Research ArticleVirology

An off-target effect of BX795 blocks herpes simplex virus type 1 infection of the eye

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Science Translational Medicine  14 Feb 2018:
Vol. 10, Issue 428, eaan5861
DOI: 10.1126/scitranslmed.aan5861
  • Fig. 1 BX795 suppresses HSV-1 infection.

    (A and B) HCE cells were infected with multiplicity of infection (MOI) 1 HSV-1(K26RFP) with BX795 at the indicated concentrations for 2 hours, and then fresh culture medium containing BX795 was added. Mock treatment was used as a control. Virus yields were determined at 24 hours post-infection (hpi) using flow cytometry (A) [represented as percentage of mock-treated mean fluorescence intensity (MFI)] and qPCR (B) (represented as percentage of mock-infected cells) (n = 3 replicates). (C) Representative micrographs (left panel) of HCE cells depicting the presence of HSV-1 (green). HCE cells were infected with HSV-1(K26GFP) at the indicated MOIs, and at 2 hpi, fresh culture medium containing mock, TFT (50 μM), or BX795 (10 μM) was added to the cells. Images were captured at 24 hpi using AxioVision 100 under 10× objectives. Scale bar, 100 μm. The number of GFP-infected cells per field was calculated using the MetaMorph imaging software (right panels, n = 3 fields). Data are represented as percentage of mock-infected cells. Significance to mock-treated cells was determined by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test. (D) HSV-1 VP16 and gD transcripts determined by qRT-PCR. Infection was performed as mentioned in (C). Cells were collected at 24 hpi to extract RNA, and viral transcripts were quantified by qRT-PCR and represented as fold change over the mock-treated cells. Significance to mock-treated cells was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test (n = 4 replicates). (E) Immunoblots of HSV-1 gB protein and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from infected HCE cells at 24 hpi. Infection was performed as mentioned in (C). Representative blots from three replicates are shown.

  • Fig. 2 BX795 is nontoxic to HCE cells at therapeutic concentration.

    (A) Apoptosis analysis [Annexin V–FITC (fluorescein isothiocyanate) versus PI] by flow cytometry. HCE cells were incubated with mock, BX795 (10 μM), or TFT (50 μM) for 24 hours and assayed for apoptosis using the Dead Cell Apoptosis Kit. The numbers shown in each quadrant indicate the percentage of cells. The numbers in bold in the top-right and bottom-right quadrants indicate the percentage of early and late apoptotic cells, respectively. Representative plots from three replicates are shown. (B) Cell viability of infected and treated HCE cells assessed by MTT assay. The cells were infected with MOI 0.1 HSV-1(KOS) and, at 2 hpi, treated with mock or BX795 (10 μM). At 24 hpi, viability was assessed by MTT assay. The data (n = 9) are represented as percentage of nontreated, noninfected cells. Significance between the mock-treated and BX795-treated cells was determined by one-way ANOVA with Tukey’s multiple comparisons test. (C and D) Human MAPK phosphorylation array (C) and a heat map (D) depicting the intensity of the spots on the array from HCE cell lysates. The cells were infected with MOI 5 HSV-1(KOS), and at 2 hpi, fresh culture medium containing mock or BX795 (10 μM) treatments was added. At 4 hpi, the cells were collected and the lysates were assayed using the Human MAPK Phosphorylation Kit. Colored boxes indicate the spots on the array, which significantly differ between the mock-treated and BX795-treated cells. Duplicate spots for each sample are shown on the array. (E) Quantification of the kinases shown in (C) relative to the mock-treated cells. Colored bars indicate the kinases that are significantly different. Representative array from two replicates is shown. One-way ANOVA with Dunnett’s multiple comparisons test was performed to determine the significance.

  • Fig. 3 BX795 blocks the synthesis of HSV-1 virions.

