Human MicroRNAs Attenuate the Expression of Immediate Early Proteins and HCMV Replication during Lytic and Latent Infection in Connection with Enhancement of Phosphorylated RelA/p65 (Serine 536) That Binds to MIEP

Attenuating the expression of immediate early (IE) proteins is essential for controlling the lytic replication of human cytomegalovirus (HCMV). The human microRNAs (hsa-miRs), miR-200b-3p and miR-200c-3p, have been identified to bind the 3′-untranslated region (3′-UTR) of the mRNA encoding IE proteins. However, whether hsa-miRs can reduce IE72 expression and HCMV viral load or exhibit a crosstalk with the host cellular signaling machinery, most importantly the NF-κB cascade, has not been evaluated. In this study, argonaute-crosslinking and immunoprecipitation-seq revealed that miR-200b-3p and miR-200c-3p bind the 3′-UTR of UL123, which is a gene that encodes IE72. The binding of these miRNAs to the 3′-UTR of UL123 was verified in transfected cells stably expressing GFP. We used miR-200b-3p/miR-200c-3p mimics to counteract the downregulation of these miRNA after acute HCMV infection. This resulted in reduced IE72/IE86 expression and HCMV VL during lytic infection. We determined that IE72/IE86 alone can inhibit the phosphorylation of RelA/p65 at the Ser536 residue and that p-Ser536 RelA/p65 binds to the major IE promoter/enhancer (MIEP). The upregulation of miR-200b-3p and miR-200c-3p resulted in the phosphorylation of RelA/p65 at Ser536 through the downregulation of IE, and the binding of the resultant p-Ser536 RelA/p65 to MIEP resulted in a decreased production of pro-inflammatory cytokines. Overall, miR-200b-3p and miR-200c-3p—together with p-Ser536 RelA/p65—can prevent lytic HCMV replication during acute and latent infection


Introduction
Lifelong latent infection with human cytomegalovirus (HCMV) can alter the host immune system through the excessive clonal inflation of highly differentiated HCMV-specific CD45RA + CD8 + effector memory T lymphocytes [1,2]. Subclinical immune senescence caused by the intermittent reactivation of latent HCMV infection can induce various chronic inflammatory morbidities and aging in individuals, causing difficulties in making a good diagnosis and that metagenomics can help in the diagnostic process [1][2][3][4][5][6][7]. Moreover, lytic HCMV replication can be-directly as well as indirectly-detrimental to the health of hematopoietic stem cell or solid organ transplant (SOT) recipients [8,9]. Such crucial effects of the HCMV latency on various populations indicate that primary infection should be controlled or antiviral strategies should be developed based on a thorough understanding of the pathophysiologic mechanisms [10][11][12][13]. candidate hsa-miRs with the most normalized transcripts per million (TPM) at 2 dpi in the 3 -UTR of HCMV UL123 were selected by subjecting AGO-CLIP-seq and mRNA-seq data to bioinformatic analysis [27]. (B) Seed sequence, matched type, and free energy value for the binding of each miRNA to the 3 -UTR of UL123 were obtained using the BiBiServ RNA hybrid program (https://bibiserv. cebitec.uni-bielefeld.de/rnahybrid, accessed on 17 January 2022). (C) Human U373MG cells seeded (1 × 10 6 /well) in 6-well plates were transfected with pEGFP-N1 plasmid vectors harboring mutant UL123 (∆UL123)-3 -UTR, UL123-3 -UTR, or UL122-UL123-3 -UTR for 5 days. GFP-positive cells were sorted from U373MG cells using FACS. We established stably transfected cells (SCs) by incubating sorted cells for 2 weeks in RPMI-1640 medium supplemented with 10% FBS. Positive control U373MG cells were stably transfected with empty pEGFP-N1 plasmid (pMOCK). Cell populations positive for GFP in the SC groups were assayed by flow cytometry and visualized using fluorescence microscopy. (D) The transfection efficiency of U373MG cells for EGFP plasmids harboring ∆UL1233 -UTR, UL123-3 -UTR, or UL122-UL123-3 -UTR was determined by counting GFP-positive cells. (E) MiRNA mimics (300 ng) for eight candidates that bind to the 3 -UTR of UL123 and negative control (NC) were transfected into U373MG SCs (pMOCK, UL122-3 -UTR-EGFP SC, and UL122-123-3 -UTR-EGFP SC; 1 × 10 6 cells/well) seeded in 6-well plates; exposure was allowed for 2 days in the absence of HCMV infection. The percentage of GFP-positive cells was quantified by flow cytometry, and GFP-positive populations were determined using the FlowJo TM software. (F) Relative GFP-positive cell numbers were normalized to those of cells transfected with NC mimics. All quantitative data were generated from triplicate experiments for each miRNA mimic-transfected SC group. Upper lines in box and error bars indicate means and SEM, respectively.

