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

In the positive-stranded RNA genome of beet yellows closterovirus (BYV), th e 5'-terminal ORF la encodes a 295 kDa polyprotein with the domains of papa in-like cysteine proteinase, methyltransferase (MT) and helicase (HEL), whe reas ORF Ib encodes an RNA-dependent RNA polymerase. Eleven and five hybrid oma cell lines secreting monoclonal antibodies (MAbs) were derived from mic e injected with the bacterially expressed fragments of the BW 1 a product e ncompassing the MT and HEL domains, respectively, On immunoblots of protein from BW-infected Tetragonia expansa plants, four MAbs against the MT recog nized a similar to 63 kDa protein, and two MAbs against the HEL recognized a similar to 100 kDa protein. Both the methyltransferase-like protein and t he helicase-like protein were found mainly in the fractions of large organe lles (P1) and membranes (P30) of the infected plants. These data clearly in dicate that (i) the BW methyltransferase-like and helicase-like proteins, l ike other related viral enzymes, are associated with membrane compartments in cells, and (ii) the la protein, apart from the cleavage by the leader pa pain-like proteinase that is expected to produce the 66 kDa and 229 kDa fra gments, undergoes additional processing by a virus-encoded or cellular prot einase.