The Requirement Of The Turnip Mosaic Virus (TuMV) CI Protein For Viral Replication And Movement And Its Interaction With Host Proteins

The multifunctional cylindrical inclusion (CI) protein of potyviruses contains ATP binding and RNA helicase activities. As part of the viral replication complex, it assists viral genome replication, possibly by binding to RNA and unwinding the RNA duplex. It also functions in viral cell-to-cell movement, likely via the formation of conical structures at plasmodesmata (PD) and the interaction with coat protein (CP). Theses processes are expected to rely on multiple interactions with host factors. However, little is known about the host genes that are involved in, or that may interfere with CI functions. To date, only three proteins were reported interacted with potyviral CI. CI of Plum pox virus (PPV) interacts with PSI-K (photosystem I protein K) protein of Nicotiana benthamiana, and virus accumulated much more in PSI-K knockout plants than in Wild-tpye Arabidopsis (Col-0). Tobacco etch virus (TEV) CI interacts with P58IPK of Arabidopsis, P58 is a double stranded RNA-dependent protein kinase(PKR) inhibitor.Viral infection of p58 silenced N.benthamiana or Irabidopsis knockout mutant leads to host death, suggesting P58IPK protein plays an important role in viral pathogenesis. CI together with VPg directly overcome the eIF4E resistance.To further understand the role of CI in the viral infection process, we employed the alanine-scanning mutagenesis approach to mutate CI in the infectious full-length cDNA clone of Turnip mosaic virus (TuMV) tagged by green fluorescent protein. A total of 40 double-substitutions were made at the clustered charged residues. The effect of these mutations on viral genome amplification was determined using a protoplast inoculation assay. All the mutants were also introduced into Nicotiana benthamiana plants to assess their cell-to-cell and long-distance movement. Three cell-to-cell movement-abolished mutants were randomly selected to determine if their mutated CI protein targets PD and interacts with CP by confocal microscopy.Twenty CI mutants were replication-defective (5 abolished and 15 reduced), one produced an elevated level of viral genome in comparison with the parental virus, and the remaining 19 retained the same replication level as the parental virus. The replication-defective mutations were predominately located in the helicase domains and C-terminal region. Fifteen replication-defective mutants showed delayed or abolished cell-to-cell movement. Nine of 20 replication-competent mutants contained infection within single cells. Five of them distributed mutations within the N-terminal 100 amino acids. Most of replication-defective or cell-to-cell movement-abolished mutants failed to infect plants systemically. Analysis of three randomly selected replication-competent yet cell-to-cell movement-abolished mutants revealed that the mutated CI failed to form regular punctate structures at PD and/or to interact with CP.Yesat two-hybrid was used to screening the cDNA library of Arabidopsis.9 host factors were identified and confirmed in yeast. BiFC was used to confirm the interaction in planta,5 of the nine candidate genes were positive including TFL2, HCF173, IPCS2, UN-2 and CYP79B2. TFL2 showed the strongest interaction with TuMV CI. Interestingly, CYP79B2-CI interactions only occur with the presence of TuMV in plants, other host proteins interact with CI with or without virus infection. Protoplast transfection assay were used to assess whether TFL2 is essential for TuMV replication and the results showed that TFL2 severely inhibits TuMV replication.In conclusion, the helicase domain and C-terminal region of TuMV CI are essential for viral genome replication, and the N-terminal sequence modulates viral cell-to-cell movement. TuMV CI plays both interlinked and distinct roles in replication and intercellular movement. The ability of CI to target PD and interact with CP is associated with its functional role in viral cell-to-cell movement. We provide the evidences for a role of the novel host factor TFL2 in poty virus replication.