Morphogenesis of a bluetongue virus variant with an amino acid alteration at a neutralization site in the outer coat protein, VP2.
Neutralization-resistant variants of bluetongue virus, selected with a monoclonal antibody to the outer coat protein VP2, have been used to delineate a neutralization epitope on the VP2 protein. Comparison of the RNA 2 sequence of four variants with that of the wild-type virus indicated that each variant contained a single nucleotide substitution which in turn resulted in a single amino acid alteration in VP2. The changes were clustered within a span of eight amino acids at positions 328 to 335 in the VP2 protein. In addition, analyses of cells infected with wild-type and a variant virus V35B2 have provided information on the site of VP2 addition to virus particles during morphogenesis. Electron microscopic examination revealed few virus-like particles around virus inclusion bodies (VIB) in wild-type virus-infected cells and cytoskeletons. In contrast, VIB in cells infected with the neutralization-resistant variant V35B2 were surrounded by particles identified as virus cores on the basis of their size and morphology. Probing of cytoskeletons with gold-labeled anti-VP2 monoclonal antibody revealed that in wild-type virus-infected cells the antibodies reacted weakly with VIB and only at locations where virus particles appeared to be leaving. The core-like particles surrounding VIB in V35B2-infected cells labeled very weakly with the anti-VP2 antibody. In contrast, wild-type and V35B2 virus particles which bound to the cytoskeleton at locations distal to VIB and those outside the infected cell bound significant amounts of antibody. These results suggest that although some VP2 may be added to developing virus particles at the periphery of VIB, the remainder of the VP2 protein is added outside the VIB either in the cytosol or following attachment of the particles to the cytoskeleton.
Gould
A R
AR
Australian Animal Health Laboratory, CSIRO, Geelong Victoria.
Hyatt
A D
AD
Eaton
B T
BT
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D001483:Base Sequence; D001820:Bluetongue virus / Q000235:genetics / Q000254:growth & development* / Q000276:immunology; D002213:Capsid / Q000235:genetics* / Q000276:immunology; D003599:Cytoskeleton / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D008969:Molecular Sequence Data; D009024:Morphogenesis; D009500:Neutralization Tests; D012087:Reoviridae / Q000254:growth & development*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral