Bluetongue virus (BTV), an insect (Culicoides sp) transmitted double-layered non-enveloped virus, is the type species virus of the genus Orbivirus, in the family Reoviridae. BTV is an economically important virus and causes hemorrhagic disease in sheep and other ruminants [1]. The virus has a genome consisting of ten double stranded (ds) RNA segments, located in a core particle made up of two major proteins (VP3 and VP7) and three minor proteins (VP1, VP4 and VP6). In the mature particle, this core is coated by two outer capsid proteins, VP2 (110 kDa) and VP5 (60 kDa) (see schematic, Fig. 1). The most exposed protein on the mature virion, VP2, is responsible for receptor binding [2-4], haemagglutinating activity [5] and elicits host specific immunity [6-8]. The smaller protein, VP5, is involved in cell penetration during the initial stages of infection [9,10]. After entry into cells, the virus is uncoated by removal of VP2 and VP5 to yield the core particle that is transcriptionally active. This complex is an end-point in virus disassembly and protects the viral dsRNA genome from intracellular surveillance mechanisms.
Like other members of Reoviridae, BTV replicates in the cytoplasm of infected cells. Electron microscopic analysis of thin sections of BTV infected cells have revealed a large numbers of virus-specific tubules, and juxtanuclear inclusions bodies containing virus-like particles in addition to small numbers of intracellular virus particles (15). While the tubules are multimers of NS1 [11], inclusion bodies are predominantly formed by NS2 [12,13]. Similar to rotavirus inclusion bodies [14], it has been speculated that during BTV replication and assembly NS2 binds core proteins, viral single-stranded (ssRNA) [15,16] and recruits these components to the inclusion bodies [17]. Additionally, it has been hypothesised that the viral genome interacts with the minor structural proteins to form the transcriptase complex which is subsequently encapsulated by a single shell of VP3 to form the subcore of the assembling virus [18]. The subcore acts as a scaffold for the deposition of VP7 trimers, thus forming a stable core structure [19-21]. However, the outer capsid proteins, VP2 and VP5 have not been localised to the viral inclusion bodies and it is still not clear when and where in the cell are they assembled on to the core. As the outer capsid proteins, particularly VP2, would effectively block the channels in the core that are used during transcription of the viral genome it is unlikely that the coating of cores would be an uncontrolled process.
Previous electron microscopy studies have indicated that BTV particles can be found attached to vimentin intermediate filaments [22]. Negative staining of intact, infected cells labelled with anti-VP2 antibody revealed that BTV particles were also present under the cell membrane as well as on the cell surface [23]. In addition, both virus aggregates and single virion particles retain an association with the cortical layer of the cytoskeleton following cell lysis. This has lead to speculation that there may be interaction of the particle with the actin-rich cortical layer underlying the cell membrane [23]. Recent studies have shown that NS3, the only virus-coded glycoprotein of BTV, interacts with VP2 as well as Tsg101 and the p11 subunit of the heterotetrameric calpactin II complex, thereby facilitating virus release [24,25]. Thus, in addition to its role in virus attachment, VP2 is emerging as a key player in the control of BTV assembly and egress from infected cells. A detailed understanding of the control of VP2 association with the newly assembled core is therefore an important step in understanding virus assembly and egress.
In this manuscript we demonstrate that normal subcellular distribution of VP2 relies on an association with vimentin intermediate filaments, and identify key residues in this interaction located in the N-terminus of the protein. Furthermore, disruption of the vimentin filament network using pharmacological inhibitors lead to a disruption of virus egress.
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