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Title:Characterization of the glycosylation and signal peptide sequences within the vaccinia virus m2 protein and influence on ERK1/2 and NF-kB activation
Author(s):Hinthong, Olivia
Director of Research:Shisler, Joanna L.
Doctoral Committee Chair(s):Shisler, Joanna L.
Doctoral Committee Member(s):Tapping, Richard I.; Wilson, Brenda A.; Whitaker, Rachel J.
Department / Program:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
signal peptide
endoplasmic reticulum
Abstract:Activation of the NF-κB transcription factor during cellular infection establishes an anti-viral state via upregulation of proinflammatory proteins. Wild-type vaccinia virus (VV) strains subvert the immune response by expressing proteins that interfere with nuclear translocation of NF-κB. In contrast, infection with an attenuated strain, MVA, results in host cell NF-κB activation. Inclusion of the M2 gene of VV into the attenuated MVA virus was shown to reduce NF-κB activation, and also ERK1/2 activation, an early event in MVA infection. Understanding the molecular basis for M2- mediated inhibition of NF-κB and ERK1/2 activation was the primary goal of this dissertation. Characterization of the M2 protein began with an examination of its expression profile during infection. Much like other immunomodulators, M2 is expressed early during infection. Analysis of the M2 amino acid sequence revealed an N-terminal signal peptide sequence, 4 putative glycosylation residues, and endoplasmic reticulum (ER) retention/retrieval sequences, indicative of an ER-localized protein. Confocal microscopy analysis confirmed localization of M2 to the ER. It was found that M2 requires a signal peptide cleavage site to confer proper localization and targeting across the ER membrane and that M2 is N-linked glycosylated. Mutant M2 proteins that are missing either a putative N-terminal signal peptide (∆sp) sequence or a C-terminal ER (∆ERret) localization sequence no longer localize to the ER, and have a reduced ability to inhibit NF-κB activation and ERK1/2 activation. As such, ER localization is critical for full NF-κB and ERK1/2 inhibitory activity for the M2 protein. An M2 mutant lacking the signal peptide lost proper ER targeting and was not glycosylated. This M2 mutant also lost NF-κB and ERK1/2 inhibitory activity. It remained unclear whether loss of NF-κB or ERK1/2 inhibitory function was the result of impaired ER localization, trafficking, or post-translational modification. Using site directed mutagenesis, mutant viruses expressing M2 proteins that were impaired in targeting or modification via the secretory pathway revealed functional consequences. ii Four mutant viruses expressing M2 with each of the four putative glycosylation sites abolished (N49Q, N79Q, N118Q, N154Q), and two mutant viruses (S17I and S17F) which prevent recognition by the signal peptidase present in the ER lumen were constructed. Of the amino acid residues tested, 17,49,79,118,154 contribute to ERK1/2 inhibitory activity, while residue 118 contributes the most for NF-κB inhibitory activity. It was also found that the level of M2 protein expression is influenced by the level of glycosylation conferred to the protein. Data presented in this dissertation establish that M2 is not only cell associated, but transits the classical secretory pathway into the cellular medium as evidenced by sensitivity to Brefeldin A treatment. Alteration to the signal peptide cleavage site impaired the time of cellular secretion of M2. Most intriguing was that a point mutation within the second glycosylation site (N79Q) resulted in a non-detectable protein which still had function, indicating that transient expression of N79Q is sufficient to inhibit cytoplasmic signaling pathways. This dissertation elaborates on M2’s molecular functioning as a viral immunomodulator to inhibit both the ERK1/2 and NF-κB pathways, which is reliant on its glycosylation status and proper cellular trafficking within the infected cell. As M2 has no homology to any known viral or cellular protein, this suggests a novel immunoregulatory function. Data contained within this dissertation provides insight into the molecular mechanism of an ER-localized protein, and its impact on cytoplasmic signaling pathways within the context of an anti-viral immune response.
Issue Date:2011-01-21
Rights Information:Copyright 2010 Olivia Hinthong
Date Available in IDEALS:2011-01-21
Date Deposited:2010-12

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