Host MicroRNA miR-197 Plays a Negative Regulatory Role in the Enterovirus 71 Infectious Cycle by Targeting the RAN Protein

Abstract

Enterovirus 71 (EV71), a member of Picornaviridae, is associated with severe central nervous system complications. In this study, we identified a cellular microRNA (miRNA), miR-197, whose expression was downregulated by viral infection in a time-dependent manner. In miR-197 mimic-transfected cells, EV71 replication was inhibited, whereas the internal ribosome entry site (IRES) activity was decreased in EV71 strains with or without predicted miR-197 target sites, indicating that miR-197 targets host proteins to modulate viral replication. We thus used a quantitative proteomics approach, aided by the TargetScan algorithm, to identify putative target genes of miR-197. Among them, RAN was selected and validated as a genuine target in a 3' untranslated region (UTR) reporter assay. Reduced production of RAN by RNA interference markedly reduced the synthesis of EV71-encoded viral proteins and virus titers. Furthermore, reintroduction of nondegradable RAN into these knockdown cells rescued viral protein synthesis. miR-197 levels were modulated by EV71 to maintain RAN mRNA translatability at late times postinfection since we demonstrated that cap-independent translation exerted by its intrinsic IRES activity was occurring at times when translation attenuation was induced by EV71. EV71-induced downregulation of miR-197 expression increased the expression of RAN, which supported the nuclear transport of the essential viral proteins 3D/3CD and host protein hnRNP K for viral replication. Our data suggest that downregulation of cellular miRNAs may constitute a newly identified mechanism that sustains the expression of host proteins to facilitate viral replication.

Importance: Enterovirus 71 (EV71) is a picornavirus with a positive-sense single-stranded RNA that globally inhibits the cellular translational system, mainly by cleaving cellular eukaryotic translation initiation factor 4G (eIF4G) and poly(A)-binding protein (PABP), which inhibits the association of the ribosome with the host capped mRNA. We used a microRNA (miRNA) microarray chip to identify the host miRNA 197 (miR-197) that was downregulated by EV71. We also used quantitative mass spectrometry and a target site prediction tool to identify the miR-197 target genes. During viral infection, the expression of the target protein RAN was upregulated considerably, and there was a parallel downregulation of miR-197. The nuclear transport of viral 3D/3CD protein and of the host proteins involved in viral replication proceeded in an RAN-dependent manner. We have identified a new mechanism in picornavirus through which EV71-induced cellular miRNA downregulation can regulate host protein levels to facilitate viral replication.