Research ArticleMICROBIOLOGY

Standalone or combinatorial phenylbutyrate therapy shows excellent antiviral activity and mimics CREB3 silencing

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Science Advances  04 Dec 2020:
Vol. 6, no. 49, eabd9443
DOI: 10.1126/sciadv.abd9443
  • Fig. 1 CREB3 is up-regulated upon HSV-1 infection.

    HCEs were either mock infected or infected with 0.1 multiplicity of infection (MOI) HSV-1 17 GFP (green fluorescent protein). At 24 hpi, cells were collected and immunoblotted for (A and C) shown proteins. (B) In a similar experiment, cells plated in a glass bottom dish were fixed with 4% paraformaldehyde (PFA) and stained for CREB3 (red), HSV-1 (green), and nucleus (blue). In a time course experiment, HCEs were infected with 0.1 MOI HSV-1 17 GFP for 0, 3, 6, 12, 24, or 48 hours and collected for (D) immunoblotting analysis or (E) quantitative reverse transcription polymerase chain reaction (qRT-PCR). (F) HSV-1–infected HCEs were collected on 24 hpi, and the downstream effectors of CREB3 were analyzed through qRT-PCR and (G) fluorescence imaging.

  • Fig. 2 Modulation of CREB3 expression effects viral life cycle.

    (A) HCEs were transfected with CREB3 siRNA for a period of 24 hours, and the cell lysates were immunoblotted for the shown antibodies. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. (B) HCEs were transfected with CREB3 siRNA for a period of 24 hours before the addition of 0.1 MOI HSV-1 17 GFP. At 24 hpi, cells were (A) imaged using a fluorescence microscope. (C) Cell lysates from the cell culture were used as inoculum for a plaque assay on Vero cells. (D) HCEs were transfected with CREB3 siRNA or scrambled siRNA for a period of 24 hours and then infected with mock or HSV-1 17 GFP (green) strain for a period of 24 hours in the presence of propidium iodide (PI) (red) to signify cell death. (E) Quantification of the number of cells stained with PI in multiple frames. (F) HCEs were transfected with CREB3 plasmid or empty vector pcDNA plasmid for a period of 24 hours and then infected with mock or HSV-1 17 GFP (green) strain for a period of 24 hours in the presence of PI (red) to signify cell death. (G) Quantification of the number of cells stained with PI in multiple frames. Nonparametric Student’s t test was conducted to determine significance. ***P < 0.001.

  • Fig. 3 ER stress alleviation through PBA reduces viral infection and induces CHOP and ATF4 expression.

    HCEs were infected with 0.1 MOI HSV-1 17 GFP (pseudocolored red) and treated with tunicamycin (1 μM), thapsigargin (1 μM), brefeldin A (1 μM), salubrinal (75 μM), or PBA (10 mM). At 24 hpi, cells were (A) imaged for the presence of fluorescent HSV-1, and (B) cells were lysed and used as inoculum on Vero cells to assess viral titer via plaque assay. Multiple Student’s t test was conducted to determine significant difference between dimethyl sulfoxide (DMSO)–treated and drug-treated samples. ****P < 0.0001. (C) HCEs plated in glass bottom dishes were treated with mock DMSO or PBA (10 mM) for 24 hours before fixing them with 4% PFA and staining them with fluorescently labeled antibodies. These dishes were imaged at 63× on a Zeiss confocal microscope. (D) HCEs were infected at MOI of 0, 1, or 0.1 for 24 hours in the presence or absence of PBA. Cells were lysed in a hypotonic buffer to remove the cytoplasmic protein portion followed by radioimmunoprecipitation assay buffer to extract nuclear proteins. The samples were separated and immunoblotted for shown proteins. GAPDH was used as loading control.

  • Fig. 4 Therapeutic efficacy of PBA in ex vivo human tissues.

