Identification and characterization of human cytomegalovirus-encoded microRNAs

Abstract

MicroRNAs (miRNAs) are an extensive class of noncoding genes that regulate gene expression through posttranscriptional repression. Given the potential for large viral genomes to encode these transcripts, we examined the human cytomegalovirus AD169 genome for miRNAs using a bioinformatics approach. We identified 406 potential stem-loops, of which 110 were conserved between chimpanzee cytomegalovirus and several strains of human cytomegalovirus. Of these conserved stem-loops, 13 exhibited a significant score using the MiRscan algorithm. Examination of total RNA from human cytomegalovirus-infected cells demonstrated that 5 of the 13 predicted miRNAs were expressed during infection. These studies demonstrate that human cytomegalovirus encodes multiple conserved miRNAs and suggest that human cytomegalovirus may utilize an miRNA strategy to regulate cellular and viral gene function.

FIG. 1.

Predicted stem-loop secondary structures of HCMV-encoded candidate miRNAs. Sequences in red indicate the miRNA sequences predicted by the MiRscan algorithm. For UL70-1, the detected miRNA is indicated in blue. Secondary structures were determined using Mfold (11).

FIG. 2.

Northern blot analysis of HCMV-expressed miRNAs. Human fibroblast cells were infected at a multiplicity of 5 PFU per cell. Total RNA was harvested and subjected to Northern blot analysis using probes specific for predicted viral miRNA sequences. (a) Expression of UL36-1 miRNA as well as the ∼80-base pre-miRNA species. (b to e) Expression of miRNAs UL70-1, US4-1, US5-1, and US5-2. Ethidium bromide staining of the tRNA band is shown as loading control for each blot. Lane U, uninfected; other lanes are from 2, 8, 24, 48, and 72 h p.i. Positions of radioactive size markers are indicated on the right of the blot.

FIG. 3.

Expression kinetics of HCMV miRNAs. Human fibroblast cells were treated with either cycloheximide, foscarnet, or with no drug and infected at a multiplicity of 5 PFU per cell. At 36 h, total RNA was harvested and subjected to Northern blot analysis. Total RNA from infected cells was harvested as a negative control. Lanes: U, uninfected; I, infected, no drug; CHX, cycloheximide treated; FOS, foscarnet treated. (a) Expression of UL36-1 miRNA and potential pre-miRNA sequence. (b to e) Expression of UL70-1, US4-1, US5-1, and US5-2. Positions of radioactive size markers are indicated on the right of the blots. tRNA band loading control was visualized by ethidium bromide staining of the polyacrylamide gel.

FIG. 4.

Northern blot analysis of HCMV-expressed miRNAs. Human fibroblast cells were infected at a multiplicity of 5 PFU per cell. Total RNA was harvested and subjected to Northern blot analysis using probes specific for predicted viral miRNA sequences. (a to d) Expression of miRNAs UL22A-1, UL112-1, US25-1, and US25-2. Ethidium bromide staining of the tRNA band is shown as a loading control for each blot. Lane U, uninfected; other lanes are from 2, 8, 24, 48, and 72 h p.i. (e to h) Expression of miRNAs in the presence of cycloheximide, foscarnet, or no drug. Uninfected controls are shown. Lanes: U, uninfected; I, infected, no drug; CHX, cycloheximide treated; FOS, foscarnet treated.