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J. Virol. doi:10.1128/JVI.01274-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Bluetongue virus outer capsid protein VP5 interacts with membrane lipid rafts via a SNARE domain

Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine

^* To whom correspondence should be addressed. Email: Polly.Roy{at}lshtm.ac.uk.

	Abstract

Bluetongue virus (BTV) is a non-enveloped double-stranded RNA virus belonging to the Reoviridae family. The two outer capsid proteins, VP2 and VP5 are responsible for virus entry. However, little is known about the role of these two proteins, particularly VP5, in virus trafficking and assembly. In this study we have used density gradient fractionation and methyl beta cyclodextrin, a cholesterol sequestering drug to demonstrate that not only does VP5 co-purify with lipid raft domains in both transfected and infected cells but also that raft domain integrity is required for BTV assembly. Previously we showed that BTV non-structural protein 3 (NS3) interacts with VP2 and also with cellular exocytosis and ESCRT pathway proteins indicating its involvement in virus egress (Beaton et al. 2002. Proc. Natl. Acad. Sci. USA 99:13154-9; Wirblich. et al. 2006. J. Virol. 80:460-73). Here we show by pull-down and confocal analysis that NS3 also interacts with VP5. Further, a conserved membrane docking domain similar to the motif in Synaptotagmin, a protein belonging to the SNARE (Soluble N-ethylmaleimide-sensitive fusion attachment protein receptor) family was identified in VP5 sequence. By site directed mutagenesis followed by floatation and confocal analysis we demonstrated that raft association of VP5 depends on this domain. Together these results indicate that VP5 possesses an autonomous signal for its membrane targeting and that the interaction of VP5 with membrane associated NS3 might play an important role in virus assembly.