Identification of miRNAs That Bind to the 3 -UTR of UL123 Based on AGO-CLIP-Seq Data
We searched for hsa-miRs that could bind to the 3 -UTR of UL123 using bioinformatic analysis. We obtained raw high-throughput AGO-CLIP-seq and mRNA-seq datasets from cultured human fibroblasts infected with the Towne strain of HCMV representing acute lytic infection (GEO accession number: GSE63797) [27]. We compared enriched regions of AGO-CLIP-seq with background mRNA-seq reads with a total of 406 base pairs (bp) within the 3 -UTR of UL123 2 days post-infection (dpi) (maximal reads: 4059 vs. 1591, respectively; Figure 1A, lower panel). The height of the mapped reads was similar between AGO-CLIP-seq and mRNA-seq at 1 (1011 vs. 1282) or 3 dpi (2713 vs. 1444) ( Figure S1). Thereafter, eight miRNAs with the most normalized transcripts per million (TPM) at 2 dpi were selected as having a high likelihood of binding to the 3 -UTR of UL123. These miRNAs harbored canonical binding sites (8mer, 7mer-A1, and 7mer-m8) that match the seed region. miR-1208/-4676-3p/-892c-3p or -662/-4716-5p had identical seed sequences ( Figure 1A,B).

Binding of the Predicted miRNAs to the 3 -UTR of UL123
We evaluated the binding of miRNAs to the 3 -UTR of mRNA by using an assay that employed the intracellular expression of green fluorescent protein (GFP) as an indicator of binding. The GFP-based cell assay allows diverse analyses of GFP expression via immunoblotting, immunofluorescence, and flow cytometry. It also enables more reproducible verification of the degrees of miRNA binding through the permanent and stable incorporation of EGFP plasmids harboring the DNA fragments of interest into cells, which facilitates GFP-positive cell sorting [70,71]. To ensure better results, we performed the GFP assay in human glioblastoma astrocytoma (U373MG) cells that exhibit a high transfection efficiency for large plasmids (pEGFP-N1 vector: 4700 bp) and small RNAs [72,73]. Flow cytometry and fluorescence microscopy revealed that U373MG cells stably transfected with EGFP plasmids (SCs) expressed GFP ( Figure 1C,D). The percentage of GFP-positive cells in UL123-3 -UTR-SC (SCs transfected with oligonucleotides corresponding to the 3 -UTR of UL123 mRNA) was as high as that in positive control cells transfected with an empty EGFP plasmid (pMOCK), even when the transfection rate of UL122-123-3 -UTR-SCs (transfected with oligonucleotides encoding the 3 -UTRs of UL122 and UL123 mRNAs) was 45% ( Figure 1C,D).
In our in-house EGFP-based assay, reduced GFP expression was observed when the miRNA bound to the 3 -UTR of mRNA in the SCs. Eight miRNAs were predicted based on the results of AGO-CLIP-seq (a negative control (NC) mimic was included in vitro to ensure the absence of off-target effects) [74]. These miRNAs were transiently transfected into UL123or UL122-123-3 -UTR-SCs for 2 days during the same period [27]. UL123and UL122-123-3 -UTR-SCs transiently transfected with the eight miRNAs expressed GFP ( Figure 1E,F). The number of GFP-positive cells normalized to that in cells transfected with the NC mimic for all eight miRNAs did not differ among pMOCK, UL123-, and UL122-123-3 -UTR-SCs ( Figure 1E,F). These findings indicated that the miRNAs identified by AGO-CLIP-seq do not bind to the 3 -UTR of UL123.
Abundant IE72 and IE86 are generally synthesized at 2-3 dpi in permissive cells infected with a laboratory-adapted HCMV strain [27,83,85,86]. We also found abundant IE72 and IE86 expression at 2 dpi in the nuclei of HFF-1 cells infected with the HCMV Towne strain at an MOI of 0.1 ( Figure S3). According to these findings, we measured IE72 and IE86 levels as well as HCMV titers at 2 dpi after transient transfection with miR-200b-3p or miR-200c-3p mimics under the same conditions in vitro [27].
Restoration of the repressed endogenous miR-200b-3p and miR-200c-3p after acute HCMV infection via mimic transfection resulted in significantly decreased IE72 and IE86 expression (all p < 0.001) compared with the immunoblots of HFF-1 cells transfected with NC mimic (Figure 3C,D). To confirm the effect of miR-200b-3p and 200c-3p on IE72 and IE86 production, we transfected the specific antisense inhibitor of each microRNA after 1 h in HFF-1 cells simultaneously infected with HCMV Towne strain and transfected with miR-200b-3p or miR-200c-3p mimics. Treatment with a miRNA inhibitor, which can downregulate the expression miR-200b-3p and miR-200c-3p in the background of exogenous microRNA mimic exposure during HCMV lytic replication, resulted in the expression of IE72 and IE86 to levels similar to those observed in HFF-1 cells infected with HCMV and transfected with the NC mimic ( Figure 3E).
The expression of HCMV IE72 and IE86 mRNAs can start 4-6 h post-infection (hpi) regardless of the differences in cell types or the size of the viral inoculum [87,88]. Decrease in the expression of miR-200b-3p and miR-200c-3p started at 2 hpi in acute lytic-infected cells, which preceded IE72 and IE86 expression ( Figure 4). This finding also showed that immediately after acute infection, HCMV induced the downregulation of miR-200b-3p and miR-200c-3p to maintain the production of lytic proteins.

