Title page for ETD etd-11242005-110136


Document Type Master's Dissertation
Author Meiring, Tracy Leonora
URN etd-11242005-110136
Document Title An Investigation into nonstructural proteins NS3 and NS3A of African Horsesickness virus
Degree MSc (Genetics)
Department Genetics
Supervisor
Advisor Name Title
Dr V Van Staden
Keywords
  • African horse sickness virus genetics
Date 2002-04-01
Availability unrestricted
Abstract
The aims of this investigation were to compare the nonstructural proteins, NS3 and NS3A, of African horse sickness virus (AHSV) and to further characterize the cytotoxic properties of these proteins.

NS3 and NS3A are encoded from two in-phase overlapping reading frames on the smallest double-stranded (ds) RNS genome segment, segment 10 (S10) of AHSV. The proteins differ only with respect to the presence of an additional 10 or 11 amino acids, depending on the serotype, at the N-terminal of NS3. All known orbiviruses have been shown to encode two closely related proteins from S10. Sequence analysis of the N-terminal region of the NS3 proteins of the different serotypes of AHSV and various orbiviruses revealed that this region is not highly conserver.

Both AHSV NS3 and NS3A are membrane-associated and cytotoxic to insect cells causing membrane permeabilisation and eventual cell death when expressed individually (Van Staden et al., 1995; Van Staden et al., 1998; Van Niekerk et al., 2001a). AHSV infection of Vero cells results in the synthesis of both proteins in equimolar amounts (Van Staden, 1993). The effect on the cytotoxic properties of these proteins when expressed together in insect cells was therefore investigated here. Whether co-expressed or expressed individually NS3 and NS3A caused a dramatic decrease in the viability of insect cells. The NS3 protein is therefore representative of the NS3 and NS3A proteins together in terms of its cytotoxic effect.

The effect of the exogenous addition of NS3 on the membrane permeability of Vero cells was also investigated. The NS3 protein was found to cause a rapid increase in the membrane permeability of Vero cells. The cytotoxic properties of NS3 appear therefore not to be limited to their endogenous effects on insect cells.

The AHSV-3 NS3 and NS3A proteins were expressed as histidine tagged recombinants in the baculovirus expression system, to allow for the purification of large quantities of protein for functional and comparative studies. The resulting NS3 histidine fusion product, however, displayed a decrease in solubility, probably as a result of incorrect folding due to the presence of this histidine tag extension at the N-terminus of the protein.

To produce antibodies that detect NS32, and not NS3A, in AHSV infected cells, the N-terminal region unique to AHSV-3 NS3 was displayed on the surface of the AHSV core protein, VPO7. The chimeric protein VP7-NS3 displayed the same structural characteristics as the wild type VP7 protein, aggregating into highly insoluble crystals. Antiserum was prepared against purified VP7-NS3 and analysed in terms of its ability to recognize denatured and non-denatured AHSV-3 NS3. Although the antiserum was shown to contain antibodies directed against VP7 epitopes no immune reaction with NS3 was observed. The use of alternate sites on the surface region of VP7 for the display of such a small peptide needs to be investigated.

Although no functional differences between NS3 and NS3A were identified in this investigation, the finding that NS3 causes membrane permeability of damage to Vero cells represents the first indication that this AHSV protein causes extra cellular membrane damage in mammalian cells. Many viral membrane damaging proteins or viroporins are thought to contribute significantly to the severity of virus-induced pathogenesis. The mechanism of membrane damage and the contribution of the membrane damaging properties of NS3 to AHSV-induced pathogenesis needs to be investigated

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