The vesicular stomatitis virus matrix protein promotes FACT subunit depletion to suppress the FEAR pathway

We recently identified the FACT-ETS-1 Antiviral Response (FEAR) pathway as an interferon-independent innate immune response that restricts DNA virus replication and that is antagonized by poxvirus-encoded A51R proteins (Rex et al., 2024, Nature Microbiology). The human FEAR pathway is mediated by the FACT complex, consisting of hSpt16 and SSRP1 subunits, that remodels chromatin to activate expression of the antiviral transcription factor, ETS-1. To counter this pathway, poxvirus A51R proteins tether SUMOylated hSpt16 subunits to microtubules to prevent ETS-1 expression. While these observations indicate a role for the FEAR pathway in DNA virus restriction, it was unclear if RNA viruses interact with this pathway. Here, we show that the RNA virus, vesicular stomatitis virus (VSV; Rhabdoviridae), is also restricted by the FEAR pathway but has evolved a distinct mechanism to block this antiviral response. Through in vitro assays and cell culture studies, we demonstrate that the VSV encoded matrix (M) protein directly interacts with SUMOylated hSpt16 to promote its ubiquitination and proteasome-dependent degradation. Using deletion mapping, we discovered a N-terminal motif in VSV M that is both required for interaction with host hSpt16 proteins and for their proteasomal degradation. This degradation effectively blocks ETS-1 induction and suppresses FEAR pathway activation during infection. Strains encoding mutant M proteins that cannot antagonize the FEAR pathway exhibit replication defects in human cells that can be rescued by hSpt16 or ETS-1 depletion, suggesting FEAR pathway evasion is critical for efficient VSV replication. Moreover, we show that the inability of VSV M to interact with and degrade SUMOylated Spt16 in lepidopteran cells results in an abortive infection, suggesting VSV-Spt16 interactions can influence virus host range. Collectively, our study illustrates that DNA and RNA viruses have independently evolved diverse mechanisms to antagonize SUMOylated host Spt16 proteins, underscoring the physiological importance of the FEAR pathway to antiviral immunity.