Study Of Cytopathology Of Broad Bean Wilt Virus 2 And Functions Of VP37 Protein

Broad bean wilt virus 2(BBWV 2)which caused severe damage to many economiccrops has been reported to be widely distributed in China.To understand the intracellular andintercellular transportation mechanism of BBWV 2,the cytopathological structure of BBWV 2was observed,the subcellular localization of movement protein VP37 and its binding abilityto coat protein were studied.The BBWV 2 isolates B935,PV131 and P158 were mechanical inoculated intoChenopodium quinoa and Vicia faba plants,respectively.The ultrastructure of these infectedcells was observed by transmission electronic microscope(EM).An extensive proliferationof membrane,which may act as"viral factory"appeared nearby the nuclei.In the cytoplasm,virions accumulated into crystals or formed into tubules.Immuno-gold labeling EM withanti-CP antibody showed that virions or coat proteins were located in the enlarged membrane,crystals and tubules,and cytoplasm near the cell wall.Three isolates induced similarcytopathological changes in different hosts although symtoms on V.faba were more severethan that on C.quinoa.In B935 infected cells,tubules formed by 21 virions were observedwith the diameter of 155 nm,while tubules formed by 9 virions were observed in P158 orPV131 infected cells with the diameter of 78 nm.Models of tubules formed by viral particleswere deduced according to the particle modality and the shape of the tubules under the EM.The virions arrange in a stagger way on the transverse section which caused an inclinedplane on the vertical section.Comparions of the coat protein amino acid(aa)sequenceamong B935 isolate and other 9 BBWV 2 isolates revealed that there were several aachanges,eg V201L,L220F,R423K,R476K,L571G and R580Q,in LCP and SCP of B935isolate which might be responsible for these special tubules formed by its particles.The GFP-VP37 fused protein was expressed in Nicotiana benthamiana epidermal leaves,BY-2 protoplasts and BY-2 suspension cells.The green fluorescence was observed byconfocal laser scan microscope(CLSM).GFP-VP37 localized around the nuclei region,accumulated at cell wall in punctuates spots,which may present plamodesmata(PD),anddistributed on some granular bodies.Plasmolysis experiments showed that the greenfluorescent spots were still attached to the surface of the retracting plasma membrane when the plasma membrane was separated from the cell wall.This conformed that the GFP-VP37proteins were associated to the plasma membrane but not the cell wall.The fusion proteinfailed to target PD after treating with brefildin A(BFA).For the proteins which have alreadylocalized at the punctuate spots,however,BFA had little effect to them.Similar to the freeGFP,the mutants ml,m2 and m7 of the VP37 proteins all distributed in the nucleus andcytoplasm.The mutant m9 could accumulate around the nuclear although it failed to targetthe peripheral punctate structure.These data suggested the C-terminal of VP37 protein playan important role in viral intracellular movement and the central region would be involved inPD targeting.Co-expression studies revealed that the lost function of mutant VP37 proteinscould be complemented by wild type.This may imply that VP37 could be dimerized ormultimerized by itself.6×His-VP37 fusion protein was expressed by Escherichia.coli BL21(DE3)PlysS/PETexpression system and was purified for polyclonal antibody preparation.Using ELISA-basedbinding assay and blot overlay assay,purified VP37 protein was shown to bind specificallyto intact BBWV 2 virions and to both the large and small CP.The association between VP37protein and small CP was absolutely tight than that between VP37 protein and large CP.Thisbinding was not observed with a 101-237 aa deletion mutant of the VP37 protein.However,yeast two hybrids system failed to confirm the interaction between VP37 protein and smallCP.At the same time,interaction between VP37 proteins was also detected by blot overlayassay and yeast two hybrids.Neither of them confirmed the association between VP37proteins,though wt-VP37 protein could support mutant VP37 to accumulate at punctuatespots in the cells.A simple viral intracellular and intercellular movement model has deduced based on theabove results.CP might be involved in viral cell-to-cell movement,but it would not be usedfor virion assembly to pass through PD.Cell-to-cell movement of BBWV 2 may perform asviral RNA/MP/CP complex.The tubules formed by virions may have no relationship withviral movement and these tubules would just be a kind of virus accumulation.Since BFA dointerfere the GFP-VP37 fusion protein target cell wall,membrane system may play animportant role in viral movement.