## 3 Results
### miRNAs miR-183, -96, and -182 Regulate a Number of Common Targets  
To determine the potential targets of miR-183, -96, and -182, we implemented TargetScan 7.1 (Bioinformatics and Research Computing, Whitehead Institute for Biomedical Research, Cambridge, MA, USA), and miRB ([PERSON]'s lab, Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO, USA), two widely used algorithms that predict mRNA targets [48; 49]. The prediction revealed a large number of highly conserved potential targets for each of these miRNAs (between 300 and 778 targets found by both prediction tools for each of the miRNAs; not shown). Notably, only 33 potential targets were identified as shared by all three miRNAs, and these largely represent genes encoding proteins involved in the regulation of gene expression (Figure 3A and Table S4). Among these, _FoxO1_ was identified as a potential target for all three miRNAs, and two other members of the Forkhead box protein family, _FoxO3_ and _FoxO4_, were found as potential targets for miR-182 and miR-96, and solely miR-96, respectively (Figure 3A). The binding sites for each miRNA of the cluster predicted by TargetScan are shown in Figure S3. Members of the FoxO protein family are well-known transcriptional factors that are regulated by the insulin/PI3K/Akt signaling pathway and are known to have many diverse roles in cell cycle progression, apoptosis, metabolism, differentiation, and oxidative stress resistance [50; 51; 52; 53] including roles in viral infections [54; 55; 56; 57; 58; 59; 60; 61; 62; 63; 64; 65]. The regulation of the _FoxO_ family by miRNAs of the mir-183/96/182 cluster is well-established and has been investigated in various cellular pathways including the modulation of the innate immune response in infections or autoimmune diseases, or in various cancers [66; 67]. Thus, we did not additionally validate this regulation in this study.  
Figure 3: The members of the FoxO family are predicted targets of miRNAs of the miR-183/96/182 cluster and are induced during productive HSV-1 infection. (**A**) The Venn diagram shows the number of conserved targets for miR-183 (blue circle), miR-96 (yellow circle), and miR-182 (green circle). Thepredicted targets of the FoxO family are indicated with arrows. Gene Ontology was determined by the Enrichr Gene Ontology analysis tool (Ma'ayan Lab, Icahn School of Medicine at Mount Sinai, NY, USA) [68]. The length of the bar and the color indicate the number of genes in the different functional categories, i.e., the longer the bar and the lighter the color, the greater the number of genes. Genes with only poorly conserved sites are not shown. (**B**) The RPE cells were mock-infected (mock), infected with HSV-1 (wt), or infected and treated with acyclovir (ACV), collected at indicated time points, and analyzed by Western blot. ICP0, immediate-early viral gene, and gc, late viral gene. (**C**) HEK293 cells were mock-infected (mock), infected with HSV-1 (wt), or infected and pretreated with indicated inhibitors: cycloheximide (CHX), actinomycin D (ActD), proteasome inhibitor MG132 (*) 30 min before infection, or (**) 2 h post-infection; or infected with UV-inactivated HSV-1 (UV-HSV-1). Proteins for the Western blot were collected at 12 h.p.i. (**D**) Levels of the _FoxO1_ and _FoxO3_ transcripts were obtained by the total RNA sequencing and bioinformatics analysis. Results were normalized for the sequencing depth and gene length and reported in fragments per kilobase million (FPKM). (**E**) The expression levels of the _FoxO1_ mRNAs was determined by RT-qPCR and normalized to 185 rRNA expression. Statistical significance is indicated with asterisks (* \(p\) < 0.05).