## 3 Results  
### miR-183/96/182 Cluster Is Upregulated in HSV-1 Infected Primary HFFs  
To comprehensively investigate the deregulation of host miRNAs during productive HSV-1 infection and their potential biological functions, we compared two independent miRNA sequencing datasets. First, we infected the primary HFFs at an MOI of 5, extracted RNAs from mock-infected cells, and cells infected with HSV-1 strain KOS at 8 and 18 h post-infection (h.p.i. ), and analyzed miRNA expression by massive parallel sequencing. The sequences obtained were analyzed using sRNAtoolbox. In each experiment, we detected about 1500 human miRNA expressed at a wide abundance range (i.e., from 0.02--279,598 reads per million (RPM)) (Table S2). On the other hand, HSV-1 encoded miRNAs represented only a minor fraction of the total miRNA in the sample (i.e., 0.24% and 0.68% at 8 and 18 h.p.i., respectively) (Figure S1), which is in accordance with similar previous studies. Our differential expression analysis showed significant deregulation (fold change \(\geq\) 2) of 134 human miRNAs at 18 h.p.i. (Figure 1A), however, only a small fraction of miRNAs were deregulated with a fold change \(\geq\)3. As expected, at 8 h.p.i., we did not observe a significant deregulation of miRNAs compared to the mock; nonetheless, a slight trend in the deregulation of the same miRNAs that were significantly deregulated at 18 h.p.i. can be observed. The second experiment represented one of our previous sequencing efforts, which was performed similarly (i.e., using the identical virus strain (KOS) infecting the matching cell type (HFFs)) under similar conditions (10 and 24 h.p.i.) and the same sequencing platform. Of note, the overall sequencing quality was comparable (i.e., total number of reads, the HSV-1 miRNA pattern, and the relative quantity) (Table S3). We hypothesized that by comparing two unrelated sequencing experiments, we would be able to identify the reproducibly deregulated miRNAs in HSV-1 infections. Surprisingly, the overlap between these two experiments was rather limited (i.e., only four miRNAs (miR-182, -183, -96, and -375) were reproducibly upregulated and two miRNAs were downregulated (miR-29 and -27a) under all experimental conditions) (Figure S2). The fold change in the reproducibly deregulated miRNAs is shown in Figure 1B. These results suggest a strong dependence on the cells used in the experiments and may explain the observed discrepancies between different studies. Nonetheless, miRNAs expressed from a single transcriptional cluster, miR-183, -96, and -182 [38] were reproducibly upregulated and showed the highest level of deregulation, indicating their importance. The same cluster was found upregulated in a limited study on two in vitro HSV-1 latency models (rat superior cervical ganglia derived neurons quiescently infected with HSV-1 GFP-US11 (Patton strain) and HFF quiescently infected with HSV-1 KOS strain) [29], but also in cells infected with other herpesvirusures [27; 28]. Importantly, we detected only minor fluctuations (<1.5\(\times\)) in the expression levels for several miRNAs that had previously been shown to be significantly deregulated in HSV-1 infected cells [21; 23; 26; 29; 39; 40; 41; 42; 43; 44; 45; 46; 47]. As above-mentioned, the use of different viruses, different cells, and conditions could explain this discrepancy. To additionally challenge the reproducibility of our sequencing results and to address the upregulation of the miR-183/96/182 cluster in different cells, we infected two primary cells (i.e., human foreskin fibroblasts HFFs and mouse bone marrow-derived macrophages BMDM), human lung fibroblasts WI38 cells), and two widely used transformed cell lines (i.e., human embryonic kidney cells HEK293 and human neuroblastoma cells SH-SYSY) and analyzed the miRNA expression by the stem-loop-RT-qPCR at 8 and 18 or 24 h.p.i. In the analysis, we included an additional four host miRNAs, miR-23a-3p, miR-101-3p, miR-132-3p, and miR-138-5p, previously reported as deregulated and/or important for HSV-1 infection [21; 24; 40; 43].