Green fluorescent protein (GFP) color reporter gene visualizes parvovirus B19 non-structural segment 1 (NS1) transfected endothelial modification

Thomas Wurster, Catharina Poelzelbauer, Tanja Schoenberger, Angela Paul, Peter Seizer, Konstantinos Stellos, Andreas Schuster, Rene M. Botnar, Meinrad Gawaz, Boris Bigalke

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

Abstract

Background: Human Parvovirus B19 ((PVB19) has been associated with myocarditis putative due to endothelial infection. Whether PVB19 infects endothelial cells and causes a modification of endothelial function and inflammation and, thus, disturbance of microcirculation has not been elucidated and could not be visualized so far.

Methods and Findings: To examine the PVB19-induced endothelial modification, we used green fluorescent protein (GFP) color reporter gene in the non-structural segment 1 (NS1) of PVB19. NS1-GFP-PVB19 or GFP plasmid as control were transfected in an endothelial-like cell line (ECV304). The endothelial surface expression of intercellular-adhesion molecule-1 (CD54/ICAM-1) and extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) were evaluated by flow cytometry after NS-1-GFP or control-GFP transfection. To evaluate platelet adhesion on NS-1 transfected ECs, we performed a dynamic adhesion assay (flow chamber). NS-1 transfection causes endothelial activation and enhanced expression of ICAM-1 (CD54: mean +/- standard deviation: NS1-GFP vs. control-GFP: 85.3 +/- 11.2 vs. 61.6 +/- 8.1; P<0.05) and induces endothelial expression of EMMPRIN/CD147 (CD147: mean +/- SEM: NS1-GFP vs. control-GFP: 114 +/- 15.3 vs. 80 +/- 0.91; P<0.05) compared to control-GFP transfected cells. Dynamic adhesion assays showed that adhesion of platelets is significantly enhanced on NS1 transfected ECs when compared to control-GFP (P<0.05). The transfection of ECs was verified simultaneously through flow cytometry, immunofluorescence microscopy and polymerase chain reaction (PCR) analysis.

Conclusions: GFP color reporter gene shows transfection of ECs and may help to visualize NS1-PVB19 induced endothelial activation and platelet adhesion as well as an enhanced monocyte adhesion directly, providing in vitro evidence of possible microcirculatory dysfunction in PVB19-induced myocarditis and, thus, myocardial tissue damage.

Original languageEnglish
Article numbere33602
Number of pages5
JournalPLoS ONE
Volume7
Issue number3
DOIs
Publication statusPublished - 15 Mar 2012

Keywords

  • Parvoviridae Infections
  • Intercellular Adhesion Molecule-1
  • Viral Nonstructural Proteins
  • Parvovirus B19, Human
  • Green Fluorescent Proteins
  • Humans
  • Microvessels
  • Recombinant Fusion Proteins
  • Endothelial Cells
  • Antigens, CD147
  • DNA, Viral
  • Platelet Adhesiveness
  • Base Sequence
  • Transfection
  • Genes, Reporter
  • Myocarditis
  • Cell Line

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