Fig 1.
A) Schematic diagram of gK constructs used for binding to SPP. Full-length gK is shown at the top. gK1, gK2, and gK3 represent the 1st, 2nd, and 3rd regions of full-length gK, respectively. gK1.1 is similar to the gK1 fragment except that it is lacking its 30 aa signal sequence. All constructs have an ATG and a termination codon (TAA) and are inserted into pcDNA3.1 with 3X in-frame Flag tags. B) Binding of gK to SPP in vitro. HeLa cells were transfected with Flag-gK1 or Flag-gK1.1 and HA-SPP plasmids at a 1:1 ratio for 48 hr. Cell lysates were incubated with anti-Flag antibody bound to IgG beads and the resulting IP was analyzed by western blot using anti-HA antibody. Lower blot shows similar SPP expression in all three samples using anti-HA antibody for the western blot.
Fig 2.
gK4 peptide inhibits the binding of gK protein to SPP protein.
A) Blocking gK binding to SPP. HEK293 cells were co-transfected with Flag-gK and HA-SPP plasmids. Cells were harvested 48 hr after transfection. Cell lysate was incubated with the indicated peptide for 2 hr at 4°C. IP using anti-Flag antibody in the presence of 5 μg/ml of indicated peptide recovered similar amounts of Flag-gK protein, but significantly less HA-SPP was recovered in the presence of peptide 4. B) Mutated form of gK4 does not precipitate SPP. HEK293 cells were transfected with HA-SPP and Flag-gK or Flag-gK mutant (amino acid RCIYA was mutated to AAAAA). Cell lysates were immunoprecipitated using anti-Flag antibody. The Flag-gK4 mutant failed to pull down HA-SPP, indicating the importance of the mutated region for gK binding to SPP. Panels: A) Mapping gK binding region to SPP; and B) Fine mapping of gK4 binding to SPP.
Table 1.
Fine mapping of the gK binding region to SPP using gK synthetic peptidesa.
Fig 3.
Inhibition of HSV-1 replication by gK4 peptide in vitro.
Vero cells were infected with 0.1 PFU/cell of HSV-1 strain McKrae in the presence of 10, 20, 30, 40, or 50 μg/ml of dTat-gK4 peptide or control dTat peptide for 1 hr. After 1hr infection, the media was replaced with fresh media containing each peptide. Infected cells were harvested 24 hr PI and virus titer was measured by standard plaque assay. Virus replication was significantly inhibited by dTat-gK4 peptide in a dose dependent manner. Half maximal effective concentration (EC 50) of dTat-gK4 peptide was around 2.1 μM (6.6 μg/ml).
Fig 4.
Analysis of infected cells in the presence of gK4 peptide.
(A) Detection of HSV-GFP± cells. Vero cells were infected with 0.1 PFU/cell of HSV-1 expressing GFP in the presence of 2, 5, 10, and 20 μg/ml of dTat-gK4 peptide or control dTat peptide for 1 hr. After 1hr infection, the media was replaced with fresh media containing each peptide. At 24 hr PI, cells were trypsinized, fixed, and the presence of GFP+ cells at each peptide concentration was determined by FACS. B) Quantitation of HSV-GFP± cells. Percentage of GFP+ cells infected as in (A) above were quantitated by FACS. Each point represents the mean ± SEM from four independent experiments.
Fig 5.
Effect of blockade of gK interaction with SPP using gK4 on gK localization in HSV-1 infected cells.
A) Detection of HSV-1 gK in infected cells. Vero cells grown to confluency on chamber slides were infected with 1 PFU/cell of VC1 virus in the presence of 20 μg/ml of dTatgK4 or dTat peptide for 1 hr. After 1hr infection, the media was replaced with fresh media containing each peptide for 16 hr. Infected cells were fixed and immunostained using anti-V5 (for gK) with anti-GM130 (for Golgi), and chicken anti-goat AlexaFluor 488 (for gK) with chicken anti-rabbit AlexaFluor 594 (for GM130) as secondary antibodies. DAPI was used for nuclear staining (blue). There were fewer gK positive cells in the dTat-gK4 treated group than in control dTat peptide-treated group. gK protein colocalized with Golgi protein GM130 in the control peptide group, indicating enrichment of gK protein in the Golgi apparatus. However, gK protein did not colocalize with GM130 in the dTat-gK4 treatment group. gK protein localization to the Golgi was inhibited by blocking the binding of gK to SPP. Photomicrographs are shown at 630X direct magnification. Experiments were repeated twice; and B) Quantification of photomicrographs from A. Different areas of 3 slides/peptide from IHC described above were imaged and the number of HSV-1 gK+ cells was counted. Each point represents the mean ± SEM of HSV-1 gK+ DCs from 7 images.
Fig 6.
Effect of dTat-gK4 peptide on virus replication, eye disease, and survival in BALB/c infected mice.
