August 19, 2013 Noon (334 JFB)
Title: Applications and Correlation of Nano Resolution Microscopy Techniques to Viral Protein Localization"
Advances in the optical realm of super-resolution microscopy techniques have enabled scientists to observe phenomena much smaller than the Abbe diffraction limit by stochastically limiting the number of molecules excited at a given instance and localizing their positions one at a time. Additionally, methods such as Atomic Force Microscopy (AFM) allow scientists to measure the topological features and material properties of samples through contact with a force probe. This thesis describes the application of these two techniques to virology in order to localize internal viral proteins of enveloped virions, and measure their effect on the elastic properties of the virion.
By utilizing super-resolution microscopy techniques such as Fluorescent Photo-Activated Localization Microscopy (fPALM) on virions, which have had their surface glycoproteins labeled with a photo-switchable label, the viral envelope may be accurately recovered. This thesis describes the development and application of this technique as it applies to envelope recovery of Vesicular Stomatitis Virus (VSV) and Human Immunodeficiency Virus-1 (HIV-1). Both of these viruses are significantly smaller than the diffraction limit. By fluorescently labeling proteins, which are internal to each of these viruses, I have been able to localize a variety of viral proteins within their recovered envelopes. This is done without significant damage to the virion, making this method a highly effective in vivo technique.
When applying the STORM/fPALM technique to VSV in order to localize its internal RNA dependent polymerase proteins, an asymmetry along the central axis became apparent and was contradictory to the widely believed model of uniform distribution. However, due to the inhomogeneity of the underlying proteins such an asymmetry would predict that the Young’s modulus would vary along the central axis of the virion. This thesis also describes utilizing AFM to explore and measure the variance in young’s modulus between the two distinct elastic regions observed in VSV virions. From these combined results I have found a strong correlation between the two methodologies in order to calculate the distribution of polymerases within VSV.