The Expression of miR-200b-3p and miR-200c-3p Is Not Downregulated during Latent HCMV Infection and Exogenous Inhibition of miRNA Expression Induced IE Expression and Virus Replication during Latency
We evaluated whether miR-200b-3p and miR-200c-3p levels are altered and similarly lead to suppressed IE gene expression during latent HCMV infection, as it is under acute lytic replication. The non-permissive human monocytic leukemia (THP-1) cell line, which is a suitable model of latent HCMV infection, was infected with the HCMV Toledo strain at an MOI of 5 [28,[89][90][91]. Previous studies detected UL123 mRNA at 1, 2, and 3 dpi, but not at 7 and 10 dpi in THP-1 cells infected with the Toledo strain at a high MOI [28,91].
We also verified early UL122 and UL123 mRNA expression in THP-1 cells infected with the Toledo strain without mimic transfection but not in latent-infected THP-1 after 7 dpi ( Figure S4) [28]. Endogenous miR-200b-3p and miR-200c-3p levels were significantly decreased at 1, 2, and 3 dpi in THP-1 cells with HCMV infection and without mimic transfection compared with THP-1 cells without HCMV infection. These results were consistent with those of acute lytic infection. However, the levels of the two miRNAs did not differ between THP-1 cells with and without HCMV infection at 7, 10, and 14 dpi ( Figure  5A). High-throughput screening of cellular miRNAs in latent-infected THP-1 cells at 10 dpi has not identified any changes in the levels of miR-200b-3p or miR-200c-3p [28]. These findings indicated that downregulated miR-200b-3p and miR-200c-3p when IE proteins

