Stacy M. Horner

Regulation of intracellular innate immunity is essential to prevent constitutive production of interferon (IFN). Intracellular innate immunity to RNA virus infection (IFN-induction) is activated by the cytosolic pattern recognition receptors RIG-I and MDA5, which sense pathogen-associated molecular patterns presented by RNA viruses to drive formation of a MAVS-signaling complex. Formation of this MAVS-signaling complex results in signal transduction that leads to the transcriptional activation of type I and type III IFN and ultimately the production of IFN-stimulated genes, which limit viral replication. Many RNA viruses, including HCV, suppress the activation of this antiviral program.

Previously, we have identified cellular proteins that relocalize to MAVS-signaling sites at ER-mitochondrial membrane contact sites at mitochondrial-associated ER membranes (MAM) during IFN induction. Further, we have found that the HCV protease, NS3-NS4A cleaves MAVS at the MAM to block this signaling. Thus, we propose that dynamic localization of host and viral proteins to the MAM helps determine the outcome of HCV and RNA virus infection. As such, current research in the lab is focused at defining how membrane-targeted and relocalized host proteins coordinate the formation of the RIG-I/MAVS-signaling complex on the MAM response and how the HCV NS3-NS4A protease is targeted to the MAM and other subcellular compartments for innate immune regulation. This work has led us to uncover new innate immune regulators, such as RAB1B and UFL1 (see below). This work has also defined specific domains and amino acids of NS3-NS4A that are essential for regulating anti-HCV immunity, leading us to uncover a new antiviral signaling pathway, independent of RIG-I/MAVS, governed by the E3 ubiquitin ligase Riplet.

One of proteins that is relocalized in response to RIG-I signaling is UFL1, an E3 scaffold-type ligase for the ubiquitin-like modifier UFM1. UFM1 is added to lysine residues on proteins by the process of UFMylation. UFMylation has a conjugation (E1, UBA5; E2, UFC1; E3, UFL1, UFBP1) and deconjugation (UFSP2, a cysteine protease) system similar to ubiquitination that allows it to be a dynamic and selective modulator of protein function. We have found that UFL1 and the process of UFMylation promote RIG-I signaling to induce type I IFN. 14-3-3 for RIG-I membrane translocation and downstream signaling Importantly, these studies revealed that 14-3-3ε, the protein that targets RIG-I to membranes, has increased UFM1 conjugation following RIG-I activation, and that the process of UFMylation (UFL1 and UFM1) is required for RIG-I to interact with 14-3-3ε.

Current projects in the lab are focused at defining how UFL1 and process of UFMylation broadly regulate RIG-I signaling and the antiviral response. Questions we are addressing include: (1) What proteins are targeted by UFMylation in response to viral infection? (2) How are UFMylation targets selected and how does UFM1 alter the function of modified proteins? (3) How does UFMylation regulate the function of proteins in the antiviral response? (4) What is the role of UFL1 during infection by flaviviruses?

The host response to RNA virus infection consists of hundreds of gene expression changes in cells that regulate viral infection. Many of these gene expression changes are antiviral and limit virus replication and spread, such as the well-known IFN-stimulated genes induced by the antiviral innate immune sensing pathways that produce IFNs (see above). Some of these genes are also pro-viral and encode proteins required to promote viral replication. A successful viral infection requires tight gene regulation to balance the induction of genes of the innate immune response with those required for viral infection. While we know many of the key protein players in the antiviral innate immune sensing pathways during viral infection, we know very little about the post-transcriptional regulation to these pathways or to the gene expression changes that occur during viral infection. Specifically, post-transcriptional RNA regulatory controls, such as base modifications and associated RNA binding proteins, likely regulate these responses during infection. Viral RNA itself can also be directly regulated by base modifications and RNA binding proteins.

Work from our lab has found that genomic RNA of viruses in the Flaviviridae family, including HCV, ZIKV, DENV, yellow fever, and West Nile virus all contain the RNA base modification m6A within their RNA genomes, and we have found that in HCV, m6A negatively regulates viral genome packaging by modulation specific RNA binding protein interactions with the viral genome.

Current projects in the lab include (1) defining how m6A is targeted to the these viral RNA genomes to regulate infection, (2) defining how m6A changes on cellular RNAs during viral infection are induced, and (3) identifying new RNA binding proteins that regulate these interactions.