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

To determine if the pH change in infected cells was caused by the E protein, HeLa cells were cotransfected with a plasmid encoding IBV E along with the pHlorin-TGN38 reporter. We used transient transfection of the reporter here to ensure the pHluorin expressing cells were also expressing the E protein. In separate cells, we included vector alone and a plasmid encoding IBV M (as another overexpressed Golgi membrane protein) as controls. As described above, transfected cells were pretreated with cycloheximde for 60 min to chase newly synthesized pHlorin-TGN38 out of the ER. A standard curve was produced in cells treated with ionophores in calibrated pH buffers. As shown in Figs 2B and C, IBV E neutralized the trans-Golgi luminal pH when over expressed in HeLa cells, whereas the IBV M protein did not.