Fig 2.
HSV-1 miRNAs and construction and characterization of HSV-1 recombinants lacking miR-H2, miR-H3, or miR-H4. (A) Schematic diagram of the HSV-1 genome and expanded view of the LAT locus. The diagram of the HSV-1 genome (∼150 kb) shows the unique long (UL) and short (US) regions, flanked by the terminal repeat long (TRL) and internal repeat long (IRL) and by the internal repeat short (IRS) and terminal repeat short (TRS), respectively. The location of the LAT within the RL is shown. The 8.5-kb LAT primary transcript is shown, with the exons and 2.0-kb stable intron indicated. Additionally, the lytic ICP0 and ICP34.5 transcripts are designated. Six miRNAs (miR-H2, -H3, -H4, -H5, -H7, and -H8) are encoded within the LAT exonic region, and two miRNAs (miR-H1 and -H6), which lie antisense to each other, are encoded within the LAT promoter (shaded arrowheads). (B) Inactivating mutations introduced into HSV-1 miR-H2 and miR-H3. Shown are the coding sequences of the HSV-1 ICP0 and ICP34.5 genes, located antisense to HSV-1 miR-H2-3p and miR-H3-3p miRNAs, respectively. In each case, the upper line gives the predicted amino acid sequence; the second line, the wild-type DNA sequence; and the third line, the mutant sequence. Changes are indicated by boldface, and the miRNA seed sequence targets are boxed. The mutations introduced do not affect the coding sequence but were predicted to disrupt the formation of the pri-miRNA stem-loop. (C) Characterization of HSV-1 mutants lacking miR-H2 (17mH2), miR-H3 (17mH3), or miR-H4 (17dH4). RS cells were infected with recombinant virus clones of 17mH2 (left), 17mH3 (center), 17dH4 (right), and 17syn+ (WT). The infected cells were harvested at 18 h postinfection, and miRNA abundance was quantified using qRT-PCR. The relative quantities were normalized to that of HSV-1 miR-H1-5p. The standard error of the mean was calculated from three independent experiments. (D) MicroRNA assay of HSV-1 mutants 17mH2, 17mH3, and 17dH4. Recombinant virus clones of 17mH2 (light green), 17mH3 (purple), 17dH4 (sky blue), and wild-type 17syn+ (blue) were used to infect RS cells. The infected cells were harvested at 18 h postinfection, and miRNA abundance was quantified using qRT-PCR. The relative quantities were normalized to the level of the viral thymidine kinase mRNA (UL23). Threshold cycle values indicated by asterisks were as high as those seen in mock infections (data not shown). The standard error of the mean was calculated from three independent experiments.