To investigate the role of muSOX-induced host shutoff during viral infection, we engineered a host shutoff defective MHV68 virus by replacing WT muSOX with muSOX R443I using bacterial artificial chromosome (BAC)-based homologous recombination [28] . The R443I mutation introduced an additional PsiI restriction site within the muSOX gene, allowing us to screen for successful recombination via restriction digest (Figure 2A) . We also generated a mutant rescue (MR) virus by replacing the R443I mutant with WT muSOX to ensure that any observed phenotypes could be attributed to the R443I mutation rather than to unexpected secondary mutations elsewhere in the viral genome. Restriction digests with PsiI and EcoRI confirmed that both recombinant viruses were successfully isolated and did not have any unexpected recombination events ( Figures 2B & 2C) . These viruses shall henceforth be referred to as MHV68.DHS (host shutoff defective virus) and MHV68.MR (mutant rescue virus). Sequencing of muSOX and the surrounding genomic regions in both recombinants confirmed that only the desired changes were introduced.