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Structure and Assembly

Analysis of the Role of Vaccinia Virus H7 in Virion Membrane Biogenesis with an H7-Deletion Mutant

Xiangzhi Meng, Xiang Wu, Bo Yan, Junpeng Deng, Yan Xiang
Xiangzhi Meng
aDepartment of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
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Xiang Wu
aDepartment of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
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Bo Yan
aDepartment of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
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Junpeng Deng
bDepartment of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma, USA
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Yan Xiang
aDepartment of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
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DOI: 10.1128/JVI.00845-13
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    Fig 1

    Characterization of a BS-C-1 cell line that stably expresses VACV H7 protein. (A) Western blot analysis of lysates from a BS-C-1 cell line that stably expresses VACV H7 (BSC-H7) and the parental BS-C-1 cells (BSC). Anti-H7 MAb 25E2 (bottom) and a MAb (top) against cellular heat shock protein 70 (Hsp70; Santa Cruz Biotechnology) were used as the primary antibodies. Molecular sizes in kilodaltons are shown on the left. Immunofluorescence analysis of uninfected BSC-H7 cells (B) and VACV-infected BHK cells (C). BHK cells were infected with WR VACV at an MOI of 0.5 PFU/cell for 8 h. The cells were fixed, permeabilized; stained with anti-H7 MAb 25E2, the DNA dye 4′,6-diamidino-2-phenylindole (DAPI), and appropriate secondary antibodies; and processed for fluorescence microscopy as described before (25). Fluorescence signals from DAPI and anti-H7 antibody staining are shown separately in white. F, viral DNA factories. The arrows point to uninfected BHK cells, which are negative for factories and H7 staining.

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    Fig 2

    Construction of the ΔH7 mutant, an H7R-deletion mutant of VACV. (A) Schematic representation of the ΔH7 mutant genome. The ΔH7 mutant was constructed through homologous recombination of WT VACV WR and a PCR product containing GUS flanked by H6R and D1R sequences. This resulted in the replacement of nucleotides 93428 to 93901 of the WT VACV WR genome (GenBank accession number NC_006998) with the GUS cassette. P11 is a VACV promoter for late gene expression. (B) Plaque morphology. BS-C-1 (BSC) or BSC-H7 cells were infected with the ΔH7 mutant or WT VACV WR for 48 h. Plaques were visualized following crystal violet staining. (C) One-step growth curve analysis. BS-C-1 or BSC-H7 cells were infected at 10 PFU per cell with either the ΔH7 mutant or WT VACV WR. At the indicated times in hours postinfection (hpi), cells were harvested and viral titers were determined by duplicate plaque assays on BSC-H7 cells. (D) Comparison of H7 expression kinetics by WT VACV and BSC-H7 cells. BS-C-1 (left) or BSC-H7 (right) cells were infected with WT VACV or the ΔH7 mutant, respectively. The cells were harvested at the indicated times postinfection and analyzed by Western blotting with MAbs against H7, D8 (24), and Hsp70. The values to the left are molecular sizes in kilodaltons.

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    Fig 3

    Construction and characterization of iH7/GFP, an IPTG-inducible H7R mutant of VACV. (A) Schematic representation of the iH7/GFP genome. iH7/GFP was constructed through homologous recombination of vT7LacOI and a PCR product containing green fluorescent protein (GFP), the T7 promoter, and the E. coli lac repressor flanked by H6R and H7R sequences. The recombination resulted in the replacement of the native promoter of the H7 gene (nucleotides 93428 to 93901 of the WT VACV WR genome) with an IPTG-inducible T7 promoter and the simultaneous insertion of a GFP cassette between the H6R gene and the T7 promoter. LacO, lac operator; T7 pol, bacteriophage T7 RNA polymerase; LacI, E. coli lac repressor; PT7, T7 promoter; IRES, the internal ribosomal entry site from encephalomyocarditis virus. (B) IPTG-dependent synthesis of H7 by iH7/GFP. BS-C-1 cells were infected with iH7/GFP at an MOI of 5 PFU/cell in the absence or presence of 100 μM IPTG. Cells were harvested at 12 h p.i. and analyzed by Western blotting with MAbs against H7, D8, and Hsp70. The values to the left are molecular sizes in kilodaltons. (C) Plaque morphology. BS-C-1 cells were infected with iH7/GFP in the presence or absence of 100 μM IPTG for 48 h. Cells were fixed and stained with crystal violet. (D) One-step growth curve analysis. BS-C-1 cells were infected at an MOI of 5 PFU/cell with iH7/GFP in either the presence or the absence of 100 μM IPTG. After 0, 12, 24, and 48 h, infected cells were harvested and virus titers were determined by plaque assay in the presence of 100 μM IPTG.

