Parameters influencing salubrinal-induced fusion.

<p><b>(A)</b> Vero cells were infected with strains KOS, F, or 17 at MOIs of 0.5, 1, or 3 and were incubated in medium containing 50 μM salubrinal. At 12 hpi, the fusion ratio (% free nuclei) was determined by flow cytometry. The mean values (±SD) from 3 independent experiments are plotted. <b>(B)</b> After infection with strain 17 (MOI = 3), cells were incubated in medium containing DMSO, and 50 μM salubrinal was added for the indicated time intervals. After a total of 12 hpi, the fusion ratios were measured by flow cytometry. Data are from 2 independent experiments. A student T-test was used to determine statistical significance for samples compared to the DMSO control. <b>(C)</b> After infection with strain 17 (MOI = 3), cells were incubated for the indicated time periods in medium containing 50 μM salubrinal, which was then replaced with control medium. At 12 hpi, the fusion ratio was determined by flow cytometry, and the data were analyzed as in (3B).</p

PTP1B inhibitor XXII blocks cell-to-cell spread.

<p><b>(A)</b> Vero cells were infected with the KOS strain (100 PFU/well in 6-well plates) and incubated in infection medium or medium containing 5 mg/ml of pooled human IgG to neutralize extracellular virions. At 18, 24, 30, and 42 hpi, the cells were immunostained for the major capsid protein, VP5, and nuclei were stained with DAPI. <b>(B)</b> To quantify virus spreading, the areas of 10–15 VP5-positive plaques were measured per sample and these were plotted. <b>(C and D)</b> Vero cells were infected (100 PFU/well) with strains (C) KOS or (D) 17 and then treated with increasing amounts of inhibitor XXII, all in the presence of 5 mg/ml of pooled IgG. At 42 hpi, the cells were immunostained for VP5, and representative plaques are shown. The areas of 10–15 plaques were measured for each drug concentration and compared to those from the DMSO control. Data from three independent experiments were combined and are represented as the mean ±SD. A student T-test was used to determine statistical significance for samples compared to the DMSO control. <b>(E and F)</b> Virus replication assays were performed in Vero cells infected (MOI = 5) with strains (E) KOS or (F) 17. The cells were incubated in medium containing DMSO or 30 μM inhibitor XXII, and at 6-hour time points, duplicate samples were collected to measure the virus titers (cell lysate + medium), which were averaged and plotted. <b>(G)</b> Representative images of plaques produced by the KOS strain on HaCaT cells treated with DMSO or 30 μM inhibitor XXII in the presence of 5 mg/ml pooled IgG. <b>(H)</b> At 42 hpi, 20 plaques from each sample in (6G) were measured, and their areas were plotted relative to the average obtained for the DMSO control.</p

Salubrinal-induced fusion of HSV-infected cells is dependent on accessory proteins.

<p><b>(A)</b> Diagram of relevant HSV-1 proteins involved in cell-to-cell spread and syncytia formation. The core fusion proteins are blue (gB, gH/gL, and gD), and proteins that can be altered to create syncytial variants are purple (gK, UL20, UL24, and gB). Two of the accessory glycoproteins are green (gE and gI), and three of the accessory tegument proteins are yellow (UL11, UL16, and UL21). <b>(B)</b> Vero cells were infected (MOI = 1) with HSV-1 strains KOS or 17 and incubated in medium containing DMSO or 50 μM salubrinal. At 12 hpi, the cells were immunostained for ZO-1 (red), and nuclei were stained with DAPI. Examples of syncytia are indicated (arrows). <b>(C)</b> Cells were infected with the KOS strain (MOI = 0.5) and incubated in the presence of salubrinal, as indicated. At 18 hpi, the cells were immunostained for ZO-1, and DAPI-stained nuclei were scored as being inside syncytia or within single cells. 1000 nuclei were scored per image for 3 replicates. Data are represented as mean ±SD, and statistical significance was determined by a student T-test. <b>(D)</b> Cells were infected (MOI = 0.5) with WT, gEΔCT, or ΔUL16 viruses and incubated in the presence of 50 μM salubrinal for 18 hours. DAPI-stained nuclei were scored as in (1C).</p

HSV-1 cell-to-cell spread is limited in PTP1B^-/- MEFs.

