The coronavirus E protein is a small membrane protein with a single predicted hydrophobic domain (HD), and has a poorly defined role in infection. The E protein is thought to promote virion assembly, which occurs in the Golgi region of infected cells. It has also been implicated in the release of infectious particles after budding. The E protein has ion channel activity in vitro, although a role for channel activity in infection has not been established. Furthermore, the membrane topology of the E protein is of considerable debate, and the protein may adopt more than one topology during infection. We previously showed that the HD of the infectious bronchitis virus (IBV) E protein is required for the efficient release of infectious virus, an activity that correlated with disruption of the secretory pathway. Here we report that a single residue within the hydrophobic domain, Thr16, is required for secretory pathway disruption. Substitutions of other residues for Thr16 were not tolerated. Mutations of Thr16 did not impact virus assembly as judged by virus-like particle production, suggesting that alteration of secretory pathway and assembly are independent activities. We also examined how the membrane topology of IBV E affected its function by generating mutant versions that adopted either a transmembrane or membrane hairpin topology. We found that a transmembrane topology was required for disrupting the secretory pathway, but was less efficient for virus-like particle production. The hairpin version of E was unable to disrupt the secretory pathway or produce particles. The findings reported here identify properties of the E protein that are important for its function, and provide insight into how the E protein may perform multiple roles during infection. Author Summary Coronaviruses are enveloped viruses that bud and assemble intracellularly, and therefore must use the host secretory pathway for release. Coronavirus E is a small protein that contains a single predicted hydrophobic domain and is targeted to the Golgi region. The E protein has been implicated in the assembly of coronavirus particles, as well as in virus release after assembly. The mechanism of action is not understood, but may involve ion channel activity. The membrane topology of the E protein is also unclear, and the protein may adopt distinct topologies that have different functions. We previously showed that the E protein from the infectious bronchitis virus could disrupt the secretory pathway to the apparent advantage of the virus. Here we have mapped this activity to a single, essential residue within the hydrophobic domain. Additionally, we developed mutant versions of IBV E that adopt a single membrane topology, and showed that a transmembrane topology is required for disruption of the secretory pathway. Our results broaden the understanding of E protein function and will impact the development of antiviral strategies.
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