Characterization of an overexpressed spindle protein during a baculovirus infection

The nucleopolyhedrovirus CfDEFNPV contains a gene encoding a viral protein, which accumulates as bipyramidal inclusion bodies (spindles) in the cytopl asm of infected cells. The spindles appear as early as 24 h postinfection, approximately 1 day earlier than viral occlusion bodies (OBs). Purification and characterization of the spindle protein was complicated by the fact. t hat the OBs copurified with the spindles. We therefore modified CfDEFNPV by replacing the polyhedrin gene (plh) with a cassette containing the green f luorescent protein (GFP) gene. The recombinant virus did not produce OBs; h owever, the synthesis and morphogenesis of the spindles were not altered: W hen analyzed by SDS-PAGE, the spindles produced a 50-kDa protein, which was termed spindlin. Tunicamycin inhibition and endoglycosidase studies showed that spindlin was glycosylated. The N-terminus of spindlin was sequenced a nd its gene (gp50) was located on the viral genome. The gene was cloned and sequenced. Homologs of gp50 were found in several baculoviruses as well as in entomopoxviruses (EPV). In the latter virus, the homologous gene is tha t of fusolin, which also encodes a protein that forms spindle-shaped inclus ion bodies in the cytoplasm of infected cells. Immunoblot analysis indicate d that spindlin and fusolin were not serologically related, even though the y share conserved polypeptide domains. Sequence analysis showed that gp50 o f CfDEFNPV contains two late promoter motifs (TTAAG) in its 5' flanking reg ion. Both were used, but the proximal motif (-14 to -18 nt relative to the ATG) was the primary sequence from which most of the mRNA was initiated. Wh en gp50 was cloned in a heterologous baculovirus expression system, spindli n was synthesized, although the spindles were irregular in shape. This sugg ested that the spindle structure may be species-specific or it may require more than one gene product for its morphogenesis. (C) 2000 Academic Press.