"Trafficking and pH-dependent fusion of enveloped viruses in the endocytic system"
Cabot, Maya, Biophysics - School of Medicine, University of Virginia
Tamm, Lukas, MD-MPHY Mole Phys & Biophysics, University of Virginia
Most enveloped viruses infect cells by binding receptors at the cell surface and undergo trafficking through the endocytic pathway to a compartment with the requisite conditions to trigger fusion with a host endosomal membrane. Both binding at the cell surface and the eventual fusion of virus and host membranes is mediated by the viral entry and fusion protein(s), proteins on the virus surface that are sometimes simply referred to as glycoproteins. Under conducive conditions the fusion protein(s) undergo conformational changes and induce the merging of the host membrane and viral membrane, allowing viral contents to enter the host cell cytoplasm initiating infection. Factors that induce conformational change to trigger viral fusion with the host membrane can be low pH, receptors in the endosomal membrane, proteases, a high concentration of a specific cation such as calcium in the endosomal lumen, a specific lipid, or some combination of these factors. These features of virus entry are reviewed in Chapter 1.
Broad categories of compartments in the endocytic pathway include early and late endosomes, which can be further categorized into subpopulations with differing rates of maturation and motility characteristics. In Chapter 2, viral particles pseudotyped with the vesicular stomatitis virus glycoprotein on their surface and equipped with a novel pH sensor and a fluorescent content marker were used to measure pH, motion, and fusion at the single particle level in live cells. We found that the VSV-G particles fuse predominantly from more acidic and more motile endosomes, and that a significant fraction of particles is trafficked to more static and less acidic endosomes that do not support their fusion. Moreover, the fusion-supporting endosomes undergo directed motion.
In Chapter 3, a correlative assay to ascertain the pH of fusion mediated by Ebola virus glycoprotein is described. Ebola virus has a complex entry mechanism and while assays reconstituting fusion have shown that pH plays a critical role, the pH at which Ebola virus fuses in intact cells has not previously been determined. A novel pH sensor, mNectarine-Lamp1 was used to specifically assess late endosomal pH in cells treated with bafilomycin to alkalinize endosomes. The inhibition of viral entry was assessed upon treatment with the same concentration range of bafilomycin. The change in late endosomal pH was then correlated with inhibition of entry. With this approach, we determined that the majority of EBOV particles fuse in U2OS cells below pH 5.7. The methodology presented could be used to determine the pH dependence of fusion in live cells for other enveloped viruses that enter cells through an endosomal pathway.
PHD (Doctor of Philosophy)
endocytosis, endosomes, organelle motility, fluorescent pH sensor, vesicular stomatitis, viral trafficking, virus entry, viral membrane fusion