Manipulation of Viral MicroRNAs as a Potential Antiviral Strategy for the Treatment of Cytomegalovirus Infection

Abstract

Cytomegalovirus (CMV) infection leads to notable morbidity and mortality in immunosuppressed patients. Current antiviral drugs are effective but seriously limited in their long-term use due to their relatively high toxicity. In the present study, we characterized the expression of murine CMV microRNAs (MCMV miRNAs) both in vitro and in vivo. Although 29 miRNAs were detectable during in vitro infection, only 11 miRNAs (classified as Group 1) were detectable during in vivo infection, and as many as 18 viral miRNAs (classified as Group 2) were less detectable (<50% of animals) in both the liver and lungs. In addition, viral miRNA profiles in the blood revealed unstable and reduced expression. We next explored the in vitro effects of viral miRNAs on MCMV replication. The inhibition of Group 1 viral miRNAs had little effect on virus production, but transfected cells overexpressing miR-m01-3-5p, miR-M23-1-5p, miR-M55-1, and miR-m107-1-5p in Group 2 showed statistically lower viral loads than those transfected with control miRNA (29%, 29%, 39%, and 43%, respectively, versus control). Finally, we performed hydrodynamic injection of viral miRNA agomirs and observed lower levels of MCMV recurrence in the livers of animals overexpressing the miR-m01-3-5p or mcmv-miR-M23-1-5p agomirs compared with those of animals transfected with control agomir, confirming the antiviral effects of viral miRNA manipulation in vivo. Therefore, the manipulation of viral miRNA expression shows great therapeutic potential and represents a novel antiviral strategy for the miRNA-based treatment of cytomegalovirus infection.

Keywords: antiviral therapy; cytomegalovirus; transfection; viral miRNA.

Figure 1

A schematic diagram depicting agomir transfection into mouse livers is shown. Briefly, BALB/c mice with latent murine cytomegalovirus (MCMV) infection were divided equally into three groups (eight per group). All mice were injected intraperitoneally with lipopolysaccharide (LPS; 15 μg/kg weekly) and cyclosporin A (CsA; 20 mg/kg/d every other day) for three weeks. To overexpress viral miRNAs, the mice received hydrodynamic injections of 2 mL phosphate-buffered saline (PBS) containing miR-m01-3-5p, miR-M23-1-5p, or negative control agomir (NC-agomir) on day 0 and day 10.

Figure 2

Expression kinetics of murine cytomegalovirus MCMV-encoded microRNAs (miRNAs) in vitro. Mouse embryonic fibroblasts (MEFs) were infected with MCMV, and miRNA profiles were determined using a revised real-time quantitative PCR (RT-qPCR) protocol at the indicated time points post-infection. MEFs with mock infection were used as negative controls. The results are shown as fold-change values relative to the expression levels at 72 h post-infection (hpi). Data presented are the result of three independent experiments and are shown as the mean ± standard deviation (SD). N.D.: not detected.

Figure 3

Examination of MCMV miRNA profiles in livers and lungs from two animal models. To test the expression of MCMV miRNAs in vivo, livers and lungs from acute infection/viral recurrence models were collected and performed to ascertain the miRNA profiles by RT-qPCR. The cycle threshold values are shown. The establishment of the two models was described in the Materials and Methods in detail.

Figure 4

The inhibition of Group 1 viral miRNAs exerts minimal effects on viral production. (A) MEF cells were cultured in 24-well plates and transiently transfected with specific inhibitors of miRNAs from Group 1 or negative control RNA (NC-RNA). After 72 h of MCMV infection, viral copies were quantified by qPCR. (B) Two inhibitors of representative miRNAs (miR-m01-4-5p and miR-m21-1) were further examined using TCID₅₀ (50% tissue culture infective dose) assays. The results are shown as the mean ± SD. The assays were performed in triplicate wells, and data were confirmed in three independent experiments. p < 0.05 for viral miRNA-transfected versus NC-RNA-treated MEFs. PFU: Plaque-forming unit.

Figure 5

The overexpression of several Group 2 viral miRNAs inhibits MCMV replication. MEF cells were transfected with specific mimics of viral miRNAs or NC-RNA, followed by MCMV infection. Viral copies were quantified at 72 hpi using qPCR (A). To further investigate the effects of viral miRNAs on MCMV replication, the mimics, inhibitors of two representative miRNAs (miR-m01-3-5p and miR-M23-1-5p), or NC-RNA were transfected into MEFs at two concentrations (30 and 100 nM), followed by MCMV infection. Viral titers were examined using TCID₅₀ assays, as described above (B,C). The results are shown as the mean ± SD. The assays were performed in triplicate wells, and all data were confirmed with three independent experiments. The different fold changes are shown above the horizontal bar. * p < 0.05 for viral miRNA-transfected versus NC-RNA-treated MEFs.

Figure 6

MCMV miRNA agomirs reduce MCMV recurrence in vivo. (A) After viral reactivation and transfection, livers were collected and analyzed to assess viral load; livers from latently-infected mice without drug administration were also measured as negative control. (B) To visualize viral reactivation in animal livers, immunohistochemical (IHC) staining targeting MCMV-gB antigen was performed (black arrow: recurrent virus; red box: the selected magnified area; magnification: 100×/200×). Data are based on two identical experiments. Dashed lines indicate the limit of detection (LOD) for the titer assay. * p < 0.05 for miRNA agomir-transfected group versus NC-agomir-transfected group.