Interaction between geminivirus replication protein and host proteins

Geminiviruses replicate their small DNA genomes in the nuclei of infected plant cells after inducing host DNA replication machinery. The viral protein AL1 initiates DNA replication, regulates viral gene expression, and induces plant gene transcription. Using site-directed mutagenesis, we generated a series of mutations in AL1 and analyzed their impact on AL1 function. Nearly all mutations in the oligomerization domain inhibited or attenuated the ability of AL1 to support DNA replication but enhanced transcriptional repression, suggesting that different AL1 complexes are used for DNA replication and repression. KEE146, a mutation inside the RBR (the plant Rb homologue) interaction domain, greatly reduced AL1 binding to RBR. Plants infected with viruses harboring this mutation developed milder symptoms. In situ analysis showed that unlike the wild type virus, which infects and causes the host DNA replication factor proliferating cell nuclear antigen (PCNA) induction in all mature tissues, the mutant virus infects and induces PCNA expression only in vascular tissue. These results demonstrated the importance of AL1-RBR interaction in host cell induction during geminivirus infection. To better understand how AL1 interacts with host proteins, we used yeast two-hybrid library screening and a baculovirus protein interaction system to identify other plant proteins that interact with AL1. These studies identified a serine/threonine kinase, a kinesin and histone H3 as AL1 partners. Both the kinase and the kinesin are phosphoproteins. Phosphorylation of the kinesin is CDK-dependent. Immunostaining showed that kinase protein level and subcellular location are regulated during plant development and geminivirus infection. In contrast, expression of the kinesin is ubiquitous and associated with the spindle apparatus in mitotic cells.