Examination of the role of early viral protein expression in MVA-induced NF-κB activation

Martin, Stefani

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https://hdl.handle.net/2142/29572 Copy

Description

Title

Examination of the role of early viral protein expression in MVA-induced NF-κB activation

Author(s)

Martin, Stefani

Issue Date

2012-02-01T00:56:08Z

Director of Research (if dissertation) or Advisor (if thesis)

Shisler, Joanna L.

Committee Member(s)

• Tapping, Richard I.
• Wilson, Brenda A.
• Kuzminov, Andrei

Department of Study

Microbiology

Discipline

Microbiology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Modified Vaccinia Virus Ankara (MVA)
• NF-kappaB
• Vaccinia

Abstract

NF-κB, a potent pro-inflammatory cellular transcription factor, is an inhibition target for many viruses, including the prototypic poxvirus member, vaccinia virus (VACV). However, infection of HEK293T cells with the attenuated Modified Vaccinia Virus Ankara (MVA) no longer inhibits NF-κB activation, due to the absence of various host range and immunosuppressive genes in this strain. The work presented here focused on investigating the viral molecular mechanism responsible for the activation of NF-κB. To that end, we found that infection with MVA in the presence of AraC, a drug that inhibits viral DNA replication and intermediate and late gene expression, results in NF-κB nuclear translocation. However, under conditions where either viral entry is blocked, the virions are inactivated, or viral mRNA synthesis is prevented, NF-κB remains inactive in virus-infected cells. Thus, early viral gene transcription is responsible for MVA-triggered NF-κB activation. To identify the viral ORF(s) whose products activate NF-κB during MVA infection, we first tested a VACV plasmid library to identify which viral ORF could activate a NF-κB-controlled firefly luciferase gene. We then utilized chemically synthesized siRNAs, testing the effect of silencing each of the viral genes individually on MVA-induced NF-κB activation in these cells. Three viral genes (C11R, E7R, and A35R) have been identified whose silencing can prevent MVA-induced ERK1/2 activation, which occurs upstream of NF-κB activation. The product of C11R, a viral growth factor protein, is capable of inducing ERK1/2 and NF-κB activation in the host 293T cells when it is expressed ectopically. Furthermore, infection with a deletion mutant virus, MVAΔC11R, resulted in reduced ERK2 phosphorylation and NF-κB activation compared to cells infected with the parental MVA strain. This reduction was observed in both 293T cells and in keratenocyte cell line, HaCaT, indicating the C11R protein product modulates viral-induced NF-κB activation in multiple cell types. Future work should focus on the molecular pathway utilized by C11 and the additional contributions the E7 and A35 proteins have in MVA-induced NF-κB activation.

Graduation Semester

2011-12

Permalink

http://hdl.handle.net/2142/29572

Copyright and License Information

Copyright 2012 Stefani Martin. Portions of Chapter 2 have been published in Virology 390:298-306 Copyright 2009 Elsevier Inc

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