Structural studies of virus-induced membranes and assembly of alphaviruses and flaviviruses

Thomas J Edwards, Purdue University

Abstract

Alphaviruses and flaviviruses are small, membrane-containing viruses that are comprised of an icosahedrally symmetric glycoprotein shell that contains a nucleocapsid core that encapsidates a single-stranded, positive-sense RNA genome of approximately 11 kb in length. During replication, these viruses induce several alterations to the architecture of host cell organelles. Alphaviruses, including Sindbis virus, induce small 50 nm pockets, termed spherules within endosomes and lysosomes. Flaviviruses, including dengue virus, induce small 100 nm pockets within the endoplasmic reticulum called vesicles and are the sites of replication. Alphaviruses also induce a double membrane structure that is derived from Golgi membranes called a CPV II, CPV II structures have been linked with transporting glycoproteins and nucleocapsid cores to the plasma membrane for final assembly and virion budding. Within this work, the structures of alphavirus CPV I and CPV II are examined in three dimensions for the first time utilizing electron tomography. Electron tomography allows for the imaging and reconstruction of asymmetric, non-unique structures into three-dimensional volumes to a nominal resolution of 4-6 nm. High pressure freezing coupled with freeze substitution have allowed for the preparation of cells for electron microscopy with increased fidelity to the natural state of the cell. From these techniques quantitative information concerning the spatial and temporal relationships between viral intermediates, such as the nucleocapsid core, virus induced structures, and cellular structures were derived from three-dimensional reconstructions of volumes of infected cells. Combining data from electron tomography, immuno-localization assays, a more accurate model of the generation and transport of nucleocapsid cores of Sindbis virus was constructed. The Sindbis virus nucleocapsid core is generated at the sites of replication on the CPV I and is transported to the plasma membrane via the CPV II. These CPV II are associated with intermediate filaments, however these filaments are not necessary for virus release. In subsequent chapters the effect of inhibition of furin cleavage on virus maturation is examined on the structures of the alphavirus Ross River virus, and the flavivirus, dengue virus utilizing single particle reconstructions of images derived from electron microscopy. The hepatitis C virus particle is isolated and characterized by electron microscopy using a novel affinity grid purification scheme. This allowed for the determination of the pleomorphic nature of HCV particles. The importance of the cytoplasmic domain of E2 is examined by electron microscopy and found to mediate nucleocapsid core attachment to CPV II structures. Additionally, the cytoplasmic domain of E2 was also found to mediate an early step in nucleocapsid core formation. A Venezuelan Equine Encephalitis virus-like particle that is deficient in genome packaging was examined by single particle reconstruction. Finally, inhibitors of the fatty acid synthase pathway were shown to have a profound effect on the dengue virus-induced replication structures.

Degree

Ph.D.

Advisors

Kuhn, Purdue University.

Subject Area

Virology|Biophysics

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