The Expression of miR-200b-3p and miR-200c-3p Is Not Downregulated during Latent HCMV Infection and Exogenous Inhibition of miRNA Expression Induced IE Expression and Virus Replication during Latency
We evaluated whether miR-200b-3p and miR-200c-3p levels are altered and similarly lead to suppressed IE gene expression during latent HCMV infection, as it is under acute lytic replication. The non-permissive human monocytic leukemia (THP-1) cell line, which is a suitable model of latent HCMV infection, was infected with the HCMV Toledo strain at an MOI of 5 [28,[89][90][91]. Previous studies detected UL123 mRNA at 1, 2, and 3 dpi, but not at 7 and 10 dpi in THP-1 cells infected with the Toledo strain at a high MOI [28,91].
We also verified early UL122 and UL123 mRNA expression in THP-1 cells infected with the Toledo strain without mimic transfection but not in latent-infected THP-1 after 7 dpi ( Figure S4) [28]. Endogenous miR-200b-3p and miR-200c-3p levels were significantly decreased at 1, 2, and 3 dpi in THP-1 cells with HCMV infection and without mimic transfection compared with THP-1 cells without HCMV infection. These results were consistent with those of acute lytic infection. However, the levels of the two miRNAs did not differ between THP-1 cells with and without HCMV infection at 7, 10, and 14 dpi ( Figure 5A). High-throughput screening of cellular miRNAs in latent-infected THP-1 cells at 10 dpi has not identified any changes in the levels of miR-200b-3p or miR-200c-3p [28]. These findings indicated that downregulated miR-200b-3p and miR-200c-3p when IE proteins are expressed return to levels equivalent to those of cells without lytic HCMV replication during latency. points until 14 dpi. Both IE72 and IE86 were undetectable in quiescent, latent-infected THP-1 cells (7, 10, and 14 dpi in Figure 5B). However, initial transfection with inhibitors resulted in abundant expression of IE72 and IE86 at 7, 10, and 14 dpi compared with that in HCMV-infected THP-1 cells without inhibitor transfection at 10 dpi ( Figure 5C). HCMV titers after 7 dpi were significantly increased in THP-1 cells that were initially transfected with inhibitors in contrast to those without inhibitor or NC mimic transfection ( Figure  5D). These findings indicated that miR-200b-3p and miR-200c-3p can regulate the shift from latent-to-lytic phase transition of HCMV. Based on this finding, we transfected THP-1 cells with miR-200b-3p or miR-200c-3p inhibitors immediately after HCMV infection to downregulate the two miRNAs during quiescence; then, we measured IE72 and IE86 levels and HCMV titers at various time points until 14 dpi. Both IE72 and IE86 were undetectable in quiescent, latent-infected THP-1 cells (7, 10, and 14 dpi in Figure 5B). However, initial transfection with inhibitors resulted in abundant expression of IE72 and IE86 at 7, 10, and 14 dpi compared with that in HCMV-infected THP-1 cells without inhibitor transfection at 10 dpi ( Figure 5C). HCMV titers after 7 dpi were significantly increased in THP-1 cells that were initially transfected with inhibitors in contrast to those without inhibitor or NC mimic transfection ( Figure 5D).
These findings indicated that miR-200b-3p and miR-200c-3p can regulate the shift from latent-to-lytic phase transition of HCMV.

IE Proteins and MiR-200b-3p or miR-200c-3p Are Associated with Phosphorylation of RelA/p65 at Ser536
The NF-κB binding to MIEP can potentially and rapidly induce the activation of genes encoding the IE72 and IE86 during acute HCMV lytic infection. Therefore, there could be a substantial relationship between the activation of NF-κB and the production of IE proteins. We confirmed that TNF-α appropriately induced RelA/p65 phosphorylation at Ser 536 (p-Ser 536 RelA/p65) within 1 h, which was consistent with previous findings ( Figure  S5) [52,53,55,56]. However, p-Ser 536 RelA/p65 levels were significantly low from 6 to 24 hpi even though the expression of RelA/p65 was maintained in HFF-1 cells infected with the HCMV Towne strain at the same time points ( Figure 6A).
MiR-200b-3p and miR-200c-3p affected IE72 and IE86 expression after binding to the 3 -UTR of UL122 and UL123 and were downregulated during acute lytic replication, and the levels of p-Ser 536 RelA/p65 continuously decreased during the extensive synthesis of IE72 and IE86. We speculated that IE proteins are associated with p-Ser 536 RelA/p65 or that p-Ser 536 RelA/p65 interacts with the HCMV MIEP, which is a major transcriptional promotor of IE protein production [13,14,43]. We transfected U373MG cells with plasmids encoding IE72 and IE86 and measured levels of RelA/p65 and p-Ser 536 RelA/p65 after 2 days. U373MG cells expressing IE72 and IE86 protein did not express p-Ser 536 RelA/p65, even though RelA/p65 was expressed in U373MG cells transfected with IE72 or IE86 plasmids ( Figure 6B). These initial findings indicated that IE72 and IE86 inhibit the phosphorylation of RelA/p65 at Ser 536 and that acute infection with HCMV does not induce RelA/p65 phosphorylation at Ser 536 (unlike the case observed in the background of infection with other microbes) [52,53].

