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

Abstract

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.

Human cytomegalovirus microRNA miR-US25-1-5p inhibits viral replication by targeting multiple cellular genes during infection

“…In addition, hcmv-miR-US25-1-5p was demonstrated to inhibit HCMV DNA replication through the reduction of IE72 and pp65 expressions (Stern-Ginossar et al, 2009). It has also been reported to target ATP6V0C, an essential host factor for HCMV replication (Pavelin et al, 2013). A recent study revealed that hcmv-miR-US25-1-5p aggravates the ox-LDL-induced apoptosis of endothelial cells via targeting and down-regulating BRCC3 (Fan et al, 2014).…”

Ranavirus Replication: Molecular, Cellular, and Immunological Events

“…For example, poxviruses encode decoy receptors for IFNα/β, IFN-γ, IL-1β, IL-18, various chemokines, and complement that bind their cognate targets and downregulate their effector functions (Johnston et al 2005 ;McFadden 2003 , 2004 ;Seet et al 2003 ). In addition, virus-encoded miRNAs may also play critical roles in blocking host responses or controlling viral replication (Babu et al 2014 ;Hook et al 2014 ;Pavelin et al 2013 ).…”

Cytomegalovirus microRNAs

“…Experimental techniques including microarray [12] and SILAC [17,18] have been used to identify targeted transcripts and proteins respectively, that are differentially expressed upon the exogenous addition of the miRNA of interest or anti-sense inhibitors to the miRNA [18]. Recently, biochemical approaches based on RISC immunoprecipitation (RIP), including RIP-Chip, PAR-CLIP, and HITS-CLIP, have been developed to directly identify targeted transcripts incorporated into the RISC complex [2,13,19-26]. These approaches usually identify transcripts containing seed matches (upwards of 75%) and experimental validation has been exceptionally high compared with bioinformatics approaches [13,27,28].…”

“…Building on the comprehensive miR-US25-1 RISC-IP analysis that led to the identification of cell cycle control genes, Pavelin et al [26] demonstrated that miR-US25-1 targets the endosomal acidification complex component ATP6V0C and validated five other targeted genes (Table 1, Fig. 1D).…”