To compare the mode of action of NVV Rev with that of HIV-1 Rev, a series of functional assays were planned. The <I>rev </I>gene of a British isolate of MVV (EV-1) was cloned and sequenced. Recombinant Rev was expressed as a fusion protein in yeast and bacterial systems. Expression in yeast was characterised by a low product yield, due to the highly toxic nature of the Rev fusion protein. Alternative expression and purification protocols were unable to greatly improve the yield and purity of product. The bacterial pGEX system, in which Rev was fused to glutathione S-transferase (GSTRev), was employed as an alternative. Purification by affinity chromatography resulted in an improved yield and purity of product. Partial instability of the fusion protein may have resulted in observed contamination. Binding of GSTRev to RNA corresponding to the predicted RRE was assayed by filter binding experiments. A specific vector context and low temperature were required to generate high quality RNA. GSTRev bound with high affinity to RRE-RNA, but not to RNA corresponding to antisense RRE. Addition of a non-specific competitor RNA reduced binding to antisense, but not sense, RNA. Rev is the least well conserved protein amongst sequenced isolates of MVV. To test for the functional conservation of the Rev/RRE axis, the cross reactivity of Rev function on heterologous RREs was examined by transient transfection assay. Whilst cross-strain functional reciprocity was observed, both the EV-1 and 1514 isolate Rev proteins demonstrated greatest activity on cognate RRE. Co-divergence of the <I>rev</I> gene and RRE structure of each strain has therefore occurred. MVV Rev was able to function through the RRE of the closely related caprine arthritis encephalitis virus. These results may have implications for the possible development of anti-lentiviral gene therapy based on <I>trans-</I>dominant, inhibitory Rev molecules.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:651057 |
| Date | January 1996 |
| Creators | Fotheringham, Michael William |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/29764 |
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