Characterisation of the herpes simplex virus type 1 mutant, ambUL12
The herpes simplex virus type 1 (HSV-1) UL12 gene encodes an alkaline nuclease. Although the UL12 gene is not absolutely essential for replication, UL12 null mutants produce 100-1000 fold less viable virus than wt HSV-1. It has been suggested that the alkaline nuclease functions to remove branched structures from high molecular weight concatemeric DNA prior to its cleavage into monomeric genomes that are packaged into viral capsids. Failure to remove the branches results in unstable packaging of DNA into capsids which fail to egress from the nucleus. This thesis describes detailed characterisation of the HSV-1 mutant, ambUL12 (Patel et al., 1996) which fails to express the alkaline nuclease due to the insertion of an amber stop codon into the UL12 open reading frame. The ability of ambUL12 to replicate and package both viral genomic DNA and amplicons (plasmids containing the HSV-1 origin of replication and DNA encapsidation signal) was examined. In contrast to results obtained with other alkaline nuclease mutants, which replicate and package DNA with close to wt HSV-1 efficiency (Shao et al., 1993; Martinez et al., 1996b), ambUL12 displayed a 3-5 fold drop in replication and a 15-20 fold drop in packaging of genomic DNA. Similar reductions were observed in the replication and packaging of amplicon DNA. The replication and packaging of amplicons by ambUL12 in these transient assays could be partially complemented when UL12 was supplied in trans. Close inspection of the DNA molecules synthesised during transient assays demonstrated that amplicon replication intermediates are complex high molecular weight concatemers that undergo intermolecular recombination, analogous to viral DNA replication intermediates.