In this application the investigators outline plans to focus on the dual specificity protein phosphatase encoded by vaccinia virus and retained by Molluscum Contagiosum (MC) virus. They have recently shown that the vaccinia phosphatase is essential for virus infectivity, affecting both virion structure and transcriptional capability, and have made significant progress in identifying authentic substrates. They plan to express recombinant MC phosphatase, characterize its enzymatic function, and determine whether it can recognize vaccinia proteins as substrates. Targeted mutagenesis will be used to engineer variant forms of the phosphatase that can be used to trap viral and cellular substrates. The investigators will also construct a vaccinia recombinant in which the MC phosphatase can substitute for the vaccinia phosphatase in a regulated fashion. These studies may provide biochemical and biological assays for MC phosphatase function. Finally, they will initiate studies to identify small molecule inhibitors which can specifically block MC phosphatase function in vitro and in vivo.