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    Fig 4

    The ΔH7 mutant shows a defect at an earlier step of virion membrane biogenesis than the IPTG-inducible H7 mutant (iH7). BS-C-1 cells were infected with iH7 in the presence (A) or absence (B) of IPTG or with the ΔH7 mutant at an MOI of 1 PFU/cell for 20 h (C and D). The cells were fixed and prepared for transmission EM. Panel D is a higher magnification of the boxed area of panel C. Note the short membranes (arrows) at the peripheries of viroplasm inclusions in panel B but not in panels C and D. N, nucleus; V, viroplasm.

  • Fig 5
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    Fig 5

    Effect of H7 gene deletion on viral protein stability. BS-C-1 and BSC-H7 cells were infected with either the ΔH7 mutant or WT VACV WR for 10 or 24 h. Proteins from the cell lysates were analyzed by Western blotting with MAbs against H7, E3, D8 (24), A11 (25), L1 (14), and D13 (24). Western blot signals were quantitated by densitometry, and the values below the lanes represent the signals relative to that of WT VACV-infected BSC-1 cells (set at 1). The values to the left are molecular sizes in kilodaltons.

  • Fig 6
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    Fig 6

    Effect of H7 gene deletion on intracellular localization of VACV proteins. BHK cells were infected with the ΔH7 mutant or WT VACV for 8 h and processed for fluorescence microscopy as described previously (25). Cells in panels A to E were stained with MAbs against the indicated proteins, while cells in panels F to I were stained with both anti-A11 MAb 10G11 (IgG2b [25]) and a MAb against A10 (BG3, IgG2a [25]), A13 (11F7, IgG2a [28]), or D8 (AB12, IgG1 [24]). The latter were further stained with isotype-specific secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 594. The areas of the cell within the white boxes are enlarged and shown in more detail in the last column. The white arrows in panels C, D, and E point to staining around viroplasmic inclusions. F, viral factory.

  • Fig 7
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    Fig 7

    Model of vaccinia virus crescent membrane formation. Endoplasmic reticulum (ER)- or ER-Golgi intermediate compartment (ERGIC)-derived membranes are recruited to viral factories in a poorly understood process (dashed arrow) that requires viral proteins H7, A6, L2, A11, and F10. Some integral viral membrane proteins (diamonds) are synthesized in the ER and may have been recruited to viral factories in the same process. The membranes and viral membrane proteins are then organized into crescents (dark lines) around a viroplasm that contains viral core proteins (black spheres with white dots). This process requires viral proteins A14, A17, and D13. The deletion of H7 from vaccinia virus results in the absence of viral membranes in viral factories and a defect in the localization of viral membrane proteins to viral factories.

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Analysis of the Role of Vaccinia Virus H7 in Virion Membrane Biogenesis with an H7-Deletion Mutant
Xiangzhi Meng, Xiang Wu, Bo Yan, Junpeng Deng, Yan Xiang
Journal of Virology Jun 2013, 87 (14) 8247-8253; DOI: 10.1128/JVI.00845-13

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Analysis of the Role of Vaccinia Virus H7 in Virion Membrane Biogenesis with an H7-Deletion Mutant
Xiangzhi Meng, Xiang Wu, Bo Yan, Junpeng Deng, Yan Xiang
Journal of Virology Jun 2013, 87 (14) 8247-8253; DOI: 10.1128/JVI.00845-13
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