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1.
Genomics ; 113(6): 3842-3850, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34547402

RESUMEN

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.


Asunto(s)
Enfermedades de los Peces , Virus de la Necrosis Pancreática Infecciosa , Salmo salar , Animales , Enfermedades de los Peces/genética , Marcadores Genéticos , Sitios de Carácter Cuantitativo , Salmo salar/genética
2.
PLoS Pathog ; 9(12): e1003820, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24385903

RESUMEN

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.


Asunto(s)
Citomegalovirus/fisiología , Interacciones Huésped-Patógeno/genética , MicroARNs/genética , ATPasas de Translocación de Protón Vacuolares/fisiología , Replicación Viral/genética , Células Cultivadas , Citomegalovirus/patogenicidad , Infecciones por Citomegalovirus/genética , Perfilación de la Expresión Génica , Células HEK293 , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Análisis por Micromatrices , Organismos Modificados Genéticamente , ARN Interferente Pequeño/farmacología , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Proteínas Virales/genética , Replicación Viral/efectos de los fármacos
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