Inhibition of Autophagy by HSV-1 Beclin-Binding Domain (BBD) Induces Apoptosis of Viral Infected Retinas

Abstract

Purpose: The autophagy response induced by HSV-1 is antagonized by the neurovirulence gene product, ICP34.5, which binds to the essential autophagy protein Beclin1 (Atg6) via Beclin-Binding Domain (BBD). The purpose of this study was to determine if inhibition of autophagy by HSV-1 BBD induces apoptosis of virus infected retinas.

Methods: Female BALB/c mice were injected with 1 x 10⁴ PFU of BBD-deficient HSV-1 (Δ68H) or its marker-rescued counterpart (Δ68HR) via the AC route. Neural retinas were removed from C57 mice, Atg5^flox/flox; nestin-Cre mice and control Atg5^+/flox; nestin-Cre mice, cultured at 37°C and infected with 5 x 10⁵ PFU of Δ68H or Δ68HR. At different intervals, the injected eyes and the retinal cultures were harvested and analysed by plaque assay, HSV-1 staining, western blot for protein light chain 3-Ι (LC3-Ι) and LC3-∥ and cleaved caspase 3.

Results: Significantly more replicating virus was recovered from Δ68HR injected eyes than from Δ68H injected eyes starting from day 3 p.i. Less cleaved caspase 3 was detected in Δ68H injected eyes, compared to the eyes infected with the rescued strain Δ68HR. HSV-1 infected and spread in the cultured retina with a similar pattern as that observed in vivo and in a similar manner to in vivo infection, less cleaved caspase 3 was detected in Δ68H infected retinal cultures than in Δ68HR infected retinal cultures. However, in contrast to in vivo infection, similar amounts of virus were recovered from both Δ68H and Δ68HR infected retinas. Our results also showed that more cleaved caspase 3 was observed in Δ68H infected Atg5^flox/flox; nestin-Cre retina than in Δ68H infected Atg5^+/flox; nestin-Cre retina.

Conclusions: More apoptosis were induced by wild type HSV-1 than by BBD-deficient HSV-1 and absence of autophagy increased apoptosis of retinas infected by BBD-deficient HSV-1. The results suggest that inhibition of autophagy by HSV-1 BBD constitutes one mechanism of HSV-1 induced apoptosis of infected retinas.

Keywords: 545 herpes simplex virus • 426 apoptosis/cell death • 694 retinal culture