Regulation and antiviral mechanism of a plant ribosome inactivating protein

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Plants have evolved several strategies to evade pathogen attack and subsequent disease. One group of proteins generally thought to be involved in defense is the ribosome inactivating proteins. These are enzymes synthesized by many plants that remove a purine base from a conserved region of ribosomal RNA. Early studies showed how this depurination slowed the rate of protein translation, and resulting toxicity was cited to explain a role in defense; that is, localized host cell death would limit pathogen spread. However, little attention has been paid to their mechanisms of action in their native plants. Interestingly, one of these ribosome inactivating proteins was first discovered because of its antiviral, rather than toxic, properties. Namely, pokeweed antiviral protein (PAP), synthesized by the pokeweed plant Phytolacca americana, inhibits the replication of several plant and animal viruses. We were the first to demonstrate that this activity is due to in vivo depurination of the viral genomic RNA; therefore, the enzyme's substrate is not limited to ribosomal RNA. The objectives of our research program will build on this important finding, to determine the molecular mechanisms by which PAP is antiviral in its native pokeweed and when expressed in other, heterologous plants. Our short-term research objectives are two-fold: 1) to determine how PAP targets viral RNAs without causing host cell death. We will co-localize PAP and viral RNAs in cells and identify proteins that may mediate PAP activity. 2) In addition, we want to determine how PAP expression is regulated in pokeweed, to understand its role in its native system. We will investigate the transcriptional and post-transcriptional control of PAP expression by characterizing its promoter and the functions of a PAP-specific small RNA and pseudogene that we have recently identified. Together, results from these studies will provide useful information to improve strategies for crop tolerance to viruses, and will contribute to the long term goal of our research program to expand knowledge of the natural defense strategies of plants.