Meganuclease-mediated Inhibition Of HSV1 Infection

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