Comprehensive analysis of small-RNA deep-sequencing libraries constructed from NIH 3T3 and S11 cells. (A) Table of sequence distribution after annotation according to the MHV-68 genome and the mouse genome. NIH 3T3−, uninfected NIH 3T3 cells; NIH 3T3+, MHV-68-infected NIH 3T3 cells (48 h after infection); S11−, untreated S11 cells; S11+, S11 cells treated with TPA (20 ng/ml for 48 h). (B) Schemes showing the abundances of MHV-68 and mouse sequences in NIH 3T3 and S11 small-RNA deep-sequencing libraries.

MHV-68 miRNAs are located directly after the vtRNA sequences. (A) Genomic locations of MHV-68 miRNAs, modified from reference 32. The first 6 kbp of the MHV-68 genome are shown. Filled triangles represent the 8 vtRNA sequences. M1 to M9 (in red) are the known MHV-68 miRNA precursors. M10 to M15 (in blue) are the 6 novel MHV-68 miRNA genes identified in this paper. ORF, open reading frame; TR, terminal repeat. (B to I) RNA folding structures of 8 vtRNA sequences and their subsequent miRNAs. Sequences are color coded as follows: green, the predicted anticodons of the vtRNA sequences (5); red, the known MHV-68 miRNAs; yellow, the novel star miRNAs of known precursors; blue, the novel miRNAs of novel MHV-68 miRNA precursors. The GenBank accession number of the MHV-68 genome and the genomic locations of the sequences are given at the left of each sequence.

Validation of novel MHV-68 miRNAs. (A) Northern blot validation of novel MHV-68 miRNAs. RNAs were extracted from S11 total-cell lysates (lane 1), a control IP with anti-BrDU (lane 2), and an IP with anti-mAgo2 (monoclonal antibody 6F4) (lane 3) and were blotted onto a nylon membrane. Probes complementary to known or novel MHV-68 miRNAs were labeled and used for detection. Novel MHV-68 miRNAs are highlighted in blue. (B) Stem-loop qRT-PCR validation of the novel MHV-68 miRNA mghv-mir-M1-13. RNAs were extracted from total-cell lysates of S11 cells and from infected (+) or uninfected (−) Ag8 cells. The latter served as a negative control. The samples were subjected to stem-loop RT-qPCR as described in Materials and Methods. Data shown are means ± standard deviations for triplicate determinations. As positive controls, the expression of mghv-mir-M1-1 and that of the cellular miRNA mmu-miR-191 are also shown. (C) Generation of MHV-68 miRNAs in MHV-68-infected Dicer^−/− MEFs. Two independent clones of Dicer^−/− MEFs (lanes 2 and 4) were infected with MHV-68 in parallel with wild-type controls (lanes 1 and 3). Total RNAs were extracted and blotted onto a nylon membrane. Probes complementary to mghv-mir-M1-1 and mmu-mir-21 were labeled and used for detection. Probing against tRNA^Met served as a loading control.

(A and D) Cellular miRNA profiling of uninfected or MHV-68-infected NIH 3T3 cells and of untreated or TPA-treated S11 cells. Cellular miRNA levels were calculated and compared in the four deep-sequencing libraries. (A) Heat map depicting MHV-68-infected versus noninfected NIH 3T3 cells. (D) Heat map depicting TPA-treated versus untreated S11 cells. Only miRNAs that were more than 0.2% abundant in at least one library are shown. (B) Northern blot validation of cellular miRNAs upregulated after infection of NIH 3T3 cells. Total RNAs were extracted from uninfected (lane 1) or MHV-68-infected (lane 2) NIH 3T3 cells. RNAs were blotted onto a nylon membrane and were detected by labeled probes complementary to mmu-mir-16, mmu-mir-15b, or mghv-mir-M1-1. Probing against tRNA^Met served as a loading control. (C) Downregulation of BRCA-1 in MHV-68-infected NIH 3T3 cells. To analyze the expression of BRCA-1, RNAs were isolated from uninfected and MHV-68-infected NIH 3T3 cells 48 h after infection and were subjected to qRT-PCR using primers specific for BRCA-1 and 18S RNA. Data shown are means ± standard deviations for triplicate determinations.