Identification and characterization of the antiviral mode of action of interferon-stimulated genes
Final Report Abstract
The innate immune system, driven by interferon (IFN), holds a vast arsenal of efficient antiviral strategies, raising roadblocks at various steps in virus life cycles. These host mechanisms can teach us new approaches for antiviral intervention, which is especially important for virus infections lacking efficient treatment options. The research objective of this proposal was to gain better insight into specific IFN- stimulated gene (ISG) effector functions – by characterizing the ISG DDX60 in mechanistic detail, and by identifying and characterizing effectors that specifically inhibit late stages of viral replication cycles. The ISG DDX60 had been recently identified as an inhibitor of hepatitis C virus (HCV) replication. Our working hypothesis was that DDX60 acts by inhibiting HCV gene expression or undermines HCV RNA stability. However, early in the project, we realized that its presumed anti-HCV function was an artifact of the HCV reporter virus used in the experiment. This HCV reporter virus carries an internal ribosomal entry site of encephalomyocarditis virus (EMCV) origin, which drives the expression of HCV genes. IRESs are distinct regions of viral RNAs with a specific secondary structure, enabling viruses to recruit ribosomes in the absence of a 5’-cap structure. We found evidence that DDX60 interferes with EMCV IRES-driven translation, as well as with the translation of closely related foot and mouth disease virus (FMDV) IRES. Future experiments will determine the mechanistic details of inhibition, but we hypothesize that DDX60 inhibits one or several IRES-specific translation initiation factors. Our finding is relevant in that the EMCV IRES is used in many engineered (not only HCV) reporter viruses, and some of these viruses have been used in large drug screens. If IRES-specific translation initiation is indeed a target for antiviral intervention, these reporter viruses might no longer be suitable for such screens. In addition to gaining mechanistic detail for a single ISG effector, such as DDX60, it is important to further dissect the multiple antiviral barriers raised upon IFN-signaling. I established and an image-based gain-offunction screen specifically geared to identify inhibitors of late stages of replication cycles: assembly, egress, and infectivity. Screening >400 ISGs for antiviral activity against influenza A virus (IAV), we identified the first ISG effector that acts extracellularly, and further characterized its molecular mechanism of action. The serpin PAI-1 inhibits influenza A virus (IAV) spread by inhibiting extracellular proteases that the virus uses for envelope glycoprotein cleavage. Our study opened up a new field of research: how the innate immune system reshapes the proteolytic environment to inhibit the last step in a virus life cycle - maturation. As many viruses rely on a similar maturation step to complete their life cycle, this antiviral host strategy might be of broad antiviral nature. Furthermore, the example of PAI-1 and other ISGs such as viperin or tetherin highlights the potency of late-acting ISG inhibitors in concert with the IFN-response. Other ISGs might also target late stages of viral replication cycles, and hold potentially novel mechanisms of action. To identify and characterize other late-acting ISGs inhibiting viruses from different families will be part of future work. Understanding the mechanisms by which individual effectors effectively inhibit viral infections may open new avenues for a targeted drug design. Our study on PAI-1 was covered in online articles by The Rockefeller University Newswire and the Medical Research Council News. http://newswire.rockefeller.edu/2015/02/12/key-to-blocking-influenza-virus-may-lie-in-a-cells-own-machinery/ http://www.nimr.mrc.ac.uk/news/host-protease-inhibitor-helps-block-influenza-virus-spread/
Publications
- (2014). Inferferon-Stimulated genes: a complex web of host defences. Annual Reviews of Immunology Vol 32, 513-545.
Schneider WM, Dittmann Chevillotte M, Rice CM
(See online at https://doi.org/10.1146/annurev-immunol-032713-120231) - (2014). Polymorphisms in MDA5 link protein function to clearance of hepatitis C virus. Hepatology. 2015 Feb;61(2):460-70
Hoffmann FS, Schmidt A, Dittmann Chevillotte M, Wisskirchen C, Hellmuth J, Willms S, Gilmore RH, Glas J, Folwaczny M, Müller T, Berg T, Spengler U, Fitzmaurice K, Kelleher D, Reisch N, Rice CM, Endres S, Rothenfusser S
(See online at https://doi.org/10.1002/hep.27344) - A serpin shapes the extracellular environment to prevent influenza A virus maturation. Cell 2015 Feb 12;160(4):631-43
Dittmann M, Hoffmann HH, Scull MA, Gilmore RH, Bell KL, Ciancanelli M, Wilson SJ, Crotta S, Yu Y, Flatley B, Xiao JW, Casanova JL, Wack A, Bieniasz PD, Rice CM
(See online at https://doi.org/10.1016/j.cell.2015.01.040) - ATP-Dependent Effector-like Functions of RIG-I-like Receptors. Molecular Cell, Volume 58, Issue 3, 7 May 2015, Pages 541-548
Yao H, Dittmann M, Peisley A, Hoffmann HH, Gilmore RH, Schmidt T, Schmidt-Burgk J, Hornung V, Rice CM, Hur S
(See online at https://doi.org/10.1016/j.molcel.2015.03.014)
