On the Importance of Host MicroRNAs During Viral Infection

Abstract

Every living organism has to constantly face threats from the environment and deal with a large number of pathogens against which it has to defend itself to survive. Among those, viruses represent a large class of obligatory intracellular parasites, which rely on their host machinery to multiply and propagate. As a result, viruses and their hosts have engaged in an ever-evolving arms race to be able to maintain their existence. The role played by micro (mi)RNAs in this ongoing battle has been extensively studied in the past 15 years and will be the subject of this review article. We will mainly focus on cellular miRNAs and their implication during viral infection in mammals. Thus, we will describe current techniques that can be used to identify miRNAs involved in the modulation of viral infection and to characterize their targets and mode of action. We will also present different reported examples of miRNA-mediated regulation of viruses, which can have a positive outcome either for the host or for the virus. In addition, the mode of action is also of a dual nature, depending on the target of the miRNA. Indeed, the regulatory small RNA can either directly guide an Argonaute protein on a viral transcript, or target a cellular mRNA involved in the host antiviral response. We will then see whether and how viruses respond to miRNA-mediated targeting. Finally, we will discuss how our knowledge of viral targeting by miRNA can be exploited for developing new antiviral therapeutic approaches.

Keywords: defense mechanism; host–pathogen interaction; microRNA; post-transcriptional regulation; virus.

FIGURE 1

miRNA canonical biogenesis and function. miRNA genes are transcribed by the RNA polymerase II into a primary precursor (pri-miRNA) which is processed in the nucleus by the Microprocessor (Drosha and its cofactor DGCR8) to produce a hairpin structure precursor (pre-miRNA) that will be exported to the cytoplasm by Exportin 5. The pre-miRNA is processed in turn by Dicer into the mature miRNA duplex that will be loaded in an Argonaute protein (AGO) within the RNA induced silencing complex (RISC). One of the strands remains bound to Ago and the complex can mediate post-transcriptional gene regulation by targeting mRNAs through binding of the miRNA seed region (nucleotides 2–8) to the target mRNA (binding site represented by a red rectangle). Adaptor protein GW182 is recruited by RISC and can interact with polyA-binding proteins (PABP) inducing recruitment of CCR4-NOT deadenylase complex. The target mRNA is destabilized by deadenylation and decapping leading to its degradation. Translation of targeted mRNAs is also repressed by inhibition of the preinitiation complex assembly.

FIGURE 2

Current approaches available for identification of miRNAs involved in the regulation of viral infection (A,B) and their targets (C–E). (A) Reciprocal regulation of miRNA expression and viral infection allows identification of candidate miRNAs upregulated (green arrow) or downregulated (red arrow) by miRNA profiling through microarray. Sequencing (next generation sequencing, NGS) provides further information on regulated targets and reveals networks of gene regulation. (B) Virus-centered phenotypic approaches are based on miRNA regulation of the infection. Screens based on the overexpression or inhibition of candidate miRNA in the context of infection, generally using a reporter virus (indicated by the green color), allow a direct observation of the effect on the viral accumulation. This approach coupled to transcriptome profiling also identifies target genes of candidate miRNA. (C) Computational analysis for target identification of a given miRNA are based on the identification of seed-matches in the 3′ UTRs of cellular mRNAs. Bioinformatic predictions rely on the use of target prediction tools such as Targetscan or miRanda for cellular targets or ViTa for viral genomes and transcripts. (D) Biochemical isolation of AGO crosslinked to the miRNA and bound target followed by deep sequencing (AGO-CLIP) allows identification of miRNA specific targets, either cellular or viral, in a genome-wide manner and reveals the precise binding sites on the target. (E) Luciferase (Luc) reporter assays allow functional validation of a miRNA binding site based on the measure of the luciferase enzymatic activity when a potential binding site is present on the 3′UTR. Variants of luciferase (F, firefly; or R, Renilla) containing or not the binding site are used to estimate differential regulation.

FIGURE 3

Mechanism of action of miRNAs which modulate viral infection. (A) miRNA direct effect on virus regulation takes place by direct targeting of viral RNAs at different regions such as 3′UTR, 5′UTR or coding sequences. Binding leads to RNA stabilization, enhanced translation or impaired replication. (B) Indirect effect involves modulation of expression of a cellular transcript encoding a host factor required for one or several steps in the viral cycle. Modulation of receptor expression regulates entry of the virus affecting tropism and cofactors required for replication complexes or translation can impair or enhance viral replication and viral protein production respectively. miRNAs also participate to enhance or restrain cell responses to the infection for instance immune response or defense mechanisms such as apoptosis induction. Viral cycle steps are represented in blue, while host factors and associated pathways are labeled in orange.