Autophagy up-regulation upon FHV-1 infection on CRFK cells

Autophagy is a highly conserved cellular pathway that contributes to the maintenance of cellular homeostasis through degradation of cytosolic material. The autophagy network is also involved during host-virus interactions, representing a cellular defense mechanism with an antiviral activity [1]. Thanks to millennia of coevolution, several viruses are able to modulate the autophagy machinery to their own advantage. It has previously been observed that different members of the Herpesvirus family (such as Human herpes simplex, HSV, and Kaposi’sarcoma human herpesvirus, KSHV) can evade autophagy through the action of virally encoded inhibitors [1]. Other Herpersvirus members, such as Varicella-zoster virus (VZV), Pseudorabies virus (PRV), Duck enteritis virus (DEV), and Bovine herpesvirus type 4 (BoHV-4) induce increased autophagic flux in order to accomplish the secondary envelopment and successfully complete their life cycle [2, 3, 4, 5]. Very little is currently known about the interaction between feline herpesvirus (FeHV-1) and autophagy. FeHV-1 is a widespread virus responsible for feline viral rhinotracheitis (FVR), which includes upper respiratory and ocular disease. The aim of this work is to investigate the relationship between this virus and autophagy and to evaluate its proviral or antiviral role. Monolayers of Crandell-Rees Feline Kidney Cell (CRFK) were infected with FeHV-1 strain Ba/91 at various time points (3, 6, 12, 24, 48, and 72 hours post infection) using MOI 1. Cell lysates were collected and electro-transferred onto polyvinylidene fluoride (PVDF) membranes after being run in SDS-page gels. Autophagy-related signaling pathways were examined by incubating each membrane with primary antibodies against specific marker involved in the autophagy process. Western blot analyses found that the level of LC3-II was significantly higher since 12 hours post infection and increased until 72 hours post infection. The conversion of LC3-I into LC3-II and the degradation of p62/SQSTM1 indicated complete autophagy flux. In the second experiment, autophagy was pharmacologically inhibited by exposing the cells to bafilomycin (BAF) for 3 hours before infection. BAF has the property of impeding acidification during the late stage of autophagic flux, thereby avoiding the fusion of autophagosomes and lysosomes. Cells viablility was assessed by the MTT assay. This showed that cytotoxicity was lower in Crfk treated with BAF before infection. The viral titer of surnatants was determinated with TCID50 assay while cell lysate was used for western blot analysis. The FeHV titers (TCID50/mL) were significantly lower in the BAF treated cells than in the mock-treated cells after 24 hours of infection. Western blot analysis revealed lower levels of LC3I conversion and p62 degradation in BAF treated cells than mock-treated cells. In addition, densitometric analysis showed a decrease in the expression of glycoprotein D and an increase of glycoprotein B in the infected cells. We hypothesize that the decrease in FeHV titers and glycoprotein sintesis is due to the apparent failure of secondary envelopment caused by BAF treatment as described for VZV infection [6]. These findings contribute to a better understanding of the biology and pathogenesis of FeHV infection, as well as new insights into the development of effective therapeutic strategies.