Characterization of virus-like particles produced by a recombinant baculovirus containing the gag gene of the bovine immunodeficiency-like virus.
The entire gag gene of the bovine immunodeficiency-like virus (BIV) was inserted behind the strong polyhedron promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). The resultant recombinant baculovirus (AcNPV-BIVgag) was used to infect insect cells in order to overexpress and characterize BIV gag gene products. The infection resulted in the high-level expression of a protein similar in size to the predicted BIV gag precursor (Pr53gag). BIV Pr53gag was detected in AcNPV-BIVgag-infected insect cells and in culture supernatants. Electron microscopy of these cells revealed an abundance of virus-like particles (VLPs) in the cytoplasm, budding from the cell membrane, and free in the culture medium. The size and morphology of the VLPs were similar to those of the immature forms of BIV observed in infected mammalian cells. The VLPs sedimented at a density of 1.16 g of sucrose per milliliter in linear gradients and were shown to contain the majority of the supernatant Pr53gag. Antigenic determinants on Pr53gag from VLPs were recognized by BIV and HIV-1 antiserum, and serum from rats immunized with VLPs reacted with recombinant and viral BIV Pr53gag and processed products. The protease (PR) activity in BIV virions was capable of processing recombinant Pr53gag; this activity was blocked by pepstatin A, a potent aspartyl PR inhibitor. Baculovirus-expressed BIV Pr53gag appears to be an excellent source of gag precursor; it may prove useful for structural studies and enable the development of assays to detect retroviral PR inhibitors. The data further suggest that unprocessed BIV Pr53gag plays a major role in the assembly of BIV particles. The expression of other BIV structural genes in insect cells may prove instructive in the study of molecular events involved in the assembly and processing of these BIV proteins.
Laboratory of Cell and Molecular Structure, Program Resources, Inc., NCI-Frederick Cancer Research Facility, Maryland 21701.
L
L
Rasmussen
J K
JK
Battles
W H
WH
Ennis
K
K
Nagashima
M A
MA
Gonda
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics* / Q000648:ultrastructure; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000378:metabolism; D000939:Epitopes / Q000235:genetics; D015683:Gene Products, gag / Q000235:genetics* / Q000276:immunology; D005814:Genes, Viral; D016087:Immunodeficiency Virus, Bovine / Q000201:enzymology / Q000235:genetics* / Q000276:immunology; D008856:Microscopy, Fluorescence; D009024:Morphogenesis / Q000235:genetics; D009036:Moths / Q000382:microbiology / Q000648:ultrastructure; D011401:Promoter Regions, Genetic; D013379:Substrate Specificity; D014771:Virion / Q000235:genetics*
D000914:Antibodies, Viral; D000939:Epitopes; D015683:Gene Products, gag; D010450:Endopeptidases