Characterization of a Novel FACT-ETS-1 Antiviral Response Pathway and Identification of RNA Virus-Encoded Antagonists of this Host Response - Project Summary Transcription of antiviral factors by the host cell is a fundamental aspect of innate immune responses to virus infection. We recently discovered a novel transcriptional response in humans termed the “FACT-ETS-1 Antiviral Response (FEAR)” Pathway that restricts the replication of vaccinia virus (VV), a large DNA virus belonging to the poxvirus family. Activation of this pathway requires the FACT complex, an ancient histone chaperone that is conserved from yeast to humans. The human FACT complex is comprised of two protein subunits, hSpt16 and SSRP1, that function together to regulate cellular gene transcription. Our work discovered that FEAR pathway activation requires a novel, SUMOylated form of hSpt16 that is normally found in the cytoplasm of cells but that translocates to the nucleus upon virus infection to form specialized FACT complexes that activate expression of ETS-1. The ETS-1 protein is a member of the ETS family of transcription factors that arose in multicellular animals ~600 million ago during evolution. Our previous work demonstrated that FACT- induced ETS-1 expression is required for the restriction of VV in human cells. However, the VV-encoded A51R protein functions as a FEAR pathway inhibitor by directly binding to SUMOylated hSpt16 and tethering it to cytosolic microtubules. More recently, we discovered that both FACT and ETS-1 are also required to restrict the replication of the RNA virus, vesicular stomatitis virus (VSV). Moreover, we found that the VSV-encoded matrix (M) protein promotes the specific depletion of SUMOylated, but not non-SUMOylated, hSpt16 subunits during infection. However, a mutant VSV strain encoding a single amino acid substitution in its M protein both fails to deplete SUMOylated hSpt16 subunits during infection and strongly induces ETS-1 expression. These data suggest that the FEAR pathway may also both restrict, and be antagonized by, RNA viruses. However, the human genes that are regulated by ETS-1 during infection that contribute to RNA virus restriction are unknown. Our preliminary data suggest that that genes upregulated by ETS-1 during VSV infection are largely distinct from the interferon response, a well-characterized antiviral transcriptional response that inhibits diverse viruses. Notably, we have also discovered that RNA viruses unrelated to VSV, such as paramyxoviruses and flaviviruses, also specifically promote depletion of SUMOylated hSpt16 subunits during infection. We hypothesize that VSV and these other RNA viruses encode FEAR pathway antagonists that deplete SUMOylated hSpt16 levels to prevent FEAR pathway activation and expression of ETS-1-regulated antiviral genes. Thus, our study goals are to: 1) Identify the human genes regulated by ETS-1 during RNA virus infection that are involved in virus restriction and 2) Determine if the FEAR pathway restricts other RNA viruses besides VSV and whether these viruses encode FEAR pathway inhibitors. Our long-term goal is to understand how the FEAR pathway broadly restricts DNA and RNA viruses and how virus-encoded FEAR pathway antagonists contribute to viral pathogenesis.
