Identification of Restriction Factors by Human Genome-Wide RNA Interference Screening of Viral Host Range Mutants Exemplified by Discovery of SAMD9 and WDR6 as Inhibitors of the Vaccinia Virus K1LC7L Mutant

ABSTRACT RNA interference (RNAi) screens intended to identify host factors that restrict virus replicationmay fail if the virus already counteracts host defense mechanisms. To overcome this limitation, we are investigating the use of viral host range mu-tants that exhibit impaired replication in nonpermissive cells. A vaccinia virus (VACV)mutant with a deletion of both the C7L and K1L genes, K1LC7L, which abrogates replication in human cells at a step prior to late gene expression, was chosen for this strategy. We carried out a human genome-wide small interfering RNA (siRNA) screen in HeLa cells infected with a VACV K1LC7Lmutant that expresses the green fluorescent protein regulated by a late promoter. This positive-selection screen had remarkably low background levels and resulted in the identification of a few cellular genes, notably SAMD9 andWDR6, from approximately 20,000 tested that dramatically enhanced green fluorescent protein expression. Replication of the mutant virus was enabled by multiple siRNAs to SAMD9 orWDR6. Moreover, SAMD9 andWDR6 clustered regularly interspaced short palin-dromic repeat (CRISPR)/Cas9 knockout HeLa cell lines were permissive for replication of the K1LC7Lmutant, in agreement with the siRNA data. Expression of exogenous SAMD9 or interferon regulatory factor 1 restricted replication of the K1LC7L mutant in the SAMD9/ cells. Independent interactions of SAMD9 with the K1 and C7 proteins were suggested by immunopre-cipitation. Knockout ofWDR6 did not reduce the levels of SAMD9 and interactions ofWDR6 with SAMD9, C7, and K1 proteins were not detected, suggesting that these restriction factors act independently but possibly in the same innate defense pathway