Figure 1.
Comparison of cDNA cloning and RNA-Seq data in relation to current genome annotation.
Comparison of poly(A) cDNA library (green arrows) and RNA-Seq analysis of murine cytomegalovirus (gray histograms). The longest clone from each group of clones in the cDNA library is shown. ELAND alignments of RNA-Seq reads were loaded in Integrative Genomics Viewer and compared to NC_004065.1, (red arrows) and GU305914.1 (blue arrows). The data range for RNA-Seq data was set to 20–5000. Data is shown in 30 kb ranges with 1 kb overlap. Data is shown for the first 120 kb of the MCMV genome and the figure legend is shown in Figure 2.
Figure 2.
Comparison of cDNA cloning and RNA-Seq data in relation to current genome annotation.
Comparison of poly(A) cDNA library (green arrows) and RNA-Seq analysis of murine cytomegalovirus (gray histograms). The longest clone from each group of clones in the cDNA library is shown. ELAND alignments of RNA-Seq reads were loaded in Integrative Genomics Viewer and compared to NC_004065.1, (red arrows) and GU305914.1 (blue arrows). The data range for RNA-Seq data was set to 20–5000. Data is shown in 30 kb ranges with 1 kb overlap. Data is shown for genomic region spanning 119–230 kB of the MCMV genome.
Figure 3.
Analysis of the novel most abundant MCMV transcript and protein.
(A) Comparison of RNA-Seq data and the longest MAT cDNA clone (E125) with current annotation (GU305914). The predicted exons are shown in white boxes. (B) Predicted amino acid sequence of the MAT protein. The first 127 residues match a truncated m169 translation and the C-terminal 20 residues highlighted in gray are derived from exon 2, mapping to the m168 gene. (C) Northern analysis of MAT RNA in MEF cells infected with various deletion mutants. Note that the single gene mutants are partial gene deletions and thus truncated transcripts accumulate. (D) Immunoblot analysis of MEF cell lysates probed with monoclonal antibody generated to the predicted m169 ORF or monoclonal antibody to actin (45 kDa band). (E) Immunoblot analysis of the time course of MAT protein accumulation in infected cells and (F) quantitation. (G) Immunoblot analysis of MAT protein from cells exposed to wild virus isolates. (H) MAT protein accumulation in WT and m168mut virus infected Balb/c MEF. Mutation of the binding site for miR27-b in MAT 3′UTR did not alter regulation of MAT protein expression.
Figure 4.
Transcriptional activity of MCMV.
(A) Whole genome visualization using IGV viewer of RNA-Seq reads mapping to the MCMV genome showing different data ranges. Row 1, range of 20–50,000 reads; Row 2, range of 20–5000 reads; Row 3, range 20–500 reads; Row 4, annotation from NC_004065. (B to D) Quantitation of transcript abundance varies with annotation. The most expressed MCMV genes (RPKM>10 000) relative to NCBI NC_004065 and (B) and GU305914.1 (C). (D) Percentage of reads mapping to coding (exon) or intergenic regions using NC_004065.1 (NC) or GU305914.1 (GU). (E) Example of a transcriptionally active region between M85 and M87.
Figure 5.
Verification of new transcripts by northern blot.
Balb/c MEF cells were infected with BAC derived Smith virus and harvested at indicated times post infection. Total RNA was separated by denaturing gel electrophoresis, transferred to nylon membrane and incubated with probes specific for S and AS transcripts. RNA integrity and loading was evaluated by inspecting 28S (not shown) and 18S rRNA bands under UV light after transfer to membrane. Transcripts in the m15–16 (A), m19-m20 (B), M116 (C) and M71-m74 (D) gene regions were analyzed (Due to smiling effects during gel electrophoresis for the image shown in 4A and C, the ladder was not accurate for inner lanes of the gel and the position of the ribosomal bands was therefore used to estimate the band sizes). Predicted genes (Rawlinson's annotation) are depicted as empty arrows, while thin black arrows show longest transcripts cloned in our cDNA library as well as clones used to generate probes (marked with *). 3′ ends of transcripts are marked with arrowheads. The nucleotide coordinates relative to Smith sequence (NC_004065.1) of isolated transcripts are given below thin arrows, while the names of the clones are written above. Thin gray lines show isolated transcripts that cannot be detected with the probe. Gray histograms showRNA-Seqreads aligned to MCMV genome. Maximal possible exposure times were used to ensure even low abundance transcripts are detected and are noted on the blots.
Table 1.
cDNA clones and PCR primers used to generate probes for Northern analyses.
Table 2.
Top 20 host genes1 induced in infection.
Table 3.
Top 20 host genes1 upregulated in infection.
Table 4.
Top 15 host genes1 repressed in infection.
Table 5.
Top 20 host genes1 downregulated in infection.
Figure 6.
Validation of RNA-Seq analysis of host genes by western blot.
(A) Immunoblot analysis of MEF.K (A) or Balb/c MEF (B–C) cell lysates infected with wild-type MCMV. Cell lysates were separated by SDS-PAGE, transferred to PVDF membrane, and probed with antibody to Jag2 (A), EN2 (B) or Trim71 (C). Monoclonal antibody to actin was used as loading control. Bar charts represent relative quantification of proteins. In the case of Trim71 (C where anti-Trim71 antibody detected multiple bands, the bars show quantification of the middle band.
Figure 7.
Gene enrichment analysis of differentially regulated mouse genes in MCMV infection.
Differentially expressed genes were identified by SAMMate and analyzed with IPA Core Analysis with fold change ratio cutoff of 2. Shown are top diseases and disorders, molecular and cellular functions, and physiological system development and functions (A) and top canonical pathways (B) of DE genes.