Fig 1: HLTF Restricts Early HCMV Infection(A) Viral proteins with the highest cellular concentration, estimated using a “proteomic ruler” approach (Wisniewski et al., 2014) with data from the 0- to 6-hr pSILAC experiment. A UL123 C-terminal GFP tag impaired viral growth. A similar problem occurred with UL135 (data not shown). The inhibitor of apoptosis UL36, which was the fourth most abundant viral protein, was therefore selected and C-terminally tagged with a self-cleaving P2A peptide followed by EGFP.(B) A C-terminal UL36-GFP tag does not impair protein UL36 function. Cells were infected with the indicated virus for 24 hr, then treated with cycloheximide (CHX) and a crosslinking Fas antibody. A viability dye was used to quantify live cells by flow cytometry. Data were normalized to the number of live cells in the absence of Fas antibody. UL36-P2A-GFP virus was comparable with wt, whereas virus lacking UL36 was significantly more sensitive to Fas-mediated apoptosis. *p < 0.05 by two-way ANOVA (n = 3).(C) Schematic of the restriction assay. HFFF-TERTs were infected at low MOI after stable knockdown for a putative restriction factor or control.(D) Validation of the restriction assay using shRNA. Representative results from one of four experiments are shown (left panel). At MOI = 0.1, a difference between shSp100 and shControl cells was no longer detectable, suggesting that the antiviral activity of Sp100 was efficiently overcome. In the same experiment, HLTF restricted infection similarly to Sp100. Values shown are mean ± SEM p values for a difference between shSp100 or shHLTF, and control cells were estimated using a two-tailed t test (n = 3). *p < 0.05 (for both Sp100 and HLTF where indicated). Immunoblot confirmed knockdown of all Sp100 isoforms (right panel) and HLTF (E).(E) HLTF restricts early HCMV infection. Application of the restriction assay at low MOI using two different shHLTF vectors suggested that HLTF restricted infection at least as potently as Sp100 (left panel). p values were estimated using a two-tailed t test (n = 3). **p < 0.005, ***p < 0.0005.(F) Confirmation that HLTF restricts early HCMV infection using five independent polyclonal CRISPR/Cas9 Sp100 and HLTF cell lines. Each employed integrated guide RNAs (gRNAs) with different target sequences within a given gene. Control cells expressing non-targeting gRNAs were generated in a similar manner (right panels; superfluous lanes from the HLTF and corresponding GAPDH gel have been digitally eliminated as indicated by the dashed line). Infection at MOI = 0.01 identified a substantial increase in viral replication in knockdown compared with control cells (left panels). p values were estimated using a two-tailed t test (n = 3). *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.00005.