59] , and BAC removal was confirmed by PCR analysis.