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 the role of IBV E oligomerization in the alteration of Golgi luminal pH, we analyzed two HD point mutants of IBV E that segregate into different oligomeric states. Our previous findings suggest that IBV E A26F is found predominantly in the LMW, likely monomeric form, and IBV E T16A is found predominantly in the HMW, higher-order oligomer (26). In addition, we analyzed the EG3 mutant of IBV E, in which the entire HD of IBV E was replaced by a heterologous HD sequence. Both IBV E T16A and IBV EG3 had pH measurements similar to the IBV M membrane protein control . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 6.95 and pH 6.87, respectively), while IBV E A26F elicited a robust pH increase similar to that of the wild-type IBV E protein (pH 7.18, Figure 3A .) This suggested that the LMW pool of IBV E correlates with the luminal pH increase of the Golgi in addition to the secretory pathway disruption demonstrated in our previous work (23, 26, 29). The same experiment was performed with a medial-Golgi pHluorin, GnT1-pHluorin (Gnt1, N-acetylglucosaminyltransferase I), to assess if the alteration in pH was specific to the TGN. Indeed, both IBV E and IBV E A26F elicited a robust pH increase ( Figure 3B ). Interesting, IBV E T16A increased the pH significantly, though not as robustly as IBV E and IBV E A26F . We found that the pH of the trans-Golgi (measured with TGN38-pHluorin) was higher than the medial-Golgi (measured with GnT1-pHluorin). This was unexpected and is addressed in the discussion. The results in transfected cells implicate the monomeric form of IBV E in neutralization of the Golgi lumen during infection and transfection.