Proteolytic Activation of the Coronavirus Fusion Protein

Coronaviruses are enveloped viruses with a positive-stranded RNA genome. They have been isolated from various mammals and birds and can cause severe diseases among farm and companion animals. Cross-species transmission of animal viruses and genuine human coronavirus infections pose a potential threat to human health. All viruses depend on their host cells for propagation. To enter a cell and deliver the viral genetic information, enveloped viruses carry highly specialized glycoproteins. These membrane anchored fusion proteins catalyze membrane merger under strict spatiotemporal control and are the main targets for virus neutralizing antibodies. Coronavirus entry is mediated by the spike (S) protein, a fusion protein that contains two subunits of similar size with the canonical structural features of class I viral fusion proteins. The N-terminal S1 subunit contains the receptor-binding domain, while the C-terminal S2 subunit comprises the fusion machinery, including a putative fusion peptide, heptad repeat regions, and transmembrane domain. To prevent premature activation after biosynthesis, viral class I fusion proteins adopt a locked conformation and require proteolytic cleavage to render them fusion-ready. In the present dissertation, we examine function and control of the porcine epidemic diarrhea virus (PEDV) and mouse hepatitis virus (MHV) S protein. MHV is a prototype coronavirus that has been well studied. In the second chapter 2 we determine the S protein cleavage product that actually mediates membrane fusion with the host cell. We developed an unbiased assay to determine the cleavage status of MHV S proteins directly after membrane fusion has occurred. Our entry assay enables the specific identification and biochemical characterization of viral S proteins of successfully fusing virions. The study in chapter 3 was designed to identify the protease(s) which cleave the MHV S protein by RNA interference technology and by using pseudotyped virus‐like particles. Besides studying the very details of MHV entry, we investigated the particular growth requirements for PEDV. The propagation of PEDV field isolates is strictly dependent on the supplementation of active trypsin to the cell culture medium. To set the stage for the investigation of PEDV, a reverse genetic system was established that allows the modification of the viral genome (Chapter 4). Based on the trypsin-independent cell culture‐adapted PEDV strain DR13, we were able to generate PEDV derivatives. Genetic manipulation of the S gene enabled us to study details of PEDV entry. Therefore, we compared the S proteins of the trypsin dependent field isolate CV777 and the trypsin independent cell culture‐adapted PEDV DR13 and were able to map the genetic determinants within the spike gene for trypsin-dependent entry (Chapter 5). In the final chapter, the current understanding of the various steps leading to coronavirus membrane fusion are discussed and compared to typical class I fusion proteins. Reviewing our results from the MHV and PEDV studies, we discuss the underlying mechanism and the putative biological role of proteolysis in the coronavirus life cycle