Biophysical Investigations of Retroviral Assembly Using Novel Quantitative Fluorescence Microscopy Techniques

Abstract

This dissertation describes biophysical investigations of retroviral assembly, a process that is driven primarily by the viral structural protein Gag. Novel quantitative fluorescence microscopy methods are developed that provide previously unavailable insights into the assembly process. First, a tractable fluorescently-labeled model system is established that recapitulates the assembly of human T-cell leukemia virus type 1 (HTLV-1) Gag. Next, a stoichiometric imaging technique is developed that utilizes the molecular brightness to infer the copy number of labeled biomolecules in complexes such as virus particles. Preliminary data from this imaging technique demonstrate its utility and accuracy for quantifying the copy number of fluorescent Gag in viral particles. In order to study the plasma membrane (PM) binding of Gag proteins during the early stages of the assembly process, z-scan fluorescence microscopy is extended to a dual color (DC) modality. The new DC z-scan technique yielded an order of magnitude improvement the PM binding limit of detection and enabled observation of cooperative binding to the PM in the case of the matrix domain of human immunodefficiency virus type 1 (HIV-1) Gag. Further application of the DC z-scan technique led to the unexpected discovery that large ribonucleoprotein complexes are partially excluded, or partitioned, from the interior of the actin cortex. This partitioning effect represents a novel mechanism through which cells, by modulating the physical properties of their interior, may impose biomolecular organization and structure within the cytoplasmic space. Because large viral RNAs must transit the cortex in order to be packaged into nascent particles at sites of viral assembly, the cortical partitioning effect is hypothesized to represent a barrier to the assembly process. This possibility is supported by preliminary data on HIV-1. The advances presented here help to inform our basic understanding of the critical assembly stage in the lifecycle of the human retroviruses HIV-1 and HTLV-1. They also provide new insights into the interplay of the cytoskeleton and large RNAs within the living cell.