Detection of beet yellows closterovirus methyltransferase-like and helicase-like proteins in vivo using monoclonal antibodies

Department of Virology and Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia 1 Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, 117871 Moscow, Russia 2 Federal Biological Research Centre for Agriculture and Forestry, Institute for Plant Protection in Fruit Crops, Schwabenheimer Str. 101, D-69221 Dossenheim, Germany 3 Author for correspondence: Alexey Agranovsky. Fax +7 095 939 31 81. e-mail aaa{at}closter.genebee.msu.su In the positive-stranded RNA genome of beet yellows closterovirus (BYV), the 5'-terminal ORF 1a encodes a 295 kDa polyprotein with the domains of papain-like cysteine proteinase, methyltransferase (MT) and helicase (HEL), whereas ORF 1b encodes an RNA-dependent RNA polymerase. Eleven and five hybridoma cell lines secreting monoclonal antibodies (MAbs) were derived from mice injected with the bacterially expressed fragments of the BYV 1a product encompassing the MT and HEL domains, respectively. On immunoblots of protein from BYV-infected Tetragonia expansa plants, four MAbs against the MT recognized a ~63 kDa protein, and two MAbs against the HEL recognized a ~100 kDa protein. Both the methyltransferase-like protein and the helicase-like protein were found mainly in the fractions of large organelles (P1) and membranes (P30) of the infected plants. These data clearly indicate that (i) the BYV methyltransferase-like and helicase-like proteins, like other related viral enzymes, are associated with membrane compartments in cells, and (ii) the 1a protein, apart from the cleavage by the leader papain-like proteinase that is expected to produce the 66 kDa and 229 kDa fragments, undergoes additional processing by a virus-encoded or cellular proteinase.