## 3 Results
### FoxO1 and FoxO3 Are Not Required for Efficient Replication of HSV-1  
To address the biological relevance of the miR-183/96/182 cluster upregulation in HSV-1 infection on one hand, and the upregulation of FoxO family members on the other, we performed several functional assays. First, we asked whether the increased levels of miRNAs of the miR-183/96/182 cluster affected the virus replication. To test this, we transfected HFFs with mimics of miR-96, miR-182, and miR-183 or negative control mimics (NC), and 24 post-transfection, we infected cells with HSV-1 at a MOI of 5 (Figure 5A) and 0.001 (Figure 5B). To our surprise, we did not observe any obvious difference in virus replication (Figure 5A,B). Similarly, these miRNAs only had minor effects on viral infection in Neuro-2a cells using a high throughput assay in which cells were transfected with individual miRNAs and infected with HSV-1-luciferase-reporter virus ([PERSON] et al., not published; personal communication). These results led us to the conclusion that the upregulation of the miR-183/96/182 cluster has, at best, a minor role in HSV-1 replication in cultured cells. Figure 4: MCMV infection, IFN, and poly (I:C) induce the FoxO family of proteins. (**A**) Mouse embryonal fibroblasts (MEF) were mock-infected or infected with MCMV and samples for protein analysis were collected at the indicated time points after infection (h.p.i.). (**B**) Cells were infected as in (**A**) and RNA samples were collected. The expression levels of _FoxO1_ and _FoxO3_ mRNAs were determined by RT-qPCR and normalized to 185 rRNA expression. The experiment was performed in triplicate and the standard deviations are indicated. (**C**) Protein samples were collected from untreated RPE1 (mock) or cells treated with poly (I:C) (**left panel**) or IFN-\(\beta\) (**right panel**) at the indicated time points after treatment and analyzed by Western blot. Next, to investigate the role of FoxO proteins in HSV-1 infection, we inactivated the _FoxO1_, _Foxo3_, and _FoxO4_ (_FoxO1 -/-_, _FoxO3 -/-_, _FoxO4 -/-_) genes in the SH-SY5Y cell using CRISPR-Cas9 technology. Briefly, we generated guides using the GPP sgRNA Designer Tool [35; 36] and selected the top three different candidate guides to target each of the genes. Several individual clones were analyzed for the expression of targeted genes  
Figure 5: FoxO proteins are not required for HSV-1 replication. (**A**) HFFs were transfected with miR-182, -96, and -183 mimics or the negative control mimic (NC) and after 24 h infected with HSV-1 at an MOI of 5 (**A**) and 0.001 (**B**). Samples for titration were collected at the indicated time points and titrated on Vero cells. The virus yield is shown as plaque-forming units per mL (Pfu/mL). (**C**) Cells were mock-infected (mock) or infected with HSV-1 at an MOI of 5 and proteins were extracted at 18 h.p.i. for Western blot analysis. SH-SY5Y—parental cells, NC- negative control gRNA, _FoxO1_\(-/-\), and _FoxO3_\(-/-\) cells. (**D**) Cells were infected at an MOI of 5 and 0.001 MOI (**E**) and the supernatants were collected at the indicated time points after infection (h.p.i.). The virus titer was determined by titration on Vero cells and shown as plaque-forming units per mL (Pfu/mL). 0 h.p.i. corresponds to the virus titer in the infectious medium. SH-SY5Y—parental cells, NC—negative control gRNA, _FoxO1_\(-/-\), and _FoxO3_\(-/-\) cells. and only the complete knock-down cells were used in further experiments. We successfully knocked out FoxO1 and FoxO3 proteins (a representative Western blot Figure 5C); however, we were not able to generate FoxO4 deficient cells, or cells deficient for all FoxO proteins (not shown). Next, FoxO1 and FoxO3 deficient cells were infected at high and low MOIs, and virus replication was monitored through the time course of infection. Interestingly, we observed a slight decrease in ICP0 and gC expression in a high MOI experiment (Figure 5C) at 18 h.p.i.