Equine herpesvirus 1 gene 12 can substitute for vmw65 in the growth of herpes simplex virus (HSV) type 1, allowing the generation of optimized cell lines for the propagation of HSV vectors with multiple immediate-early gene defects

Herpes simplex virus (HSV) has often been suggested for development as a ve ctor, particularly for the nervous system. Considerable evidence has shown that for use of HSV as a vector, immediate-early (IE) gene expression must be minimized or abolished, otherwise such vectors are likely to be highly c ytotoxic. Mutations of vmw65 which abolish IE promoter transactivating acti vity may also be included to reduce IE gene expression generally. However, when vmw65 mutations are combined with an IE gene deletion, such viruses ar e hard to propagate, even on cells which otherwise complement the IE gene d eletion effectively. We have found that vmw65 mutants can be effectively gr own on cell lines expressing equine herpesvirus 1 gene 12, a non-HSV homolo gue of vmw65 with little sequence similarity to its HSV counterpart. This p revents repair by homologous recombination of vmw65 mutations in the virus, which would occur if mutations were complemented by vmw65 itself. The gene 12 protein is not packaged into HSV virions, which is important if viruses grown on such cells are to be used as vectors. These results not only furt her strengthen the evidence for direct functional homology between and simi lar modes of action of the two proteins but have allowed the generation of gene 12-containing cell lines in which ICP4 and ICP27 expression is induced by virus infection (probably by ICP0) and which give efficient growth of v iruses deficient in ICP27, ICP4, and vmw65 (the viruses also have ICP34.5/O RFP deleted). Efficient growth of such viruses has not previously been poss ible. As these viruses are highly deficient in IE gene expression generally , such virus-cell line combinations may provide an alternative to HSV vecto rs with deletions of all four of the regulatory IE genes which, for optimal growth, require cell lines containing all four IE genes but which are hard to generate due to the intrinsic cytotoxicity of each of the proteins.