In this study, expression kinetics of hcmv-miR-US5-1-5p was determined in U373 cells at different time points during HCMV infection. As shown in Figure 1, hcmv-miR-US5-1 was highly expressed 24 hpi and then accumulated gradually along with prolonged HCMV infection.
Figure 1. Expression kinetics of hcmv-miR-US5-1 in HCMV infected U373 cells. U373 cells were infected with HCMV at 3 MOI. Expression levels of hcmv-miR-US5-1 in HCMV infected cells were quantified by RT-qPCR at 0, 24, 48, 72, 96, 120 and 144 hpi. Data from three independent repetitions were used for statistical analysis.
Hybrid-PCR was used to identify putative targets of hcmv-miR-US5-1. Specific sequences of 23 candidate target mRNAs were obtained from 116 sequenced clones (Table 1). Corresponding mRNAs were identified by blast in GenBank database (http://www.ncbi.nlm.nih.gov/GenBank). All identified putative target mRNAs of hcmv-miR-US5-1 were located in host genome. We then validated a number of mRNAs by luciferase reporter assay. Eleven putative target mRNAs were chosen from our results above, including three mRNAs whose target sites located in coding regions. As shown in Figure 2, led to a diversity decrease of luciferase activity was observed in samples transfected with hcmv-miR-US5-1 and pMIR vectors containing candidate target sequences compared to the miRNA negative control group (NC). These results demonstrate that hcmv-miR-US5-1 can directly bind to these putative target sequences that obtained from hybrid-PCR results. Notably, the putative target mRNAs NUDT5, GMNN and RPL5 whose target sites located in CDS were also recognized and combined by hcmv-miR-US5-1. Among these target genes, GMNN, the cellular DNA replication inhibitor was previously reported to accumulate during HCMV infection. We further investigate the regulation between hcmv-miR-US5-1 and GMNN.
Putative target mRNA Accession Number Position of binding site Nudix (nucleoside diphosphate linked moiety X)-type motif 5 (NUDT5) NM_014142.2 CDS Geminin, DNA replication inhibitor (GMNN) NM_001251989.1 CDS Ribosomal protein L5 (RPL5) NM_000969.3 CDS Polycomb group ring finger 2 (PCGF2) NM_007144.2 3′ UTR Serine/threonine/tyrosine interacting-like 1 (STYXL1) NM_016086.2 3′ UTR Deleted in liver cancer 1 (DLC1) NM_182643.2 3′ UTR SIN3 transcription regulator family member B (SIN3B) NM_015260.2 3′ UTR Ribosomal protein S18 (RPS18) NM_022551.2 3′ UTR Ornithine decarboxylase antizyme 1 (OAZ1) NM_004152.2 3′ UTR Nuclear factor I/X (CCAAT-binding transcription factor) (NFIX) NM_002501.3 3′ UTR Apoptotic peptidase activating factor 1 (APAF1) NM_013229.2 5′ UTR ATPase, H+ transporting, lysosomal 14kDa, V1 subunit F (ATP6V1F) NM_001198909.1 3′ UTR Transmembrane protein 47(TMEM47) NM_031442.3 3′ UTR Transmembrane protein 109 (TMEM109) NM_024092.2 3′ UTR Inner membrane protein, mitochondrial (IMMT) NM_006839.2 3′ UTR Ferritin, heavy polypeptide 1 (FTH1) NM_002032.2 CDS Tripartite motif containing 26 (TRIM26) NM_003449.4 3′ UTR Ral guanine nucleotide dissociation stimulator-like 1 (RGL1) NM_015149.3 3′ UTR Zinc finger, SWIM-type containing 7 (ZSWIM7) NM_001042697.1 3′ UTR Ribosomal protein L41 (RPL41) NM_021104.1 3′ UTR RAD51 paralog D (RAD51D) NM_002878.3 3′ UTR KIAA1462 NM_020848.2 3′ UTR Chromosome 20 open reading frame 194 (C20orf194) NM_001009984.2 3′ UTR
Table 1. Putative targets of hcmv-miR-US5-1 identified by hybrid-PCR
Figure 2. Hcmv-miR-US5-1 mediated repression of target luciferase activity. Dual luciferase assays in HEK293 cells transfected respectively with the empty PMIR reporter vector (pMIR), the reporter vectors containing the putative target sequences together with miRNA negative control (NC) or hcmv-miR-US5-1 (US5-1). Results were shown as percentage expression of negative control sample (NC) following correction for transfection levels according to control renilla luciferase expression. Data from three independent repetitions were used for statistical analysis. ** indicates P < 0.01, * indicates P < 0.05, by comparing to data from cells transfected with miRNA negative control.