    (A) HSV-1 viral entry in the presence and absence of BX795 assessed by qPCR (left panel) and flow cytometry (right panel). HCE cells were infected with MOI 5 HSV-1(KOS). Mock or BX795 (10 μM) treatments were added along with the virus. At 2 hpi, the cells were collected and genome levels were detected by qPCR. For flow cytometry, HCE cells were infected with MOI 1000 HSV-1(K26GFP). Mock or BX795 (10 μM) treatments were added along with the virus. At 45 min after infection, the cells were washed and processed for flow cytometry. Data (n = 3 replicates) are represented as percentage of mock-treated MFI. (B) Immunoblots of HSV-1 VP16, human GAPDH, and human histone H3 in the organelle fractions of infected HCE cells. HCE cells were infected with MOI 5 HSV-1(KOS), and 15 min later, mock or BX795 (10 μM) treatments were added. The cells were collected at the indicated times and fractioned. The lysates were then electrophoresed to detect HSV-1 VP16, human GAPDH, and human histone H3. The numbers shown below the blot are the intensities of the VP16 band relative to GAPDH in the cytoplasmic fraction and relative to H3 in the nuclear fraction. (C) Representative confocal microscopy images of infected and mock- or BX795-treated HCE cells, indicating the presence of virus (green, white arrows) at different times after infection. The cells were infected with MOI 5 HSV-1(K26GFP) with or without BX795 (10 μM), and at the indicated times, actin (red) and the nuclei (blue) were stained and imaged under 63× objectives. Magnified images of the cells highlighted in yellow boxes and labeled with numbers, for each treatment, are shown on the right. Scale bars, 10 μm. (D) Representative images showing the presence of ICP0-GFP (green) and gC-RFP (red) HSV-1 in the mock- and BX795-treated HCE cells. The cells were infected with MOI 0.1 dual-fluorescent HSV-1(KOS), after which the mock and BX795 (10 μM) treatments were added. The cells were imaged 24 hpi. Scale bars, 100 μm. (E) Immunoblots of the total and phosphorylated Akt and human GAPDH in noninfected and infected HCE cell lysates. The cells were either noninfected or infected with MOI 5 HSV-1(KOS), and at 2 hpi, fresh culture medium containing the mock or BX795 (10 μM) treatments was added to both the noninfected and the infected cells. At the indicated times, the cells were collected and the lysates were electrophoresed. The numbers shown below are the intensities of the phosphorylated Akt band relative to GAPDH. (F) Representative confocal images of phosphorylated Akt in the infected and mock- or BX795-treated HCE cells. The cells were infected as in (E), and at 6 hpi, the cells were stained for actin (red), nuclei (blue), and phosphorylated Akt (green) and imaged under 63× objectives. Scale bars, 10 μm. (G) Immunoblots of the total and phosphorylated 4E-BP1 in infected HCE cell lysates. The cells were infected as in (E), and at the indicated times, the cells were collected and the lysates were electrophoresed. α, β, and γ indicate the phosphorylation status of 4E-BP1, with γ being hyperphosphorylated. Representative blots from three replicates are shown.

  • Fig. 4 BX795 suppresses HSV-1 corneal infection in vivo.

    BALB/c mice were infected on the right eye with 106 plaque-forming units (PFU) of HSV-1(McKrae), and at 24 hpi, mock, TFT (50 μM), or BX795 (10 μM) was topically added to the eyes. (A) Representative micrographs of right eyes from infected and treated mice. Scale bars, 2 mm. (B) Secreted virus titers assessed from the swabs of right eyes (n = 7 per treatment group). Significance between the mock-treated and TFT/BX795-treated mice was determined by two-way ANOVA followed by Dunnett’s multiple comparisons test. (C) Representative corneal histology sections taken from the right eye at 9 dpi. Scale bars, 100 μm. (D) Thickness of the corneal epithelium assessed from histology. Data were obtained from the mice (n = 3 per treatment group) in (C). Significance between the mock-treated and TFT/BX795-treated mice was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test. (E) HSV-1 genomes and titers in the TG assessed by qPCR and plaques assays, respectively, at 9 dpi. Mice (n = 3 per treatment group) were euthanized to extract the TGs. Homogenized TG lysates were used for qPCR to detect HSV-1 genomes (left panel) and were also used to titer HSV-1 (right panel) on Vero cells. Significance between the mock-treated and TFT/BX795-treated mice was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test.

  • Fig. 5 BX795 suppresses HSV-1–associated corneal disease pathologies.

