Detection and subcellular localization of the turnip yellow mosaic virus 66K replication protein in infected cells

Turnip yellow mosaic virus (TYMV) encodes a 206-kDa (206K) polyprotein with domains of methyltransferase, proteinase, NTPase/helicase, and RNA-depende nt RNA polymerase (RdRp). In vitro, the 206K protein has been shown to unde rgo proteolytic processing, giving rise to the synthesis of 140-kDa (140K) and 66-kDa (66K) proteins, the latter comprising the RdRp protein domain. A ntibodies were raised against the 66K protein and were used to detect the c orresponding viral protein in infected cells; both leaf tissues and protopl asts were examined. The antiserum specifically recognized a protein of simi lar to 66 kDa, indicating that the cleavage observed in vitro is also funct ional in vivo. The 66K protein accumulates transiently during protoplast in fection and localizes to cellular membrane fractions. Indirect immunoflores cence assays and electron microscopy of immunogold-decorated ultrathin sect ions of infected leaf tissue using anti-66K-specific antibody revealed labe ling of membrane vesicles located at the chloroplast envelope. (C) 2001 Aca demic Press.