SONCHUS YELLOW NET RHABDOVIRUS NUCLEAR VIROPLASMS CONTAIN POLYMERASE-ASSOCIATED PROTEINS

We have initiated a study of the cytopathology of nucleorhabdoviruses by analyzing the subcellular localization of sonchus yellow net virus (SYNV) genomic and antigenomic RNAs and the encoded polymerase protein s. In situ hybridizations demonstrated that the minus-strand genomic R NA sequences are restricted to the nuclei of infected cells, while the complementary plus-strand antigenomic RNA sequences are present in bo th the nuclei and the cytoplasm. Immunofluorescence and immunogold lab eling experiments also revealed that the nucleocapsid (N) protein and phosphoprotein (M2) are primarily localized to discrete regions within the nuclei and in virus particles that accumulate in perinuclear spac es. The N protein antiserum specifically labeled the nuclear viroplasm s, whereas the M2 antiserum was more generally distributed throughout the nuclei. Antibody detection also indicated that the polymerase (L) protein is present in small amounts in the viroplasm, When the N and M 2 proteins were expressed individually from the heterologous potato vi rus X (PVX) vector, both proteins preferentially accumulated in the nu clei. In addition, viroplasm-like inclusions formed in the nuclei of c ells infected with the PVX vector containing the N gene. Fusions of th e carboxy terminus of beta-glucuronidase to N and M2 resulted in stain ing of the nuclei of infected cells following expression from the PVX vector. Deletion analyses suggested that multiple regions of the N pro tein contain signals that are important for nuclear localization.