IE Proteins and MiR-200b-3p or miR-200c-3p Are Associated with Phosphorylation of RelA/p65 at Ser536
The NF-κB binding to MIEP can potentially and rapidly induce the activation of genes encoding the IE72 and IE86 during acute HCMV lytic infection. Therefore, there could be a substantial relationship between the activation of NF-κB and the production of IE proteins. We confirmed that TNF-α appropriately induced RelA/p65 phosphorylation at Ser 536 (p-Ser 536 RelA/p65) within 1 h, which was consistent with previous findings (Figure S5) [52,53,55,56]. However, p-Ser 536 RelA/p65 levels were significantly low from 6 to 24 hpi even though the expression of RelA/p65 was maintained in HFF-1 cells infected with the HCMV Towne strain at the same time points ( Figure 6A).  MiR-200b-3p and miR-200c-3p affected IE72 and IE86 expression after binding to the 3′-UTR of UL122 and UL123 and were downregulated during acute lytic replication, and the levels of p-Ser 536 RelA/p65 continuously decreased during the extensive synthesis of IE72 and IE86. We speculated that IE proteins are associated with p-Ser 536 RelA/p65 or that p-Ser 536 RelA/p65 interacts with the HCMV MIEP, which is a major transcriptional promotor of IE protein production [13,14,43]. We transfected U373MG cells with plasmids encoding IE72 and IE86 and measured levels of RelA/p65 and p-Ser 536 RelA/p65 after 2 days. U373MG cells expressing IE72 and IE86 protein did not express p-Ser 536 RelA/p65, even though RelA/p65 was expressed in U373MG cells transfected with IE72 or IE86 plasmids ( Figure 6B). These initial findings indicated that IE72 and IE86 inhibit the phosphorylation of RelA/p65 at Ser 536 and that acute infection with HCMV does not induce RelA/p65 phosphorylation at Ser 536 (unlike the case observed in the background of infection with other microbes) [52,53].

Phosphorylated RelA/p65 (Ser 536 ) Binds to MIEP Region
We used cross-linking chromatin immunoprecipitation (ChIP) assays to investigate whether RelA/p65 phosphorylated at Ser 536 interacts with the enhancer region containing NF-κB-binding sites in MIEP to uncover how the expression of IE proteins is correlated with the reduction in p-Ser 536 RelA/p65 levels. We found that the RelA/p65 and p-Ser 536 RelA/p65 bound to the HCMV MIEP within 1 h of Towne strain infection ( Figure 7A). The relative amount of RelA/p65 bound to MIEP was continuously increased compared with that of p-Ser 536 RelA/p65 ( Figure 7A). The ratio of RelA/p65 to p-Ser 536 RelA/p65 increased over time ( Figure 7C). In contrast to natural acute HCMV infection, transfection with miR-200b-3p and miR-200c-3p mimics induced the binding of p-Ser 536 RelA/p65 to MIEP to a greater extent than that observed with RelA/p65 ( Figure 7B). The ratio of RelA/p65 to p-Ser 536 RelA/p65 at MIEP decreased over time in HFF-1 cells transfected with miR-200b-3p/200c-3p mimics with significant changes at 6 and 24 hpi for both miR-200b-3p and miR-200c-3p. Opposite results were observed in cells without mimic transfection ( Figure 7C). We also found that the upregulation of miR-200b-3p and miR-200c-3p results in decreased levels of pro-inflammatory interleukin (IL)-1β, IL-6, and RANTES (CCL5), all of which are induced in response to NF-κB activation ( Figure 7D). These data suggested that downregulating the expression of IE proteins by upregulating the levels of miR-200b-3p and miR-200c-3p can In contrast to natural acute HCMV infection, transfection with miR-200b-3p and miR-200c-3p mimics induced the binding of p-Ser 536 RelA/p65 to MIEP to a greater extent than that observed with RelA/p65 ( Figure 7B). The ratio of RelA/p65 to p-Ser 536 RelA/p65 at MIEP decreased over time in HFF-1 cells transfected with miR-200b-3p/200c-3p mimics with significant changes at 6 and 24 hpi for both miR-200b-3p and miR-200c-3p. Opposite results were observed in cells without mimic transfection ( Figure 7C). We also found that the upregulation of miR-200b-3p and miR-200c-3p results in decreased levels of proinflammatory interleukin (IL)-1β, IL-6, and RANTES (CCL5), all of which are induced in response to NF-κB activation ( Figure 7D). These data suggested that downregulating the expression of IE proteins by upregulating the levels of miR-200b-3p and miR-200c-3p can result in the induction of p-Ser 536 RelA/p65 expression. Thereafter, instead of RelA/p65, p-Ser 536 RelA/p65 binds to the MIEP owing to its abundance and finally results in the inhibition of NF-κB activity and pro-inflammatory cytokine production.

