Journal: Journal of Virology

Article Title: Reactive oxygen species promotion drives auranofin’s antiviral activity against hepatitis E virus

doi: 10.1128/jvi.01917-25

Figure Lengend Snippet: Auranofin displays dose-dependent antiviral activity at non-toxic concentrations against two genotypes of HEV and a ribavirin treatment failure-associated HEV mutant. ( A ) The effect of auranofin on cell viability in Huh7-S10-3 (human hepatocyte) cells, measured via MTS assay. Data was collected 72 h after the addition of the compound, background-subtracted, and normalized to the DMSO vehicle control. X-axis values are log-transformed. The lines represent non-linear regression curves, and data points represent means ± SD; n = 6 performed in two independent experiments. ( B and C ) Antiviral activity of auranofin against the HEV-1 Sar55 Gluc replicon ( B ) and HEV-1 Sar55 (Hib) infectious reporter virus ( C ). ( D–F ) Antiviral activity of auranofin against the HEV-3 P6 Gluc replicon ( D ), HEV-3 P6 (Hib) infectious reporter virus ( E ), and HEV-3 G1634R P6 Gluc ribavirin resistance mutant ( F ). For antiviral assays, data were collected 72 h post-transfection for Gluc replicons and the HEV-3 P6 (Hib) infectious reporter virus, and 7 days post-transfection for the Sar55 (Hib) infectious reporter virus. Data from panels B to F were background-subtracted, normalized to the DMSO level in the 2 μM auranofin group, and analyzed by one-way ANOVA with Dunnett’s multiple comparisons test (single pooled variance). Error bars represent ± SD; n = 6 performed in two separate experiments. ns = P > 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001.

Article Snippet: Additional auranofin stocks (Selleckchem) were also prepared to 10 mM in DMSO.

Techniques: Activity Assay, Mutagenesis, MTS Assay, Control, Transformation Assay, Virus, Transfection

Journal: Journal of Virology

Article Title: Reactive oxygen species promotion drives auranofin’s antiviral activity against hepatitis E virus

doi: 10.1128/jvi.01917-25

Figure Lengend Snippet: Auranofin’s antiviral activity is dependent on the promotion of reactive oxygen species. ( A–C ) The effect of 2 μM auranofin in the presence of 10 mM NAC against HEV-3 P6 Gluc replicon ( A ), HEV-3 P6 HiBiT infectious virus ( B ), and ROS production as quantified by the H 2 DCFDA fluorescent probe ( C ). ( D–F ) The effect of 2 μM auranofin in the presence of 500 μM DTT against HEV-3 P6 Gluc replicon ( D ), HEV-3 P6 HiBiT infectious virus ( E ), and ROS production as measured by the H 2 DCFDA fluorescent probe ( F ). Single-cell suspensions were analyzed by flow cytometry, and the median fluorescent peak was recorded. Data is background-subtracted, normalized to DMSO vehicle control, and analyzed by one-way ANOVA with Dunnett’s multiple comparisons test (single pooled variance). Data points represent means ± SD; n = 6 performed in two independent experiments. **** = P < 0.0001.

Article Snippet: Additional auranofin stocks (Selleckchem) were also prepared to 10 mM in DMSO.

Techniques: Activity Assay, Virus, Flow Cytometry, Control

Journal: Journal of Virology

Article Title: Reactive oxygen species promotion drives auranofin’s antiviral activity against hepatitis E virus

doi: 10.1128/jvi.01917-25

Figure Lengend Snippet: In the presence of HEV-3, auranofin triggers ROS-dependent antioxidant, antiviral, and ER stress responses. ( A ) The effect of 2 μM auranofin in the presence of 10 mM NAC on HEV-3 P6 Gluc production as a proxy for HEV-3 P6 replication 24 h post-compound application. Data is background-subtracted and normalized to the DMSO control. ( B–I ) Transcriptional modulations measured by RT-qPCR following 24-hour treatment with 2 μM auranofin and/or 10 mM NAC during HEV-3 P6 Gluc infection. Data are presented as fold change of ROS- and Nrf2-related genes (NQO1 and HMOX1) ( B and C ), IFN-stimulated genes (GBP5 and ISG15) ( D and E ), ER stress response genes (GRP78 and Xbp1) ( F and G ), and ERAD-associated genes (HERPUD1 and EDEM1) ( H and I ). Data presented as fold change and normalized to the housekeeping gene (GAPDH) and vehicle (DMSO) control. Data were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test (single pooled variance), with data points representing means ± SD. n = 6 performed in two independent experiments. * = P < 0.05, **** = P < 0.0001.

Article Snippet: Additional auranofin stocks (Selleckchem) were also prepared to 10 mM in DMSO.

Techniques: Control, Quantitative RT-PCR, Infection

Journal: Journal of Virology

Article Title: Reactive oxygen species promotion drives auranofin’s antiviral activity against hepatitis E virus

doi: 10.1128/jvi.01917-25

Figure Lengend Snippet: Auranofin and ribavirin combined treatment in vitro displays synergistic antiviral activity against HEV-3. ( A ) The effect of ribavirin on cytotoxicity of Huh7-S10-3 cells and antiviral activity against the HEV-3 P6 Gluc replicon. Data collected 72 h post-compound application is background-subtracted and normalized to the vehicle control. X-axis values are log-transformed. The lines represent non-linear regression curves, and data points represent means ± SD; n = 3 performed in two independent experiments. ( B and C ) Huh7-S10-3 cells were transfected with HEV-3 P6 Gluc RNA, compounds were applied, and Gluc was quantified from supernatants 72 h post-complication. Resulting data were generated via the SynergyFinder web application (version 3.0). ( B ) Dose-response HEV-3 P6 Gluc inhibition matrix of the combined concentrations of auranofin and ribavirin tested at a range between 0–1.5 μM and 0–25 μM, respectively. The average inhibition of HEV replication is presented for all tested compound combinations and for auranofin and ribavirin treatment alone. ( C ) Synergy map generated via the HSA model. The overall HSA synergy score is depicted as 4.034 ± 2.72. ( D–F ) Graphs depicting the concentration combinations that displayed synergy (synergy scores over 10): ( D ) auranofin 0.5 μM + ribavirin 5 μM, ( E ) auranofin 0.5 μM + ribavirin 10 μM, and ( F ) auranofin 1 μM + ribavirin 20 μM. Data in panels B–F are background-subtracted and normalized to the appropriate vehicle control. A one-way ANOVA with Dunnett’s multiple comparisons test (single pooled variance) was performed for panels D–F. The data points represent means ± SD; n = 6, performed in two independent experiments. ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001.

Article Snippet: Additional auranofin stocks (Selleckchem) were also prepared to 10 mM in DMSO.

Techniques: In Vitro, Activity Assay, Control, Transformation Assay, Transfection, Generated, Inhibition, Concentration Assay