VARICELLA-ZOSTER VIRUS ORF61 DELETION MUTANTS REPLICATE IN CELL-CULTURE, BUT A MUTANT WITH STOP CODONS IN ORF61 REVERTS TO WILD-TYPE VIRUS

Varicella-zoster virus (VZV) ORF61 encodes a phosphoprotein that trans activates VZV promoters. Transfection of cells with cosmid DNAs, inclu ding a cosmid with a large deletion in ORF61, resulted in a VZV ORF61 deletion mutant that was impaired for growth in vitro and could be par tially complemented by growth in neuroblastoma or osteosarcoma cell li nes. Cells infected with the VZV ORF61 deletion mutant expressed norma I levels of an immediate-early VZV protein, but had reduced levels of a late protein and showed abnormal syncytia. Carboxy terminal truncat ion mutants of VZV ORF61 protein have a transrepressing phenotype and inhibit the infectivity of cotransfected wild-type viral DNA. Transfec tion of cells with cosmid DNAs, including a cosmid with stop codons th at should result in an ORF61 truncation mutant expressing a transrepre ssing protein that retains the RING finger domain, resulted in a viral genome which reverted back to the wild-type sequence. BAL-31 exonucle ase was used to produce deletions at the site of the stop codons in OR F61 of the cosmid, resulting in loss of the RING finger domain. Transf ection of tissue culture cells with the ORF61 BAL-31 deletion mutants and other cosmid DNAs yielded viable viruses. Thus, while deletion mut ants lacking the RING finger domain of ORF61 replicate in cell culture , a mutant with stop codons that retains this domain could not be prop agated and reverted to wild-type virus.