(A) Virus replication in the eye of infected mice. Ten BALB/c mice from two separate experiments were infected ocularly with 1 X 105 PFU/eye of HSV-1 strain McKrae. One day before infection and on days 1–5 PI, mice received 20 μg of dTat-gK4 or dTat control peptide as an eye drop twice daily in 5 μl of 1xPBS. Eye swabs were collected each day for 6 days and virus titer in tear films were measured by standard plaque assay. Each point represents mean ± SEM from 20 eyes; (B) Blepharitis in ocularly infected mice. Blepharitis in infected mice was measured on day 4 PI. Each point is mean ± SEM from 20 eyes; and (C) Survival in ocularly infected mice. Survival of the above mice was followed for 28 days. Survival is based on ten mice per treatment group.
Fig 7.
Expression of gK, UL20, ICP0, and gB transcripts in cornea and TG of ocularly infected mice in the presence of dTat-gK4.
BALB/c mice were ocularly infected with 1 X105 PFU/eye of HSV-1 strain McKrae and treated with dTat-gK4 or dTat control peptide as described in Materials and Methods. Levels of gK, UL20, ICP0, and gB transcripts in the cornea and TG were determined on day 4 PI by qRT-PCR. Estimated relative copy number of HSV-1 UL20, gK, ICP0 and gB was calculated using standard curves generated from pUL20 [14], pAC-gB1 [65], pAc-gK1 [6], and pcDNA3.1-ICP0 [61]. Briefly, a DNA template, serially diluted 10-fold such that 1 μl contained from 103 to 1011 copies of each plasmid, was amplified by TaqMan PCR with the same primer set. Copy number of each reaction was determined by comparing the CT of each sample to the threshold cycle of the standard. GAPDH expression was used to normalize relative expression of each transcript in the cornea and TG of infected mice. Each bar represents mean ± SEM from five mouse corneas or TG. Panels: (A) gK expression in cornea; (B) gB expression in cornea; (C) ICP0 expression in cornea; (D) UL20 expression in cornea; (E) gK expression in TG; (F) gB expression in TG; and (G) ICP0 expression in TG.
Fig 8.
Effect of dTat-gK4 treatment on expression of CD4, CD8, IFNα2, IFNβ, and IFNγ transcripts in cornea and TG of ocularly infected mice.
BALB/c mice were infected as described in Fig 7. Levels of CD4, CD8, IFNα2, IFNβ, and IFNγ transcripts in cornea and TG were determined on day 4 PI by qRT-PCR. The ratio of expression of each mRNA in the cornea and TG was normalized to its expression in the cornea and TG of uninfected control mice. GAPDH expression was used to normalize relative expression of each transcript in the cornea and TG of infected mice. Each bar represents the mean ± SEM from five mouse corneas or TG. Panels: (A) gK expression in cornea; (B) gB expression in cornea; (C) ICP0 expression in cornea; (D) UL20 expression in cornea; (E) gK expression in TG; (F) gB expression in TG; and (G) ICP0 expression in TG.
Fig 9.
Effect of dTat-gK4 peptide on virus replication and CS in C57BL/6 infected mice.
C57BL/6 mice from two separate experiments were infected ocularly with 2 X 105 PFU/eye of HSV-1 strain McKrae. One day before infection and on days 1–5 PI, mice received 20 μg of dTat-gK4 or dTat control peptide twice daily as an eye drop in 5 μl of 1xPBS. (A) Virus replication in the eye of infected mice. Eye swabs were collected each day for 6 days and virus titer in the tear film was measured by standard plaque assay. Each point is mean ± SEM from 26 eyes for dTat-gK4 and 22 eyes for dTat control. (B) CS in ocularly infected mice. CS in treated mice was measured on days 14 and 28 PI. Each point is mean ± SEM from 26 eyes for dTat-gK4 and 20 eyes for dTat control. Panels: A) Virus titer in the eye; B) CS on day 14 PI; and C) CS on day 28 PI.
Fig 10.
Effect of dTat-gK4 peptide on latency and exhaustion marker levels in C57BL/6 infected mice.
TG from mice described in Fig 9 legend were harvested on day 28 PI. Total RNA from each TG was isolated as described in Materials and Methods. A) Quantitation of LAT RNA in TG of immunized mice. Quantitative RT-PCR was performed on each mouse TG. In each experiment, an estimated relative copy number of HSV-1 LAT was calculated using standard curves generated from pGem5317. Briefly, DNA template was serially diluted 10-fold such that 1 μl contained 103 to 1011 copies of LAT, then subjected to TaqMan PCR with the same primer set. By comparing the normalized threshold cycle (CT) of each sample to the threshold cycle of the standard, copy number for each reaction was determined. GAPDH expression was used to normalize relative expression of viral LAT RNA in TG. Each bar represents the mean ± SEM from 22 TG for dTat-gK4 treated mice and 18 TG for dTat control group. B) qRT-PCR analyses of IFNγ, PD-1, Tim-3, and TNF-α transcripts in TG of latently-infected mice. Total RNA described above from each individual TG was used to estimate relative expression of IFNγ, PD-1, Tim-3, and TNF-α transcripts in TG of mice in dTat-gK4 treated or dTat control group. The expression ratio of each mRNA in the TG was normalized to its expression in TG of uninfected control mice. GAPDH expression was used to normalize relative expression of each transcript in TG of immunized mice. Each bar represents the mean ± SEM from 12 TG for each group. Panels: A) LAT; and B) Exhaustion markers.