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The Modulation of Autophagy in Mammalian Cells by Vaccinia Virus

by
Joseph Gennaro Moloughney
M.S., Montclair State University - 2004





Thesis Advisor: Shengkan (Victor) Jin, Ph.D.
Graduate Program in Cellular & Molecular Pharmacology

Pharmacology Department Conference Room
5th floor, RWJMS Research Tower
Piscataway

Thursday, March 3, 2011
3:30 p.m.


Abstract

Vaccinia virus is a large and complex enveloped virus belonging to the Orthopoxivirus genus of the family Poxviridae. Vaccinia was employed as the vaccine that eradicated variola virus, the causative agent of the smallpox disease which was responsible for an estimated 300-500 million deaths during the 20th century. The morphogenesis of vaccinia virus from a non-infectious immature virus (IV) to the extracellular enveloped virus (EEV) involves the formation of two separate membranes and several distinct forms of virion. Even though much of the life cycle of vaccinia has been described, the mechanisms by which the virus evades the defense of the host cell and the origin of the IVís core membrane are issues that are still poorly understood.
Autophagy is an evolutionarily conserved intracellular process serving as both a means to maintain cellular homeostasis and for cell survival under various stress conditions. It has been demonstrated that autophagy plays a significant role in all aspects of the immune system (intrinsic, innate and adaptive) against intracellular pathogens, including viruses. The hallmark of autophagy is the formation of autophagosomes, and similar to the core membrane of vaccinia, are double-membraned structures that are believed to be derived by a controversial de novo synthesis process. Because of similar morphological characteristics, intense scrutiny, and controversial nature surrounding the origin and composition of both the core membrane of vaccinia virus and the autophagosome, the premise that autophagy may be subverted by vaccinia virus for membrane biogenesis was investigated.
To test this hypothesis we examined the response of various cell lines to vaccinia virus infection. It was determined that significant LC3 lipidation, the molecular marker for membrane formation during autophagy, occurs post vaccinia virus infection and is independent of the autophagy related genes atg5 and atg7. Mass spectrometry analysis accompanied by western blot data revealed that the viral infection generates a novel protein conjugate between two essential autophagy related proteins, ATG12 and ATG3. Subsequent, viral growth studies demonstrated that autophagy does not play a role in vaccinia virus membrane biogenesis; in contrast, electron microscopy studies reveal the presence of no autophagosome or autophagosome-like membrane structures post vaccinia virus infection.
The results demonstrated that vaccinia virus does not employ the autophagosome membrane as its source for the IV membrane, but rather the virus actively interferes with the cellular autophagy machinery, thereby inhibiting autophagy and potentially providing the virus with a countermeasure to a potent antiviral defense mechanism of the host cell.


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