Discussion
The discovery of new biological agents targeting IE protein synthesis during acute HCMV replication and immediately after the lytic switch from latent infection is essential to improve early detection, prognostic prediction, design preventive strategies, and ultimately control the harmful effects from HCMV in the general population as well as critically ill or severe immunocompromised patients [13,15,34,92]. MicroRNAs and short hairpin (sh) RNAs are promising candidates for silencing gene expression via post-transcriptional regulation [24,29,31,35,66,93]. The exploration of miRNAs generally requires the prediction of the seed sequences that bind to the 3 -UTR using various algorithms and de novo or machine learning tools, but various computational analyses could not identify enough binding ability or downstream biological roles of the predicted miRNAs in vitro or in vivo. Accordingly, we selected new potential miRNAs that can bind to the 3 -UTR of HCMV UL123, which downregulates IE72 expression, using high-throughput sequencing data from AGO-CLIP-seq instead of traditional algorithms in silico, because cellular miRNAs interacting with UL123-3 -UTR have not been evaluated despite the crucial role of IE72 during early infection [13,27,59,63,87,94,95]. However, sophisticated SC-based flow cytometry of GFP expression revealed that miRNAs with the most abundant transcripts in AGO-CLIP-seq from HCMV-infected cells did not bind to the 3 -UTR of UL123.
Thus, the inability of high-throughput sequencing to predict the roles of miRNAs was verified, and new high AGO-CLIP-seq transcripts for miR-200b-3p and miR-200c-3p that are capable of binding to the 3 -UTR of UL123 were identified. An association between miR-200b-3p and miR-200c-3p and IE86 suppression through binding to the 3 -UTR of UL122 supported the hypothesis that miR-200b-3p and/or 200c-3p function during the IE stage of HCMV replication by targeting the 3 -UTR of UL123 and are associated with IE72 expression [34,35,40]. The transient transfection of SCs, flow cytometric measurements of GFP, immunoblotting, and fluorescence imaging showed that miR-200b-3p and miR-200c-3p bind to the 3 -UTR of UL123. The binding affinity of miR-200b-3p and miR-200c-3p for the 3 -UTR of UL122 and UL123 was consistently similar. We and others have also found that fluorescence emission is reduced by 50% in luciferase reporter assays with the MIEP promoter [34,35]. These findings provide tangible evidence of the interactions between miR-200b-3p as well as miR-200c-3p and the 3 -UTR of UL122-123 (exons 4 and 5).
The results of the reporter assay using the GFP-expressing SC system correlated with a decrease in IE72 and IE86 protein levels as well as HCMV titers after treatment with miR-200b-3p and miR-200c-3p. In addition, miR-200b-3p and miR-200c-3p inhibitors that counterbalanced the effects of exogenously supplied miRNA returned the expression of IE72 and IE86 to initial levels. These data support the hypothesis that upregulated miR-200b-3p and miR-200c-3p downregulate the expression of IE72 and IE86 after binding to the 3 -UTR of UL122-123 and finally inhibit HCMV replication. In contrast, HCMV can trigger the downregulation of miR-200b-3p and miR-200c-3p to facilitate the expression of IE72 and IE86 during early acute infection. The levels of miR-200b-3p or miR-200c-3p were inversely correlated with the expression of IE72 and IE86 and HCMV replication in latent-infected THP-1 cells.
The site-specific post-translational phosphorylation of RelA/p65, RelB, and c-Rel subunits after the degradation of inhibitor of NF-κB (IκBα) in the canonical NF-κB pathways plays an important role in modulating the downstream effects of NF-κB in response to a wide range of inflammatory stimuli [67,68,96]. Researchers have found a connection between IE proteins and MIEP and the canonical NF-κB signaling cascade, and they identified that this phenomenon involves the regulation of the activities of the IKK complex (IκB kinase) or modulation of NF-κB activity [49,97,98]. The I kappa kinases IKKα, IKKβ, or IKKε also phosphorylate the Ser 468 or Ser 536 residues in the RelA/p65 subunit of NF-κB, which is an event that results in an improved