Journal: Journal of Virology

Article Title: Human cytomegalovirus attenuates AKT activity by destabilizing insulin receptor substrate proteins

doi: 10.1128/jvi.00563-23

Figure Lengend Snippet: Human cytomegalovirus (HCMV) prevents AKT activation in response to serum. (A) Stimulation of various receptor tyrosine kinases (RTKs) and other growth factor receptors causes the recruitment of the scaffolding adaptor insulin receptor substrate 1 (IRS1) to the receptor complex. IRS1 is then phosphorylated, which provides a docking site for recruitment of class IA phosphoinositol 3-kinases (PI3K). PI3K phosphorylates the lipid substrate phosphoinositol 4,5 diphosphate (PIP2) to generate phosphoinositol 3,4,5 triphosphate (PIP3). The pleckstrin homology (PH) domain of AKT binds to PIP3, which drives its localization to membranes where mTORC2 and phosphoinositol-dependent kinase 1 (PDK1) are available to phosphorylate AKT at Ser473 and Thr308, respectively, activating its kinase activity. Once activated, AKT phosphorylates many substrates, such as the proline-rich AKT substrate of 40 kDa (PRAS40) at Thr246, Forkhead box class O (FoxO) family transcription factors, and the tuberous sclerosis complex 2 (TSC2) protei. Phosphorylation of TSC2 leads to activation of mTORC1, which in turn phosphorylates and activates S6 kinase (S6K). S6K phosphorylates S6 to increase protein synthesis. (Schematic generated in BioRender.) (B) Serum-starved fibroblasts were stimulated with insulin or 5% newborn calf serum for the indicated times (min) prior to lysis. Protein extracts were then resolved by SDS-PAGE, and transferred to nitrocellulose membranes for Western blot analysis using a phospho-specific antibody to detect AKT phosphorylated at Ser473 compared with AKT (pan) antibody. (C–G) Fibroblasts were serum starved overnight and either mock infected or infected with HCMV strain Towne at multiplicity of infection (MOI) of 2 (MOI = 2 TCID50/cell) for the indicated times, hours post-infection (hpi). Cells were then either treated (or mock treated) with serum for 10 min prior to lysis for Western blot analysis. (E–G) Fluorescent signals from dye-labeled secondary antibodies were quantified, and phospho-specific antibody detection of pAKT Thr308, pAKT Ser473, and PRAS40 Thr246 was normalized to AKT (pan) or PRAS40. The arithmetic mean was calculated and graphed. Error bars indicate standard error of the mean (SEM) from three independent biological replicates. Where indicated, Ponceau staining was used to visualize protein loading and confirm efficient transfer.

Article Snippet: Stocks of HCMV strains Towne (ATCC VR-977) and parental AD169rv ( 99 ) were produced by low MOI infection of HFFTs (MOI of 0.01 TCID50/cell).

Techniques: Activation Assay, Scaffolding, Activity Assay, Generated, Lysis, SDS Page, Western Blot, Infection, Labeling, Staining

Journal: Journal of Virology

Article Title: Human cytomegalovirus attenuates AKT activity by destabilizing insulin receptor substrate proteins

doi: 10.1128/jvi.00563-23

Figure Lengend Snippet: Membrane recruitment of AKT is defective in HCMV-infected cells. (A–B) Serum-starved fibroblasts were either mock infected or infected with HCMV strain Towne (MOI= 2 TCID50/cell) for 12 h and either mock treated or treated with 5% newborn calf serum (NCS) for 10 min. Samples were mechanically lysed and differentially centrifuged to separate the membrane (M) fraction from the cytoplasmic (C) fraction and subjugated to Western blot analysis of indicated proteins alongside total (T) lysate. Ponceau staining was used as a loading control. (B) Detection signals from AKT (pan) bands were quantified for each condition, and bands from membrane fractions were normalized to signal cognate “total lysate” bands. Results were then normalized to the “No Serum” condition, the arithmetic mean was calculated, and values were graphed. A one-way ANOVA was run using Tukey’s multiple comparison post-test for statistical analysis (*P < 0.05, ****P < 0.0001). Error bars indicate standard error of the mean (SEM). n = 4 independent biological replicates.

Article Snippet: Stocks of HCMV strains Towne (ATCC VR-977) and parental AD169rv ( 99 ) were produced by low MOI infection of HFFTs (MOI of 0.01 TCID50/cell).

