Serological analysis of the two RNA2-encoded proteins of barley mild mosaic virus

Although the genomic structure of bymoviruses resembles in many aspects tha t of potyviruses, the biological function of several non-structural protein s is still unknown. To improve the experimental basis for the investigation of their role in the life cycle of barley mild mosaic virus (BaMMV), the R NA2 specific sequences coding for the polypeptides pi and P2 were cloned an d expressed in Escherichia coli. The recombinant proteins containing thiore doxin as a fusion partner were separated in polyacrylamide gels and injecte d into rabbits. In Western blot analyses, the obtained polyclonal antisera reacted specifically with the homologous antigens in total protein extracts of virus-infected barley plants. The same results were obtained for crude sap in a plate-trapped antigen (PTA) ELISA format but only in the case when the final substrate incubation was performed overnight at 4 degrees C. Thi s clearly indicates a very low concentration of the two proteins in vivo. A ll attempts to detect P2 as a putative integral component of the virus caps id that might be responsible for the transmissibility of BaMMY by the natur al vector Polymyxa graminis failed. Neither Western blot analyses using pur ified virus preparations nor electron microscopy of purified samples and cr ude sap from infected barley Leaves revealed any result chat could support this hypothesis. To prove the proteolytic activity predicted for pi on the basis of the nucleotide sequence homology with potyviruses, a fragment repr esenting nearly the entire open reading frame of the RNA2 was cloned in an overexpression system. The results of Western blotting utilizing antisera s pecific to pi, P2 and the RNA1-encoded P3 clearly demonstrated that the cod ing region as a whole is translated in vitro and the product is processed a fterwards into two proteins with the expected molecular weight for P1 and P 2.