Expression and functional characterisation of the Rev gene product of maedi visna virus EV- 1

The ovine Maedi Visna Virus (MVV) and the Human Immunodeficiency Virus Type 1 (HIV-1) are members of the lentiviririae retrovirus subfamily. Lentiviruses possess a complex genomic organisation, encoding several genes with a regulatory or auxiliary function. Alternative splicing of the genomic-length primary transcript is used to express this complexity. mRNA expression is subject to temporal regulation in both MVV and HIV-1. In HIV-1 a highly basic protein. Rev, mediates this regulation. Rev function requires binding to a highly structured RNA target, the Rev responsive element (RRE), and may involve facilitation of RNA nucleocytoplasmic export. Rev/RRE interaction is essential for virus replication. There is little overall sequence homology between lentivirus Rev proteins. However, functionally important basic and leucine-rich motifs are conserved. These domains are found within the putative MVV Rev protein, and a predicted RRElike structure is present in a similar genomic location to that in HIV-1.To compare the mode of action of MVV Rev with that of HIV-1 Rev, a series of functional assays were planned. The rev 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. A polyclonal antiserum directed against a synthetic Rev polypeptide was generated to aid purification. 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 rev 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 frans-dominant, inhibitory Rev molecules