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Journal of Virology, April 2001, p. 3937-3947, Vol. 75, No. 8
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.8.3937-3947.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

RGD Tripeptide of Bluetongue Virus VP7 Protein Is Responsible for Core Attachment to Culicoides Cells

Boon-Huan Tan,1,2 Emma Nason,2 Norbert Staeuber,1,2 Wenrong Jiang,2 Katherine Monastryrskaya,1,2 and Polly Roy1,2,3,*

Department of Biochemistry, University of Oxford, Oxford OX1 3QU,1 and Institute of Virology & Environmental Microbiology, Oxford OX1 3SR,2 United Kingdom, and Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 352943

Received 9 October 2000/Accepted 19 January 2001

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.

* Corresponding author. Present address: Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel St., London WC1E 7HT, United Kingdom. Phone: 44 20 7927 6239. Fax: 44 20 7636 8739. E-mail: polly.roy{at}lshtm.ac.uk.

Journal of Virology, April 2001, p. 3937-3947, Vol. 75, No. 8
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.8.3937-3947.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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