NF-?B is a conserved eukaryotic transcription factor that controls proinflammatory and immune genes. Vaccinia virus (VV) inhibits NF-?B activation through a number of viral immunevasion proteins. Infection with Modified Vaccinia Virus Ankara (MVA), an attenuated W strain missing many viral immunomodulators, leads to NF-?B activation. The event(s) during MVA infection responsible for this activation have not been determined. The goal of this project is to identify the MVA gene(s) whose product(s) activate NF-?B. Current work suggests an event prior to viral genomic replication, but after viral entry, signals through the MEK/ERK pathway to cause the NF-?B activation. Additionaly, I found that a viral gene transcribed early in infection is responsible for the NF-?B activation observed during infection. Testing of a VV plasmid library revealed twenty-five "early" viral encoded ORFs whose products could activate NF-?B independent of infection. To identify the viral ORF(s) whose products activate NF-?B during MVA infection, siRNAs specific for each ORF have been generated and are being tested to determine the effect of silencing each viral gene individually during an infection on NF-?B activation. Deletion mutant virus(es) lacking the gene(s) of interest will be constructed to confirm the identity of the viral genes involved in activating NF-?B during MVA infection. Finally, the molecular mechanism utilized by the viral gene product will be characterized by confocal microscopy and immunoprecipitations. Identification of these genes will lead to a greater understanding of poxviral regulation of this cellular transcription factor both in cell culture and in vivo. Understanding the properties of the MVA virus is important as MVA is currently being studied as a safe vaccine vector for smallpox and additional viruses, including HIV and malaria. Furthermore, characterization of these viral products will give insights into the function of orthologous proteins produced by variola virus (the causative agent of smallpox) as well.