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Virus-Cell Interactions

Long-Term Infection and Transformation of Dermal Microvascular Endothelial Cells by Human Herpesvirus 8

Ashlee V. Moses, Kenneth N. Fish, Rebecca Ruhl, Patricia P. Smith, Joanne G. Strussenberg, Liangjin Zhu, Bala Chandran, Jay A. Nelson
Ashlee V. Moses
Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, Oregon 97201, and
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Kenneth N. Fish
Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, Oregon 97201, and
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Rebecca Ruhl
Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, Oregon 97201, and
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Patricia P. Smith
Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, Oregon 97201, and
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Joanne G. Strussenberg
Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, Oregon 97201, and
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Liangjin Zhu
Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas 66160
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Bala Chandran
Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas 66160
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Jay A. Nelson
Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, Oregon 97201, and
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DOI: 10.1128/JVI.73.8.6892-6902.1999
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    Fig. 1.

    DNA and RT-PCR analyses of HHV8-infected DMVEC. (a) DNA PCR amplification of the HHV8-specific KS330233 sequence from serial dilutions of HHV8-exposed DMVEC (+HHV8). DNA was amplified from 2 × 103, 4 × 102, and 2 × 102 and cell equivalents at 7 and 14 days PI (d7 PI and d14 PI, respectively). For positive and negative controls, PCR was performed on genomic DNA prepared from 2 × 103 BCBL-1 cells and mock-infected DMVEC. (b) RT-PCR detection of ORF 29 mRNA using cDNA from 2 × 103 HHV8-exposed DMVEC (+HHV8) at 7 and 14 days PI (d7 PI and d14 PI, respectively) to verify the authenticity of HHV8 infection. cDNA prepared from BCBL-1 cells was used as a positive control. No signal was obtained from mock-infected cells or samples prepared in the absence of RT (−RT). Cellular HPRT was simultaneously amplified from all +RT samples as a control for cDNA synthesis (data not shown). Amplification products from reverse-transcribed BCBL-1 cell cDNA and genomic DNA (BCBL-1 gDNA) demonstrate that the RT-PCR product from spliced mRNA is smaller than the product from genomic DNA. Products amplified from HHV8-infected DMVEC show only the smaller band, indicating lack of contamination from the viral inoculum or replicated virus.

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    Fig. 2.

    Expression of latency-associated genes in HHV8-infected DMVEC. (a) RT-PCR detection of HHV8 kaposin (ORF K12) mRNA by RT-PCR from HHV8-infected (+HHV8) but not mock-infected DMVEC. cDNA from uninduced BCBL-1 cells was included as a positive control. Products amplified from cDNA prepared from 2 × 103, 4 × 102, and 2 × 102 and cell equivalents at day 14 PI are shown. No signal was detected in samples prepared in the absence of RT (−RT). Cellular HPRT was simultaneously amplified from all +RT samples as a control for cDNA synthesis (data not shown). (b) Nuclear expression of latent antigen ORF 73 in HHV8-infected DMVEC at day 7 PI visualized with a polyclonal antibody against ORF 73 and a goat anti-rabbit FITC conjugate.

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    Fig. 3.

    Expression of lytic-cycle-associated proteins in HHV8-infected DMVEC. (a) Nuclear expression of ORF 59 visualized with MAb 11D1 and a goat anti-mouse FITC conjugate at 1, 4, and 8 weeks PI in HHV8-infected DMVEC cultures that were not induced (No TPA). Cells were subcultured at weekly intervals. The increase in ORF 59-positive cells indicates maintenance of the HHV8 genome and a complete viral replication cycle allowing infection spread. (b) ORF 59 expression in duplicate DMVEC cultures at 1, 4, and 8 weeks PI that had been pretreated with TPA for 48 h (+TPA). (c) ORF 59 expression in HHV8-infected DMVEC at 4 weeks PI without (No Dex) or with (+Dex) pretreatment with dexamethasone for 5 days. Treatment with exogenous induction stimuli (TPA or dexamethasone) increased the percentage of ORF 59-positive cells approximately 10-fold. (d) Expression of glycoprotein ORF K8.1A/B, a late gene product, on the surface of HHV8-infected DMVEC visualized with a MAb against K8.1A/B and a goat anti-mouse FITC conjugate.

