Viruses are pathogens in every kingdom of life. They are neither truly living nor truly dead, as they rely on their host cell for replication. Because viruses rely on hosts for survival, they are orders of magnitude simpler than any truly living system. This simplicity allows us a glimpse into the complicated biological world. Proteins, the essential building blocks of biological systems, form the structure of viruses. Quantifying protein-protein interactions is necessary to understand viral assembly, but it is notoriously difficult. Fluorescence fluctuation spectroscopy (FFS) is a promising experimental technique that determines the interactions of proteins and other biomolecules from fluorescence signal fluctuations with single molecule sensitivity. FFS can directly quantify protein interactions inside a living cell, giving it a distinct advantage over previously used techniques. In FFS, proteins of interest are labeled with a fluorescent tag, often a fluorescent protein. Every time a labeled protein passes through a tiny optical observation volume, it creates an observable fluctuation in fluorescence signal. Statistical analysis extracts information about the sample from these fluctuations. Through statistics, we can determine information about the transport properties, the concentration, and the oligomerization state of the labeled protein.
FFS is most often used to study interactions of a small number of proteins directly inside cells or in aqueous solution. In this dissertation, I will step outside the cell and explore FFS as a tool to study the protein copy number of released viral particles containing hundreds to thousands of labeled proteins. The protein copy number provides implicit information on the assembly of the virus which is discussed in further detail in this dissertation. This dissertation will first introduce FFS and cover the analysis methods used, focusing on autocorrelation and photon counting histogram (PCH), along with the important details of applying these analysis methods to study viruses. Next, the extension of FFS to study the human retrovirus HTLV-1 will be covered. Studying HTLV-1 and comparing the results to our studies of HIV-1 provides hints about which features are common among retroviruses and which features are virus specific.
In previous FFS studies of VLPs, we focused on a single protein, Gag, as this is the only protein required to produce VLPs. However, complex viruses such as HIV-1 contain many other proteins including both viral and cellular proteins. Studying the relationship between the copy number of two proteins within VLPs can provide us with additional information about the assembly process. We expand our VLP FFS studies to the characterization of two proteins using dual color labeling. We explore the application of two-dimensional photon counting histogram (2D-PCH) analysis to determine the average copy number of two proteins in VLPs. Additionally, we present a new method called dual-color intensity fraction plots (IFP) that takes advantage of the low concentration of viral samples and allows for quick illumination of the qualitative distribution of protein copy number ratios. Many virus studies are difficult due to low sample concentrations and the time consuming concentration steps that are necessary to prepare the sample for measurement. We introduce flow-FFS as one method to overcome these limitations. Flow-FFS is a powerful technique which combines hydrodynamic flow through a microfluidic device and PCH analysis. This combination allows us to increase the accessible concentration range of FFS experiments, decrease the measurement time per sample, and measure samples with very high fluorescence background. These experiments expand the range of feasible VLP studies and open up a lower concentration range than was previously inaccessible by FFS.
University of Minnesota Ph.D. dissertation. Ph.D. October 2012. Major: Physics. Advisor:Dr. Joachim Mueller. 1 computer file (PDF); ix, 181 pages, appendices A-B.
Johnson Armstrong, Jolene Lois.
Studying viral-like particles using fluorescence fluctuation spectroscopy.
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