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Genome Replication and Regulation of Viral Gene Expression

Genetic Analysis of Varicella-Zoster Virus ORF0 to ORF4 by Use of a Novel Luciferase Bacterial Artificial Chromosome System

Zhen Zhang, Jenny Rowe, Weijia Wang, Marvin Sommer, Ann Arvin, Jennifer Moffat, Hua Zhu
Zhen Zhang
1Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, 225 Warren Street, Newark, New Jersey 07101
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Jenny Rowe
2Department of Microbiology and Immunology, State University of New York Upstate Medical University, Syracuse, New York 13210
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Weijia Wang
1Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, 225 Warren Street, Newark, New Jersey 07101
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Marvin Sommer
3Department of Pediatrics and Microbiology and Immunology, Stanford University, 300 Pasteur Drive, Stanford, California 94305-5208
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Ann Arvin
3Department of Pediatrics and Microbiology and Immunology, Stanford University, 300 Pasteur Drive, Stanford, California 94305-5208
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Jennifer Moffat
2Department of Microbiology and Immunology, State University of New York Upstate Medical University, Syracuse, New York 13210
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Hua Zhu
1Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, 225 Warren Street, Newark, New Jersey 07101
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  • For correspondence: zhuhu@umdnj.edu
DOI: 10.1128/JVI.02666-06
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  • FIG. 1.
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    FIG. 1.

    Construction of VZVBAC. (A) A BAC vector, pUSF-6, contains the prokaryotic replication origin (ori) gene, the replication and partition (repE, parA, and parB) genes, the Camr gene, a green fluorescent protein (gfp) gene, two 500-bp VZV fragments (gray bars a and b), and two loxP sites (two white bars). To insert this BAC vector into a VZV cosmid, pUSF-6 was digested by BamHI, resulting in a linear fragment. (B) Schematic diagram of the VZV pOka genome shows that VZV contains a 125-kb genome with unique long (UL) and unique short (US) segments. Four cosmids containing overlapping VZV genomic segments are shown below the diagram. pUSF-6 was inserted between ORF60 and ORF61 in a VZV cosmid, pvSpe23, by homologous recombination. (C) The pvSpe23 containing a BAC vector was cotransfected with three other VZV cosmids into MeWo cells, and the resulting recombinant virus was replicated and produced a green fluorescent plaque. (D) The growth curve of VZVBAC was compared to that of wild-type pOka. PFU/ml at each dpi were recorded. This result indicates that VZVBAC (green) has no detectable growth defect; it grows as well as the parental virus (red) in vitro.

  • FIG. 2.
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    FIG. 2.

    Generation and analysis of the VZVLuc strain. (A) Luciferase assay. MeWo cells were infected with VZVLuc or VZVBAC for 2 days, and luciferase activity was measured. The cells infected with VZVLuc showed a high level of luciferase activity, while the parental VZVBAC strain had no activity. (B) Bioluminescence measurement. Two wells of a six-well dish of MeWo cells were infected with VZVBAC (upper left), and two were infected with VZVLuc (upper right). Two days postinfection, d-luciferin substrate was added to the cultured wells, and bioluminescence was measured using IVIS imaging. Bioluminescence can be detected only in VZVLuc-infected cells. The intensities were indicated as pseudocolors, as shown by an intensity scale bar at the top; higher intensity is represented by a warmer color, and lower intensity is represented with a cooler color. The infection of these wells was verified by showing GFP-positive plaques (bottom panel). (C) Correlation of luminescence and plaque numbers. Growth curves generated by an infectious center assay (black curve and left scale) and a bioluminescence assay (green curve and right scale) were compared. (D) The growth curve comparison of VZVLuc with and without the BAC vector. MeWo cells were infected with VZV with the BAC vector (green) and without the BAC vector (blue). Their grow curves were generated by bioluminescence assay. (E) Monitoring VZV replication in vivo. SCID mice with human thymus-liver implants were inoculated with 4 × 103 PFU VZV-infected cells. At 7 days postinfection, a luciferase substrate, d-luciferin, was injected, and bioluminescence was observed 10 min later using IVIS imaging and overlaid on the mouse images.

  • FIG. 3.
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    FIG. 3.

    Measuring VZVLuc replication in SCID-hu mice. (A) Replication and progression of VZVLuc in human thymus-liver implants in SCID mice. Three SCID-hu mice implanted with thymus-liver implants were inoculated with VZVLuc. Using IVIS imaging, each mouse was scanned daily (from day 0 to day 8). Measurements were taken 10 min after intraperitoneal injection with d-luciferin substrate. Only images from mouse C are shown. Warmer colors indicate higher viral load; cooler colors indicate lower viral load. (B) VZV growth curves in vivo. Bioluminescence in three SCID-hu mice from the above experiment was measured, and VZV growth curves in human thymus-liver implants were generated.

  • FIG. 4.
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    FIG. 4.

