Synthesis of the virus-specified tubules of epizootic haemorrhagic disease virus using a baculovirus expression system.
The formation of virus-specific tubules is one of the most characteristic features in the orbivirus infection cycle, yet little is known about their role in virus replication. The tubuli are composed of a major nonstructural protein, NS1. We have investigated the expression of the NS1-encoding gene of epizootic haemorrhagic disease virus serotype 2 (Alberta-strain) by producing a recombinant Autographa californica nuclear polyhedrosis virus (AcNPV). Prior to cloning in the baculovirus transfer vector, pAcYM1 and cotransfection with AcNPV DNA, the NS1 gene was tailored by means of a polymerase chain reaction method to remove G/C tails. The baculovirus recombinant, AcNPV-EHDV2 NS1, expressed large amounts of a 55 K protein which could be purified by sucrose gradient sedimentation as a tubular complex. It appeared that the tubules could break up into 50 nm diameter circular units, which in turn were composed of approximately 16 subunits. The circular units appeared to be hollow and stacked on top of one another (100 units/micron tubule length), giving the tubules a segmented, ladderlike appearance. A large excess of EHDV2-specific tubuli could also be demonstrated in AcNPV-EHDV2 NS1-infected Spodoptera frugiperda cells by electron microscopic examination of thin sections. With pulse-labelling experiments it was shown that, regardless of the level of NS1 expression, the majority of NS1 synthesized in a 30 min period could only be recovered in a particulate form.
Nel
L H
LH
Department of Microbiology and Plant Pathology, University of Pretoria, South Africa.
Huismans
H
H
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D015870:Gene Expression; D005814:Genes, Viral; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000648:ultrastructure; D012087:Reoviridae / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure
D004279:DNA, Viral; D011994:Recombinant Proteins