Functional Analysis Of Barley Stripe Mosaic Virus TGB1 Protein In Nuclear-cytoplasmic Trafficking And Hijacking Of The Nucleolar Protein Fibrillarin

Barley stripe mosaic virus, a (+) ssRNA virus, contains three segmented genomic (g) RNAs designated?, ?, and ?. RNAa directly encodes aa, the large subunit of RdRp complex, while RNAy directly encodes the replicase protein 7a. yb, the RNA silencing suppressor of BSMV, encoded by sgRNA?. RNA? directly encodes CP and indirectly encodes the triple gene block protein TGB1, TGB2, and TGB3. BSMV replicates in the chloroplasts, and TGB1 binds the progeny RNA with TGB2 and TGB3 to form the viral ribonucleoprotein (vRNP) to perform cell-to-cell movement. TGB1 locates to the cytoplasm when expressed alone, and mainly to the plasmodesmata in the context of viral infection. So far, the nuclear localization and function of TGB1 and the involvement of host factors in BSMV vRNP have not been reported. In this study, the nuclear and nucleolar localization of TGB1 and the involvement of Fib2 in vRNP were studied. In addition,the relationship between TGBI and viral replication was preliminary explored.In our previous study, we have found that TGB1 could be phosphorylated by CK2a, and colocalized to the cytoplasm, nucleus, and nucleolus with CK2a. LSCM (laser scanning confocal microscopy),immunogold labelling,and nuclear isolation experiments verified the nuclear and nucleolus localization of TGB1. Moreover, TGB1 was transported to the nucleus via the well characterized importin ?/? pathway by directly binding Impa. To identify the effects of nuclear-cytoplasmic trafficking of TGBI on virus infection, the imposed nuclear localization signal and nuclear export signal were fused to TGB 1 in RNA?.Detection of the mutated viruses accumulation on Nicotiana benthamiana and barley showed that the nuclear-cytoplasmic trafficking of TGBI is required for BSMV movement. Predication analyses and LSCM experiments verified that TGB1 has a nucleolar localization signal (NoLS) (aa 95-104) and a nuclear localization signal (NLS) (aa 227-238). NLS mutation inhibited viral cell-to-cell and long-distance movement in both Nicotiana benthamiana and barley, whereas NoLS mutation reduced viral cell-to-cell and long-distance movement in Nicotiana benthamiana, but not in barley, showing the difference between N. benthamiana and barley being BSMV hosts.In addition, the expression of nucleolar protein Fib2 was upregulated upon BSMV infection, and downregulated fib2 reduced viral accumulation and cell-to-cell movement. BiFC, Co-IP, and MBP pull-down experiments verified that TGB1 interacts directly with Fib2 GAR domain, whereas TGB1 NoLS and NLS mutation abolished this interaction, indicating that TGB1 nucleolus localization is required for the interaction between TGB1 and Fib2. Fib2 colocalized with TGB1 and BSMV RNA to the cytoplasm next to the PD, implying that Fib2 was hijacked and redirected to the plasmodesmata by TGB1 to be a component of BSMV vRNP and contributed to cell-to-cell movement. For the first time that we have showed the involvement of Fib2 in viral cell-to-cell movement but not long-distance movement.TGB 1, one of the core components of vRNP, binds BSMV RNA to perform cell-to-cell and long-distance movement, but little is known about where TGB1 picks up BSMV RNA. Immunoprecipitation experiments found that TGB1 may associate with BSMV replication system. LSCM experiments found that TGB 1 may localized to the periphery of chloroplasts mediating by virus RNA but not by viral proteins.Moreover,TGB1 unexpressed promoted viral replication, indicating that TGB1 may downregulate BSMV replication. It is hypothesized that TGB1 couples the viral replication and movement by localizing to the periphery of chloroplasts, and contributes to the transition from replication to movement by inhibiting replication.The new discovery of TGB1 nuclear-cytoplasmic trafficking and the effect on BSMV cell-to-cell and long-distance movement,and the nucleolar protein Fib2 was hijancked by TGB1 to be a component of vRNP and involved in BSMV cell-to-cell movement. These studies contribute to our knowledge of the nuclear-cytoplasmic-trafficking proteins encoded by RNA viruses, and also increase our understanding of the involment of Fib2 in viral momovement and the multifunction of TGB1.