All ETDs from UAB

Advisory Committee Chair

James W Noah

Advisory Committee Members

Louise Chow

Sadis Matalon

Diana Noah

Hengbin Wang

Document Type

Dissertation

Date of Award

2014

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Influenza A Virus is a respiratory pathogen of the family Orthomyxoviridae and causes significant worldwide morbidity and mortality during seasonal epidemics and periodic pandemics. As with all viruses, influenza's limited coding capacity requires the use of host proteins and processes in its replication and release cycle. Phosphorylation, mediated by cellular kinases and phosphatases, represents a significant mechanism of post-translational modification of cellular products, and is responsible for regulating the activi-ty, lifespan and localization of many lipids and proteins in host-cells. Several cellular kinases have been associated with various steps of the Influenza lifecycle, including PKC-ßII (viral entry), PKR (host defense), and the MEK/ERK path (nuclear export and packaging). These, and other, kinase targets represent targets for anti-viral drug design. This work describes a multidirectional approach to drug discovery combining a systems approach to novel target identification in addition to traditional phenotypic high through-put screening of a targeted compound library. Using a probe capable of cross-linking a biotin group to the active site lysine of cellular kinases and ATPases we cataloged the active kinome of influenza virus infected cells. Profiling of cells infected by both seasonal and pandemic isolates emphasizes the differences in host state during infections of dif-fering pathogenicity. Kinases which showed reproducible dysregulation during infection were evaluated by siRNA analysis for their effect on replication and release. These studies identified both canonical influenza related kinases in addition to novel targets which regulate replication both negatively and positively. Concurrently, a commercial available library of computational designed putative kinase inhibitors were screened in both seasonal and pandemic influenzas for their ability to inhibit influenza virus induced cytopathic effect in vitro. Several compounds are identified with minimal toxicity to the host cell and strong inhibitory effects. These data provide novel drug targets and clarify the mechanisms by which influenza virus affects host cell processes to promote its own replication. They also provide further validation for inhibiting viral replication and pathogenicity by the modulation of cellular pathways.

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