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 Culicoide s species to vertebrate hosts. The double-capsid virion is infectious for C ulicoides vector and mammalian cells, while the inner core is infectious fo r only Culicoides-derived cells. The recently determined crystal structure of the BTV core has revealed an accessible RGD motif between amino acids 16 8 to 170 of the outer core protein VP7, whose structure and position would be consistent,vith a role in cell entry. To delineate the biological role o f the RGD sequence within VP7, we have introduced point mutations in the RG D 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 struc ture of each was compared with those of the wild-type (wt) VP7 molecule. Ea ch 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 mt CLPs . Twelve monoclonal antibodies (MAbs) was generated using purified VP7 or C LPs as a source of antigen and were utilized for epitope mapping with avail able chimeric VP7 molecules and the RGD mutants. Several MAbs bound to the RGD motif on the core, as shown by immunogold labeling and cryoelectron mic roscopy. 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 sur face. Together, these data indicate that the RGD motif present on BTV VP7 i s responsible for Culicoides cell binding activity.