A Human Cytomegalovirus-Encoded microRNA Regulates Expression of Multiple Viral Genes Involved in Replication

Abstract

Although multiple studies have documented the expression of over 70 novel virus-encoded microRNAs (miRNAs), the targets and functions of most of these regulatory RNA species are unknown. In this study a comparative bioinformatics approach was employed to identify potential human cytomegalovirus (HCMV) mRNA targets of the virus-encoded miRNA miR-UL112-1. Bioinformatics analysis of the known HCMV mRNA 3′ untranslated regions (UTRs) revealed 14 potential viral transcripts that were predicted to contain functional target sites for miR-UL112-1. The potential target sites were screened using luciferase reporters that contain the HCMV 3′UTRs in co-transfection assays with miR-UL112-1. Three of the 14 HCMV miRNA targets were validated, including the major immediate early gene encoding IE72 (UL123, IE1), UL112/113, and UL120/121. Further analysis of IE72 regulation by miR-UL112-1 with clones encoding the complete major immediate early region revealed that the IE72 3′UTR target site is necessary and sufficient to direct miR-UL112-1-specific inhibition of expression in transfected cells. In addition, miR-UL112-1 regulation is mediated through translational inhibition rather than RNA degradation. Premature expression of miR-UL112-1 during HCMV infection resulted in a significant decrease in genomic viral DNA levels, suggesting a functional role for miR-UL112-1 in regulating the expression of genes involved in viral replication. This study demonstrates the ability of a viral miRNA to regulate multiple viral genes.

The Cell Biology of Cytomegalovirus: Implications for Transplantation

“…This tissue specificity suggests that CMV may activate multiple transcriptional programs depending on the The mechanisms underlying tissue specificity of viral gene expression may also relate to recently described and generally conserved microRNAs (miRNAs) of CMV (77)(78)(79)(80)(81). These small, noncoding RNA species have diverse effects including regulation of viral replication and immune evasion, which appear to vary by the cell type infected (77)(78)(79)(80)(81)(82)(83). HCMV-miR-UL112-3p, for example, appears to affect both the stress-induced ligand MICB and the antiviral interferon-response element IRF-1 (80,84).…”

The immunology of human cytomegalovirus latency: could latent infection be cleared by novel immunotherapeutic strategies?

“…50 During lytic infection, HCMV expresses approximately 24 miRNAs derived from 13 pre-miRNAs which have been shown to target both viral and cellular RNAs. Viral targets include IE72 as well as a number of viral genes involved in DNA synthesis and it has been suggested that these targets might play some role in latency establishment and reactivation, [51][52][53] although, as yet, there is no direct evidence for this. Cellular targets of HCMV-encoded miRNAs include gene products with functions relating to control of cell cycle, secretory cellular pathways and, of particular interest, immune evasion.…”

MicroRNAs in the regulation of TLR and RIG-I pathways

“…69 For instance, miR-UL112 is encoded by HCMV and targets transcriptional factor IE1, which controls the expression of many viral genes and is vital for viral replication at the onset of infection. [114][115][116] HCMV miR-US4-1 specifically downregulates ERAP1, which participates in trimming MHC class I-presented peptide precursors to mature epitopes. 117 Accordingly, the trimming of HCMV-derived peptides is inhibited, leading to decreased susceptibility of infected cells to HCMV-specific cytotoxic T lymphocytes, thus helping identify a previously unknown viral miRNA-based cytotoxic T lymphocyte-evasion mechanism.…”