The coronavirus spike protein : mechanisms of membrane fusion and virion incorporation

The coronavirus spike protein is a membrane-anchored glycoprotein responsible for virus-cell attachment and membrane fusion, prerequisites for a successful virus infection. In this thesis, two aspects are described regarding the molecular biology of the coronavirus spike protein: its membrane fusion mechanism and its assembly into the virion. Chapter 2 and 3 deal with the role in membrane fusion of two heptad repeat regions (a structural motif with a high propensity to form an alpha-helical coiled coil) in the spike protein of the mouse hepatitis virus (MHV) and the recently discovered SARS coronavirus (SARS-CoV) respectively. The membrane-distal (HR1) and the membrane-proximal (HR2) heptad repeat domain of the spike protein were biochemically and functionally characterized. Peptides corresponding to the HR1 and HR2 regions, when mixed together, assembled into a thermostable, rod-like six-helix bundle consisting of three HR1 and three HR2 helices oriented in an antiparallel fashion. This six-helix bundle complex supposedly represents a postfusion conformation that is formed after insertion of the fusion peptide, proposed here for coronaviruses to be located immediately upstream of HR1, into the target membrane. The resulting close apposition of fusion peptide, and spike transmembrane domain facilitates membrane fusion. The HR2 peptide of the MHV and SARS-CoV was found to be a potent inhibitor of the MHV and SARS-CoV infection respectively, presumably by binding to the HR1 region in the spike protein in a dominant-negative manner, thereby preventing membrane fusion. Chapter 4 deals with the cleavage necessity of the MHV spike protein for infection and cell-cell fusion, a requirement for the membrane fusion capacity of similar membrane fusion proteins of different virus families. Cleavage of the MHV spike protein was blocked in a concentration-dependent manner by a peptide furin inhibitor, indicating that furin or a furin-like enzyme, is responsible for this process. While cell-cell fusion was clearly affected by preventing spike protein cleavage, virus-cell fusion was not, indicating that these events have different requirements. Chapters 5 and 6 focus on the assembly requirements of the spike protein. In chapter 5 the flexibility of the virion was examined with respect to the incorporation of a recombinant spike protein extended at its cytoplasmic domain with the green fluorescent protein. The spike protein is incorporated into the assembling virion by interactions with the viral membrane (M) protein. The extension of the spike cytoplasmic domain did neither abrogate its interaction with the M protein nor its incorporation into virus-like particles. Moreover, a recombinant virus with the spike gene replaced by the S-GFP chimeric gene was viable and resulted in fluorescent virions bearing the S-GFP chimeric spikes. However, viruses carrying the S-GFP gene were genetically instable. Virion incorporation of spikes carrying the large GFP moiety was impaired and selected against during the assembly of virions. Chapter 6 describes a mutational analysis of the spike virion incorporation requirements. We show that the cytoplasmic domain, not the transmembrane domain, determines the association with the membrane protein M protein, and is sufficient to effect the incorporation into viral particles of chimeric spikes as well as of foreign viral glycoproteins