We have characterized the interaction and nuclear localization of the nucleocapsid (N) protein and phosphoprotein (P) of sonchus yellow net nucleorhabdovirus. Expression studies with plant and yeast cells revealed that both N and P are capable of independent nuclear import. Site-specific mutagenesis and deletion analyses demonstrated that N contains a carboxy-terminal bipartite nuclear localization signal (NLS) located between amino acids 465 and 481 and that P contains a karyophillic region between amino acids 40 and 124. The N NLS was fully capable of functioning outside of the context of the N protein and was able to direct the nuclear import of a synthetic protein fusion consisting of green fluorescent protein fused to glutathione S-transferase (GST). Expression and mapping studies suggested that the karyophillic domain in P is located within the N-binding domain. Coexpression of N and P drastically affected their localization patterns relative to those of individually expressed proteins and resulted in a shift of both proteins to a subnuclear region. Yeast two-hybrid and GST pulldown experiments verified the N-P and P-P interactions, and deletion analyses have identified the N and P interacting domains. N NLS mutants were not transported to the nucleus by import-competent P, presumably because N binding masks the P NLS. Taken together, our results support a model for independent entry of N and P into the nucleus followed by associations that mediate subnuclear localization. Plant-infecting members of the family Rhabdoviridae have been classified into two genera (56). Members of the genusCytorhabdovirus 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 pur...