    Analysis of VZV ORF0 to ORF4. (A) Schematic diagram of the genomic organization of VZV ORF0 to ORF5 (1). Each ORF, from 0 to 4, was replaced by a Kanr expression cassette to generate the deletion mutant as indicated for ORF4 (2). To create a rescue virus for the ORF4 deletion, ORF4 was cloned into pGEM-zeo-loxP, and the loxP-zeo-ORF4 cassette was amplified by PCR and used to replace the Kanr cassette by homologous recombination (3). The zeocin-resistant gene (Zeor) can be removed from the viral genome by introducing Cre recombinase (4). (B) Digestion analysis of VZV wild-type and recombinant viral BAC clones. VZV DNA from the wild type (WT), the ORF0 to ORF4 deletion clones (0D to 4D), and the ORF0D and ORF4D rescue clones (0R and 4R) was digested with HindIII and loaded onto a 0.5% agarose gel. In order to clearly show both smaller fragments and large fragments, two photos were taken. The lower panel was taken at an earlier time, and the upper panel was taken at a later time. Sizing markers (MR) are indicated at the left. (C) Growth curve analysis of VZV deletion mutants in vitro. One hundred PFU of each deletion mutant and VZVLuc (WT) were used to infect MeWo cells in six-well dishes in triplicate. Luminescence was measured using IVIS every day for 7 days after d-luciferin was applied to the cultured media. Total photon flux in each well (photons/s/cm2/steradian) was measured, and the values from triplicates were averaged. Growth curves were generated when the averaged photon counts for each day were plotted. (D) Growth curve analysis of VZV deletion mutants in vivo. SCID-hu mice with thymus-liver implants were inoculated with 4 × 103 PFU VZVLuc or VZV deletion mutant strains as indicated. VZV replication was measured by IVIS daily for 1 week as photon flux in the regions of interest above the thymus-liver implants. Each line represents an average of data from 2 or 3 mice. An uninfected SCID-hu thymus-liver mouse was injected with d-luciferin, and the background luminescence was measured (pink line). (E) Growth curve analysis of VZV ORF0 and ORF4 deletion rescue viruses in vitro. The conditions in this assay were same as those described for panel B. (F) Growth curve comparison of measurements by luminescence assay and infectious center assay. MeWo cells were grown in six-well dishes in triplicate. The cells were infected with 100 PFU of wild-type (WT), ORF0 deletion (ORF0D), and ORF0D rescue (ORF0R) viruses, respectively. Growth curve analyses were carried out by luminescence assays (LA) and infectious center assays (IA) as described above. Total photon counts in each well were measured using IVIS, averaged, and plotted on the left. Total plaques per well were counted, averaged, and plotted on the right.

  • FIG. 5.
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    FIG. 5.

    Analysis of VZV ORF0D and ORF0R viruses in SCID mice with human skin implants. (A) The three panels show human fetal skin implants infected by VZVLuc and imaged at 15 dpi. ORF0D right (R), the right implant was infected with the ORF0 deletion mutant. The bioluminescence signal is significantly lower than that of its rescue virus. ORF0R left (L) and R, left and right implants were infected with the rescue virus of the ORF0 deletion mutant. High luciferase activities were detected in these implants. (B) Growth curve analysis of ORF0D (red circles), ORF0R (brown circles), and wild-type (WT, blue circles) viruses in the human skin tissues implanted in SCID mice. The implants without inoculation of VZV were used as a negative control (Mock, green triangles). Each curve was generated by averaging data from three implants. One set of data points is shown for a control of WT and Mock infection.

Tables

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  • TABLE 1.

    Summary of genetic analysis of VZV ORF0 to ORF4

    ORFLocationSize (bp)Gene productHSV-1 orthologueMutant growth property
    In vitroSCID-huRescue
    0173-562390Putative transmembrane proteinUL56SlowSlowNormal
    1589-915a327Putative transmembrane proteinNoneNormalNormalN/Ab
    21134-1850717UnknownNoneNormalNormalN/A
    31908-2447a540UnknownUL55NormalNormalN/A
    42783-4141a1,359Posttranscriptional regulatorUL54EssentialN/ANormal
    • ↵ a Encoded on the bottom strand.

    • ↵ b N/A, not applicable.

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Genetic Analysis of Varicella-Zoster Virus ORF0 to ORF4 by Use of a Novel Luciferase Bacterial Artificial Chromosome System
Zhen Zhang, Jenny Rowe, Weijia Wang, Marvin Sommer, Ann Arvin, Jennifer Moffat, Hua Zhu
Journal of Virology Aug 2007, 81 (17) 9024-9033; DOI: 10.1128/JVI.02666-06

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Genetic Analysis of Varicella-Zoster Virus ORF0 to ORF4 by Use of a Novel Luciferase Bacterial Artificial Chromosome System
Zhen Zhang, Jenny Rowe, Weijia Wang, Marvin Sommer, Ann Arvin, Jennifer Moffat, Hua Zhu
Journal of Virology Aug 2007, 81 (17) 9024-9033; DOI: 10.1128/JVI.02666-06
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KEYWORDS

Gene Deletion
Herpesvirus 3, Human
Viral Proteins
virus replication

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