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 degrees C, this mutant fails to f orm plaques upon infection of Sf-21 cultured insect cells; infection i s limited to a single cell, even though the infection proceeds through the very late phase. Marker rescue mapping and DNA sequencing identif ied the gene, ORF 54 , which was altered by a single nucleotide substi tution in tsN1054, Transcriptional analysis of the ORF 54 region ident ified 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 prote in, VP1054, in infected-cell lysates. VP1054 was found to be a compone nt of both budded virus and occlusion-derived virions. The level of VP 1054 mas dramatically reduced in tsN1054-infected Sf-21 cells propagat ed at 33 degrees C, and electron microscopic analysis of these cells s howed that nucleocapsids failed to form in the nuclei of these infecte d cells, Instead, novel round, electron-dense bodies were found associ ated with the virogenic stroma in tsN1054-infected cells. Therefore, V P1054 is a virus structural protein required for nucleocapsid assembly .