<p><b>(A)</b> Virus replication assays were performed in PTP1B+ and PTP1B^-/- MEFs infected with strain 17 (MOI = 5). At 6-hour time points, duplicate samples were collected for measurements of the virus titers (cell lysate + medium), which were averaged and plotted. <b>(B)</b> To measure syncytia formation, PTP1B+ MEFs or PTP1B^-/- MEFs were infected (MOI = 3) with strain 17 and treated with DMSO, 50 μM salubrinal, or 50 μM salubrinal + 30 μM inhibitor XXII. At 24 hpi, cells were immunostained for ZO-1 and DAPI-stained nuclei in syncytia were manually counted. 1000 nuclei were scored per image, and 2 replicates were averaged. <b>(C)</b> To assay for cell-to-cell spread, PTP1B+ MEFs or PTP1B^-/- MEFs were infected (100 PFU/well) with strains KOS or 17, and the cells were incubated in medium containing 5 mg/ml pooled IgG. At 42 hpi, the cells were immunostained for VP5, and the areas of 10–15 plaques per sample were measured. Data are represented as mean ±SD from 3 independent experiments. A student T-test was used to determine statistical significance for the PTP1B^-/- samples compared to the PTP1B+ control. <b>(D and E)</b> PTP1B+ MEFs or PTP1B^-/- MEFs were infected with strain 17 (100 PFU/well) and incubated in medium containing 5 mg/ml of pooled human IgG along with either DMSO or 30 μM inhibitor XXII. At 42 hpi, cells were immunostained for VP5. (D) Representative plaques are shown. (E) To quantify the results, 10–15 plaques per sample were measured, and the mean plaque area was plotted from 2 independent experiments. A student T-test was used to determine statistical significance for samples compared to the PTP1B+ DMSO control. <b>(F)</b> To ascertain their ability to respond to salubrinal, uninfected PTP1B+ MEFs or PTP1B^-/- MEFs were incubated in medium containing DMSO, 50 μM salubrinal, or 1 μM thapsigargin for 2 hours. Cell lysates were harvested in the presence of phosphatase inhibitors and probed via western blotting for total eIF2α and phosphorylated eIF2α. Duplicate samples were analyzed in the same western blot and band intensities were quantified.</p

Differential effects of salubrinal and inhibitor XXII on HSV-1 syncytial variants.

<p><b>(A and B)</b> Vero cells were infected with mutant gB.A855V at MOIs of (A) 1 or (B) 0.1. Since this mutant prefers to drive fusion with neighboring uninfected cells, the baseline level is higher at the lower MOI. Cells were treated with increasing amounts of (A) salubrinal or (B) inhibitor XXII, and at 12 hpi (A) or 24 hpi (B) the fusion ratios were determined by flow cytometry. The data are represented as the mean ±SD from 3 independent experiments, and a student T-test was used to determine statistical significance for samples compared to the DMSO control. <b>(C and D)</b> Cells were infected (MOI = 1) with mutants gK.L118Q or gK.A40V and treated with (C) salubrinal or (D) inhibitor XXII. At 12 hpi, syncytia were analyzed as in (5A). <b>(E and F)</b> Cells were infected (MOI = 1) with mutant UL20.F222A and treated with (E) salubrinal or (F) inhibitor XXII. At 12hpi, syncytia were analyzed as in (5A).</p

Host protein tyrosine phosphatase 1B is important for salubrinal-induced fusion.

<p><b>(A)</b> Vero cells were infected with strain 17 (MOI = 3) and incubated in DMSO, 50 μM salubrinal, or 50 μM guanabenz (GBZ) for 12 hours. Cell lysates were prepared in the presence of phosphatase inhibitors and probed via western blotting for total eIF2α and for phosphorylated eIF2α. <b>(B)</b> Cells were infected with KOS (MOI = 3) and incubated in DMSO, salubrinal, or guanabenz. At 24 hpi, cell lysates and media were harvested, and viral titers (cell lysates + media) were measured. Data are the averages of 2 replicates with statistical significance determined by a student T-test. <b>(C)</b> Cells were infected with strain 17 (MOI = 3) and treated with 50 μM salubrinal and increasing amounts of PTP1B inhibitors TCS-401 or XXII. At 12 hpi, the fusion ratio was determined by flow cytometry. Data are represented as the mean ±SD from 3 independent experiments, and a student T-test was used to determine statistical significance for samples compared to the salubrinal-only control. <b>(D)</b> C10 cells were transfected with plasmids encoding gB, gD, gH, and gL in a 3:1:1:1 ratio and treated with DMSO, 50 μM salubrinal, or 50 μM inhibitor XXII for 16 hours. The 10 largest syncytia per sample were measured by counting the number of nuclei per syncytium. A student T-test was used to compare samples to the DMSO-only control.</p

Flow cytometry-based method to measure cell fusion.

<p><b>(A)</b> Vero cells were infected with strain 17 (MOI = 3) and incubated in media containing DMSO or 50 μM salubrinal. At 12 hpi, the samples were trypsinized, harvested in 2% PFA, and stained with PI to label nuclei. Samples were run through a cell analyzer and PI-positive events were gated. <b>(B)</b> Cells were infected with strain 17 (MOI = 3) and incubated for 12 hours in medium containing DMSO or 50 μM salubrinal. The salubrinal sample was processed as in (2A), and the DMSO-treated cells were lysed with buffer containing Triton X-100 to release all the nuclei, which were stained with PI. <b>(C)</b> Cells were infected with strain 17 (MOI = 3) and treated with DMSO or 50 μM salubrinal in duplicate. At 12 hpi, one replicate (blue bars) was harvested and processed for flow cytometry as in (2A), and the other (pink bars) was immunostained for ZO-1, and DAPI-stained nuclei in syncytia were manually scored as in (1C). The averages of two independent experiments are shown. <b>(D)</b> Vero or BHK21 cells were infected (MOI = 3) with strain 17 and treated with DMSO or 50 μM salubrinal. At 12 hpi, the cells were harvested and analyzed by flow cytometry as in (2A).</p