Involvement of p63 in the herpes simplex virus-1-induced demise of corneal cells.
BACKGROUND
The transcription factor p63 plays a pivotal role in the development and maintenance of epithelial tissues, including the ocular surface. In an effort to gain insight into the pathogenesis of keratitis caused by HSV-1, we determined the expression patterns of the p63 and Bax proteins in the Staatens Seruminstitute Rabbit Cornea cell line (SIRC).


METHODS
SIRC cells were infected with HSV-1 at various multiplicities and maintained for different periods of time. Virus replication was measured by indirect immunofluorescence assay and Western blot analysis. Cell viability was determined by MTT assay. The apoptotic response of the infected cells was quantified by ELISA detecting the enrichment of nucleosomes in the cytoplasm. Western blot analysis was used to determine the levels of p63 and Bax proteins.


RESULTS
Indirect immunofluorescence assays and Western blot analyses demonstrated the presence of HSV-1 glycoprotein D (gD) in the infected SIRC cell line, and the pattern of gD expression was consistent with efficient viral replication. The results of MTT and ELISA assays showed that HSV-1 elicited a strong cytopathic effect, and apoptosis played an important role in the demise of the infected cells. Mock-infected SIRC cells displayed the constitutive expression of DeltaNp63alpha. The expressions of the Bax-beta and TAp63gamma isoforms were considerably increased, whereas the level of DeltaNp63alpha was decreased in the HSV-1-infected SIRC cells. Experiments involving the use of acyclovir showed that viral DNA replication was necessary for the accumulation of TAp63gamma.


CONCLUSIONS
These data suggest that a direct, virus-mediated cytopathic effect may play an important role in the pathogenic mechanism of herpetic keratitis. By disturbing the delicate balance between the pro-survival DeltaN and the pro-apoptotic TA isoforms, HSV-1 may cause profound alterations in the viability of the ocular cells and in the tissue homeostasis of the ocular surface.
Department of Medical Microbiology and Immunobiology, University of Szeged, Dóm tér 10, H-6720 Szeged, Hungary.
László
L
Orosz
Eva
E
Gallyas
Lajos
L
Kemény
Yvette
Y
Mándi
Andrea
A
Facskó
Klára
K
Megyeri
D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D002470:Cell Survival; D003588:Cytopathogenic Effect, Viral; D004261:DNA Replication; D018259:Herpesvirus 1, Human / Q000472:pathogenicity* / Q000502:physiology; D016849:Keratitis, Herpetic / Q000209:etiology* / Q000378:metabolism / Q000473:pathology / Q000821:virology; D011817:Rabbits; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000378:metabolism; D014779:Virus Replication; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D025521:Tumor Suppressor Proteins; D014759:Viral Envelope Proteins; D051028:bcl-2-Associated X Protein; C515577:glycoprotein gD, herpes simplex virus type 1