The predicted binding site for hcmv-miR-US5-1 was located in nucleotides (nt) 128–148 in GMNN coding region. This binding site was also predicted by online software RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/submission.html) with minimum free energy (mfe) of -29.8kcal/moL (Figure 3A). To further validate whether GMNN is a direct target of hcmv-miR-US5-1, luciferase reporter assay and western blot were performed. As shown in Figure 3B, compared those negative control, cells transfected with pMIR-GMNN-Wt and hcmv-miR-US5-1 mimics resulted in a significant decrease (~75%) of luciferase activity; however, mutation in the hcmv-miR-US5-1 binding site could abolish the effect of hcmv-miR-US5-1 (Figure 3B). Ectopic expression of hcmv-miR-US5-1 markedly reduced the endogenous GMNN protein levels in U373 cells at the same level of those treated with specific siRNA for GMNN. Silencing the over expressed hcmv-miR-US5-1 by its specific inhibitor counteracted the inhibition effect of hcmv-miR-US5-1 on GMNN expression (Figure 3C). These data demonstrated that hcmv-miR-US5-1 could specifically inhibit GMNN protein expression through binding to the coding sequence of GMNN.
Figure 3. Hcmv-miR-US5-1 mediated inhibition of GMNN expression by targeting GMNN coding sequence. Schematic diagram of predicted binding site of hcmv-miR-US5-1 in the GMNN coding domain sequence (CDS). The binding site is at nucleotide 128–148 of the GMNN CDS. (B) Dual luciferase assay were performed in HEK293 cells transfected respectively with the empty PMIR reporter vector (PMIR), the reporter vectors containing the wild type GMNN CDS (GMNN-Wt) and mutant type GMNN CDS (GMNN-Mut) together with miRNA negative control (NC) or hcmv-miR-US5-1 (US5-1). (C) Expressions of GMNN in U373 cells transfected respectively with miRNA negative control (NC), hcmv-miR-US5-1 mimics (US5-1), hcmv-miR-US5-1 mimics and its inhibitor (US5-1+Inhi), and siRNA for GMNN (Si-G) were detected by western blot. Data from three independent repetitions were used for statistical analysis.* indicates P < 0.05, by comparing to data from cells transfected with miRNA negative control.
GMNN proteins were detected in U373 cells mock or infected with HCMV in different conditions. As shown in Figure 4, the expression of GMNN was increased in HCMV infected cells compared with mock cells, and its level accumulated as the infection progressed. However, after transfection of hcmv-miR-US5-1 mimics, the accumulation of GMNN was blocked.
Figure 4. Reduction of GMNN by hcmv-miR-US5-1 during infection. (A) Expression levels of GMNN in U373 cells mock or infected by HCMV during natural infection or after transfection of hcmv-miR-US5-1 mimics were detected by western blot at 24, 48, 72 hpi. Data from three independent repetitions were used for statistical analysis. * indicates P < 0.05, by comparing to data from the mock infected and the hcmv-miR-US5-1 mimics transfected cells at the same time points.
