Cellular localisation of the proteins of region 3 of feline enteric coronavirus

Abstract

Feline enteric coronaviruses have three open reading frames (ORFs) in region 3 (3a, 3b, and 3c). All three ORFs were expressed with C-terminal eGFP and 3xFLAG tags in different cell lines and their localisation was determined. ORF 3a is predicted to contain DNA-binding and transcription activator domains, and it is localised in the nucleus and in the cytoplasm. ORF 3b is also predicted to contain DNA-binding and activator domains, and was found to localise in the mitochondrion. Besides that, in some of the non-infected and FIPV-infected cells nucleolar, perinuclear or nuclear membrane accumulation of the eGFP-tagged 3b was observed. The exact compartmental localisation of ORF 3c is yet to be determined. However, based on our co-localisation studies 3c does not seem to be localised in the ER-Golgi network, ERGIC or peroxisomes. The expression of 3c-eGFP is clearly cell type dependent, it is more stable in MARC 145 cells than in Fcwf-4 or CrFK cells, which might reflect in vivo stability differences of 3c in natural target cells (enterocytes vs. monocytes/macrophages).

Identification of peptide domains involved in the subcellular localization of the feline coronavirus 3b protein

“…1b). These results are in line with the observations from Meszaros and colleagues, who investigated the subcellular localization of all FCoV ORF3-encoded proteins [25]. Interestingly, fusion of 3b at the C-terminus of EGFP abolished mitochondrial translocation and 3b expression was restricted to the nucleoli (Fig.…”

“…From these results, we can conclude that the 3b localization is not influenced by the fusion protein or tag to which it was fused nor by the cell type in which the protein is expressed, and thus most likely represents the actual subcellular localization of FCoV 3b. Furthermore, it was shown by Meszaros and colleagues that the 3b expression pattern did not change when transfection was followed by infection with FIPV DF-2, although 3b accumulation in the nucleolus was more frequently observed in infected cells compared to transfection only [25].…”

“…Five isolates (2.81 %) seemed to lack a Tom20(1) motif and two isolates (1.12 %) a Tom20(2) motif ( Table 2). Comparison of the 3b sequence of isolate HU2009FC, which is known to locate to mitochondria [25], and FECV UCD showed that the Tom20(1) and Tom20(2) recognition motifs were identical. In addition to these Tom20 recognition motifs, two overlapping hexamers (amino acids 24 to 29 and 25 to 30) with a ϕϕϕβσϕ and ϕϕβσϕϕ pattern, respectively, and potentially associated with mitochondrial targeting, were identified by MitoFates.…”

“…The FCoV 3b amino acid sequence comprises about 72 amino acid residues and is well conserved within pathotypes [24]. It was recently reported that 3b localizes to mitochondria and nucleoli, both in the presence and absence of other viral proteins [25]. However, the function(s) of this accessory protein remain unknown.…”