Expression kinetics of MCMV miRNAs. Murine fibroblast cells were infected at a multiplicity of 10 PFU per cell in the presence of either cycloheximide (CHX), foscarnet (PAA), or no drug. RNA was harvested at multiple time points (in hours) throughout infection and after 24 h of CHX or PAA treatment and subjected to Northern blot analysis. The expression of mature miRNAs and corresponding pre-miRNAs is shown. Northern blot analysis for cellular miR-16 and U6 snRNA served as loading controls. Lanes M, RNA from mock-infected cells.

In vivo detection of MCMV miRNAs by nuclease protection assay. (A) Five micrograms of RNA extracted from the livers, lungs, and spleens of two noninfected mice or three mice i.v. infected with 5 × 10⁶ PFU MCMV for 3 days (lanes A, B, and C) or 5 days (lanes D, E, and F) were hybridized to a radiolabeled probe complementary to miR-m01-4 or miR-M44-1, digested with nuclease S1, and loaded on a denaturing acrylamide gel. The arrows indicate the expected positions of the protected fragment. ND, not digested; Neg, negative-control yeast RNA; M1 and M2, RNA from mock-infected mouse 1 and mouse 2, respectively; Pos, RNA from NIH 3T3 cells infected by MCMV; dpi, days postinfection. (B) Time course of infectious virus present in different organs of BALB/c mice i.v. infected with 1 × 10⁶ PFU MCMV. Five mice were infected per group. The livers, lungs, and spleens were taken at 1, 3, 5, and 7 days postinfection. The titers of infectious virus were determined by titrating organ homogenates using centrifugal enhancement (30 min. at 800 × g) and a standard plaque assay. The broken lines indicate the detection limit, and the short black lines indicate the median value.

MCMV-encoded miRNAs. (A) Predicted stem-loop secondary structures of MCMV-encoded miRNAs. Mature miRNA sequences are shown in bold type, and miRNA* sequences are underlined. Nucleotides indicated by a black arrowhead (miR-108-1-3p [m108-1]) were changed from A to G compared to the available MCMV genome sequence. Secondary structures were determined using mFold. (B) Northern blot analysis of MCMV-expressed miRNAs. Murine fibroblast cells were infected at a multiplicity of 10 PFU per cell. Total RNA was harvested from mock-infected cells (M) and in MCMV-infected cells at 4, 14, and 26 hpi and subjected to Northern blot analysis using probes specific for the cloned viral miRNA sequences. Northern blot analysis of cellular miR-21 and of U6 snRNA served as loading controls.

MCMV miR-M23-1-5p and -3p are polyuridylated. (A) Schematic representation of the m21-M23 loci in the genome. Known or predicted ORFs are indicated by white open arrows, and miRNAs are indicated by gray arrowheads. (B) Northern blot analysis of miR-M23-1-5p and miR-M23-1-3p at 24, 48, and 72 hpi (MOI of 10) showed the appearance of an RNA ladder, a feature distinct from all other miRNAs. Lanes M, RNA from mock-infected cells. (C) Sequence clones representing miR-M23-1-5p. Nongenomic nucleotides are indicated by bold type. The number of clones of the sequence at 24, 48, and 72 hpi is indicated to the right of the sequence.

Characterization of miRNA deletion mutants. (A) Expression of viral miRNAs in NIH 3T3 cells infected (MOI of 10) with wild-type MCMV (wt) or with deletion mutants for miR-m01-4, miR-M23-2/m21-1, and miR-M44-1 or in mock-infected cells (M). Total RNA was harvested at 24, 48, and 72 hpi and subjected to Northern blot analysis. The expression of viral miRNAs and corresponding pre-miRNAs is shown. Northern blot analysis for cellular miR-16 and ethidium bromide staining for U6 snRNA served as loading controls. m01-4, MiR-m01-4. (B) Relative quantification of indicated miRNAs in ΔmiR-m01-4 versus wild-type (wt) MCMV-infected cells after normalization to U6 signals. The broken line indicates a relative value of 1. (C) Growth curves of wild-type MCMV (closed circles) and MCMV deletion mutants MCMV-ΔmiR-m01-4 (open squares), MCMV-ΔmiR-M23-2/m21-1 (open circles), and MCMV-ΔmiR-M44-1 (open triangles) on murine embryonic fibroblasts. MEFs were infected at an MOI of 0.1. Supernatants (from duplicate samples) were harvested daily over a 6-day period and titrated on MEFs by the standard plaque assay. Combined data of two independent experiments are shown. (D) Relative quantification of m01 transcripts in NIH 3T3 cells infected with wild-type (wt) MCMV (open bars) and MCMV-ΔmiR-m01-4 (gray bars) at an MOI of 10 for 24, 48, and 72 h. After RNA extraction, viral DNA was efficiently removed by DNase I treatment, and cDNA was synthesized using oligo(dT) primers. Relative quantification of m01 and IE1 transcripts was performed by real-time PCR based on standard curves prepared from BAC DNA (data not shown). The values are shown as means ± standard errors of the means (error bars).