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 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.
Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
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JI
Cohen
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KE
Seidel
D000212:Acyclovir / Q000494:pharmacology; D001483:Base Sequence; D003001:Cloning, Molecular; D003360:Cosmids; D004279:DNA, Viral / Q000235:genetics; D005814:Genes, Viral; D014645:Herpesvirus 3, Human / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D013329:Structure-Activity Relationship; D013940:Thymidylate Synthase / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured; D015678:Viral Structural Proteins / Q000235:genetics; D014779:Virus Replication
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D015678:Viral Structural Proteins; D013940:Thymidylate Synthase; D000212:Acyclovir