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

the cell surface. The IBV S protein is cleaved by a furin-like protease generating the S1 and S2 subunits during trafficking through the Golgi, and at a second site (S2') that primes the protein for fusion with the host cell (30) .The S protein of IBV-EG3 is subject to further proteolysis near the junction of the protein with the viral envelope, resulting in a C-terminal fragment we term the 'stub' (22). We predicted that neutralization of Golgi pH by IBV E during infection protects the IBV S protein from premature proteolysis at the normal acidic pH of the trans-Golgi. We also The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 presence of M2 were also observed at the cell surface, though the differences were not significant. Values for the TGN38-pHluorin in different cell types range from pH 6.2-6.7 (28, 31, 32) . We believe that the slightly higher baseline measurement we observed is likely due to some plasma membrane cycling of the TGN38-pHluorin, which would increase the average pH in a given cell since the pH of the extra cellular buffer was pH 7.3. The cycling of TGN38-pHlorin is likely to be the predominant reason for a higher than expected TGN pH, since the medial-Golgi pHluorin reported a pH of ~6.4. We believe that the consistent shift in the Golgi pH in cells expressing IBV E is more important than the actual baseline pH value we observed with the TGN38-pHlorin. Previously, the TGN38-pHluorin construct was used to demonstrate that However, the IBV E T16A or IBV EG3 HD mutants did not cause a statistically significant shift (0.20 and 0.12 pH units, respectively). We previously showed that the LMW pool of IBV E correlates with the secretory pathway disruption, including Golgi disassembly and slow trafficking of model cargo proteins, observed when the wild-type IBV E protein is expressed (23, 29) . We hypothesized that the Golgi disruption was likely occurring in an IBV E ion channelindependent manner (26, 29). This study corroborates our hypothesis and suggests that the that proteolytic processing at two cleavage sites (S1/S2 and S2') releases the protein from its prefusion conformation and allows for a conformational change that exposes the fusion peptide (33) . This change in conformation may also release the S1 subunits from The S2 subunits of the CoV S trimer (33) . A possible detrimental consequence of the normal acidic Golgi pH to the virus could be that the S protein is subject to a conformational change and premature and possibly excessive proteolytic processing, resulting in release of the S1 subunit prior to receptor binding. This would result in noninfectious or impaired virions. Here we demonstrate that when IBV S is overexpressed in cells in the presence of EG3, the levels of IBV S cleavage species are increased significantly as compared to IBV S in the presence of IBV E or IAV M2, lending support to the hypothesis that the neutralization of Golgi pH by IBV E protects IBV S from premature cleavage. Additionally, when over expressed in cells IBV S is present to a greater extent at the cell surface, corroborating previous data observed during IBV EG3 infection (23).