Ultrastructural localization and epitope mapping of the methyltransferase-like and helicase-like proteins of Beet yellows virus

Monoclonal antibodies (MAbs) specific to the methyltransferase (MT) and hel icase (MEL) domains of the closterovirus Beet yellows virus (BYV) were used for immunogold labelling of ultrathin sections of virus-infected Tetragoni a expansa plants, MAbs 4A2 and 4A5 from the MT panel, and 1C4 from the HEL panel, specifically labelled distinct closterovirus-induced membranous stru ctures, the 'BYV-type vesicles', thus suggesting that the closterovirus MT- like and MEL-like proteins co-localize in these structures. Probing of the MT and HEL MAbs with synthetic octapeptides spanning the sequences of the r ecombinant MT and HEL fragments that had been used as immunogens showed tha t 4A5 and 4A2 recognized a single epitope, SRLLENET (aa 686-692 in the BW l a protein), and 1C4 reacted with the DDPF epitope (aa 2493-2496). These epi topes apparently reside on the exposed parts of the membrane-associated mol ecules of the closterovirus MT-like and MEL-like proteins. Two other epitop es determined for the MT MAbs that were nonreactive in the immunogold label ling, namely TMVTPGEL (aa 750-757; MAbs 3C5, 4B4 and 4C5) and SREQLVEA (aa 806-813; MAb 2A4), are possibly buried in the MT domain fold or shielded by membranes or other proteins involved in the viral replicative complex.