    Human corneas that were infected with HSV-1 17 GFP for 3 days before ACV (50 μM), PBA (10 mM), or DMSO were added to the cornea culture media. (A) Images were procured using a stereoscope on 3 and 7 days postinfection (dpi) to visualize viral spread. (B) Swabs taken from the corneal tissue were titrated using plaque assays. (C) Primary epithelial cells were isolated from human corneas and infected with HSV-1 17 GFP followed by treatment with PBA (10 mM) or DMSO control. At 24 hpi, images were taken at 10× using a fluorescence microscope. (D) Cell lysates were immunoblotted for the presence of viral proteins. (E) Human skin samples procured from Genoskin were cultured according to the manufacturer’s protocol. Skin epithelium was abraded using a sharp blade and infected with HSV-1 17 GFP. At 3 dpi, fluorescence image of the skin was acquired using a Zeiss stereoscope. PBA was administered to the skin systemically by adding PBA (10 mM) to the growth media provided by the manufacturer. At 7 dpi, fluorescence images were taken again to assess extent of viral spread in the skin samples. Two-way analysis of variance (ANOVA) was used to assess significant differences. ***P < 0.001.

  • Fig. 5 Antiviral activity of PBA in murine models of ocular and genital infection.

    (A) Eight-week-old male C57BL6 mice (n = 5 per group) were infected with 5 × 105 PFU HSV-1 McKrae after corneal epithelial debridement with a 30-gauge needle. At 1 dpi, ACV (5 mg/kg), PBA (50 mg/kg), or PBS mock was administered to mice twice a day intraperitoneally with volume not exceeding 300 μl per dosing. Murine eyes were imaged using a Zeiss stereoscope to assess the extent of disease progression on 7 dpi. (B) Ocular swabs were collected on 2 and 4 dpi and used as inoculum on Vero cells to determine viral titers using a plaque assay. (C) The menstrual cycle of 8-week-old female BALB/c mice was synchronized using medroxy-progesterone 5 days before infecting them with 5 × 105 PFU HSV-2 333. ACV (5 mg/kg), PBA (50 mg/kg), or PBS mock was administered to mice twice a day intraperitoneally with volume not exceeding 300 μl per dosing. Infected genitalia were imaged using a Zeiss stereoscope on 7 dpi. (D) Vaginal swabs collected on 2, 4, and 7 dpi were used as inoculum on Vero cells to assess the viral titer using plaque assay. Two-way ANOVA was used to assess significant differences. *P < 0.05, ****P < 0.0001.

  • Fig. 6 PBA synergizes with ACV analogs to reduce effective antiviral concentration.

    HCEs infected with HSV-1 17 GFP (pseudocolored red) were treated with PBA alone, TFT alone, or PBA + TFT cocktail at shown concentrations. (A) The cells were imaged using a fluorescence microscope at 10× to assess the extent of viral spread. (B) Cell lysates were used as inoculum on Vero cells to assess the viral titer through plaque assay. PBA concentration was varied from 20 to 1.25 mM with twofold dilution per step, while TFT was used from 6.25 to 0.8 μM with twofold dilution per step. (C) Eight-week-old male C57BL6 mice were infected with 5 × 105 PFU HSV-1 McKrae after corneal epithelial debridement. Starting 1 dpi, murine eyes were dosed with TFT (25 μM), PBA (5 mM), PBA (5 mM) + TFT (25 μM) cocktail, or DMSO control, three times a day for 7 days. Murine eyes were imaged using a Zeiss stereoscope to assess the extent of disease progression on 7 dpi. (D) Ocular swabs taken 2 and 4 dpi were used as inoculum on Vero cells to assess the viral titer using plaque assay. Two-way ANOVA was used to assess significant differences. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 7 PBA reduces effective ACV concentration to treat HSV encephalitis.

    Eight-week-old female BALB/c mice were infected with 1 × 105 PFU HSV-1 McKrae through intranasal route to induce encephalitis. (A) Infected mice when treated with ACV look healthier with no behavioral changes (left) compared with those left untreated (right). (B) Infected mice were administered ACV (50 and 10 mg/kg), PBA (100 and 400 mg/kg), or a cocktail of PBA (100 mg/kg) + ACV (10 mg/kg). (C) Percentage survival was calculated 10 dpi for each group based on survival criteria (no more than 20% weight loss). (D) Animal weights were measured daily, and animals showing acute weight loss or behavioral changes were euthanized for humane reasons. (E) A blinded reviewer was used to assess the disease progression and score the mice on each day.

Supplementary Materials

  • Supplementary Materials

    Standalone or combinatorial phenylbutyrate therapy shows excellent antiviral activity and mimics CREB3 silencing

    Tejabhiram Yadavalli, Rahul Suryawanshi, Raghuram Koganti, James Hopkins, Joshua Ames, Lulia Koujah, Aqsa Iqbal, Krishna Raju, Alex Agelidis, Deepak Shukla

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