ability of NF-κB to bind the DNA in response to inflammatory stimuli [52,53,68,99,100]. Therefore, phosphorylation at the Ser 536 residue in the transactivation domain of RelA/p65 can facilitate the transcriptional activity of NF-κB [52][53][54]57,101,102]. However, others have found an antagonistic role of IKK-mediated phosphorylation of Ser 536 ; they indicate that this even contributes to the suppression of NF-κB activity by accelerating the turnover of the RelA/p65 or promoting the proteosomal degradation of the RelA/p65 [103][104][105]. The negative regulation of NF-κB by p-Ser 536 RelA/p65 might protect from exaggerated inflammation and a detrimental cytokine storm caused by NF-κB hyperactivation [103,105,106].
This study on acute HCMV infection showed that the p-Ser 536 RelA/p65 negatively affects IE protein levels and NF-κB activity by binding to MIEP. The expression of IE proteins is associated with inhibition of the phosphorylation of RelA/p65 at Ser 536 , and the levels of p-Ser 536 RelA/p65 are decreased during acute lytic infection of HCMV when IE proteins are expressed. Contrary to these findings, the upregulation of miR-200b-3p and miR-200c-3p which helps downregulate the expression of IE72 and IE86 resulted in increased p-Ser 536 RelA/p65 expression. This could competitively interrupt the binding of NF-κB to MIEP and then inhibit the production of pro-inflammatory cytokines. Therefore, we propose that increasing the levels of miR-200b-3p and miR-200c-3p can result in the regulation of lytic replication of HCMV (Figure 8).
Our findings are supported by the published results and methodology. The correlation between the expression of miR-200b-3p and miR-200c-3p was constant (miR-200c-3p > miR-200b-3p) [35,40], and there was a close correlation between the downregulated levels of miR-200b-3p and miR-200c-3p. The SC-based system of using vectors expressing GFP to score for the 3 -UTR binding activity of miRNA was sensitive. The numbers were sufficient to provide statistical power and reproducibility. We attenuated the effects of miR-200b-3p and miR-200c-3p using antisense inhibitors.
Our experiments have the following limitation. There would be conceptual debates as to whether THP-1 cells represent a true HCMV latency model, even though many studies used THP-1 cells for an experimental latent-reactivation model [28,89,91,107,108]. It would be quite informative to evaluate the effect of miR-200b-3p/-200c-3p and their regulation in other models of latency as well as in normal human bone marrow cells (or more specifically CD34 + HPCs). If we could confirm the role of two microRNAs on HCMV lytic and latent infection from various human cells, we will be able to secure more evidence to apply the research results to humans.
In summary, the following findings of this study were novel. MiR-200b-3p and miR-200c-3p inhibited IE72 production after binding to the 3 -UTR of UL123 and decreased the HCMV titers. In summary, the following findings of this study were novel. MiR-200b-3p and miR-200c-3p inhibited IE72 production after binding to the 3′-UTR of UL123 and decreased the HCMV titers. The downregulation of miR-200b-3p and miR-200c-3p can result in IE72 and IE86 production in the latent-infected cells. The levels of phosphorylated Ser 536 RelA/p65 are decreased during lytic replication, and IE proteins alone can inhibit the phosphorylation of p-Ser 536 RelA/p65. Phosphorylated RelA/p65(Ser 536 ) binds to MIEP. Inhibiting the expression of IE proteins by upregulating the expression of miR-200b-3p and miR-200c-3p results in increased levels of p-Ser 536 RelA/p65 and prevents hyperinflammation. In conclusion, attenuation of the expression of IE72 and IE86 proteins and decreased HCMV titers in response to the binding of miR-200b-3p and miR-200c-3p to the 3′-UTR of UL122 and UL123 is associated with the binding of p-RelA/p65 Ser 536 to MIEP. Accumulating information about the functions and effects of miR-200b-3p and miR-200-3p might support a potential clinical role for the use of these miRNAs as biomarkers or therapeutic targets and contribute to our understanding of the role of hsa-miRs during the immediate early period of HCMV lytic and latent infection.

