Generation of Varicella-Zoster Virus (VZV) and Viral Mutants from Cosmid DNAs: VZV Thymidylate Synthetase is not Essential for Replication in vitro

Four overlapping cosmid clones were constructed that contain the complete genome of the attenuated Oka strain of VZV. Transfection of human melanoma cells with the four cosmids resulted in production of infectious VZV. A double-stranded oligonucleotide, encoding a stop codon in all three open readin...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1993-08, Vol.90 (15), p.7376-7380
Main Authors: Cohen, Jeffrey I., Seidel, Karen E.
Format: Article
Language:English
Subjects:
Acyclovir - pharmacology
Base Sequence
Biological and medical sciences
Cloning, Molecular
Cosmids
DNA
DNA, Viral - genetics
Fundamental and applied biological sciences. Psychology
Genes, Viral
Genetics
Genomes
Herpes viruses
Herpesvirus 3, Human - enzymology
Herpesvirus 3, Human - genetics
Herpesvirus 3, Human - growth & development
Humans
In Vitro Techniques
Microbiology
Molecular Sequence Data
Mutagenesis, Site-Directed
Oligodeoxyribonucleotides - chemistry
Oligonucleotides
Plasmids
Structure-Activity Relationship
T lymphocytes
Thymidylate Synthase - genetics
Transfection
Tumor Cells, Cultured
Vaccines
Varicella zoster encephalitis
varicella-zoster virus
Viral DNA
Viral Structural Proteins - genetics
Virology
Virus Replication
Viruses
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Summary:Four overlapping cosmid clones were constructed that contain the complete genome of the attenuated Oka strain of VZV. Transfection of human melanoma cells with the four cosmids resulted in production of infectious VZV. A double-stranded oligonucleotide, encoding a stop codon in all three open reading frames, was inserted into one of the cosmids at the 5' end of the viral thymidylate synthetase gene. Transfection of melanoma cells with the mutant cosmid, along with the other three cosmids, resulted in VZV that does not express the viral thymidylate synthetase protein. The mutant virus grew at a rate similar to that of the parental Oka strain virus. Production of recombinant VZV using cosmid DNAs will be useful for studying the function of viral genes in VZV replication and establishment of latency. Furthermore, manipulation of the Oka strain of VZV might allow one to produce a vaccine virus that does not establish latency in the central nervous system or a virus that encodes foreign antigens for use as a polyvalent live virus vaccine.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.90.15.7376