African horsesickness virus (AHSV) is a member of the Orbivirus genus of the Reoviridae family, being a double-layered capsid virus with a genome of ten double-stranded RNA segments. The outer capsid consists of two proteins, VP2 and VP5, which play essential roles in respectively host cell entry and viral release from the endosome into the host cytoplasm for replication. These proteins have best been characterized in the prototype virus Bluetongue virus (BTV), especially with regards to structural and functional characteristics. A cytotoxic effect for BTV VP5 was observed in insect cells and the approximate region conferring this nature identified. For AHSV VP5, a cytotoxic nature has also been observed in bacterial cells in preliminary studies but no region has thus far been mapped for mediating this activity. The main aim of this investigation was thus to map this region of AHSV VP5 using a bacterial expression system. A further aim was to investigate the localization of VP5 within infected cells using the BAC-TO-BACexpression system and the eGFP marker protein fused to VP5. In this way, protein expression could easily be detected and a possible association with cell membranes investigated. Initial cytotoxicity studies in these insect cells were also done to determine if the cytotoxic effect was also present in different host cells. To determine the region conferring the cytotoxic effect, genes encoding full-length VP5 and four truncation mutants of VP5 were cloned into the inducible pET system for expression as GSTfusion proteins within bacterial cells. After confirming protein expression, kinetic studies on the various VP5-fusion proteins were performed. Each protein increased in concentration with time post induction, except for the full-length VP5. Results from the cytotoxicity assay correlated with the expression patterns observed from the kinetic studies. Only GST-VP5 was cytotoxic. The VP5 truncation mutants lacking various N-terminal domains were all non-cytotoxic. Seeing that the only difference between GST-VP5 and GST-VP5Δ1-20 was the presence of amphipathic helix one, the results indicated that it is amphipathic helix one that plays a major role in conferring cytotoxicity. Amphipathic helix two, that is situated directly downstream of amphipathic helix one, seem to still be involved but require the presence of the amphipathic helix one and be expressed in the correct conformation. The role of amphipathic helix two in cytotoxicity could therefore not be inferred from this investigation. To determine its role, further studies involving more truncation mutants would be required. Solubility studies on all bacterial expressed proteins were performed to investigate whether the observed non-cytotoxicity of the truncated mutants might have been influenced by protein aggregation and hence not give a true reflection of the functional properties. Results indicated that a substantial portion of each mutant protein was. However, present in a soluble form and hence expected to be in a functional form. To study protein localization in insect cells using the BAC-TO-BACsystem, genes encoding VP5 and VP5 1-39 were cloned into pFB-eGFP and expressed as eGFP-fusion proteins. The recombinant baculoviruses Bac-VP5-eGFP and Bac-VP5 1-39-eGFP were used to infect insect cells at a high MOI and the green fluorescence signal of the marker monitored using confocal or fluorescent microscopy. No localization to particular cell structures was observed for either proteins and thus no specific association with membranes identified. Initial studies of cytotoxicity within insect cells were performed and the preliminary results indicate that the first two amphipathic helices are responsible for the cytotoxic effect in these cells, correlating with the results obtained in the bacterial system. This study provides the first evidence for the mapping of regions conferring a cytotoxic nature to AHSV VP5. Further characterization of this protein is necessary to obtain a better understanding of its’ role in the viral life cycle and the pathogenesis of AHS.
Dissertation (MSc (Genetics))--University of Pretoria, 2009.