Identification and characterization of a baculovirus structural protein, VP1054, required for nucleocapsid formation.

The defect in a temperature-sensitive mutant of Autographa californica nuclear polyhedrosis virus, tsN1054, was mapped and characterized. At the nonpermissive temperature of 33°C, this mutant fails to form plaques upon infection of Sf-21 cultured insect cells; infection is limited to a single cell, even though the infection proceeds through the very late phase. Marker rescue mapping and DNA sequencing identified the gene, ORF 54, which was altered by a single nucleotide substitution in tsN1054. Transcriptional analysis of the ORF 54 region identified multicistronic RNAs, from early to very late times of infection, that potentially encode the ORF 54 gene product. Polyclonal antiserum raised to a TrpE-VP1054 fusion protein recognized a 42-kDa late protein, VP1054, in infected-cell lysates. VP1054 was found to be a component of both budded virus and occlusion-derived virions. The level of VP1054 was dramatically reduced in tsN1054-infected Sf-21 cells propagated at 33°C, and electron microscopic analysis of these cells showed that nucleocapsids failed to form in the nuclei of these infected cells. Instead, novel round, electron-dense bodies were found associated with the virogenic stroma in tsN1054-infected cells. Therefore, VP1054 is a virus structural protein required for nucleocapsid assembly. Baculoviruses are large, double-stranded, circular DNA viruses which are infectious to arthropods, primarily insects. Autographa californica nuclear polyhedrosis virus (AcMNPV) is the most well characterized baculovirus, and its genome has been entirely sequenced (3). AcMNPV replication produces two forms of progeny virus, an occluded form, which allows the virus to be transmitted from insect to insect through ingestion of contaminated food, and a budded form, which is responsible for dissemination of infection throughout the insect host (9, 15). The production of budded virus (BV) and occluded virus (OV) is the result of a complex cascade of gene regulation events (5, 28). Although the two viral forms are genetically identical, they have different morphologies, tissue tropisms, envelope sources, protein compositions, and temporal production (8, 32). Baculovirus infection of cultured insect cells progresses in three phases, early, late, and very late. The transition from the early stage to late stage of infection is dependent upon viral DNA replication and occurs between 6 and 12 h after the initiation of infection. During the late phase of infection, newly replicated viral DNA is condensed and packaged within the nucleus, in association with the virogenic stroma, into capsid structures to form nucleocapsids. From about 12 to 20 h, these nucleocapsids leave the nucleus, travel through the cytoplasm,