The Infectious Bronchitis Virus Coronavirus Envelope Protein Alters Golgi pH to Protect Spike Protein and Promote Release of Infectious Virus

Jason W. Westerbeck; Carolyn E. Machamer

Evidence suggests that CoV E proteins interact with host cell proteins to influence pathogenicity. Interaction of SARS-CoV E with the PDZ (post-synaptic density protein-95/discs Large/zonula occludens-1) domain containing scaffolding protein, syntenin, and with the tight The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/440628 doi: bioRxiv preprint with the large amount of IBV E known to reside at the ERGIC could disrupt the pH directly or indirectly by interfering with normal secretion or architecture of the Golgi. In summary, studies addressing CoV E protein-protein and protein-lipid interactions, and the development of tools to evaluate ion-channel activity in vivo, will go a long way in elucidating the precise mechanisms of the multifunctional family of viroporin proteins. Antibodies. The rabbit polyclonal antibodies recognizing the C-terminus of IBV E and the Nterminal head of golgin-160 have been previously described (50, 51) . The antibody generated to the C-terminus of IBV S has also been described (52) .The mouse monoclonal antibody recognizing the N-terminus of influenza A M2 has been previously described and was a generous gift from Dr. Andrew Pekosz (53). The mouse anti-GFP antibody was from Roche (Mannheim, Germany). The rabbit anti-GFP antibody was from Thermo Fisher Scientific The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/440628 doi: bioRxiv preprint glycerol containing 0.1M N-propylgallate. Images were captured using an Axioskop microscope (Zeiss) equipped for epifluorescence with an ORCA-03G charge-coupled-device camera (Hamamatsu, Japan) and iVision software (Bio Vision Technologies). The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/440628 doi: bioRxiv preprint pH were used to generate a linear calibration curve (Microsoft Excel) with which to calculate the luminal Golgi pH in infected and uninfected cells. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/440628 doi: bioRxiv preprint (Millipore). After blocking in 10 mM Tris-HCl pH 7,4, 0.15 M NaCl (TBS) plus 5% nonfat milk, membranes were probed with rabbit anti-IBV S diluted 1:5000 in TBS/0.05% Tween (TBS/Tw).