To determine whether hcmv-miR-US5-1 could influence HCMV DNA replication by repressing GMNN, HCMV DNA copies as well as the expressions of hcmv-miR-US5-1 and GMNN were detected in U373 cells infected by HCMV after transfection of miRNA negative control, hcmv-miR-US5-1 mimics, hcmv-miR-US5-1 inhibitor or GMNN specific siRNA, respectively. As expected, transfection of hcmv-miR-US5-1 into U373 cells resulted in substantially increase of hcmv-miR-US5-1 expression compared to miRNA negative control-transfected cells, while, hcmv-miR-US5-1 expression was effectively blocked in hcmv-miR-US5-1 inhibitor-transfected cells (Figure 5A). Our data showed that GMNN was accumulated during HCMV infection in U373 cells (Figure 5B); meanwhile, hcmv-miR-US5-1 expression (Figure 5A) and HCMV copy number (Figure 5C) were also exhibited time-dependent increase. However, decrease of GMNN expression caused by hcmv-miR-US5-1 and GMNN siRNA (Figure 5B) markedly reduced HCMV DNA levels (Figure 5C). Moreover, hcmv-miR-US5-1 inhibitor mediated hcmv-miR-US5-1 silence in infected U373 cells increased GMNN expression at 24, 48, 72 hpi compared with miRNA negative control-transfected cells (Figure 5B), and the corresponding HCMV copy number was also increased (Figure 5C). These results indicated that hcmv-miR-US5-1 may reduce HCMV DNA replication by regulating expression kinetics of GMNN during HCMV infection.
Figure 5. Reduction of HCMV DNA replication by hcmv-miR-US5-1 over-expression. U373 cells transfected with miRNA negative control (NC), hcmv-miR-US5-1 mimics (US5-1) or hcmv-miR-US5-1 inhibitor (Inhi) were infected with HCMV at 3 MOI 24 hours after transfection. At 24, 48, 72 hpi, expression levels of hcmv-miR-US5-1 were measured by RT-qPCR, and normalized to that of cells transfected with miRNA negative control at 24hpi. (B) U373 cells transfected with miRNA negative control, hcmv-miR-US5-1 mimics, hcmv-miR-US5-1 inhibitor or siRNA for GMNN (Si-G) were infected with HCMV at 0.5 MOI 24 hours after transfection. At 24, 48, 72 hpi, GMNN protein expressions were detected by western blot. (C) Viral DNA copies in cells treated as above were measured by real-time PCR at 24, 48, 72 hpi. The assays were performed in triplicate. Data from three independent repetitions were used for statistical analysis. * indicates P < 0.05 by comparing to data from miRNA negative control transfected cells at the same time points.
To determine whether decrease of endogenous GMNN protein level by hcmv-miR-US5-1 could influence the host cell cycle and proliferation, the cell cycle and cell number were measured as described above. As shown in Figure 6A, more cells progressed into the S and G2 cell cycle phases when transfected with hcmv-miR-US5-1 mimics or GMNN specific siRNA. Meanwhile, host cell proliferation also increased in these cells (Figure 6B). Silencing of hcmv-miR-US5-1 by its inhibitor counteracted these effects caused by the miRNA.
Figure 6. Modulation of host cell cycle and cell proliferation by hcmv-miR-US5-1 inhibiting GMNN expression. Cellular DNA contents of U373 cells transfected respectively with miRNA negative control (NC), hcmv-miR-US5-1 mimics (US5-1), hcmv-miR-US5-1 mimics and its inhibitor (US5-1+Inhi) or siRNA for GMNN (Si-G) were quantified by flow cytometry (FACS) analysis after stained with propidium iodide. (B) Cell proliferation was detected by CCK8 analysis. Data from three independent repetitions were used for statistical analysis. * indicates P < 0.05 by comparing to that of cells treated with miRNA negative control.
Table S1. Primer sequences used in plasmid construction
Human cytomegalovirus miR-US5-1 inhibits viral replication by targeting Geminin mRNA
- Received Date: 14 August 2017
- Accepted Date: 26 October 2017
- Published Date: 31 October 2017
Abstract: Viruses commonly create favorable cellular conditions for their survival through multiple mechanisms. MicroRNAs (miRNAs), which function as post-transcriptional regulators, are utilized by human cytomegalovirus (HCMV) in its infection and pathogenesis. In the present study, the DNA replication inhibitor Geminin (GMNN) was identified to be a direct target of hcmv-miR-US5-1. Overexpression of hcmv-miR-US5-1 could block the accumulation of GMNN during HCMV infection, and the decrease of GMNN expression caused by hcmv-miR-US5-1 or GMNN specific siRNA reduced HCMV DNA copies in U373 cells. Meanwhile, ectopic expression of hcmv-miR-US5-1 and consequent lower expression of GMNN influenced host cell cycle and proliferation. These results imply that hcmv-miR-US5-1 may affect viral replication and host cellular environment by regulating expression kinetics of GMNN during HCMV infection.