    The right eyes of BALB/c mice were infected with 106 PFU of HSV-1(McKrae), and at 24 hpi, mock (n = 12), 1% TFT (n = 10), and 10 μM BX795 (n = 13) were added topically to both the eyes. (A) Kaplan-Meier survival curves. Significance between the mock-treated and BX795-treated mice was calculated by log-rank test followed by Bonferroni post hoc test. (B) Corneal sensitivity measured using an electronic von Frey esthesiometer scores in mice (n = 7 per treatment group). Higher scores indicate loss in corneal sensitivity. Significance between the mock-treated and TFT/BX795-treated mice was determined by two-way ANOVA with Dunnett’s multiple comparisons test. (C) Ocular disease scores (0 to 5, 5 being severe) in mice (n = 10 per treatment group). Significance between the mock-treated and BX795-treated mice was determined by two-way ANOVA with Sidak’s multiple comparisons test. (D) Body weights (n = 10 per treatment group) for the female (left panel) and male mice (right panel) were recorded. Significance between the mock-treated and BX795-treated mice was determined by two-way ANOVA with Dunnett’s multiple comparisons test.

  • Fig. 6 BX795 blocks HSV-1 infection in primary HCE cells and organ cultures of the human cornea.

    (A) Primary HCE cells were infected with MOI 0.1 HSV-1(K26GFP), and at 2 hpi, fresh culture medium containing various concentrations of BX795 was added. At 24 hpi, the amount of GFP present in the cells was quantified by flow cytometry. Individual data points from two replicates are shown and are represented as percentage of mock-related MFI. (B) Cell viability of primary HCE cells assessed by MTT assay (n = 8 replicates). Primary HCE cells were incubated with mock or BX795 (10 μM) for 24 hours, and cell viability was assessed. (C) Representative confocal images of human corneal tissue sections depicting the presence of virus (red) in the corneal epithelium cells (blue). Human corneas were infected with 107 PFU of HSV-1 (K26RFP). One-half of the corneal tissue was treated with mock, and the other half was treated with BX795 (10 μM). At 48 hpi, the tissues were frozen, sectioned, and stained. Images were captured under 63× objectives. Scale bar, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole. (D) Percentage of fluorescent RFP virus present in (C) (n = 10 corneal sections) via MetaMorph imaging software and is represented as percentage of mock-treated tissues. Significance was determined by t test (two-tailed). (E and F) HSV-1 genome and VP16 protein levels assessed by qPCR (E) and immunoblotting (F), respectively. Human corneas (n = 3) were infected as in (C), and at 48 hpi, the corneal epithelium was isolated and used to detect genomes by qPCR or used to detect VP16 protein by immunoblotting. Significance was determined by t test (two-tailed). (G and H) HSV-1 genome and protein levels assessed by qPCR (G) and immunoblotting (H), respectively. Human corneas (n = 3) were infected with 106 PFU of virulent HSV-1(McKrae and 17syn+), and at 48 hpi, the corneal epithelium was isolated and used to detect genomes by qPCR or used to detect VP16 protein by immunoblotting. Significance was determined by t test (two-tailed).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/428/eaan5861/DC1

    Materials and Methods

    Fig. S1. BX795 blocks virus secretion and shows antiviral activity in other cell types.

    Fig. S2. BX795 shows higher potency compared to existing antivirals.

    Fig. S3. Therapeutic concentration of BX795 is nontoxic in HCE cells.

    Fig. S4. BX795 does not mediate antiviral activity through TBK1.

    Fig. S5. Antiviral activity of BX795 in the in vivo and ex vivo models of corneal infection.

    Fig. S6. Uncropped blots for figures.

    Fig. S7. Uncropped blots for supplementary figures.

    Table S1. Primary data.

    References (6268)

  • Supplementary Material for:

    An off-target effect of BX795 blocks herpes simplex virus type 1 infection of the eye

    Dinesh Jaishankar, Abraam M. Yakoub, Tejabhiram Yadavalli, Alex Agelidis, Neel Thakkar, Satvik Hadigal, Joshua Ames, Deepak Shukla*

    *Corresponding author. Email: dshukla{at}uic.edu

    Published 14 February 2018, Sci. Transl. Med. 10, eaan5861 (2018)
    DOI: 10.1126/scitranslmed.aan5861

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. BX795 blocks virus secretion and shows antiviral activity in other cell types.
    • Fig. S2. BX795 shows higher potency compared to existing antivirals.
    • Fig. S3. Therapeutic concentration of BX795 is nontoxic in HCE cells.
    • Fig. S4. BX795 does not mediate antiviral activity through TBK1.
    • Fig. S5. Antiviral activity of BX795 in the in vivo and ex vivo models of corneal infection.
    • Fig. S6. Uncropped blots for figures.
    • Fig. S7. Uncropped blots for supplementary figures.
    • References (6268)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Primary data.

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