Discrete clusters of virus-encoded micrornas are associated with complementary strands of the genome and the 7.2-kilobase stable intron in murine cytomegalovirus

Abstract

The prevalence and importance of microRNAs (miRNAs) in viral infection are increasingly relevant. Eleven miRNAs were previously identified in human cytomegalovirus (HCMV); however, miRNA content in murine CMV (MCMV), which serves as an important in vivo model for CMV infection, has not previously been examined. We have cloned and characterized 17 novel miRNAs that originate from at least 12 precursor miRNAs in MCMV and are not homologous to HCMV miRNAs. In parallel, we applied a computational analysis, using a support vector machine approach, to identify potential precursor miRNAs in MCMV. Four of the top 10 predicted precursor sequences were cloned in this study, and the combination of computational and cloning analysis demonstrates that MCMV has the capacity to encode miRNAs clustered throughout the genome. On the basis of drug sensitivity experiments for resolving the kinetic class of expression, we show that the MCMV miRNAs are both early and late gene products. Notably, the MCMV miRNAs occur on complementary strands of the genome in specific regions, a feature which has not previously been observed for viral miRNAs. One cluster of miRNAs occurs in close proximity to the 5' splice site of the previously identified 7.2-kb stable intron, implying a variety of potential regulatory mechanisms for MCMV miRNAs.

FIG. 1.

Secondary structures of MCMV precursor miRNAs. The predicted stem-loop structures from the mfold program are depicted with cloned miRNAs indicated in brackets. Two mature miRNAs were cloned from the stem-loop precursors for mir-m01-4, mir-m01-3, mir-m01-2, and mir-M23-2; the asterisk indicates the sequence that is less likely to be incorporated into the RNA-induced silencing complex. We depict only one precursor for mir-m108-2-3p and mir-m108-2-5p.2, although the organization of these mature miRNAs within the precursor (5′ overhangs) is not characteristic of a typical intermediate.

FIG. 2.

Northern blots of MCMV miRNAs. RNA was extracted from NIH 3T3 cells 72 h after viral infection (V+). RNA from mock-infected cells (M) is included for comparison. Twenty micrograms of RNA was loaded onto each lane, and an RNA marker was included for size reference (the unlabeled, leftmost lane of each blot). Probes directed against mouse mir16 and 5S RNA were used as controls. The positions of molecular size markers (in nucleotides) are shown to the left of the blots. Mus musculus (mmu) mir-16 was also included in the analysis. (A) Blots of miRNAs validated in this report; (B) blots of RNAs cloned but not scored as miRNAs in this report.

FIG. 3.

Genomic organization of MCMV miRNAs. (A) Diagram depicting the regions of the MCMV genome that contain MCMV miRNAs. Rulers indicate the genomic position in kilobases; broken lines are used to indicate a break in the scale. The predicted open reading frames in the vicinity of the miRNAs are noted with open triangles and are above the genome line if they are on the sense strand and below the genome line if they are on the antisense strand (the same format is used for depicting the miRNAs). The black rectangles are the predicted miRNA precursors from Table 1, overlaid on the miRNAs cloned in this study. The oval inset depicts the spatial relationship (not to scale) between mir-m108-2-3p, mir-m108-2-5p.2, and mir-m108-1 (drawn as black triangles) and the two introns previously identified (depicted from 3′ to 5′, to be consistent with the diagram of the whole genome above); both the 5′ splice sites are noted as well as the 3′ splice site. (B) Northern blot of mir-m108-2-3p at the indicated time points (hours) after infection. Lanes: M, mock-infected cells; V+, virus-infected cells. The positions of molecular size markers (in nucleotides) are shown to the left of the blot. The asterisk indicates the faint precursor band near the 50-nt RNA marker.

FIG. 4.

Detection of MCMV miRNAs in bone marrow-derived macrophages. RNA was extracted from NIH 3T3 (3T3) or BMM cells 72 h after infection with MCMV (MOI of 1). RNA from mock-infected cells (M) and virus-infected (V+) cells was poly(A) tailed, reverse transcribed, and subjected to RT-PCR. The RT-PCR product (0.5 μl) was loaded on the Lab901 D800 ScreenTape, which automatically separated and sized the amplified products by gel electrophoresis and integrated software. The bands indicated with an asterisk were all sized between 64 and 70 nt, as expected from the RT-PCR protocol; additional bands (band with two asterisks) were observed for mir-m108-2-3p that corresponded to primer-dimer products with distinguishable dissociation curves.

FIG. 5.

Kinetic classes of MCMV miRNAs. (A) Relative values of each miRNA from RNA samples extracted from NIH 3T3 cells infected with MCMV (MOI of 2) in the presence or absence of cycloheximide (CHX) (100 μg/ml) or phosphonoacetic acid (PAA) (250 μg/ml) after 24 h. The RNA was from mock-infected cells (M) or virus-infected cells (V+). Data shown are the averages for two biological replicates (each of which was an average from three technical replicates); error bars represent the standard deviations. Data were normalized for each miRNA by setting the quantity of the miRNA in untreated, but infected, samples at 1.0. Mus musculus (mmu) mir-16 was also included in the analysis. (B) Northern blots of mir-m01-2 and mir-m108-2-3p (for validation of the qRT-PCR results) are shown on the left. Blots of mir-M23-2 and mir-m107-1-3p shown on the right are included for kinetic class characterization of these miRNAs. The positions of molecular size markers (in nucleotides) are shown to the left of the blots.