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

Abstract

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.Orbiviruses (within the family Reoviridae), are vectored to vertebrate species by arthropods (e.g., gnats, mosquitoes and ticks) and are able to replicate in both hosts. Bluetongue virus (BTV) is the prototype virus of the genus and is transmitted by gnats (Culicoides species), causing diseases of economic importance in ruminants in many parts of the world. Vector-virus interactions play a crucial role in vector-borne disease epidemiology. The spread of Culicoides species from BTV-endemic to non-BTV (or related African horsesickness virus, AHSV, and epizootic hemorrhagic disease virus EHDV, of deer) regions of the world in the past highlights the concern that these viruses are a threat to regions of the world that are presently free from them.The initiation of a virus infection involves virus binding to ligands on the cell surface prior to cell entry by a number of mechanisms (depending on the virus). Like many other viruses, BTV appears to utilize a protein molecule(s) of mammalian cells as a receptor (20); however, it is also possible that alternative receptors may be utilized in different tissues and in different species and as accessory molecules.BTV has a genome composed of 10 segments of doublestranded RNA packaged within a double icosahedral capsid. The outer capsi...

Three-Dimensional Localization of pORF65 in Kaposi's Sarcoma-Associated Herpesvirus Capsid

“…In these micrographs, bound antibodies are present as a faint ring of density protrusions extending radially from the capsids (Fig. 1f), similar to micrographs of other antibody-bound viruses (11,18). Such rings are not present in control micrographs obtained from the capsid preparation prior to antibody labeling (Fig.…”

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“…Polyclonal anti-VP5 (gp639), monoclonal anti-VP7 (H1-5), and polyclonal anti-NS2 (R658) were used as previously described (10,26,44). Monoclonal anti-BTV 10 VP2 was a gift from J. J. MacLachlan (University of California).…”

Bluetongue virus: virology, pathogenesis and immunity

“…VP7 can mediate attachment and penetration of insect cells in the absence of either VP2 or VP5, a process that may involve an arginine-glycine-aspartate (RGD) tripeptide motif present at amino acid residues 168 to 170 on the outermost surface of the VP7 trimers on the BTV core [97]. VP7 can bind to glycosaminoglycans, although it appears likely that other specific receptors are also involved in cell attachment and penetration.…”