March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Non-Muscle Myosin IIA Mediates HSV-1 Entry Into the Cells of the Human and Pig Corneas
Author Affiliations & Notes
  • Thessicar E. Antoine
    Ophthalmology and Visual Sciences,
    Microbiology and Immunology,
    University of Illinois at Chicago, Chicago, Illinois
  • Deepak Shukla
    Ophthalmology and Visual Sciences,
    Microbiology and Immunology,
    University of Illinois at Chicago, Chicago, Illinois
  • Footnotes
    Commercial Relationships  Thessicar E. Antoine, None; Deepak Shukla, None
  • Footnotes
    Support  NIH grant RO1 AI057860
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 6153. doi:
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    • Get Citation

      Thessicar E. Antoine, Deepak Shukla; Non-Muscle Myosin IIA Mediates HSV-1 Entry Into the Cells of the Human and Pig Corneas. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6153.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Herpes Simplex Virus type 1 (HSV-1) is the most frequent cause of infectious blindness worldwide. Through complex multistep processes the virus is able to infect host cells through this usage various cellular proteins and structures. The goal of the present study was to establish a role for Non-muscle myosin IIA (NM-IIA) as an important mediator of HSV-1 infection in corneal epithelial cells.

Methods: : Cultured human corneal epithelial (HCE) cells and organotypic cultures of the pig corneas were used to examine the role of NM-IIA in HSV-1 entry. A beta-galactosidase expressing reporter virus, HSV-1(KOS) gL86 was used for entry assays and X-gal staining. The ability of NM-IIA to facilitate virus spread was examined by plaque formation assay and a virus-free cell fusion assay. Flow cytometry, immunohistochemistry and immunofluorescence were utilized to determine the expression of WT HSV-1 within cells following treatment with NM-IIA inhibitors. The significance of NM-IIA was tested by its knockdown using RNA interference (RNAi). Additional immunochemical and biochemical assays including antibody blocking and inhibitors of NM-IIA functions were used to probe the significance of NM-IIA in HSV-1 entry and cell-to-cell spread. Western blot analysis was utilized to compare the protein expression of tegument protein VP16.

Results: : Upon treatment with NM-IIA inhibitors, ML-7, ML-9 and Blebbistatin, HCE cells, which are highly susceptible to HSV-1 infection, became resistant, as viral entry was significantly decreased following treatment. Further investigation highlighted NM-IIA’s role in cell-to-cell fusion, as a dramatic impairment of cell fusion was seen when cells were treated with siRNA against NM-IIA, and NM-IIA kinase inhibitors, implicating an important role of NM-IIA in cell-to cell fusion and plaque formation. In addition, through our organotypic corneal culture system, we were able to highlight a role for NM-IIA in the infection of the pig cornea, as infection was decreased following topical application of NM-IIA inhibitors. The decreased susceptibility to HSV-1 infection in HCE cells and pig corneas allows us to indentify a new role for NM-IIA in HSV-1 pathogenesis.

Conclusions: : While we have yet to determine the exact mechanism by which NM-IIA plays a role in the infection process, we can conclude that its presence promotes viral infectivity in the corneas.

Keywords: herpes simplex virus • cornea: endothelium 
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