Background:

Human microRNAs (hsa-miRNA) bind to target-mRNAs inhibiting protein translation. We hypothesized that the host response to CMV includes hsa-miRNAs that specifically down- or up-regulate virus replication by targeting viral proteins (antiviral) or host innate-immune proteins (proviral). Identification of these miRNAs and their targets were evaluated using clinical cohorts combined with in vitro assessment.

Methods:

We determined expression profiles of 847 hsa-miRNA in peripheral blood using microarray in 24 transplant recipients with CMV-viremia vs 11 controls. We then conducted in-vitro testing on 144 differentially expressed hsa-miRNAs to determine pro- and anti-viral effects using CMV plaque assays. Computational prediction of targets of hsa-miRNAs against CMV and host genes was done. Predicted targets were confirmed using luciferase constructs and western blots. Subsequently, the most promising miRNAs underwent quantitative PCR testing in a cohort of 250 transplant recipients with CMV-viremia (day 0 and 21 of therapy). Findings were correlated with CMV-replication dynamics.

Results:

In vitro screening of 144 hsa-miRNAs using plaque assays identified 14miRNAs with a significant proviral- (n=8) or antiviral-effect (n=6). Computational analysis indicated several potential binding sites of antiviral miRNAs to critical CMV genes (e.g. CMV-UL52, UL70) and pro-viral miRNAs to important innate immune genes (e.g. IRAK1, TRAF6, IL1-receptor). Luciferase constructs and western blots confirmed several of these targets (e.g. miRNA 185* reduced UL52 and host-NFkB p50 by 50% and 80% in western blot). In a second cohort of 250 patients with CMV disease, quantitative PCR of whole blood of these 14 miRNAs demonstrated detectable transcripts in most patients. MiRNA levels showed dynamic changes with several showing significant (p<0.001) declines or increases over time, suggesting active modulation during CMV viremia. Four miRNAs, in particular showed a consistent 2-fold or higher change from day 0 to day 21. Of these four, miRNA 185* (an antiviral miRNA) dynamics were associated with CMV relapse after discontinuation of antiviral therapy (p=0.035).

Conclusions:

We identify a complex and biologically relevant host miRNA response against CMV. These include host miRNAs that likely play antiviral and proviral roles by targeting both viral and host genes and may have potential as biomarkers.

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