Cell Culture and HCMV Infection
We obtained materials from the listed suppliers as follows: HFF-1 and THP-1 cell lines (ATCC, Manassas, VA, USA); U373MG cell lines (Korean Cell Line Bank, Seoul, Korea). HFF-1 cells were cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific Inc., Waltham, MA, USA Fisher, Seoul, Korea) supplemented with 15% heat-

Evaluation of MicroRNA Expression and HCMV Viral Loads
Total small RNA (50 ng) was purified using mirVana TM miRNA isolation kits (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed in a thermal cycler using TaqMan TM MicroRNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA, USA). Complementary DNA was amplified by real-time polymerase chain reaction (PCR) using stem-loop primers (Table S1) designed for specific miRNAs (TaqMan TM MicroRNA Assays, Applied Biosystems, Foster City, CA, USA) and a LightCycler ® 480 (Roche Life Science, Penzberg, Germany) under the following conditions 95 • C for 10 min followed by 45 cycles of 15 s at 95 • C and 60 sec at 60 • C. For absolute quantitation, seven-point standard curves of serial dilutions (from 10 1 to 10 7 ) were prepared from a mirVana TM miRNA mimic with known copy numbers of miR-200b-3p/200c-3p (Invitrogen, Carlsbad, CA, USA). HCMV titers (IU/mL) were measured by performing quantitative real-time PCR for UL83 using the LightCycler ® 480 (Table 1). Standard curves were constructed from serial dilutions of the National Institute for Biological Standards and Control 09/162, which is the first WHO international standard of HCMV for nucleic acid amplification [112]. All quantitative real-time PCR results are expressed as copy numbers/µL of total input RNA or DNA mass.

Cross-Linking Chromatin Immunoprecipitation (ChiP)
We infected HFF-1 cells (1 × 10 6 /well) with HCMV Towne strain, incubated them with formaldehyde for 15 min, and then quenched cross-linking with glycine. The crosslinked HFF-1 cells were harvested and lysed in immunoprecipitation (IP) buffer containing protease inhibitors. After washing and resuspension, cell pellets were sonicated in IP buffer (Appendixes , Table S1) and then incubated with rabbit anti-RelA/p65 (Cell Signaling, Danvers, MA, USA) or mouse anti-phospho-RelA/p65 (Ser 536 ) (Santa Cruz., Dallas, TX, USA) in an ultrasonic water bath for 15 min at 4 • C. Protein A-agarose beads were washed; then, IP samples were boiled in Chelex 100 for 10 min and centrifuged. We purified the DNA present in the supernatants after centrifugation and amplified the MIEP containing the NF-κB-binding motifs (proximal 550 bp) using PCR (Table 1) [13,43].

Statistical Analysis
All flow cytometry, immunoblotting, and real-time PCR experiments were conducted in triplicate under the same conditions, and data are shown as means ± standard error of the means (SEM) or means ± standard deviation. Flow cytometry results were analyzed using FlowJo TM (version 10.7, BD Biosciences). All other graphs were constructed using Prism Software version 8.0 (GraphPad Software Inc., San Diego, CA, USA). Two groups were compared using unpaired independent t-tests. Values were analyzed using SPSS version 25.0 (IBM Corp., Armonk, NY, USA) and those with p < 0.05 (two-tailed) were considered statistically significant.

Conclusions
The human microRNAs, miR-200b-3p, and miR-200c-3p bind the 3 -UTR of HCMV UL123 mRNA-that encodes IE72-with non-canonical 5-mer matching. We found that the expression of these two miRNAs at sufficient levels can result in the downregulation of IE72/IE86 and eventually inhibition of HCMV replication during acute lytic and latent infection stages. HCMV by itself can downregulate the expression of miR-200b-3p and miR-200c-3p during lytic replication, and conversely, the downregulated miRNAs can induce IE72/86 expression and virus replication in latent infection. In addition, two miRNAs enhanced the phosphorylation of RelA/p65 (Ser 536 ) and decreased the expression of proinflammatory cytokines, suggesting a negative regulation of NF-κB activity. These findings highlight the important role played by hsa-miRs during the IE period of HCMV lytic replication and latent infection.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author.