Abstract:
The highly infectious vector-borne disease African horse sickness (AHS) is caused by African horse sickness virus (AHSV), a non-enveloped dsRNA virus of major economic importance in South Africa. The AHSV virion has a double-layered protein capsid comprised of a diffuse outer layer housing an inner icosahedral core. Viral protein (VP) 7 is the major core particle surface protein and forms trimers that can either be incorporated into the core or can self-assemble into flat hexagonal crystalline-like particles, a characteristic unique to AHSV VP7. Little is known about the self-assembly and particle formation of this highly hydrophobic and insoluble protein, and in particular what drives the stable layering of VP7 into these particles.
In this study, the effect of various minor and major top domain modifications on the self-assembly and particle formation was investigated by comparing the previously constructed AHSV VP7 vector (VP7-144, VP7-177 and VP7-200) and VP7-eGFP fusion proteins (VP7-144-eGFP, VP7-177-eGFP and VP7-200-eGFP), known to differ with regard to solubility and trimerisation, to wild type (WT) AHSV VP7. The dual confocal and transmission electron microscopy approach showed that self-assembly was unaffected by all of the modifications. Particle formation was however affected, as the morphology of the protein structures formed by all six proteins differed from the hexagonal particles formed by WT VP7.
Sucrose gradient sedimentation analysis was used to investigate the kinetics of the VP7-eGFP aggregation observed in previous studies. These results showed that VP7-144-eGFP is an inherently soluble and fluorescing protein, the spontaneous aggregation of which is L-arginine reversible. VP7-200-eGFP, however, is a largely misfolded protein with no evidence of protein aggregation. Immunofluorescence and confocal microscopy was used to investigate the localisation of misfolded, non-fluorescing VP7-eGFP fusion proteins in relation to their correctly folded, fluorescing versions. No such proteins were differentially detected using this approach thereby suggesting that both versions are located in the same area, either due to both versions being transported in a similar manner or due to correctly folded proteins becoming trapped in misfolded protein. Alternatively, the approach itself may be unsuccessful in detecting misfolded VP7-eGFP fusion proteins.
Although the role of AHSV VP7 particles in the replication cycle and disease pathogenesis remains to be investigated, this study provides insights into the self-assembly and particle formation thereof. In particular, it is the top domain in combination with a group of associated factors that strongly influences the stable layering of VP7 to form the characteristic particles.
