A Geminivirus DNA Replication Protein - Programming Its Plant Host

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9809953 Hanley-Bowdoin Geminiviruses are a family of eukaryotic DNA viruses with single-stranded genomes that replicate through a rolling circle mechanism in nuclei of infected plant cells. They have small genomes, encode one protein that is essential for their replication, and rely on the replication machinery of their hosts. The overall goal of this research is to determine the mechanisms whereby geminiviruses recruit host DNA replication machinery. The investigator showed that geminivirus infection induces the accumulation of the host DNA synthesis protein, proliferating cell nuclear antigen (PCNA), in terminally differentiated plant cells. PCNA RNA levels and promoter activity are also elevated in infected tissue, demonstrating that geminiviruses can activate transcription of genes encoding DNA synthesis proteins in quiescent plant cells. The essential viral replication protein, AL1, is sufficient to induce the accumulation of PCNA in differentiated cells of transgenic plants, and thus may be analogous to the tumor antigen proteins of mammalian DNA viruses. In animal systems, DNA tumor viruses induce replication machinery in their host through a combination of common mechanisms. They encode proteins that directly activate transcription of genes encoding DNA synthesis proteins and/or interact with cell cycle regulatory factors to render quiescent cells competent for DNA replication. The investigator will determine whether AL1 uses similar or different mechanisms to induce PCNA transcription in differentiated plant cells by 1) determining whether interaction between AL1 and the cell cycle regulatory factor, retinoblastoma protein, is required for PCNA transcription, 2) asking whether multiple domains of AL1 contribute to PCNA transcriptional activation, and 3) identifying other plant proteins that interact with AL1 for PCNA induction. Geminiviruses are excellent model systems for studying eukaryotic DNA replication and cell cycle regulation, areas of study that have not been pursued extensively in plant systems. The ultimate g oals of the project are to elucidate the pathway whereby geminiviruses modify and maintain their host plants in an optimal state for viral DNA replication and to relate this pathway to plant cell cycle and transcriptional regulatory processes. The research will identify and begin to characterize components of the geminivirus/host regulatory network. The long term objectives are to understand how each component functions during geminivirus infection and in normal cellular processes. These experiments, which are based entirely on plant virus and host components, afford a rare opportunity to study plant cell cycle regulation directly. They will also increase our understanding of the basic mechanisms whereby eukaryotic viruses modify cellular processes and may illustrate fundamental similarities and/or differences between plants and animals.