## 3 Results
### HSV-1 Induces the Expression and Post-Translational Modifications of FoxO Protein Family Members  
Based on the target prediction, we hypothesized that the increase in miRNAs miR-96, -182, and -183 will consequently lead to decreased levels of the FoxO family members. To address this, we infected various cells with HSV-1 at an MOI of 1 and collected samples for the Western blot and RNA analysis at different time points after infection. We observed an initial increase in the FoxO1 and FoxO3 levels between 1 h.p.i. and 12 h.p.i. and a slight decrease later in infection (Figures 3B and S5), which could be explained by the increase in miRNAs late in infection (Figure 2A). FoxO4 levels were steadily increased through the time of infection (Figure 3B). Of note, the same dynamic pattern of the FoxO proteins (first upregulation followed by downregulation) was observed in all cell lines tested (HEK293, RPE1, HFF, SH-SYSY; Figures 3B and S5). However, surprisingly, we observed FoxO1 and FoxO3 downregulation in cells in which we did not observe upregulated levels of miR-183/96/182 during the time course of infection (e.g., HEK293 (Supplemental Figure S5A)), indicating additional mechanisms other than miRNAs for the depletion of FoxO family members late in infection. Moreover, in cells infected and treated with different inhibitors including cycloheximide (CHX, protein synthesis inhibitor) and actinomycin D (ActD, RNA synthesis inhibitor), or in cells infected with UV inactivated virus, we did not observe the downregulation of FoxO proteins (Figure 3C). These results indicate that IE/E viral gene expression may be required for the depletion of FoxO proteins. Interestingly, infection in the presence of acyclovir (i.e., a condition that allows for the expression of IE but prevents the expression of L proteins), the depletion was not fully achieved (Figure 3B). On the other hand, the addition of a proteasome inhibitor (MG132) 30 min prior to infection (*), but not 2 h.p.i. (**), successfully prevented the depletion (Figure 3C). Taken together, these results may point to the role of the IE protein ICP0, a well-characterized ubiquitin ligase, but also to additional mechanisms such as miRNAs and late viral functions. Furthermore, by carefully analyzing the expression of FoxO protein members by Western blot, we observed the appearance of double bands (i.e., a shift of a few kilodaltons (kDa) during the time course of infection (Figure 3B)), which might represent phosphorylated forms of proteins (i.e., inactive forms). Of note, this phenomenon was observed in most of the cell lines tested, but was particularly apparent in RPE cells (representative Figures 3B and S5). Indeed, using phospho-specific antibodies, we confirmed the phosphorylation of FoxO1 and FoxO3. Interestingly, the addition of acyclovir, an inhibitor of viral DNA replication and late gene expression, prevented the shift of a few kDa of FoxO proteins (Figure 3B). The observed phosphorylation is consistent with the previous study by [PERSON] et al., who found that FoxO1 can be phosphorylated by Akt kinase and viral kinase Us3. Taken together, we show that the virus is equipped with multiple mechanisms to control the expression and function of FoxO proteins and that miRNAs might have only a minor contribution to this. Poly (I:C), IFN-\(\beta\) Treatment, and Infection with Other Herpesviruses Increase the Level of FoxO1 and FoxO3 Proteins  
We were intrigued by the increased levels of proteins of the FoxO family in cells infected with HSV-1, which indicates the possible transcriptional activation of _FoxO_ genes. To address this, we analyzed the transcriptome of HSV-1 infected cells. Briefly, we infected HEK293 cells with strain KOS at MOI 10 and collected the total RNA at 1, 4, 8, and 12 h.p.i. from two biological replicates for each time point. The sequencing libraries were prepared using the TruSeq Stranded Total RNA Sample Preparation Kit and sequenced on the NextSeq 500 Illumina platform.