June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Cytokeratin 6A (K6A) and its E3 ligase Tripartitle Motif-Containing (TRIM) 21 promote antiviral activities against Herpes Simplex Virus (HSV)-1 in human corneal epithelial cells
Author Affiliations & Notes
  • Jonathan K Chan
    Department of Ophthalmic Research, Cleveland Clinic Cole Eye Institute and Lerner Research Institute, Cleveland , Ohio, United States
  • K P Connie Tam
    Department of Ophthalmic Research, Cleveland Clinic Cole Eye Institute and Lerner Research Institute, Cleveland , Ohio, United States
    Department of Ophthalmology, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland , Ohio, United States
  • Footnotes
    Commercial Relationships   Jonathan Chan, None; K P Connie Tam, US Patent No. 9,187,541 B2 - “Anti-Microbial Peptides and Methods of Use Thereof” (P)
  • Footnotes
    Support  NIH-NEI R01EY023000 and Cleveland Clinic Seed Fund (K. P. C. Tam) NIH-NEI P30 Core Grant (IP30EY025585-01A1) and Unrestricted Grant from The Research to Prevent Blindness, Inc. (Cole Eye Institute)
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 4296. doi:
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    • Get Citation

      Jonathan K Chan, K P Connie Tam; Cytokeratin 6A (K6A) and its E3 ligase Tripartitle Motif-Containing (TRIM) 21 promote antiviral activities against Herpes Simplex Virus (HSV)-1 in human corneal epithelial cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4296.

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

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Abstract

Purpose : Emerging evidences have underscored the importance of select cytokeratin members including cytokeratin 6A (K6A), 16 and 17 in shaping the innate immunity and cytokine responses at mucosal epithelial tissues. We previously have showed that a subset of cytosolic K6A is polyubiquitinated by the E3 ligase TRIM21 and further processed by the proteasome into keratin-derived antimicrobial peptides (KAMPS). These KAMPS have potent bactericidal and cytoprotective activities, but whether K6A has additional antimicrobial activities beyond KAMPS remains unexplored. Of note, in human TRIM21’s paralog TRIM5a restricts N-tropic murine leukemia virus but not Herpes simplex virus 1 (HSV-1), the latter a leading cause of keratitis and corneal blindness worldwide. Here, we test the hypothesis that K6A and TRIM21 have antiviral activities against HSV-1.

Methods : Telomerase-immortalized human corneal epithelial (hTCEpi) cells were transduced with non-targeting control, K6A- or TRIM21-specific shRNA lentiviral particles at a multiplicity of infection (MOI) of 1. RT-qPCR and Western blotting were performed to monitor the expression and knockdown of K6A and TRIM21. Cells were infected with HSV8GFP; a derivative of HSV-1 KOS strain that encodes infected cell protein 8 fused with GFP (ICP8-GFP) at various MOI ranging from 0.01 to 0.1. ICP8, an early expressed DNA-binding protein, is essential for HSV-1 genome replication and gene expression. Flow cytometry and fluorescent microscopy were employed to assess the expression and cellular localization of ICP8-GFP.

Results : mRNA and protein expression of K6A and TRIM21 were significantly reduced in hTCEpi cells day 3 after transduction with shRNA-lentiviral particles. ICP8-GFP could be detected in the cell nucleus as early as 6 h after infection with HSV-1. Knockdown of K6A or TRIM21 markedly increased the susceptibility of hTCEpi cells to infection by HSV-1 at the MOI tested (p <0.05).

Conclusions : Our data support the idea that the immunoregulatory K6A and its E3 ligase TRIM21 play important roles in restricting HSV-1 during the early step(s) of the viral life cycle in the human corneal epithelium. Future work will focus on the molecular mechanisms of how K6A and TRIM21 carry out their anti-HSV-1 effects.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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