Interactions and Nuclear Import of the N and P Proteins of Sonchus Yellow Net Virus, a Plant Nucleorhabdovirus

Plant-infecting members of the family Rhabdoviridae have been classified into two genera (56). Members of the genus Cytorhabdovirus replicate in the cytoplasm of infected cells, a trait they share with all rhabdoviruses infecting animal hosts. In contrast, members of the genus Nucleorhabdovirus, of which Sonchus yellow net virus (SYNV) is the most extensively characterized species, appear to replicate in the nucleus and undergo morphogenesis at the inner nuclear envelope. SYNV is transmitted by an aphid (Aphis coreopsidis) in nature, is widespread in sowthistle (Sonchus oleraceus L.) and beggar ticks (Bidens pilosa) in the southern United States, and occurs sporadically in lettuce (Lactuca sativa) in Florida (7, 20). The bacilliform particles of SYNV are composed of a nucleocapsid core surrounded by a phospholipid membrane. The membrane fraction contains a glycoprotein (G) that protrudes from the surface of the virion (11) and an associated protein designated sc4 (46). The membrane fraction also contains an additional protein that is thought to be an analogue of the matrix protein (M) of the animal-infecting prototype rhabdovirus, Vesicular stomatitis virus (VSV) (16), which associates with the G protein and the nucleocapsid core during morphogenesis to stabilize the virus particle. The infectious SYNV core, which can be purified by density gradient centrifugation of nonionic-detergent-treated virions (53, 54), consists of the negative-strand genomic RNA (19) encapsidated by three core polymerase proteins. Structural studies reveal that purified nucleocapsid cores contain the nucleocapsid (N) protein (60), the phosphoprotein (P), formerly designated M2 (14), and the polymerase (L) protein (4). Thus, in many respects, the SYNV proteins correspond to those of the cytoplasmically replicating animal rhabdoviruses typified by VSV and Rabies virus. However, unlike its animal-infecting counterparts, SYNV replicates in virus-induced viroplasms located in the nuclei of infected cells (32). Data consistent with the formation of a nuclear viroplasm include the isolation of an active polymerase complex from the nuclei of infected cells that is capable of transcribing a polyadenylated leader RNA and the six SYNV mRNAs (53, 54). In situ hybridization studies have revealed that the minus-strand genomic RNA of SYNV is restricted to the nucleus while the plus-strand antigenomic RNAs are present in both the nucleus and the cytoplasm (32). Additional evidence consistent with a nuclear site for replication is the accumulation of the N and P proteins in the nuclei of virus-infected cells or in the nuclei of protoplasts infected with a plus-strand RNA virus vector that replicates in the cytoplasm (32). Treatment of SYNV-infected protoplasts with tunicamycin prevents morphogenesis and also results in the accumulation of viral core particles in the nucleus (52). These studies raise a number of questions relevant to the mechanisms involved in formation of viroplasms during nucleorhabdovirus infections. To begin to address these questions, we have analyzed the nuclear import of the N and P proteins in plant cells and have also found that Saccharomyces cerevisiae provides a useful model for refined analysis of nuclear import.