Nucleocytoplasmic Shuttling Of The Tobacco Necrosis Virus A Coat Protein And Its Functional Analysis

Tobacco necrosis virus A Chinese isolate (TNV-AC), a new strain first found in Chinese soybean fields, belongs to the Necrovirus genus in the Tombusviridae family, which causes severe damage to many economic crops. Our previous studies demonstrated that the TNV-AC coat protein (CP) localizes in host nuclei during infection. However, replication of positive strand RNA viruses, like TNV-AC, occurs in cytoplasm, which does not require CP nuclear transport, so it is important to study the biological functions of the CP nuclear localization. A series of deletion and substitution mutants in the N-terminal basic region were constructed based an infectious cDNA clone of TNV-AC. From western blot analysis of the nuclear and the cytoplasm fractions of infected N. benthamiana leaves, I concluded that amino acids16-36(RTPEQQVIEDQRDARRLARGR), especially the arginine residues, are required for nuclear localization of the CP. This finding was further demonstrated in N. bethamiana cells by analysis of the localization of CP-GFP fusion proteins harboring a series of mutations within the16-36residue region. Bimolecular fluorescence complementation (BiFC) assays also revealed that CP residues16-36function as an importin α/β pathway nuclear localization signal. More interestingly, substitution of leucines with alanines in a leucine-rich sequence within aa122-139restricted the CP to the nucleus, indicating that aa122-139have key roles in CP nuclear export. A Leptomycin experiment also confirmed that aa122-139contain a CRM1mediated NES signal. In addition, aa75-80was shown to be a NES-related sequence because deletions and substitutions within this region restrict the CPto the nucleus. Since TNV-AC CP is a determinant of systemic infection in N. benthamiana plants, we examined the long distance movement of mutant viruses whose CP were concentrated in the cytoplasm (M16, M16RA) or the nucleus (M13, M23, M7, M24), and found although local infections occurred, none of the mutant viruses were able to establish systemic infections. To maintain the integral function of the CP, two new mutant viruses, TNV-AC:PNLS and TNV-ACPNES, were designed by fusing exogenous nuclear import or export signals to the CP C-terminus. TNV-ACP:NES induced more severe necrotic symptoms on upper N.benthamiana leaves than wtTNV-AC, and these symptoms were accompanied by higher accumulation of TNV-ACP:NES sgRNAs, whereas TNV-ACP:NLS was unable to mediate systemic infections. In my TNV-AC studies, mutants M13, M23, M7, M24, and TNV-ACP:NLS CPs were confined to the nucleus, and this probably disrupted the packaging of viral RNA. Mutations in the basic region (M16, M16RA whose CP mostly is localized in the cytoplasm) probably also impaired virion formation and/or stability. All these mutant viruses failed to move systemically. Furthermore, the results strongly indicate that altered subcellular localization of the CP leads to defects in systemic infection. Taken together, my results demonstrate that the CP nucleocytoplasmic distribution has a crucial role in systemic infections of TNV-AC.Subcellular localization of the TNV-AC movement protein (MP) P8, P6and, the P23small subunit of the RNA dependent RNA polymerase (RdRp), was also investigated by transient expression of GFP fusion proteins. Our results show that GFP-P8localizes in the nuclei of N. bethamiana and onion epidermal cells, and that arginines and lysines in the15RGRARSSEGKK25N-terminal sequence are essential for nuclear targeting. P6-GFP appears to be associated with the endoplasmic reticulum, the nuclear envelope and the plasma membrane of N. bethamiana leaves, onion epidermal cells and BY-2suspended cells. However, BiFC assays failed to reveal interactions between CP and P8, CP and P6. GFP-P23is associated with the cellular membrane structure, and aggregates on the membrane and vesicles in cells of N. bethamiana leaves. These findings suggest that subcellular localization of the P6and P8movement proteins may have some as yet undefined role in the pathogenesis of TNV-AC.High-throughput analysis via microarray, RNA-seq and virus-induced gene silencing have used for screening genes interacting with virus. One of the subsequent procedures that must be performed is the estimation of the transcript abundance of the genes found to be involved in viral pathogenesis as accurately as possible. Quantitative real-time PCR (qPCR) is the most widely adopted technique for mRNA quantification. In order to obtain reliable quantification of transcripts, identification of the best reference genes forms the basis of the preliminary work. Information about the reference gene selection for virus-infected monocot plants remains largely unclear. In this work, the suitability of ten housekeeping genes (ACT, EF1α, FBOX, GAPDH, GTPB, PP2A, SAND, TUBβ UBC18and UK) for potential use as reference genes in qPCR were investigated in five different monocot plants (Brachypodium, barley, sorghum, wheat and maize) under infection with different viruses including Barley stripe mosaic virus (BSMV), Brome mosaic virus (BMV), Rice black-streaked dwarf virus (RBSDV) and Sugarcane mosaic virus (SCMV). These data indicate that each tested experimental condition demanded a specific set of reference genes, so validation of reference genes prior to their use is indispensable. By using three different algorithms, geNorm, NormFinder and BestKeeper, the most appropriate reference genes or their combinations were identified for different experimental sets, and their effectiveness for the normalization of expression studies were further validated by quantitative analysis of a well-studied PR-1gene. These results facilitate the selection of desirable reference genes for more accurate gene expression studies in virus-infected monocots.