April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
Meganuclease-mediated Inhibition Of HSV1 Infection
Author Affiliations & Notes
  • Marc Labetoulle
    Ophthalmology, Hopital Bicetre, South Paris University, Le Kremlin Bicetre, France
    Laboratoire de Virologie Moléculaire et Structurale, CNRS, Gif sur Yvette, France
  • Ayla Ergani
    Laboratoire de Virologie Moléculaire et Structurale, CNRS, Gif sur Yvette, France
  • Julianne Smith
    Cellectis, Romainville, France
  • Stephanie Grosse
    Cellectis, Romainville, France
  • Sebastien Barradeau
    Genomic Vision, Paris, France
  • Sylvain Arnould
    Cellectis, Romainville, France
  • Carole Desseaux
    Cellectis, Romainville, France
  • Eric Gabison
    Institut de la Vision, Paris, France
  • Frédéric Paques
    Cellectis, Romainville, France
  • Nicolas Huot
    Laboratoire de Virologie Moléculaire et Structurale, CNRS, Gif sur Yvette, France
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 5801. doi:
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      Marc Labetoulle, Ayla Ergani, Julianne Smith, Stephanie Grosse, Sebastien Barradeau, Sylvain Arnould, Carole Desseaux, Eric Gabison, Frédéric Paques, Nicolas Huot; Meganuclease-mediated Inhibition Of HSV1 Infection. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5801.

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

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Abstract

Purpose: : Current anti-viral treatments of Herpes simplex virus type 1 (HSV1) based on the inhibition of the viral replication impair neither the viral cycle at its early stages nor the latent form of the virus. Latent HSV1 virus could be addressed by rare cutting endonucleases, such as meganucleases. The aim of this study was to demonstrate the antiviral activity of several meganucleases recognizing HSV1 sequences in cultured cells.

Methods: : Co-transfections with plasmid expressing I-CreI variants cleaving target sequences and GFP were performed using Lipofectamine on BSR cells for 48 hours. After infection for 8h with SC-16 at different MOIs (Multiplicity Of Infection) ranging from 0.1 to 8, cells were incubated with i) HSV1 rabbit antibodies and ii) with phycoerytrine (PE) conjugated goat anti-rabbit IGg secondary antibodies. Fluorescence-activated cell sorting (FACS) analysis was performed. The ratio of infected cells among transfected (GFP+) and non transfected (GFP-) cells were counted and an antiviral index was calculated.

Results: : GFP+ and GFP- cells were both well represented in all experiments, with an average GFP+ cell frequency of 0.63 over 90 transfections (standard deviation: 0.20). A strong inhibitory effect was observed at MOIs of 0.1 and 0.5, with 4 to 7 times less infection among GFP+ cells than among GFP- ones. This effect was however not detected by a MOI of 2.

Conclusions: : This study demonstrated that expression of meganuclease proteins in BSR cells inhibits the replication of HSV1, at low and moderate MOIs (Multiplicity Of Infection), inducing a significant reduction of the viral particules. This specific mechanism of action qualifies meganucleases as a new class of antiviral agent, with the potential to address replicative as well as latent DNA viral forms. Specific meganucleases may thus be used in the future with the aim of controlling recurrent herpetic keratitis.

Keywords: herpes simplex virus • keratitis • antiviral drugs 
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