Figure 5: Significance of HPSE and HSV egress in vivo.

(a) Knockdown of HPSE in mouse eyes decreases viral spread through the cornea. Mouse corneas were injected with shRNA against HPSE or with scrambled shRNA and infected with GFP-KOS. Left—fluorescence detection 72 h post infection. Arrowhead indicates GFP-HSV. Right—knockdown of HPSE in mouse cornea demonstrated by reverse transcriptase PCR. (b) Left—corneal eye swabs collected at 24 and 72 hpi for plaque assays show decreased HSV titre in HPSE knockdown corneas (n=5 for each group). Right—decreased corneal tissue damage in HPSE knockdown mouse corneas after HSV-1 infection. HPSE was knocked down in mouse corneas as described above, and 7 days post infection with KOS-WT, fluorescein was applied to mouse eyes to highlight corneal damage. (c) Left—mouse corneas were transfected with HPSE plasmid in the corneal epithelium and infected with KOS-WT. Corneal swabs collected at 72 hpi for plaque assays show an increase in HSV titre in the cornea transfected with HPSE plasmid (n=3 for each group). Right—increased corneal tissue damage in HPSE-overexpressing mouse corneas after HSV-1 infection. Fluorescein was applied to mouse eyes to highlight tissue damage 7 days post infection. (d) 7 days post ocular infection, mouse corneas were examined for tissue damage using slit lamp biomicroscope and scored. Left—decreased tissue damage in HPSE shRNA knockdown corneas when compared with scrambled shRNA controls. Right—increased tissue damage in HPSE-overexpressed corneas when compared with vector-transfected controls (n=3 for each group). (e) Decreased viral egress after heparin treatment, measured using the plaque assay. Mouse corneas were infected with KOS-WT at 10⁵ p.f.u. After infection, 5 μl of 25 μg μl⁻¹ heparin solution was applied directly to corneas at 24 and 48 hpi, and corneal swabs for plaque assay were collected at 72 hpi. (f) Human corneoscleral buttons from the same subject were cultured and infected with KOS-WT at 10⁵ p.f.u. The epithelium was extracted at 24 hpi and an increase in HSPE (50 kDa) was observed. The experiment was repeated three times and representative western blot analysis is shown. All data are presented as mean±s.e.m. of three independent experiments (n=3). Asterisks denote a significant difference as determined by Student’s t-test; *P<0.05. See Supplementary Fig. 8 for full-length images of gels and blots.