Inhibition of Virally-Induced TFEB Proteasomal Degradation as a Host-Centric Therapeutic Approach for Coronaviral Infection

The endo-lysosomal pathway plays an evolutionarily conserved role in pathogen clearance, and both bacteria and viruses have evolved complex mechanisms to evade this host defense system. Here, we describe a novel aspect of coronaviral infection, whereby the master transcriptional activator of lysosomal homeostasis – TFEB – is targeted for proteasomal-mediated degradation upon viral infection. Through mass spectrometry analysis and an unbiased siRNA screen, we identify that TFEB protein stability is coordinately regulated by the E3 ubiquitin ligase subunit DCAF7 and the PAK2 kinase. In particular, viral infection triggers marked PAK2 activation, which in turn, phosphorylates and primes TFEB for ubiquitin-mediated protein degradation. Deletion of either DCAF7 or PAK2 blocks virally-stimulated TFEB degradation and protects against virally-induced cytopathic effects. We further derive a series of novel small molecules that interfere with the DCAF7-TFEB interaction. These agents inhibit virus-induced TFEB degradation and demonstrate broad anti-viral activities including attenuating SARS-CoV-2 infection in two animal models. Together, these results delineate a virally-triggered pathway that impairs lysosomal homeostasis in the host. Small molecule E3 ubiquitin ligase DCAF7 inhibitors that restore lysosomal function represent a novel class of host-directed, anti-viral therapies useful for current and potentially future coronaviral variants.