title: Proteomic analysis of Pteropus alecto kidney cells in response to the viral mimic, Poly I:C.
abstract: BACKGROUND
  Bats are recognised as an important reservoir for a number of highly pathogenic zoonotic viruses. While many of these viruses cause severe and often fatal disease in humans, bats are able to coexist with these viruses without clinical signs of disease. The mechanism conferring this antiviral response is not fully understood. Here, we investigated the differential protein expression of immortalised Pteropus alecto kidney cells (PaKiT03) following transfection with the viral mimic, Poly I:C. Two complementary proteomic approaches, difference gel electrophoresis (DIGE) and isobaric tagging for relative and absolute quantitation (iTRAQ) were used to quantify changes in protein expression following Poly I:C stimulation at 4, 8 and 20 hr post treatment (hpt).
  
  
  RESULTS
  The expression of ISG54 gene, a known responder to virus infection and Poly I:C treatment, was significantly induced in transfected cells compared with mock-transfected cells. Through iTRAQ analysis we show that Poly I:C up-regulates key glycolytic enzymes at 4 hpt within PaKiT03 cells. In contrast, at 20 hpt PaKiT03 cells down-regulated ribosomal subunit proteins. The analysis with DIGE of Poly I:C transfected PaKiT03 cells showed over 215 individual spots differentially regulated, however only 25 spots could be unambiguously identified by LC-MS/MS. Immunoblotting confirmed the up-regulation of Eno1 and Tpi1 in PaKiT03 cells following Poly I:C transfection. A comparison with human cells (HEK293T and HeLa) and one additional bat cell line (PaLuT02), demonstrated that glycolytic pathways are also induced in these cell types, but at different intensities.
  
  
  CONCLUSIONS
  The two techniques, DIGE and iTRAQ identified largely overlapping sets of differentially expressed proteins, however DIGE unambiguously identified significantly less proteins than iTRAQ. Poly I:C induced a rapid metabolic shift towards glycolysis within the PaKiT03 cells at 4 hpt, presumably as a consequence of increased energy requirements. On the other hand ribosomal subunit proteins were seen as down-regulated by iTRAQ, these proteins may be the limiting factors in the translational machinery available for virus replication. This study provides new insight into the antiviral response of bat cells, highlighting the importance of energy metabolism.
authors.affiliation: CSIRO, Australian Animal Health Laboratory, East Geelong, 3219 VIC Australia ; ARC Centre of Excellence in Plant Cell Walls, School of BioSciences, The University of Melbourne, Parkville, VIC Australia.
authors.forename: Lawrence
authors.initials: L
authors.lastname: Mok
authors.affiliation: CSIRO, Australian Animal Health Laboratory, East Geelong, 3219 VIC Australia.
authors.forename: James W
authors.initials: JW
authors.lastname: Wynne
authors.affiliation: ARC Centre of Excellence in Plant Cell Walls, School of BioSciences, The University of Melbourne, Parkville, VIC Australia.
authors.forename: Kris
authors.initials: K
authors.lastname: Ford
authors.affiliation: CSIRO, Australian Animal Health Laboratory, East Geelong, 3219 VIC Australia.
authors.forename: Brian
authors.initials: B
authors.lastname: Shiell
authors.affiliation: ARC Centre of Excellence in Plant Cell Walls, School of BioSciences, The University of Melbourne, Parkville, VIC Australia ; Bio21 Institute for Molecular Science and Biotechnology, The University of Melbourne, Parkville, VIC Australia.
authors.forename: Antony
authors.initials: A
authors.lastname: Bacic
authors.affiliation: CSIRO, Australian Animal Health Laboratory, East Geelong, 3219 VIC Australia.
authors.forename: Wojtek P
authors.initials: WP
authors.lastname: Michalski
keywords: Bat cells; DIGE; Poly I:C; Quantitative proteomics; Viral mimic; dsRNA analogue; iTRAQ