Abstract
Purpose :
Ocular infection by herpes simplex virus-1 (HSV-1) is the leading causes of infectious blindness. This study describes for the first time a virus degradative as well as a virus supportive role for autophagy in HSV-1 infection of the cornea and corneal cells.
Methods :
Studies were performed using human corneal epithelial (HCE) cells, cultured human and porcine corneas, mouse embryonic fibroblasts (MEF) from ATG5 knockout animals, and the murine models of corneal infection. Various HSV-1 strains that include beta-galactosidase-expressing KOS, wild-type McKrae, 17, K26GFP, or K26RFP as well HSV mutant strains were used. Flow cytometry, fluorescence microscopy, ELISA, and confocal fluorescence microscopy were employed for monitoring and quantitatively analyzing changes in the levels of HSV-1 entry, replication, and release. Murine corneas (with or without fluorescein staining) were examined for tissue damage using slit lamp biomicroscope and scored using a four point scale. Quantitative RT PCR (qRT-PCR) was used for mRNA quantification and immunoblotting was used to assess viral protein levels. Live cell imaging was performed to track virions in phago-lysosomes.
Results :
We found compelling evidence that cells with natural ability to induce and perform autophagy restrict a significant fraction of entering virions from reaching the nucleus for initiating a productive infection. By contrast, cells with natural inability (e.g. ATG5 knockout MEFS) or pharmacologically induced inability to perform autophagy failed to restrict virions from reaching the nuclei. However, the trend was reversed during the productive phase of infection. Autophagy was needed in a supportive role for optimal growth of virions over longer periods of time. Higher virus yields were reported from cells with normal ability to perform autophagy. Very similar results were observed with cultured corneas and murine models of corneal HSV-1 infection. Our findings suggest that an autophagy adapter protein of host origin, optineurin, may be the regulatory molecule for the switch from virus degradative to virus supportive roles.
Conclusions :
For the first time, we provide intriguing evidence and suggest a mechanism for a dual role for autophagy during HSV-1 infection of the cornea. Autophagy initially degrades incoming virions and then later during the infection, it is needed for optimal growth of the virions.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.