Abstract
Feline coronavirus (FCoV) has been identified as the aetiological agent of feline infectious peritonitis (FIP), a highly fatal systemic disease in cats. FCoV open reading frame 3 (ORF3) encodes accessory proteins 3a, 3b and 3 c. The FCoV 3b accessory protein consists of 72 amino acid residues and localizes to nucleoli and mitochondria. The present work focused on peptide domains within FCoV 3b that drive its intracellular trafficking. Transfection of different cell types with FCoV 3b fused to enhanced green fluorescent protein (EGFP) or 3×FLAG confirmed localization of FCoV 3b in the mitochondria and nucleoli. Using serial truncated mutants, we showed that nucleolar accumulation is controlled by a joint nucleolar and nuclear localization signal (NoLS/NLS) in which the identified overlapping pat4 motifs (residues 53–57) play a critical role. Mutational analysis also revealed that mitochondrial translocation is mediated by N-terminal residues 10–35, in which a Tom20 recognition motif (residues 13–17) and two other overlapping hexamers (residues 24–30) associated with mitochondrial targeting were identified. In addition, a second Tom20 recognition motif was identified further downstream (residues 61–65), although the mitochondrial translocation evoked by these residues seemed less efficient as a diffuse cytoplasmic distribution was also observed. Assessing the spatiotemporal distribution of FCoV 3b did not provide convincing evidence of dynamic shuttling behaviour between the nucleoli and the mitochondria.
Highlights
Coronavirus infections have emerged in various species of mammals and birds and are generally associated with a wide spectrum of respiratory, intestinal and systemic infections
Crandell Rees feline kidney (CRFK) cells were transfected with these constructs and the subcellular distribution pattern was evaluated by confocal microscopy (Fig. 1c)
Visualization of the mitochondria using MitoTracker Red CMXRos clearly demonstrated that Feline coronaviruses (FCoV) 3b–enhanced green fluorescent protein (EGFP) colocalizes with this organelle (Fig. 2)
Summary
Coronavirus infections have emerged in various species of mammals and birds and are generally associated with a wide spectrum of respiratory, intestinal and systemic infections. The feline enteric coronavirus (FECV) is an enzootic enteropathogenic virus that replicates in intestinal epithelial cells after oral uptake [1,2,3,4,5]. FIPV spreads systemically and causes the highly fatal disease known as feline infectious peritonitis virus (FIP), which is characterized by a fibrinous and granulomatous serositis, fibrinous effusions in the affected body cavities and/or multifocal granulomatous vascular lesions in several organs [10,11,12]. Besides these two pathotypes, two FCoV serotypes can be distinguished. The less prevalent serotype II FCoV arose by double recombination between serotype I FCoV and canine coronavirus (CCoV), resulting in a FCoV with spike and open reading frame 3 (ORF3) sequences of CCoV origin [20, 21]
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