Systems biology approaches to dissect virus-host interactions to develop crops with broad-spectrum virus resistance

Abstract

More than 60% of plant viruses are positive-strand RNA viruses that cause billion-dollar losses annually and pose a major threat to stable agricultural production, including cucumber mosaic virus (CMV) that infects numerous vegetables and ornamental trees. A highly conserved feature among these viruses is that they form viral replication complexes (VRCs) to multiply their genomes by hijacking host proteins and remodeling host intracellular membranes. As a conserved and indispensable process, VRC assembly also represents an excellent target for the development of antiviral strategies that can be used to control a wide-range of viruses. Using CMV and a model virus, brome mosaic virus (BMV), and relying on genomic tools and tailor-made large-scale resources specific for the project, our original objectives were to: 1) Identify host proteins that are required for viral replication complex assembly. 2) Dissect host requirements that determine viral host range. 3) Provide proof-of-concept evidence of a viral control strategy by blocking the viral replication complex-localized phospholipid synthesis. We expect to provide new ways and new concepts to control multiple viruses by targeting a conserved feature among positive-strand RNA viruses based on our results. Our work is going according to the expected timeline and we are progressing well on all aims. For Objective 1, among ~6,000 yeast genes, we have identified 96 hits that were possibly play critical roles in viral replication. These hits are involved in cellular pathways of 1) Phospholipid synthesis; 2) Membrane-shaping; 3) Sterol synthesis and transport; 4) Protein transport; 5) Protein modification, among many others. We are pursuing several genes involved in lipid metabolism and transport because cellular membranes are primarily composed of lipids and lipid compositional changes affect VRC formation and functions. For Objective 2, we have found that CPR5 proteins from monocotyledon plants promoted BMV replication while those from dicotyledon plants inhibited it, providing direct evidence that CPR5 protein determines the host range of BMV. We are currently examining the mechanisms by which dicot CPR5 genes inhibit BMV replication and expressing the dicot CPR5 genes in monocot plants to control BMV infection. For Objective 3, we have demonstrated that substitutions in a host gene involved in lipid synthesis, CHO2, prevented the VRC formation by directing BMV replication protein 1a (BMV 1a), which remodels the nuclear membrane to form VRCs, away from the nuclear membrane, and thus, no VRCs were formed. This has been reported in Journal of Biological Chemistry. Based on the results from Objective 3, we have extended our plan to demonstrate that an amphipathic alpha-helix in BMV 1a is necessary and sufficient to target BMV 1a to the nuclear membrane. We further found that the counterparts of the BMV 1a helix from a group of viruses in the alphavirus-like superfamily, such as CMV, hepatitis E virus, and Rubella virus, are sufficient to target VRCs to the designated membranes, revealing a conserved feature among the superfamily. A joint manuscript describing these exciting results and authored by the two labs will be submitted shortly. We have also successfully set up systems in tomato plants: 1) to efficiently knock down gene expression via virus-induced gene silencing so we could test effects of lacking a host gene(s) on CMV replication; 2) to overexpress any gene transiently from a mild virus (potato virus X) so we could test effects of the overexpressed gene(s) on CMV replication. In summary, we have made promising progress in all three Objectives. We have identified multiple new host proteins that are involved in VRC formation and may serve as good targets to develop antiviral strategies; have confirmed that CPR5 from dicot plants inhibited viral infection and are generating BMV-resistance rice and wheat crops by overexpressing dicot CPR5 genes; have demonstrated to block viral replication by preventing viral replication protein from targeting to the designated organelle membranes for the VRC formation and this concept can be further employed for virus control. We are grateful to BARD funding and are excited to carry on this project in collaboration.