Figure 1.
Systematic analysis of RISC-IP from HCMV infected fibroblast cells.
(A) Schematic representation of RISC-IP procedure in HCMV infected and uninfected fibroblast cells. (B) Enrichment profile of all genes from AD169 infected cells. Genes were binned according to the enrichment ratio of infected vs uninfected. For example genes with an enrichment ratio from supplemental table 1 of between 0.5 and <2.0 were binned to 1 whereas genes with an enrichment profile of <0.5 but >0.33 were binned to −2. Total number of genes are shown above each bar. Values are skewed towards positive enrichment indicating effective enrichment of HCMV miRNA targets (C). Enrichment profile of the top 100 genes from cells infected with AD169 or TR. (D) Overlap of genes enriched greater than two fold between AD169 infected cells and TR infected cells. Correlation between the enriched profiles was highly significant as determined by Chi Squared test.
Table 1.
Summary table of the top 30 most enriched genes following infection with AD169.
Figure 2.
Top 30 enriched genes contain multiple target sites for HCMV miRNAs.
Sequences for the top 30 enriched transcripts were analysed for HCMV miRNA targets using RNA Hybrid algorithm. Figure shows hit matrix where a yellow square indicates at least one target site for the indicated HCMV miRNA. Independent hit matrices shown for targets either within the whole transcript, or within the CDS, 5′UTR and 3′UTR. Total number of potential miRNAs targeting a transcript shown in the far left column.
Figure 3.
Comparison of RISC-IP profiles from infected fibroblast cells and HEK293 cells transfected with US25-1.
(A) Heat map comparing the enrichment levels from fibroblast cells infected with HCMV or HEK293 cells transfected with either a plasmid expressing US25-1 (pUS25-1) or a US25-1 mimic (bUS25-1). (B) Venn diagram showing the overlap between the top 30 enriched genes from AD169 infected cells and combined enrichment data from cells transfected with US25-1. List of overlapping genes shown in (C).
Figure 4.
Deletion of US25-1 from HCMV results in loss of enrichment of identified targets.
Enrichment of the six US25-1 targets was determined by RT-PCR following RISC-IP from cells infected with wild type virus or cells infected with US25 knock out viruses. Enrichment levels converted to percentage with enrichment from AD169 infected cells set at 100%. Raw enrichment values shown above each bar. Expression levels were corrected against GAPDH and each bar represents 3 biological repeats. * = statistically significant difference (p = <0.01). Statistics were performed on raw data using the Mann-Whitney non-parametric U-test.
Figure 5.
Deletion of US25-1 results in increased expression of identified targets in context of virus infection.
(A) Fibroblast cells were infected at an MOI of 3 with either wild type virus or US25-1 knockout virus and harvested 72 hours post infection. Western blot analysis was performed using antibodies against identified US25-1 targets. Uninfected cells and cells transfected with siRNA against the gene being analysed are included for comparison. (B) Band intensities for three independent biological repeats were determined and corrected for GAPDH levels with relative intensities shown as a percentage with the uninfected value set to 100%. As data represents the ratio between wild type and infected protein levels the error of the ratio is incorporated into the error bars shown for the KO virus protein levels. (C) Fibroblast cells were transfected with either US25-1 mimic or a negative control mimic (40 nM). Cells were harvested 48 h post infection and RNA levels for each of the indicated transcripts determined by real time RT-PCR analysis. Relative levels of RNA for US25-1 mimic transfected cells compared to negative control transfected cells are shown with results normalized to GAPDH. Results represent 3 biological repeats.
Figure 6.
US25-1 targeting of ATP6V0C occurs through the predicted target site within the ORF.
(A) The predicted US25-1 target site from ATP6V0C was cloned downstream of the luciferase reporter construct psiCheck2. The schematic shows the cloning strategy for ATP6V0C. (B) Schematic representation of sequence changes within the target site of ATP6V0C and the corresponding mutation in US25-1 mimic (sequence alterations in the US25-1 mimic are shown in red). (C) Constructs were co-transfected into HEK293 cells with US25-1 mimic, negative control siRNA or a US25-1 mimic with a mutated seed sequence at 40 nM. Data represents 3 biological replicates with standard deviation.
Figure 7.
ATP6V0C is an essential host factor for HCMV replication.
(A) Human Fibroblast cells were transfected with siRNAs (40 nM) against each of the six HCMV miRNA targets previously identified. Cells were then infected with a GFP tagged HCMV virus at an MOI of one, 16 hours post transfection. GFP levels were monitored during the course of infection and compared to determine the effect of knock down of the miRNA targets on HCMV replication. Data represents 4 biological repeats. (B) Fibroblast cells were transfected with three independent siRNAs targeting ATP6V0C (stl1, 2 and 3) as above. Each siRNA resulted in reduced virus growth as determined by GFP fluorescence. Data represents 4 biological repeats. (C) Cells were transfected with pooled siRNAs and infected as above with cells and supernatant harvested at the indicated time points and analysed for infectious virus by plaque assay. Data represents 3 biological repeats (D) Fibroblast cells were transfected with siRNAs as above targeting ATP6V1A or ATP6V1H, components of the V-ATPase complex and HCMV replication analysed by GFP fluorescence. Replication was inhibited by similar levels as observed for knockdown of ATP6V0C. Data represents 4 biological repeats.