Binding of monoclonal antibodies to the movement protein (MP) of Tobacco mosaic virus: influence of subcellular MP localization and phosphorylation.
Monoclonal antibodies (mAbs) to recombinant movement protein (MP(REC)) of Tobacco mosaic virus (TMV) were used to reveal the dependence of MP epitope accessibility to mAbs on subcellular MP localization and post-translational MP phosphorylation. Leaves of Nicotiana benthamiana or N. tabacum were inoculated mechanically with TMV or agroinjected with an MP expression vector. At different time post-inoculation, ER membrane- and cell wall-enriched fractions (ER-MP and CW-MP, respectively) were isolated and analysed. The N-terminal region (residues 1-30) as well as regions 186-222 and 223-257 of MP from the CW and ER fractions were accessible for interaction with mAbs. By contrast, the MP regions including residues 76-89 and 98-129 were not accessible. The C-terminal TMV MP region (residues 258-268) was inaccessible to mAbs not only in CW-MP, but also in ER-MP fractions. Evidence is presented that phosphorylation of the majority of TMV MP C-terminal sites occurred on ER membranes at an early stage of virus infection, i.e. not after, but before reaching the cell wall. C-terminal phosphorylation of purified MP(REC) abolished recognition of C-proximal residues 258-268 by specific mAbs, which could be restored by MP dephosphorylation. Likewise, accessibility to mAbs of the C-terminal MP epitope in ER-MP and CW-MP leaf fractions was restored by dephosphorylation. Substitution of three or four C-terminal Ser/Thr residues with non-phosphorylatable Ala also resulted in abolition of interaction of mAbs with MP.
Tyulkina
Lidia G
LG
Department of Virology, Moscow State University, Vorobjevi Gori, Moscow 119992, Russia. Lydya_Tyulkina@hotmail.com
Karger
Elena M
EM
Sheveleva
Anna A
AA
Atabekov
Joseph G
JG
D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D000914:Antibodies, Viral / Q000276:immunology* / Q000378:metabolism; D002473:Cell Wall / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000737:chemistry; D010766:Phosphorylation; D018515:Plant Leaves / Q000821:virology; D053830:Plant Viral Movement Proteins / Q000276:immunology*; D011485:Protein Binding; D014026:Nicotiana / Q000821:virology; D014027:Tobacco Mosaic Virus / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D053830:Plant Viral Movement Proteins