Abstract

DHODH (dihydroorotate dehydrogenase (quinone)) has been demonstrated as a critical regulator of programmed cell death, yet its role in macroautophagy/autophagy remains poorly defined. Flaviviridae pose a significant threat to global public health, and their replication is closely associated with autophagy. Building upon our previous findings that DHODH was a broad-spectrum target for Flaviviridae and a key regulator of Pestiviruses replication, this study employed RNA-seq screening coupled with functional validation to demonstrate that DHODH affected Pestiviruses replication by regulating mitophagy. Notably, we observed remarkable virus genus specificity in this regulatory mechanism. For autophagy-dependent Pestiviruses, DHODH deficiency impaired autophagosome-lysosome fusion, thereby suppressing viral replication. Conversely, in autophagy-inhibiting Flaviviruses, the blockade of autophagy flux facilitated viral replication. These observations underscore the specificity of DHODH-mediated viral replication regulation. Additionally, compound supplementation assays indicated that DHODH regulated autophagy via pyrimidine nucleotide metabolism, as exogenous pyrimidine precursors restored autophagosome-lysosome fusion. Furthermore, our research uncovered a novel mechanism whereby classical swine fever virus (CSFV) non-structural protein 4A (NS4A) recruited DHODH to mitochondria, facilitating its interaction with MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) through the LC3-interacting region (LIR) domain to activate mitophagy. Collectively, our findings highlight DHODH as a promising antiviral target within the metabolism-autophagy axis, providing novel insights for antiviral drug development.Abbreviation: AMPK: AMP-activated protein kinase; ATF4: activating transcription factor 4; ATG5: autophagy related 5; BafA1: bafilomycin A1; BNIP3L/NIX: BCL2 interacting protein 3 like; BVDV: bovine viral diarrhea virus; CALCOCO2/NDP52: calcium binding and coiled-coil