Virally encoded microRNAs (miRNAs) have recently been discovered in herpesviruses. However, their biological roles are mostly unknown. We developed an algorithm for the prediction of miRNA targets and applied it to human cytomegalovirus miRNAs, resulting in the identification of the major histocompatibility complex class I-related chain B (MICB) gene as a top candidate target of hcmv-miR-UL112. MICB is a stress-induced ligand of the natural killer (NK) cell activating receptor NKG2D and is critical for the NK cell killing of virus-infected cells and tumor cells. We show that hcmv-miR-UL112 specifically down-regulates MICB expression during viral infection, leading to decreased binding of NKG2D and reduced killing by NK cells. Our results reveal a miRNA-based immunoevasion mechanism that appears to be exploited by human cytomegalovirus.MiRNAs constitute a large family of small noncoding RNAs that regulate gene expression posttranscriptionally, affecting mRNA degradation and translation by base-pairing with the 3′ untranslated regions (3′UTRs) (1). The recent discovery of virally encoded miRNAs, mostly in herpesviruses, intriguingly suggests that miRNAs may function in interspecies Copyright © 2007 We applied RepTar and subsequently cRepTar to all human 3′UTRs, searching for potential binding sites of the 11 HCMV miRNAs listed in miRBase 7.0 (9). MICB, an immunorelated gene, was among the highest ranking predicted targets and the top prediction for hcmv-miR-UL112 (Fig. 1A). MICB is a stress-induced ligand of NKG2D, a natural killer (NK) activating receptor expressed on almost all human NK cells and activated cytotoxic T lymphocytes (CTLs) (10). The importance of MICB in the immune response against HCMV infection is substantiated by the specific down-regulation of MICB surface expression via the UL16 protein of HCMV (11,12). MICA, another stress-induced ligand of NKG2D, was also ranked among the top predicted targets of hcmv-miR-UL112 (Fig. 1A). The hcmv-miR-UL112 putative binding sites of both genes are almost identical and are located within a highly similar but not evolutionarily conserved (7) 150-nucleotide (nt) region of their 3′UTRs.To assess the function of hcmv-miR-UL112, we expressed this miRNA in various human tumor cell lines that endogenously express MICA and MICB with the use of recombinant lentiviral vectors: hcmv-miR-UL112 and two control vectors, a non-miRNA sequence (miRcontrol) and hcmv-miR-US5-1. The expression of hcmv-miR-UL112 was confirmed by quantative real-time polymerase chain reaction (qPCR) ( fig. S1). The vectors contained green fluorescent protein (GFP) for monitoring the infection efficiency (7). No difference in the transduction efficiency of the different lentiviral vectors was measured ( fig. S2). Analysis of the various tumor cells transduced with hcmv-miR-UL112 revealed a specific and extensive reduction of MICB and little or no reduction of MICA (Fig. 1B). The downregulation was specific to MICB and to hcmv-miR-UL112, because no change in the level of major histocompati...

Micro (mi)RNAs are small non-coding RNAs that regulate the expression of their targets' messenger RNAs through both translational inhibition and regulation of target RNA stability. Recently, a number of viruses, particularly of the herpesvirus family, have been shown to express their own miRNAs to control both viral and cellular transcripts. Although some targets of viral miRNAs are known, their function in a physiologically relevant infection remains to be elucidated. As such, no in vivo phenotype of a viral miRNA knock-out mutant has been described so far. Here, we report on the first functional phenotype of a miRNA knock-out virus in vivo. During subacute infection of a mutant mouse cytomegalovirus lacking two viral miRNAs, virus production is selectively reduced in salivary glands, an organ essential for virus persistence and horizontal transmission. This phenotype depends on several parameters including viral load and mouse genetic background, and is abolished by combined but not single depletion of natural killer (NK) and CD4+ T cells. Together, our results point towards a miRNA-based immunoevasion mechanism important for long-term virus persistence.