Single Virion Fusion Studies Of Membrane-Enveloped Viruses To Biomimetic Membranes

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SINGLE VIRION FUSION STUDIES OF MEMBRANE-ENVELOPED VIRUSES TO CELL-DERIVED MEMBRANES Deirdre Ann Costello, Ph. D. Cornell University [2014] Understanding the mechanisms involved in the viral entry process is key for developing effective anti-viral therapies and vaccines. Entry of membrane-enveloped viruses typically involves two key processes mediated by a dual-function viral glycoprotein. During influenza infection hemagglutinin (HA) binds to sialic acid moieties on epithelial cell membranes, triggering engulfment into the cell via endocytosis. As the endosome matures and becomes more acidic, HA undergoes a conformational change, which induces fusion of the viral and endosomal membranes, releasing viral RNA into the cell. Quantitative kinetic studies of binding and fusion are often conducted in-vitro to obtain high-resolution measurements. Total internal reflection microscopy combined with microfluidics and supported bilayers is a powerful, single particle tracking (SPT) platform for host-pathogen membrane fusion studies. One inadequacy of the aforementioned SPT platform has been capturing the complexity of the cell membrane, including membrane proteins. Sialic acid receptors are easily integrated into the platform using glycolipids. However, viruses that bind proteinaceous receptors for cell entry have been precluded from study. We have developed a general method to integrate proteinaceous receptors and cellular membrane components into supported lipid bilayers for SPT fusion studies of feline coronavirus (FCoV). Supported lipid bilayers are formed from mammalian cell membrane vesicles that express the FCoV receptor, aminopeptidase N (APN), using a cell blebbing technique. SPT is then used to identify fusion intermediates and measure membrane fusion kinetics of FCoV, which to our knowledge have not been obtained before. Overall, the fusion results recapitulate what is observed in-vivo, that coronavirus required binding to specific receptors, a low-pH trigger, and that membrane fusion is receptor and protease-dependent. We also observe that small populations of FCoV viruses are capable of fusing at neutral pH. This phenomenon was previously un-reported in-vivo for this strain of FCoV and may have implications for explaining the infection efficiency and cellular tropism of some coronaviruses. This platform also provides a new route to study how viruses rapidly adapt to other hosts, and to identify the factors that led to the emergence of other zoonotic coronaviruses, such as SARS-CoV and the new emerging MERS-CoVe. In this thesis I will also present our advances on using the cell blebbing technique to create simple virus-like particles (VLPs). VLPs can be used to study the highly pathogenic coronaviruses such as SARS-CoV and avian influenzas such as H5N1 in a biosafety level 2 laboratory

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Virus fusion; single virion; biomimetic membranes


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Union Local


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Daniel, Susan

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Pollack, Lois
Whittaker, Gary R
Putnam, David A.

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Chemical Engineering

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Ph. D., Chemical Engineering

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Doctor of Philosophy

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Government Document




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dissertation or thesis

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