The specific roles of the two proteins in the orbivirus outer capsid in the processes of virus attachment and membrane penetration have now been elucidated [4,9,10]. Intriguingly, one of these proteins, VP2, also plays a role in virus egress from infected cells as it binds a viral non-structural protein that acts as a molecular bridge between the virus particle and cellular export and release factors [24,25,34]. Thus, VP2 is important to both the infectivity and egress of BTV particles. The proteins that are required to form the transcriptionally active orbivirus core structure have all been localised to inclusion bodies within virus infected cells [17,35,36]. However, the outer capsid proteins do not localise to inclusions and therefore the site of assembly of mature virus particles before transport out of infected cells is unknown. Members of other genera in the Reoviridae cannot be used as a model for BTV in outer capsid assembly, as this information is either not available, or the outer capsid and assembly process is quite distinct. For example, unlike BTV, significant steps in the outer capsid assembly of rotavirus are sequestered in the endoplasmic reticulum [37]. Thus, in order to gain further insight into the assembly and intracellular trafficking of BTV, the localisation of VP2 in virus infected cells was followed using immunofluorescence microscopy. In virus infected cells VP2 was located in punctuate distribution throughout the cytoplasm even at quite early times post infection (Fig. 2). This distribution could be mimicked in cells transfected with plasmid expressing VP2 protein, full-length VP2 fused at its C-terminus to GFP, or the N-terminal 118 amino acids of VP2 fused to GFP (Fig. 2; Fig. 3). Furthermore, it was found that VP2 co-fractionated with the intermediate filament protein vimentin in fractionation studies, and co-localised with vimentin in immunofluorescence of VP2 in transfected and virus infected cells (Fig. 3). These data are consistent with earlier electron microscopy studies that have found mature virus particles in linear arrays along intermediate filaments in infected cells [26]. Furthermore they demonstrate for the first time that the determinant for BTV-vimentin association is the VP2 protein.
The vimentin associated localisation of VP2 in virus infected and VP2 transfected cells was quite distinct from the aggresome structure that has been noted following the overexpression of certain proteins. There was no evidence of the cage-like vimentin arrangement around VP2 which is observed with aggresomes [38,39]. Nor was there any evidence that ubiquitin co-localised with VP2 in transfected cells (Fig. 3).
Using a deletion mutagenesis strategy we were able to localise the vimentin localisation signal within VP2 to the region encompassing the amino acids 65–114 (Fig. 4). This was confirmed by systematic site-directed mutagenesis of conserved amino acids within this region to identify key residues in VP2-vimentin association. Of five sets of mutants, which changed a total of 13 conserved amino acids, only one, mutation of GVV to DVD at postions 70–72, prevented co-localisation of VP2 with vimentin (Fig. 5). This mutant was specifically designed to alter the hydrophobicity profile of the VP2 region from amino acids 63–80 to make it more hydrophilic. Thus, it was initially unclear whether the disruption of the association between VP2 and vimentin was due to global changes in the folding of the protein. However, when the DVD mutation was introduced into full-length VP2 the ability of the recombinant protein to assemble into trimeric VP2 structures was equivalent to that of unmodified protein. Thus, if there are changes to the structure of the protein induced by the DVD mutation, they occur at a local as opposed to a global level within the protein. These data indicate the DVD mutation either disrupts vimentin-VP2 association because the conserved GXV motif at position 70–72 is necessary for vimentin interaction or, like the deletions of aminio acids 65–92 or 93–114, mutation of this motif results in local rearrangement of the structure of VP2. At a primary sequence level, there is no apparent homology between this region of VP2 and vimentin, or any other known vimentin interacting protein. Therefore, in combination with the identity of the completely conserved amino acid (Glycine) and our mutagenesis data it is likely that VP2-vimentin interaction involving amino acids 65–114 is conformationally dependent.
Within the cell vimentin is emerging as a key player in intracellular trafficking. Vimentin structures have been shown to move along microtubules, and two candidate motors had been identified for this motility: conventional kinesin, and more recently, cytoplasmic dynein [29,31-33]. Significantly for VP2-vimentin interactions, the motility of vimentin intermediate filaments is principally from the nucleus towards the cell surface in BHK-21 cells [33]. Indeed, in this study when vimentin intermediate filaments were disrupted pharmacologically either directly, or indirectly by disrupting microtubules, there was a dramatic reduction in the amount of virus released from infected cells (Fig. 6). This reduction in release was not entirely due to a decrease in overall virus synthesis as, particularly in the case of acrylamide treatment, decrease in BTV release corresponded to an increase in cell associated virus (Fig. 6B). A plausible explanation for these observations would be that VP2-vimentin interaction is involved in the transport of mature virus particles. This hypothesis would correlate well with recent evidence that the viral non-strucutral protein NS3, which interacts with VP2 at its C-terminus [24], interacts at its N-terminus with the cellular release factor Tsg101 [25]. In this model, mature virus particles would be assembled from cores released from juxtanuclear inclusions, trafficked to the cell surface through vimentin association and released through the interaction of NS3 and Tsg101. Future work will focus on testing the validity of this model.
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