Activation of Checkpoint Kinase 2 Is Critical for Herpes Simplex Virus Type 1 Replication in Corneal Epithelium

Abstract

Background/Aims: Herpes simplex virus (HSV) type I keratitis remains a leading cause of corneal morbidity, despite the availability of effective antiviral drugs. Improved understanding of virus-host interactions at the level of the host DNA damage response (DDR), a known factor in the development of HSV-1 keratitis, may shed light on potential new therapeutic targets. This report examines the role of checkpoint kinase 2 (Chk2), a DDR mediator protein, in corneal epithelial HSV-1 infection. Methods: A small-molecule inhibitor of Chk2 (Chk2 inhibitor II) was applied to HSV-1-infected cultured human corneal epithelial cells (hTCEpi and HCE) as well as to explanted and organotypically cultured human and rabbit corneas. Infection levels were assessed by plaque assay and real-time PCR. RNAi-mediated depletion of Chk2 was performed to confirm the effect of the inhibitor. Results: Inhibition of the Chk2 kinase activity greatly suppresses the cytopathic effect, genome replication and infectious progeny production in vitro and ex vivo. Conclusion: This report demonstrates the critical role of Chk2 kinase in the establishment of HSV-1 corneal epithelial infection. These data contribute to our understanding of herpesvirus-host interactions and underscore the significance of DDR activation in HSV-1 keratitis.

Phosphoproteomic Profiling Reveals Epstein-Barr Virus Protein Kinase Integration of DNA Damage Response and Mitotic Signaling

“…Our study suggests an extensive participation of DDR proteins in viral life cycle. The importance of our finding is further supported by the fact that DDR signaling also plays a key role in the replication of a variety of viruses [ 81 – 84 ] and that viral latency proteins tend to block the DDR to facilitate latency establishment [ 6 , 85 – 87 ].…”

New Paradigms for the Study of Ocular Alphaherpesvirus Infections: Insights into the Use of Non-Traditional Host Model Systems

“…Corneas may or may not be scarified prior to infection with herpesvirus and are either covered with media and cultured for up to 8 days, or placed in a rocking air-liquid interface and cultured for up to three weeks. Thus far, corneoscleral explants have been described to model HSV-1 infection using human, rabbit and pig corneas [ 103 , 104 , 105 , 106 ], CHV-1 infection using canine corneas [ 8 ] and FHV-1 infection using feline corneas [ 72 , 107 ]. The infection in these cornea models was shown to be similar across the different viruses and is depicted schematically in Figure 2 .…”

“…Human and rabbit corneal explants have been used to study the inhibition of herpes viral replication using phosphonoacetic acid (PAA) and to demonstrate the role of Checkpoint Kinase 2 (Chk2) in promoting HSV-1 replication in the cornea, suggesting that Chk2 inhibitors could be used to treat HSV-1 ocular infection [ 103 , 104 ]. Our group has used canine corneal explants to assess the local innate immune response and inflammation associated with CHV-1, similar to what has been done for HSV-1 infection of human corneal explants [ 8 , 105 ].…”

Human Kinase/Phosphatase-Wide RNAi Screening Identified Checkpoint Kinase 2 as a Cellular Factor Facilitating Japanese Encephalitis Virus Infection

“…Herpes simplex virus (HSV) induces an ATM-damage response that is essential for viral replication (Lilley et al, 2005 ; Shirata et al, 2005 ). Inhibition of CHK2 kinase activity by the CHK2 inhibitor II significantly reduces the CPE and genome replication of HSV-1 in corneal epithelium (Alekseev et al, 2015 ). Hepatitis C virus (HCV), an RNA virus belonging to Flaviviridae , induces G2/M phase arrest (Wu et al, 2008 ) and activates the dsDNA damage response pathway by causing DSBs and enhancing the mutation of cellular genes (Machida et al, 2004 ).…”