LS-6-P-2223 An investigation into the association of African horse sickness virus protein VP7 with host trafficking pathways and the role of trimer-trimer interactions in VP7 crystalline particle formation
African horse sickness (AHS) is a highly infectious and deadly vector-borne disease of Equidae with a mortality rate of up to 90% in susceptible horses. The etiological agent for AHS is an orbivirus from the Reoviridae family known as African horse sickness virus (AHSV). The AHSV virion is composed of two protein layers that are organized into an outer capsid and an icosahedral core particle made up of major core protein VP7 and subcore protein VP3. A unique characteristic of AHSV VP7 is that it is highly insoluble and, unlike any of the cognate orbivirus proteins, forms large flat crystals in AHSV-infected cells. The impact of the formation of these crystals or their role in AHSV replication remains to be discovered. The aim of this study was to investigate the process of AHSV VP7 crystal formation by immunofluorescence microscopy. We first characterized the localization of AHSV VP7 in different systems and studied the association of VP7 crystals with virus factories during infection. Co-localization data revealed that only a small amount of VP7 was associated with virus factories, while the majority of VP7 was sequestered into crystals (Fig. 1A). This is likely to have a negative impact on virus assembly. Next, we set out to investigate whether VP7 crystal formation resulted from interaction with host trafficking pathways or, alternatively, whether the crystals were a product of VP7 self-assembly. We investigated the co-localization of a VP7-eGFP fusion protein (Fig. 1B and C) with components of three host cellular trafficking pathways, i.e. the cytoskeleton, the aggresomal pathway, and protein degradation pathways. We then selectively blocked each of these pathways by studying the effect of chemical pathway inhibitors on VP7 distribution. We found that VP7 forms crystals in a host-independent manner and manages to evade host defences against protein aggregation (Fig. 2, 3, and 4). These results implied that the unique ability of VP7 to form crystals was driven by VP7 self-assembly. During core assembly, VP7 assembles into trimers that form a lattice that surrounds the inner VP3 subcore. We therefore suggest that VP7 crystal formation may be driven by trimer-trimer interactions. We set out to abolish VP7 self-assembly by targeting key residues that drive VP7 trimer-trimer interactions. Upon examination of the intracellular distribution of modified VP7 proteins, we found that the disruption of trimer-trimer interactions successfully abolished the formation of VP7 crystals thus proving that VP7 self-assembly drives crystal formation. Investigating the role of trimer-trimer interactions in core assembly will provide further insight into the formation of crystals as well as their role in AHSV replication.
This work was financially supported by BioPad, PRF, NRF, MSSA Trust and University of Pretoria. We thank Flip Wege for technical support with cell culture and both Alan Hall (University of Pretoria) and Sone Ungerer for technical support with confocal microscopy.
Fig. 1: Distribution of VP7 within AHSV- and recombinant baculovirus infected cells by confocal microscopy. AHSV-infected BSR cells (A), Sf9 cells expressing wild-type VP7 (B) and VP7-144-eGFP (C) with 3D stacking image of VP7 crystal on the right. Wild-type VP7 was detected with anti-VP7 (green) and VP7-144-eGFP was detected by eGFP auto-fluorescence. |
Fig. 2: VP7 is not associated with microtubules in mammalian cells. Mock cells and cells expressing VP7-144-eGFP (green) were labelled with antibody against tubulin (red). Cells were untreated (top panel) or treated with colchicine, a microtubule depolymerizing drug (bottom panel). Nuclei were stained with DAPI. The same results were seen in insect cells. |
Fig. 3: VP7 is not associated with the 26S proteasome in insect cells. Mock cells and cells expressing VP7-144-eGFP (green) were labelled with antibody against the 26S proteasome (red). Cells were untreated (top panel) or treated with proteasome inhibitor MG132 (bottom panel). Nuclei were stained with DAPI. The same results were seen in mammalian cells. |
Fig. 4: VP7 is not ubiquinated or associated with the lysosome in insect cells. Mock cells and cells expressing VP7-144-eGFP (green) were labelled with antibody against ubiquitin (A) or incubated in the presence of LysoTracker Red Dye (Invitrogen) (B) (red). Nuclei were stained with DAPI. The same results were seen in mammalian cells. |