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    Fig. 4.

    Detection of HHV8 particles in infected DMVEC by electron microscopy. (a) View of a DMVEC nucleus showing herpesvirus-like capsid structures. The insert shows an enlarged view of a single virion (bar = 100 nm). (b) View of a cytoplasmic vesicle containing mature virions sectioned within the nuclear plane (bar = 120 nm).

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    Fig. 5.

    Induction of spindle morphology in DMVEC cultures following HHV8 infection. (a) Phase-contrast microscopy of mock-infected DMVEC, demonstrating the typical cobblestone appearance. (b) Phase-contrast microscopy of HHV8-infected DMVEC at 1 week PI, demonstrating the appearance of cells with a spindle shape within the monolayer. (c) Phase-contrast microscopy of HHV8-infected DMVEC at 4 weeks PI, demonstrating the increase in morphologic change with time PI. (d) Fluorescence (left) and phase-contrast (right) microscopy fields of HHV8-infected DMVEC, demonstrating a correlation between ORF 59 expression and severity of phenotypic change. (e) Fluorescence (left) and phase-contrast (right) microscopy fields, demonstrating strong expression of ORF K8.1A/B in DMVEC exhibiting cell rounding.

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    Fig. 6.

    Expression of endothelial cell phenotypic markers by HHV8-infected DMVEC. (a) IFA demonstrating maintenance of VE-cadherin at cell junctions in spindle-shaped, HHV8-infected DMVEC (+HHV8) and mock-infected, cobblestone DMVEC. CD31 expression was similarly retained following HHV8 infection (data not shown). (b) IFA demonstrating loss of vWF expression in DMVEC cultures following HHV8 infection (+HHV8). In contrast, mock-infected cultures express high levels of vWF in characteristic rod-shaped Weibel-Palade bodies. Cultures were uninduced and were stained at 4 weeks PI when approximately 80% of cells expressed ORF 73 (data not shown).

  • Fig. 7.
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    Fig. 7.

    Loss of contact inhibition and growth in soft agar by HHV8-infected DMVEC. (a) Phase-contrast microscopy of an HHV8-infected DMVEC monolayer demonstrating loss of contact inhibition and piling up of cells into foci following culture postconfluency for 5 and 10 days. (b) Phase-contrast microscopy of cultures grown on soft agar 2 weeks after seeding of cells from mock-infected and HHV8-infected (+HHV8) DMVEC. Colonies were formed exclusively by HHV8-infected cells.

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Long-Term Infection and Transformation of Dermal Microvascular Endothelial Cells by Human Herpesvirus 8
Ashlee V. Moses, Kenneth N. Fish, Rebecca Ruhl, Patricia P. Smith, Joanne G. Strussenberg, Liangjin Zhu, Bala Chandran, Jay A. Nelson
Journal of Virology Aug 1999, 73 (8) 6892-6902; DOI: 10.1128/JVI.73.8.6892-6902.1999

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Long-Term Infection and Transformation of Dermal Microvascular Endothelial Cells by Human Herpesvirus 8
Ashlee V. Moses, Kenneth N. Fish, Rebecca Ruhl, Patricia P. Smith, Joanne G. Strussenberg, Liangjin Zhu, Bala Chandran, Jay A. Nelson
Journal of Virology Aug 1999, 73 (8) 6892-6902; DOI: 10.1128/JVI.73.8.6892-6902.1999
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KEYWORDS

Cell Culture Techniques
Cell Transformation, Viral
Herpesvirus 8, Human

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