GAPDH sign was overlapping with MAVS-specific music group (indicated by asterisk) and was subtracted preceding normalization. (C) RPTECs were mock-treated (best panels, still left) or contaminated with BKPyV Dun-bottom sections) and were set following 72?h for confocal microscopy for DNA (blue), Vp1 (cyan), MAVS (crimson), and phosphorylated Drp1-S616 (magenta) (bottom level panels, still left). allograft failing in kidney transplant sufferers. Despite virus-specific T?cells and neutralizing antibodies, BKPyV persists in kidneys and evades defense control seeing that evidenced by urinary shedding in immunocompetent people. Here, we report that BKPyV disrupts the mitochondrial membrane and network potential when expressing the 66aa-long agnoprotein during past due replication. Agnoprotein is enough and required, which consists of amino-terminal and central area for mitochondrial network and concentrating on disruption, respectively. Agnoprotein impairs nuclear IRF3-translocation, interferon-beta appearance, and promotes p62/SQSTM1-mitophagy. Agnoprotein-mutant infections struggling to disrupt mitochondria present decreased replication and elevated interferon-beta appearance but could be rescued by type-I interferon blockade, TBK1-inhibition, or CoCl2-treatment. Mitochondrial fragmentation and p62/SQSTM1-autophagy take place in allograft biopsies of kidney transplant sufferers with BKPyV nephropathy. JCPyV and SV40 infections disrupt mitochondrial systems likewise, indicating a conserved system facilitating polyomavirus persistence and post-transplant disease. and but generally ignored by the adaptive immunity (Leuenberger et?al., 2007, Rinaldo et?al., 1998). BKPyV agnoprotein co-localizes with lipid droplets (LD) (Unterstab et?al., 2010) and membranous 4-Azido-L-phenylalanine structures of the ER (Unterstab et?al., 2013). We now report that the BKPyV agnoprotein also targets mitochondria and subverts interferon- induction by disrupting the mitochondrial network and its membrane potential and promotes p62/SQSTM1 mitophagy in cell culture and in kidney allograft biopsies. Results BKPyV Agnoprotein Colocalizes with Mitochondria and Induces Mitochondrial Fragmentation To elucidate the function of BKPyV agnoprotein in the absence of LD, we noted that the N-terminal amino acid (aa) sequence had similarity to mitochondrial targeting sequence (MTS) found in cytochrome oxidase cox8 (Figure?S1). To investigate the potential mitochondrial localization, we infected primary human renal proximal tubular epithelial cells (RPTECs) with the agnoprotein wild-type BKPyV-Dunlop (Dun-and mutant Dun-viruses, immunofluorescent staining identified infected cells in the late viral 4-Azido-L-phenylalanine replication phase by detecting both the early viral protein large T-antigen (LTag) and the late viral protein Vp1 capsid in the nucleus and agnoprotein in the cytoplasm (Figure?S2). Using the mitochondrial outer membrane protein Tom20 as a marker, its specific colocalization with both the wild-type and mutant protein was found, demonstrating agnoprotein colocalization to mitochondria (Figure?1A). However, the mitochondria of Dun-protein colocalized with the ER marker calreticulin (Figure?1C). However, whereas the mitochondrial colocalization of the protein appeared in network strings, the ER colocalization with calreticulin was patchy and reminiscent of the contact sites with the mitochondria-associated membranes CLTB (MEMs) (Figure?1C). The patchy ER-colocalization pattern was independently confirmed using protein disulphide isomerase (PDI), another ER marker protein (Figure?S2C). The results indicated that targeting of ER and mitochondria remained intact and implicated the amphipathic character of the central -helix of the wild-type agnoprotein in the disruption of the mitochondrial network. Open in a separate window Figure?1 Agnoprotein Colocalizes with Mitochondria and Induces Mitochondrial Fragmentation in the 4-Azido-L-phenylalanine Late Replication Phase of BKPyV Infection (A) Z-stacks of RPTECs infected with BKPyV Dun-(top row) or with Dun-(bottom row, large replicating cell next to small non-replicating cell) at 48?hpi, stained for Tom20 (red), agnoprotein (green), and DNA (blue). Colocalizing voxels are shown in yellow. (B) Quantification of mitochondrial morphology in six fields of two independent experiments using Fiji software (mean? SD, two-way ANOVA). (C) Z-stacks of BKPyV Dun-at indicated times post-infection. Cells were stained for LTag (red), agnoprotein (green), mitochondrial marker Tom20 (cyan), and DNA (blue). White arrows indicate cells magnified (scale bar, 20?m). Video S1. Dun-Agnoprotein Colocalizes with Mitochondria and Induces Mitochondrial Fragmentation, Related to Figure?1: Cells were infected with the indicated viral strains and fixed at 48?hpi as described in Transparent Methods. Z-stacks of BKPyV Dun-Mutant 4-Azido-L-phenylalanine Agnoprotein Colocalizes with Mitochondria, but Does Not Induces Fragmentation of Mitochondrial Network, Related to Figure?1: Z-stacks of BKPyV Dun-infected cells, stained for mitochondrial marker Tom20 (red), agnoprotein (green), and DNA.