| I focused on elucidating unresolved issues of enveloped Coronavirus entry using Murine Hepatits Virus as a model system. Like other enveloped viruses, glycoproteins, Spikes (S), project from the MHV surface. After binding to cellular CarcinoEmbryonic Antigen-related Cell Adhesion Molecule (CEACAM) receptors, spikes are triggered to initiate the membrane fusion reaction. An aim of my research was to more clearly define the mechanism of spike-induced membrane fusion reactions. I first asked at what point following their synthesis do spikes acquire CEACAM-binding competence, and what architectural components of S dictate CEACAM binding competence? Using biochemical approaches, I determined that Spikes exist as dimers and peripheral (N-terminal) regions of S contain “oligomerization determinants”. I also found that only oligomeric spikes have acquired CEACAM binding competence. Additionally, I determined that while each spike monomer (of an oligomer) contains an independent CEACAM binding site, only a single CEACAM binds each spike dimer. I also revealed that CEACAM binding induces structural rearrangements in peripheral regions of S. This binding may significantly decrease the affinity of the other S monomer (within a dimer) for CEACAM, consistent with a view of “negative cooperativity”.; I next noted that across many species of coronaviruses, the length of the cytoplasmic region of S is relatively conserved, suggesting that the length of their cytoplasmic domain of Spikes may modulate membrane fusion and/or S incorporation into virions. To address these issues, I appended Enhanced Green Fluorescent Protein (EGFP) tags to the cytoplasmic tails of several spike variants and evaluated their effects on Spike fusogenicity and incorporation into virion particles. I found that large EGFP appendages did not alter Spike biosynthesis, processing, or CEACAM binding competence. However, I found that Spikes-EGFP chimeras exhibited slightly-delayed membrane fusion kinetics. Next, using reverse genetics to engineer recombinant viruses I found that Spike-EGFP could assemble into recombinant MHV particles. However, these viruses invariably lost green fluorescence after two to four passages, due to deletions of a central region of EGFP. (Abstract shortened by UMI.)... |
