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Journal of Virology, December 2007, p. 13200-13208, Vol. 81, No. 23
0022-538X/07/$08.00+0     doi:10.1128/JVI.01148-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

A Self-Excisable Infectious Bacterial Artificial Chromosome Clone of Varicella-Zoster Virus Allows Analysis of the Essential Tegument Protein Encoded by ORF9{triangledown}

B. Karsten Tischer,1,2,# Benedikt B. Kaufer,1,# Marvin Sommer,3 Felix Wussow,2 Ann M. Arvin,3 and Nikolaus Osterrieder1*

Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853,1 Institute for Infection Medicine, University Medical Center, Campus Kiel, 24105 Kiel, Germany,2 Departments of Pediatrics and of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 943053

Received 25 May 2007/ Accepted 17 September 2007

In order to facilitate the generation of mutant viruses of varicella-zoster virus (VZV), the agent causing varicella (chicken pox) and herpes zoster (shingles), we generated a full-length infectious bacterial artificial chromosome (BAC) clone of the P-Oka strain. First, mini-F sequences were inserted into a preexisting VZV cosmid, and the SuperCos replicon was removed. Subsequently, mini-F-containing recombinant virus was generated from overlapping cosmid clones, and full-length VZV DNA recovered from the recombinant virus was established in Escherichia coli as an infectious BAC. An inverted duplication of VZV genomic sequences within the mini-F replicon resulted in markerless excision of vector sequences upon virus reconstitution in eukaryotic cells. Using the novel tool, the role in VZV replication of the major tegument protein encoded by ORF9 was investigated. A markerless point mutation introduced in the start codon by two-step en passant Red mutagenesis abrogated ORF9 expression and resulted in a dramatic growth defect that was not observed in a revertant virus. The essential nature of ORF9 for VZV replication was ultimately confirmed by restoration of the growth of the ORF9-deficient mutant virus using trans-complementation via baculovirus-mediated gene transfer.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853. Phone: (607) 253-4045. Fax: (607) 253-3384. E-mail: no34{at}cornell.edu

{triangledown} Published ahead of print on 3 October 2007.

# These authors contributed equally to this work.

Journal of Virology, December 2007, p. 13200-13208, Vol. 81, No. 23
0022-538X/07/$08.00+0     doi:10.1128/JVI.01148-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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