The nedd-8 activating enzyme gene underlies genetic resistance to infectious pancreatic necrosis virus in Atlantic salmon

Genetic resistance to infectious pancreatic necrosis virus (IPNV) in Atlantic salmon is a rare example of a trait where a single locus (QTL) explains almost all of the genetic variation. Genetic marker tests based on this QTL on salmon chromosome 26 have been widely applied in selective breeding to markedly reduce the incidence of the disease. In the current study, whole genome sequencing and functional annotation approaches were applied to characterise genes and variants in the QTL region. This was complemented by an analysis of differential expression between salmon fry of homozygous resistant and homozygous susceptible genotypes challenged with IPNV. These analyses pointed to the NEDD-8 activating enzyme 1 (nae1) gene as a putative functional candidate underlying the QTL effect. The role of nae1 in IPN resistance was further assessed via CRISPR-Cas9 knockout of the nae1 gene and chemical inhibition of the nae1 protein activity in Atlantic salmon cell lines, both of which resulted in highly significant reduction in productive IPNV replication. In contrast, CRISPR-Cas9 knockout of a candidate gene previously purported to be a cellular receptor for the virus (cdh1) did not have a major impact on productive IPNV replication. These results suggest that nae1 is the causative gene underlying the major QTL affecting resistance to IPNV in salmon, provide further evidence for the critical role of neddylation in host-pathogen interactions, and highlight the value in combining high-throughput genomics approaches with targeted genome editing to understand the genetic basis of disease resistance

Systematic MicroRNA Analysis Identifies ATP6V0C as an Essential Host Factor for Human Cytomegalovirus Replication

Recent advances in microRNA target identification have greatly increased the number of putative targets of viral microRNAs. However, it is still unclear whether all targets identified are biologically relevant. Here, we use a combined approach of RISC immunoprecipitation and focused siRNA screening to identify targets of HCMV encoded human cytomegalovirus that play an important role in the biology of the virus. Using both a laboratory and clinical strain of human cytomegalovirus, we identify over 200 putative targets of human cytomegalovirus microRNAs following infection of fibroblast cells. By comparing RISC-IP profiles of miRNA knockout viruses, we have resolved specific interactions between human cytomegalovirus miRNAs and the top candidate target transcripts and validated regulation by western blot analysis and luciferase assay. Crucially we demonstrate that miRNA target genes play important roles in the biology of human cytomegalovirus as siRNA knockdown results in marked effects on virus replication. The most striking phenotype followed knockdown of the top target ATP6V0C, which is required for endosomal acidification. siRNA knockdown of ATP6V0C resulted in almost complete loss of infectious virus production, suggesting that an HCMV microRNA targets a crucial cellular factor required for virus replication. This study greatly increases the number of identified targets of human cytomegalovirus microRNAs and demonstrates the effective use of combined miRNA target identification and focused siRNA screening for identifying novel host virus interactions

US25-1 targeting of ATP6V0C occurs through the predicted target site within the ORF.

<p>(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.</p

Systematic analysis of RISC-IP from HCMV infected fibroblast cells.

<p>(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.</p

Comparison of RISC-IP profiles from infected fibroblast cells and HEK293 cells transfected with US25-1.

<p>(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).</p

Summary table of the top 30 most enriched genes following infection with AD169.

<p> Corresponding enrichment levels are shown for TR infected cells.</p

Top 30 enriched genes contain multiple target sites for HCMV miRNAs.

<p>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.</p

Deletion of US25-1 results in increased expression of identified targets in context of virus infection.

<p>(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.</p