All ETDs from UAB

Advisory Committee Chair

Ilya Frolov

Advisory Committee Members

William Britt

Elena Frolova

Olaf Kutsch

Allan Zajac

Document Type

Dissertation

Date of Award

2012

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Venezuelan equine encephalitis virus (VEEV) is a re-emerging virus that causes a severe and often fatal disease in equids and humans. To date, despite a continuous public health threat, no vaccines or antiviral drugs against VEEV infection have been developed for human use. In this study we took advantage of accumulated knowledge of capsid protein function in viral replication, assembly and virus-host interactions. Besides being an integral part of the virion, capsid protein is the major determinant of VEEV pathogenesis. This protein is cytotoxic and induces cellular transcriptional shutoff as a countermeasure to the cellular antiviral response. We introduced targeted modifications into the VEEV capsid protein and thus, changed VEEV phenotype to fit our requirements. First, we developed a VEEV variant that contained a set of mutations in the region that is responsible for capsid-mediated cellular transcriptional shutoff. The mutant virus had no defects in replication or infectious virus production, but was incapable for transcription inhibition. As a result, infected cells induced expression of a wide variety of effector antiviral genes. By applying microarray-based technology and bioinformatic analysis, we identified a novel antiviral gene (PARP12L) that demonstrated very broad antiviral effect. The mechanism of inhibitory activity of PARP12L most likely relies on its ability to downregulate cellular translation. Furthermore, we modified capsid protein more extensively and mutated almost all of the positively charged amino acids. Thus, we pushed over the edge VEEV capsid's ability to bind viral RNA, which resulted in the developed a pseudoinfectious VEEV (VEE PIV). VEE PIV efficiently replicated its genome, expressed all of the viral structural proteins and released viral particles at levels similar to those found in wt VEEV-infected cells. However, the mutations introduced into the capsid protein made this protein almost incapable of packaging the viral genome, and thus, the released virions lacked genetic material and did not produce spreading infection. Taken together, rational modifications of the VEEV capsid protein allowed us to find a novel effector antiviral protein and to develop a new vaccine candidate that combines efficiency of live attenuated vaccine and safety of a subunit vaccine.

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