title: The membrane trafficking protein calpactin forms a complex with bluetongue virus protein NS3 and mediates virus release.
abstract: Bluetongue virus, an arbovirus of the Orbivirus genus, infects and replicates in both insect and mammalian cells. However, the cytopathic effect (cpe) on each host is very different. Mammalian cells show substantial cpe, most likely a result of the mechanism of virus release, whereas insect cells show little cpe and appear to release virus without cell lysis. Expression analysis of each infected cell type shows one protein, the nonstructural (NS) protein NS3, to be differentially expressed in the different cell types, suggesting it may act in the virus egress pathway. The molecular basis of such an interaction, however, has never been clear. Here, by using yeast two-hybrid analysis, we show that NS3 interacts with a cellular protein p11 (calpactin light chain), part of the annexin II complex that is involved in exocytosis. We map the NS3 region of interaction with p11 to a 13-residue peptide found at the N terminus of the protein and show it effectively competes with p36 (annexin II heavy chain) for p11 ligand binding. Further, we show that the C-terminal domain of NS3 interacts with VP2, the outermost protein of the fully assembled virus particle, suggesting that NS3 forms a bridging molecule that draws assembled virus into contact with the cellular export machinery. Our data describe the first host protein involvement in orbivirus egress and provide new insights into understanding arbovirus interactions with their hosts.
authors.affiliation: Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Beaton
authors.forename: Javier
authors.initials: J
authors.lastname: Rodriguez
authors.forename: Y Krishnamohan
authors.initials: YK
authors.lastname: Reddy
authors.forename: Polly
authors.initials: P
authors.lastname: Roy
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D017306:Annexin A2 / Q000737:chemistry; D017302:Annexins / Q000737:chemistry* / Q000378:metabolism; D001820:Bluetongue virus / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D005089:Exocytosis; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D007313:Insecta; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D017306:Annexin A2; D017302:Annexins; D018076:DNA, Complementary; D010455:Peptides; D011993:Recombinant Fusion Proteins; C075080:S10 protein, Bluetongue virus; D017361:Viral Nonstructural Proteins