Abstract
Malignant catarrhal fever is a fatal lymphoproliferative disease of domestic and wild
ruminants. The disease is characterised by lymphocyte migration and proliferation with
degeneration of various tissues. This is thought to be responsible for the death of the
affected animal. There are two viral causal agents of the disease, Alcelaphine
herpesvirus-l (A1HV-1) and Ovine herpesvirus-2 (OvHV-2). A1HV-1 is found naturally
in Blue Wildebeest while OvHV-2 is found in sheep. Both cause no apparent disease in
the carrier species, however, when they infect a susceptible species, animals develop the
disease which is usually fatal. The virulent strain of A1HV-1 has been sequenced and
was found to encode eleven putative pathogenesis-associated herpesvirus genes. One of
these is contained within open reading frame A9. This encodes a protein that shares a
limited amino acid homology to the bcl-2 family of apoptosis regulatory proteins.
Apoptosis is a major form of cell death and is characterised by a series of morphological
and biochemical changes that take place in the cell. A number of bcl-2 family members
have been identified, that can be either anti- or pro-apoptotic. The proteins all possess at
least one bcl homology (BH) domain that determines the capacity of the bcl-2 proteins
to form homo- or hetero-dimers and/or to bind to proteins not in the bcl-2 family. ORF
A9 possesses only one of the BH domains, BH1, which in other proteins has antiapoptotic activity. The overall aim of this study was to characterise A1HV-1 A9. The
specific aims were to determine: ifthe A9 gene product is functional; when in the viral
life cycle A9 is expressed; where in the cell the protein is localised; whether any cellular
or viral binding partners can be defined; is the A9 gene expressed in large granular
lymphocyte (LGL) T cell lines? Northern blot analyses showed that A9 mRNA was
expressed as an early gene in the productive phase of the virus life cycle. The A9 gene
product was also shown to protect CHO cells from cis-platin induced apoptosis. The
protein encoded by the A9 gene was successfully expressed in E. coli and was used to
immunise mice and rabbits. Antibodies specific for the A9 protein were generated.
However, using these antibodies, the localisation ofA9 in CHO cells was inconclusive.
A9 mRNA was expressed in LGL T cells from MCF-affected rabbits. To determine
binding partners, an expression system involving a tandem affinity purification (TAP)
technique was used. Various cDNAs related to the bcl-2 family of proteins were
successfully transfected into CHO cells as shown by RT-PCR and immunofluorescence
analysis. However, MADLI analysis of samples failed to identify specific binding
partners for A9. A9 vbcl-2 is anti-apoptotic and may function to allow viral replication
in infected cells in the productive phase of the life cycle.
