An Interferon Regulated MicroRNA Provides Broad Cell-Intrinsic Antiviral Immunity through Multihit Host-Directed Targeting of the Sterol Pathway

Abstract

In invertebrates, small interfering RNAs are at the vanguard of cell-autonomous antiviral immunity. In contrast, antiviral mechanisms initiated by interferon (IFN) signaling predominate in mammals. Whilst mammalian IFN-induced miRNA are known to inhibit specific viruses, it is not known whether host-directed microRNAs, downstream of IFN-signaling, have a role in mediating broad antiviral resistance. By performing an integrative, systematic, global analysis of RNA turnover utilizing 4-thiouridine labeling of newly transcribed RNA and pri/pre-miRNA in IFN-activated macrophages, we identify a new post-transcriptional viral defense mechanism mediated by miR-342-5p. On the basis of ChIP and site-directed promoter mutagenesis experiments, we find the synthesis of miR-342-5p is coupled to the antiviral IFN response via the IFN-induced transcription factor, IRF1. Strikingly, we find miR-342-5p targets mevalonate-sterol biosynthesis using a multihit mechanism suppressing the pathway at different functional levels: transcriptionally via SREBF2, post-transcriptionally via miR-33, and enzymatically via IDI1 and SC4MOL. Mass spectrometry-based lipidomics and enzymatic assays demonstrate the targeting mechanisms reduce intermediate sterol pathway metabolites and total cholesterol in macrophages. These results reveal a previously unrecognized mechanism by which IFN regulates the sterol pathway. The sterol pathway is known to be an integral part of the macrophage IFN antiviral response, and we show that miR-342-5p exerts broad antiviral effects against multiple, unrelated pathogenic viruses such Cytomegalovirus and Influenza A (H1N1). Metabolic rescue experiments confirm the specificity of these effects and demonstrate that unrelated viruses have differential mevalonate and sterol pathway requirements for their replication. This study, therefore, advances the general concept of broad antiviral defense through multihit targeting of a single host pathway.

Oxysterol Restraint of Cholesterol Synthesis Prevents AIM2 Inflammasome Activation

“…We show that induction of Ch25h by type I IFN plays a key role in restricting the SREBP2 pathway and cholesterol biosynthesis in the context of bacterial infection and LPS exposure. These observations are consistent with a number of recent studies that have demonstrated the ability of type I IFNs to repress cholesterol metabolism (Robertson et al, 2016)(York et al, 2015). However, our study suggests that Ch25h is not required to initiate suppression of sterol biosynthesis, but is instead required to maintain this effect over time.…”

The Intracellular Cholesterol Landscape: Dynamic Integrator of the Immune Response

“…In a landmark paper, Ghazal and colleagues showed that virus-infected cells undergo Ifnar1-dependent reduction of mevalonic acid pathway flux, and that pathway inhibition impairs cytomegalovirus replication in a manner that can be reversed by metabolic rescue with geranylgeraniol (cell-permeant version of GGPP)[49]. By contrast, interferon-induced reduction in sterols mediates suppression of influenza A virus [50]. Interestingly, reduction in the pool size of newly synthesized cholesterol, either by interferon treatment or genetic intervention on the mevalonate pathway, promotes further interferon induction by activating the ER-resident immune receptor, STING [50].…”

“…By contrast, interferon-induced reduction in sterols mediates suppression of influenza A virus [50]. Interestingly, reduction in the pool size of newly synthesized cholesterol, either by interferon treatment or genetic intervention on the mevalonate pathway, promotes further interferon induction by activating the ER-resident immune receptor, STING [50]. This finding suggests that the sterol and interferon networks communicate as a metabolic-immune circuit, and that ER cholesterol is detected not just by the classical SCAP-SREBP system, but also by innate immune receptors.…”

“…Viperin inhibits the synthetic enzyme farnesyl diphosphate synthase through direct interaction [52]. Ch25h likely inhibits the mevalonic acid pathway through its SREBP-inhibitory product, 25HC [50]. In the case of hepatocytes, 25HC also induces expression of the SREBF2 - and LDLR -targeting microRNA, miR-185, thereby inhibiting formation of the HCV membranous web [53].…”

“…In the case of hepatocytes, 25HC also induces expression of the SREBF2 - and LDLR -targeting microRNA, miR-185, thereby inhibiting formation of the HCV membranous web [53]. miR-342-5p, an interferon-induced microRNA, exerts antiviral effects against multiple pathogenic viruses ranging from cytomegalovirus to influenza A through direct, multi-hit targeting not only of SREBF-2, but also IDI1 and SC4MOL , two mevalonic acid pathway enzymes [50]. …”