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 cytoplasm 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 that the OBs copurified with the spindles. We therefore modified CfDEFNPV by replacing the polyhedrin gene (plh) with a cassette containing the green fluorescent protein (GFP) gene. The recombinant virus did not produce OBs; however, the synthesis and morphogenesis of the spindles were not altered. When 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 and 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 that of fusolin, which also encodes a protein that forms spindle-shaped inclusion bodies in the cytoplasm of infected cells. Immunoblot analysis indicated that spindlin and fusolin were not serologically related, even though they share conserved polypeptide domains. Sequence analysis showed that gp50 of CfDEFNPV contains two late promoter motifs (TTAAG) in its 5' flanking region. 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. When gp50 was cloned in a heterologous baculovirus expression system, spindlin was synthesized, although the spindles were irregular in shape. This suggested that the spindle structure may be species-specific or it may require more than one gene product for its morphogenesis.
Li
X
X
Laboratory for Molecular Virology, Great Lakes Forestry Centre, 1219 Queen St. East, Sault Ste. Marie, Ontario, P6A 5M7, Canada.
Barrett
J
J
Pang
A
A
Klose
R J
RJ
Krell
P J
PJ
Arif
B M
BM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006031:Glycosylation; D015151:Immunoblotting; D009036:Moths / Q000821:virology; D017924:Nucleopolyhedroviruses / Q000235:genetics / Q000502:physiology*; D000077183:Occlusion Body Matrix Proteins; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015678:Viral Structural Proteins; D014779:Virus Replication
D000077183:Occlusion Body Matrix Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C093475:fusolin protein, entomopoxvirus; C009829:polyhedrin protein, Nucleopolyhedrovirus