Travis Lear (University of Pittsburgh), Mads Larsen (University of Pittsburgh), Bo Lin (University of Pittsburgh), Qing Cao (University of Pittsburgh), Irene Alfaras (University of Pittsburgh), Jason Kennerdell (University of Pittsburgh), Laura Salminen (University of Pittsburgh), Daniel Camarco (University of Pittsburgh), Karina Lockwood (University of Pittsburgh), Áine Boudreau (University of Pittsburgh), Jing Ma (University of Pittsburgh), Jie Liu (University of Pittsburgh), Jay Tan (University of Pittsburgh), Michael Myerburg (University of Pittsburgh), Yanwen Chen (University of Pittsburgh), Claudette St Croix (University of Pittsburgh), Yusuke Sekine (University of Pittsburgh), John Evankovich (University of Pittsburgh), Toren Finkel (University of Pittsburgh), Bill Chen (University of Pittsburgh), Yuan Liu (University of Pittsburgh)
The endo-lysosomal pathway plays an important role in pathogen clearance and both bacteria and viruses have evolved complex mechanisms to evade this host system. Here, we describe a novel aspect of coronaviral infection, whereby the master transcriptional regulator of lysosome biogenesis – 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 viral-mediated TFEB degradation and protects against viral-induced cytopathic effects. We further derive a series of small molecules that interfere with the DCAF7-TFEB interaction. These agents inhibit viral-triggered TFEB degradation and demonstrate broad anti-viral activities including attenuating in vivo SARS-CoV-2 infection. Together, these results delineate a viral-triggered pathway that disables the endogenous cellular system that maintains lysosomal function and suggest that small molecule inhibitors of the E3 ubiquitin ligase DCAF7 represent a novel class of endo-lysosomal, host-directed, anti-viral therapies.
This work was supported by NIH grants to TBL (T32 HL110849), JWE (1K08HL144820), TF (1R01 HL142663, 1R01HL142589 and P30 AG024827), BBC (5R35HL139860 and 5R01HL133184), and YL (5R01HL142777), support from the University of Pittsburgh Aging Institute seed fund to BBC, TF and YL and a grant from Jewish Healthcare Foundation (TF).
Viral infection activates PAK2 kinase to create a TFEB phospho-degron motif. Phosphorylated TFEB is recognized as a substrate for the CRL4-DCAF7 E3 ligase complex, in which DCAF7 facilitates TFEB ubiquitination and degradation. Inhibition of DCAF7 preserves TFEB protein, and increases expression of key TFEB transcription targets, enhancing lysosomal biogenesis and activity. Inhibiting pathogen-induced TFEB protein degradation maintains endo-lysosomal activity thereby limiting viral infectivity.
