Date of this Version
Nouhou Ibrahim, Functional Implications of the BAF-B1 axis during the vaccinia virus life cycle, Doctoral Dissertation March 2014.
Vaccinia virus is the prototypic member of the Poxviridae family, which includes variola virus, the agent of smallpox. Poxviruses encode their own transcriptional machinery and a set of proteins to evade the host defense system, and thus are able to replicate entirely in the cytoplasm of their host. The poxvirus life cycle occurs in sequential stages: early gene expression, DNA replication, intermediate gene expression and then late gene expression and morphogenesis. The temporally staged poxvirus life cycle makes viral DNA replication a required event for post-replicative events to occur. However, viral DNA replication itself depends on an early viral Ser/Thr kinase B1, which inactivates the cellular DNA-binding protein, the barrier-to-autointegration factor or BAF. BAF is a well-conserved protein with essential roles in mitotic nuclear reassembly. In the absence of B1, BAF colocalizes with viral replication sites and inhibits viral DNA replication.
Results from these studies demonstrate that BAF relocalizes to any cytosolic dsDNA to form BAF-DNA nucleoprotein complexes. Further, DNA-binding and homodimerization properties but not LEM-domain interaction are essential for BAF’s activity. Several cellular proteins are present at BAF-DNA complexes, but their contribution to BAF’s activity remains to be elucidated. Also, a previous report showed that B1 regulates viral intermediate gene expression through an unknown mechanism. Our studies showed that in the absence of B1, BAF inhibits viral intermediate gene expression at the transcriptional level. Surprisingly, BAF modestly inhibits reporter gene activity under non-vaccinia nuclear promoters and has no apparent effect on T7 promoter. Further, the absence of active B1 can lead to defects in morphogenesis and/or viral release /spread independently of B1’s role in DNA replication. Collectively, studies presented in this dissertation highlight the significance of the BAF-B1 axis during vaccinia life cycle.