Senecavirus a 3C protease cleaves RETREG1 to antagonize ER-phagy and promote viral replication via endoplasmic reticulum calcium signaling.

Co-evolution between viruses and autophagy has led to the emergence of viral strategies that manipulate host endoplasmic reticulum (ER) homeostasis, ultimately promoting viral replication. ER turnover is achieved through selective autophagy, also referred to as ER-phagy, which is regulated by the RETREG1/FAM134B (reticulophagy regulator 1) family of reticulon proteins. Nevertheless, how viruses target RETREG1, a receptor for ER-phagy, remains largely unclear. In this study, we demonstrate that infection with Senecavirus A (SVA), an emerging picornavirus, triggers the cleavage of RETREG1, which functions as a negative regulator of viral replication. By screening viral proteins, we identified the SVA 3C protease (3C[pro]) as the executor of this cleavage event. Detailed mapping revealed that residues Q428, E430, and G431 of RETREG1 are involved in its cleavage by the 3C[pro], and the resulting two fragments fail to suppress viral replication. Furthermore, proteolytic cleavage of RETREG1 by 3C[pro] impairs its ability to relieve ER stress and mediate ITPR1 degradation via RETREG1-dependent ER-phagy. This disruption leads to increased ER calcium (Ca2+) release and subsequent activation of autophagy through the CAMKK2-PRKAA2-MTOR axis, which ultimately facilitates SVA replication. Taken together, these findings indicate that SVA antagonizes the antiviral function of RETREG1-mediated ER-phagy via its 3C[pro], highlighting RETREG1 as a potential therapeutic target for combating SVA infection.Abbreviations: 2-APB: 2-aminoethyl diphenylborinate; PRKAA2/AMPK: protein kinase AMP-activated catalytic subunit alpha 2; ATL3: atlastin GTPase 3; BHK-21: baby hamster kidney-21; CAMKK2: calcium/calmodulin dependent proteinkinase kinase2; CCPG1: cell cycle progression 1; CKAP4/CLIMP63: cytoskeleton associated protein 4; co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole; DM: double mutant; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; eGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; GFP: green fluorescent protein; HSPA5/GRP78/BiP: heat shock protein family A (Hsp70) member 5; HA: hemagglutinin; HDAC4: histone deacetylase 4; HEK-293T: human embryonic kidney 293T; hpi: hours post-infection; IFA: indirect immunofluorescence assay; ITPR1/IP3R1: inositol 1,4,5-trisphosphate receptor type 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LIR: LC3-interacting region; mCherry: monomeric cherry; MTOR: mechanistic target of rapamycin kinase; REEP5: receptor accessory protein 5; RETREG1/FAM134B: reticulophagy regulator 1; RTN3: reticulon 3; SD: standard deviation; SEC61B: SEC61 translocon subunit beta; SEC62: SEC62 preprotein translocation factor; SERP1/RAMP4: stress associated endoplasmic reticulum protein 1; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; ST: swine testis; SVA: Senecavirus A; TEM: transmission electron microscopy; TEX264: testis expressed 264, ER-phagy receptor; Tm: tunicamycin; U2OS: human osteosarcoma epithelial cells; UV: ultraviolet; ZVAD-FMK: benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone; μg: microgram; μm: micrometer; μM: micromole.