miR-US25-1 targets 5′UTR's in context of viral infection.

<p>(a) RISC-IP analysis was conducted on uninfected human primary fibroblast cells or cells infected with HCMV using a direct Ago2 antibody. Results show levels of enrichment of cyclin E2 or TRIM28 transcript from infected cells compared to uninfected cells. RISC-IP was also conducted using pre-bleed antibody derived from rabbits before antigen inoculation. (b) miR-US25-1 was deleted from HCMV. Levels of miR-US25-1 and miR-UL112-1 were determined by RT-PCR analysis following infection of human primary fibroblast cells with either wild type HCMV or the knock out virus. RNA from uninfected cells is used as a negative control. (c) Viral growth of miR-US25-1 knock out virus was compared to wild type HCMV following low (MOI of 0.5) or high (MOI of 10) multiplicity infection of human primary fibroblast cells. Cells plus supernatant were collected at indicated times and assayed on primary human fibroblast cells by limiting dilution (d) Levels of cyclin E2 and TRIM28 protein were determined following high multiplicity infection (MOI of 10) of human primary fibroblast cells with either wild type virus or miR-US25-1 knock out virus. Cells were either grown in normal serum conditions, serum starved conditions or serum starved cells with serum replaced 10 hours prior to harvest. Cells were harvested 72 hours post infection. Relative densities of bands normalized to GAPDH are shown below each lane. Total RNA was also isolated and transcript levels for cyclin E2 (e) and TRIM28 (f) determined by RT-PCR. Transcript copy number was normalized to GAPDH levels.</p>