We therefore designed an unbiased screen to investigate a large number of mutants following the previous methodology used to identify the single-function SOX variants ( Figure 1A ) [26] . Briefly, the muSOX gene was amplified by error-prone PCR under conditions designed to introduce 1-4 mutations per gene. The PCR product was then cloned into a mammalian expression vector, and clones were screened for the ability to repress expression of a GFP reporter in a fluorescence-based assay in 293T cells. Mutants that did not deplete GFP fluorescence were categorized as host shutoff defective and further analyzed for wildtype protein expression levels by Western blotting, as even minor changes to the muSOX primary amino acid sequence often significantly reduced its protein expression. Finally, candidate mutants were tested for retention of DNase activity using an in vitro assay established previously for SOX and BGLF5 [25, 26] . After screening approximately 150 candidates, we identified a clone that lacked mRNA degradation activity of the GFP reporter, but retained wild-type protein expression levels and DNase activity. Sequencing revealed this clone to have only a single non-silent mutation, the amino acid substitution R443M, which we modified to R443I for purposes of screening by restriction digest. MuSOX R443I failed to suppress GFP protein expression in the fluorescent-based assay for host shutoff ( Figure 1B) , which was a consequence of its inability to degrade GFP mRNA ( Figure 1C ). R443I is expressed at slightly higher levels than wild-type (WT) muSOX ( Figure 1D ), as expected of a mutant that cannot promote turnover of its own mRNA. Importantly, WT and R443I muSOX both degrade linear DNA with very similar kinetics ( Figure 1E ). We conclude that the amino acid substitution R443I produces a single-function muSOX mutant selectively defective for host shutoff activity.