Mutational analysis of the repeated open reading frames, ORFs 63 and 70 and ORFs 64 and 69, of varicella-zoster virus

Varicella-zoster virus (VZV) open reading frame 63 (ORF63); located between nucleotides 110581 and 111417 in the internal repeat region, encodes a nuc lear phosphoprotein which is homologous to herpes simplex virus type 1 (HSV -1) ICP22 and is duplicated in the terminal repeat region as ORF70 (nucleot ides 118480 to 119316). We evaluated the role of ORFs 63 and 70 in VZV repl ication, using recombinant VZV cosmids and PCR-based mutagenesis to make si ngle and dual deletions of these ORFs. VZV was recovered within 8 to 10 day s when cosmids with single deletions were transfected into melanoma cells a long with the three intact VZV cosmids. In contrast, VZV was not detected i n transfections carried out with a dual deletion cosmid. Infectious virus w as recovered when ORF63 was cloned into a nonnative AvrII site in this cosm id, confirming that failure to generate virus was due to the dual ORF63/70 deletion and that replication required at least one gene copy. This require ment may be related to our observation that ORF63 interacts directly with O RF62, the major immediate-early transactivating protein of VZV. ORF64 is lo cated within the inverted repeat region between nucleotides 111565 and 1121 07; it has some homology to the HSV-1 Us10 gene and is duplicated as ORF69 (nucleotides 117790 to 118332). ORF64 and ORF69 were deleted individually o r simultaneously using the VZV cosmid system. Single deletions of ORF64 or ORF69 yielded viral plaques with the same kinetics and morphology as viruse s generated with the parental cosmids. The dual deletion of ORF64 and ORF69 was associated with an abnormal plaque phenotype characterized by very lar ge, multinucleated syncytia. Finally, all of the deletion mutants that yiel ded recombinants retained infectivity for human T cells in vitro and replic ated efficiently in human skin in the SCIDhu mouse model of VZV pathogenesi s.