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

Given that the effects of IBV E overexpression on the Golgi are similar to those in cells where the luminal pH is neutralized, we first measured luminal pH in IBV-infected cells. We used flow cytometric analysis of a ratiometric pHluorin molecule targeted to the Golgi with a reporter consisting of the green fluorescent protein (GFP) pHluorin molecule fused to the membrane targeting sequence of the TGN38 trans-Golgi network resident protein (28) . We chose to use the trans-Golgi network pHluorin because the TGN is the most acidic compartment of the Golgi and thus any alteration in pH would likely be most detectable in this compartment. We generated a clonal Vero cell line that stably expressed the TGN38-pHluorin ( Figure 1A ). The generation of this cell line allowed us to ensure that all infected cells were expressing the TGN38-pHluorin. The cells were treated with cycloheximide for 60 minutes to chase newly synthesized TGN38-pHluorin from the ER. To generate a pH calibration curve, cells expressing the Golgi pHluorin molecule were subjected to treatment with buffers ranging from pH 5.5 to 7.5 in the presence of the ionophores monensin and nigericin prior to flow analysis ( Figure 1B) . The emission ratios of the biphasic pH-sensitive pHluorin at these known pH values can then be used to predict the Golgi luminal pH in cells The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 infected with IBV in buffer at physiological pH and lacking ionophores ( Figure 1C ). Infection resulted in a robust increase in the Golgi luminal pH.