## 4 Discussion
First, productive HSV-1 infection is rather rapid, in contrast to protracted CMV replication, and inevitably leads to the destruction of the infected cell. Second, it has been shown that HSV-1 interferes with miRNA function and biogenesis at the stage of nuclear export [80; 81], which limits the potential of miRNA regulation in general. Thus, it would be rather surprising that miRNAs, which accumulate late in infection, can exert a relevant control of the genes. Indeed, in our study, we show that the protein levels of members of the FoxO protein family, the confirmed targets of miRNAs of the upregulated cluster, increased early in infection followed by a decrease late in infection. The late phenomenon might be contributed to the regulation of miRNAs; however, the same observation was true in cells in which we did not detect an increase in miRNAs after infection. Thus, we can conclude that miR-183, -96, and -182, at best, have a minor contributing role in this process. Nonetheless, although we were not able to establish a hypothesized regulatory feedback loop, we were very intrigued with the expression of the FoxO protein family members. In this study, we showed that in cells devoid of FoxO1 or FoxO3 expression, HSV-1 replication is as efficient as it is in WT cells, indicating that individual FoxO proteins are not required for efficient replication. Potential compensatory/overlapping functions of other members might explain the lack of a phenotype, or these proteins might exert their role in latency. It is important to mention that we were not able to generate cells in which FoxO4 or all FoxO proteins were inactivated. In contrast to our observation, roles for individual FoxO proteins have been shown for EBV and KSHV. For example, it has been shown that in EBV-positive advanced nasopharyngeal carcinoma cells, which exhibit type II latency, EBV LMP1 modulates the PI3K/AKT/FoxO3 pathway, resulting in the accumulation of FoxO3 phosphorylation. This inactivation of FoxO3 led to the induction of miR-21, which in turn downregulated programmed cell death 4 (PDCD4) and Fas ligand and reduced apoptosis [82]. In addition, [PERSON] et al. showed that in EBV-associated gastric cancer cells that showed type I/II latency, the FoxO1, FoxO3, and FoxO4 protein levels were significantly lower when compared to the control cells. Interestingly, FoxO1 mRNA was downregulated, but not FoxO3 or FoxO4 mRNAs, which might suggest diverse regulatory mechanisms by various EBV latent genes [57]. Similarly, FoxO1 has been shown to sufficiently suppress [PERSON]'s sarcoma-associated herpesvirusuirus (KSHV) lytic replication and maintain latency by controlling the levels of reactive oxygen species (ROS), since the disruption of FoxO1 could trigger KSHV reactivation and induce lytic infection by increasing the cellular ROS level [60]. On the other hand, a recent study investigating human cytomegalovirus (HCMV) has shown that FoxO1 and FoxO3 positively regulate virus lytic gene expression by activating alternative major immediate-early gene promoters and promoting IE re-expression and virus reactivation [61]. Taken together, our study shows that individual FoxO proteins are not required for efficient virus replication; however, more research, and likely, an adequate in vitro latency model, is needed to understand their roles in HSV-1 latency. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/v14081661/s1](https://www.mdpi.com/article/10.3390/v14081661/s1)]([https://www.mdpi.com/article/10.3390/v14081661/s1](https://www.mdpi.com/article/10.3390/v14081661/s1)), Figure S1: HSV-1 miRNAs represent only a small fraction of miRNAs in infected cells; Figure S2: miRNAs found reproducibly deregulated between two datasets; Figure S3: miRNAs miR-182, -96, -183 target 3 UTRs of FoxO genes; Figure S4: The host miRNA cluster miR-183/96/182 is upregulated in lung fibroblasts; Figure S5: FoxO1 and FoxO3 proteins are upregulated in HEK293, SH-SY5Y, and HFF cells.