RGD tripeptide of bluetongue virus VP7 protein is responsible for core attachment to Culicoides cells.
Bluetongue virus (BTV) is an arthropod-borne virus transmitted by Culicoides species to vertebrate hosts. The double-capsid virion is infectious for Culicoides vector and mammalian cells, while the inner core is infectious for only Culicoides-derived cells. The recently determined crystal structure of the BTV core has revealed an accessible RGD motif between amino acids 168 to 170 of the outer core protein VP7, whose structure and position would be consistent with a role in cell entry. To delineate the biological role of the RGD sequence within VP7, we have introduced point mutations in the RGD tripeptide and generated three recombinant baculoviruses, each expressing a mutant derivative of VP7 (VP7-AGD, VP7-ADL, and VP7-AGQ). Each expressed mutant protein was purified, and the oligomeric nature and secondary structure of each was compared with those of the wild-type (wt) VP7 molecule. Each mutant VP7 protein was used to generate empty core-like particles (CLPs) and were shown to be biochemically and morphologically identical to those of wt CLPs. However, when mutant CLPs were used in an in vitro cell binding assay, each showed reduced binding to Culicoides cells compared to wt CLPs. Twelve monoclonal antibodies (MAbs) was generated using purified VP7 or CLPs as a source of antigen and were utilized for epitope mapping with available chimeric VP7 molecules and the RGD mutants. Several MAbs bound to the RGD motif on the core, as shown by immunogold labeling and cryoelectron microscopy. RGD-specific MAb H1.5, but not those directed to other regions of the core, inhibited the binding activity of CLPs to the Culicoides cell surface. Together, these data indicate that the RGD motif present on BTV VP7 is responsible for Culicoides cell binding activity.
Tan
B H
BH
Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.
Nason
E
E
Staeuber
N
N
Jiang
W
W
Monastryrskaya
K
K
Roy
P
P
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D000914:Antibodies, Viral / Q000276:immunology / Q000494:pharmacology; D001820:Bluetongue virus / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D002460:Cell Line; D002519:Ceratopogonidae / Q000166:cytology / Q000378:metabolism* / Q000821:virology*; D020285:Cryoelectron Microscopy; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D009154:Mutation; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014758:Viral Core Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D019065:Virus Assembly
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C074954:VP3 protein, Bluetongue virus; D014758:Viral Core Proteins; C066862:VP7 protein, orbivirus; C047981:arginyl-glycyl-aspartic acid