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 transactivates VZV promoters. Transfection of cells with cosmid DNAs, including 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 partially complemented by growth in neuroblastoma or osteosarcoma cell lines. Cells infected with the VZV ORF61 deletion mutant expressed normal levels of an immediate-early VZV protein, but had reduced levels of a late protein and showed abnormal syncytia. Carboxy terminal truncation mutants of VZV ORF61 protein have a transrepressing phenotype and inhibit the infectivity of cotransfected wild-type viral DNA. Transfection of cells with cosmid DNAs, including a cosmid with stop codons that should result in an ORF61 truncation mutant expressing a transrepressing protein that retains the RING finger domain, resulted in a viral genome which reverted back to the wild-type sequence. BAL-31 exonuclease was used to produce deletions at the site of the stop codons in ORF61 of the cosmid, resulting in loss of the RING finger domain. Transfection of tissue culture cells with the ORF61 BAL-31 deletion mutants and other cosmid DNAs yielded viable viruses. Thus, while deletion mutants lacking the RING finger domain of ORF61 replicate in cell culture, a mutant with stop codons that retains this domain could not be propagated and reverted to wild-type virus.
Cohen
J I
JI
Medical Virology Section, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland 20892, USA.
Nguyen
H
H
D018388:Codon, Terminator; D005814:Genes, Viral; D015726:Giant Cells; D014645:Herpesvirus 3, Human / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D016296:Mutagenesis; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D017384:Sequence Deletion; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D014779:Virus Replication
D018388:Codon, Terminator; D012097:Repressor Proteins; D014764:Viral Proteins; C073411:protein 61, Varicella-zoster virus