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Journal of Virology, September 2001, p. 8224-8239, Vol. 75, No. 17
Department of Pediatrics, Stanford University
School of Medicine, Stanford, California1;
Department of Pediatrics, University of Iowa, Iowa City,
Iowa2; and Department of
Microbiology, State University of New York at Buffalo, Buffalo, New
York3
Received 15 February 2001/Accepted 4 June 2001
Varicella-zoster virus (VZV) open reading frame 63 (ORF63), located
between nucleotides 110581 and 111417 in the internal repeat region,
encodes a nuclear phosphoprotein which is homologous to herpes simplex
virus type 1 (HSV-1) ICP22 and is duplicated in the terminal repeat
region as ORF70 (nucleotides 118480 to 119316). We evaluated the role
of ORFs 63 and 70 in VZV replication, using recombinant VZV cosmids and
PCR-based mutagenesis to make single and dual deletions of these ORFs.
VZV was recovered within 8 to 10 days when cosmids with single
deletions were transfected into melanoma cells along with the three
intact VZV cosmids. In contrast, VZV was not detected in transfections
carried out with a dual deletion cosmid. Infectious virus was recovered
when ORF63 was cloned into a nonnative AvrII site in this
cosmid, confirming that failure to generate virus was due to the dual
ORF63/70 deletion and that replication required at least one gene copy.
This requirement may be related to our observation that ORF63 interacts
directly with ORF62, the major immediate-early transactivating protein of VZV. ORF64 is located within the inverted repeat region between nucleotides 111565 and 112107; it has some homology to the HSV-1 Us10
gene and is duplicated as ORF69 (nucleotides 117790 to 118332). ORF64
and ORF69 were deleted individually or simultaneously using the VZV
cosmid system. Single deletions of ORF64 or ORF69 yielded viral plaques
with the same kinetics and morphology as viruses generated with the
parental cosmids. The dual deletion of ORF64 and ORF69 was associated
with an abnormal plaque phenotype characterized by very large,
multinucleated syncytia. Finally, all of the deletion mutants that
yielded recombinants retained infectivity for human T cells in vitro
and replicated efficiently in human skin in the SCIDhu mouse model of
VZV pathogenesis.
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.17.8224-8239.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Mutational Analysis of the Repeated Open Reading Frames, ORFs 63 and 70 and ORFs 64 and 69, of Varicella-Zoster Virus
*
Corresponding author. Mailing address: 300 Pasteur Dr.,
Rm. G312, Stanford University School of Medicine, Stanford, CA
94305-5208. Phone: (650) 723-5682. Fax: (650) 725-8040. E-mail:
marvman{at}stanford.edu.
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