Date of Award

Summer 8-2016

Embargo Period

10-5-2016

Degree Type

Dissertation (CMU Access Only)

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

Advisor(s)

Alex Evilevitch

Abstract

Viruses typically consist of three fundamental components, a genetic material, either DNA or RNA; a protein shell, termed as the capsid; and in the case of many eukaryotic viruses, a third part made of lipid-coat and glycoproteins, known as the envelope. Viral replication cycles start with the delivery of its genetic material into the host cell cytoplasm or nucleoplasm, thus viral infectivity largely depends on its ability to: (i) effectively and efficiently translocate the genome into the host and (ii) protect its genome prior to infection and between rounds of replication. The main goal of this doctoral thesis is to understand how viral particles regulate its infectivity through altering its genome’s physical states under different conditions, i.e. temperatures, ionic strength, osmotic pressure. First, it was investigated that ejection of genome into its host is controlled by encapsidated genome states. In many double-stranded DNA bacterial viruses and herpesviruses, the tightly packaged and highly condensed DNA molecule is trapped in a glassy state with restricted molecular motion. The viral genome undergoes a solid-to-fluid-like disordering transition to increase its mobility or fluidity, which facilitates genome release. Second, it was shown that this structural transition strongly depends on physical parameters such osmotic pressure, temperature and ionic condition. Thus viral particle only gains the infectivity under right physical surroundings. There is striking evidence that the intra-capsid DNA transition can be switched “on” and “off” by mimicking those medium conditions optimized and non-optimized in vivo. All work in the first two steps was done on bacteriophages or type-1 herpes simplex virus (HSV-1) and viral DNA ejection was triggered by incubation with purified portal-binding receptor proteins or portal-targeted trypsin solutions. Last, the idea of tuning viral DNA states by physical parameters was applied to an advanced system reconstituted with isolated cellular nuclei and cytosol, in which HSV-1 were shown be docked onto nuclear membrane through nuclear pore complexes (NPC). DNA translocation into nucleoplasm was effectively inhibited by increasing osmotic pressure in the surrounding solution. These experiments entailed quantifying t he thermodynamics of DNA ejections from viral particles via calorimetric assays, structural studies of viral genome states via solution smallangle x-ray scattering, visualizing and quantifying DNA translocation process via fluorescence and electron microscopy imaging, in combination with real-time quantitative PCR methods. Results of these studies shed light on how viral particles regulate its genome iinfectivity and stability, as references for broadspectrum anti-viral medicine research and development.

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