Techniques: Membrane, Infection, Western Blot, Staining, Comparison

Journal: Journal of Virology

Article Title: Human cytomegalovirus attenuates AKT activity by destabilizing insulin receptor substrate proteins

doi: 10.1128/jvi.00563-23

Figure Lengend Snippet: UV-treated virus fails to inactivate AKT. (A) HCMV strain Towne was mock treated or treated with 125 mJ of UV light for indicated seconds (sec) and then used to infect fibroblasts (MOI = 2 TCID50/cell) for 12 h. A Western blot was performed to monitor for IE1 expression, using Ponceau S stain to indicate total protein loading. n = 1. (B–C) Fibroblasts were infected with mock-treated or UV-treated HCMV strain Towne virus (MOI = 2 TCID50/cell) in the presence of serum, and lysates were taken at the indicated times post-infection (in hours), hours post-infection (hpi). A Western blot was performed probing for the indicated proteins; additionally, Ponceau S staining is shown as a readout for total protein loading. (C) Detection signals from 24 hpi bands were quantified, normalized to signals from 1 hpi bands, and the arithmetic means from three independent biological replicates were plotted. A one-way ANOVA with Tukey’s multiple comparison test was used to compare each condition with the control. Comparisons labeled with a single asterisk (*) indicate P < 0.05. Error bars indicate SEM, n=3. (D) Serum-starved fibroblasts were either mock infected or infected with UV-treated or untreated HCMV strain Towne (MOI = 2 TCID50/cell) , and either mock treated or treated for 10 min with 5% newborn calf serum (NCS). Samples were mechanically lysed and differentially centrifuged to separate membrane (M) and cytoplasmic (C) fractions, where subjected to Western blot analysis to monitor the expression of the indicated proteins alongside total (T) lysate.

Article Snippet: Stocks of HCMV strains Towne (ATCC VR-977) and parental AD169rv ( 99 ) were produced by low MOI infection of HFFTs (MOI of 0.01 TCID50/cell).

Techniques: Virus, Western Blot, Expressing, Staining, Infection, Comparison, Labeling, Membrane

Journal: Journal of Virology

Article Title: Human cytomegalovirus attenuates AKT activity by destabilizing insulin receptor substrate proteins

doi: 10.1128/jvi.00563-23

Figure Lengend Snippet: HCMV causes degradation of insulin receptor substrate 1. (A) Model for AKT inactivation in HCMV-infected cells. HCMV UL38-mediated activation of mTORC1 leads to phosphorylation of insulin receptor substrate 1 (IRS1), inducing its degradation. Destabilization of IRS1 in turn prevents phosphoinositide 3-kinase (PI3K) recruitment to growth factor receptors (not shown), preventing AKT membrane recruitment and activation. Cartoon was generated using BioRender. (B–C) Fibroblasts were mock infected or infected with HCMV strain Towne (MOI = 2 TCID50/cell) in the presence of serum and lysates were taken at the indicated hours post-infection (hpi). Western blots were performed to monitor levels of the indicated proteins, (C) bands were quantified, and detection signals were normalized to total protein in 11 independent biological replicates; the arithmetic means of the combined results were graphed. The dotted line indicates phosphorylation levels detected from mock-infected controls at 1 hpi, the setting against which all infected timepoints are normalized. Error bars indicate SEM. n = 11. (D–E) Similar to the experiment in Fig. 1C, fibroblasts were serum starved overnight and either mock infected or infected with HCMV strain Towne multiplicity of infection (MOI = 2 TCID50/cell). At 3 hpi, cells were mock treated or treated with 50 nM rapamycin. At 24 hpi, cells were then either treated or mock treated with serum for 10 min, and lysates were harvested for Western blot analysis. Ponceau S staining was used to monitor total protein loading. (E) Bands were quantified, and phospho-specific antibody signals were normalized to detection signal for cognate total protein and then further normalized to “no drug” control conditions. The arithmetic mean was calculated and graphed. A one-way ANOVA with Tukey’s multiple comparison test was used to assess statistical significance, wherein *P < 0.05 and **P < 0.01. Error bars indicate SEM. n = 3 independent biological replicates.

Article Snippet: Stocks of HCMV strains Towne (ATCC VR-977) and parental AD169rv ( 99 ) were produced by low MOI infection of HFFTs (MOI of 0.01 TCID50/cell).

Techniques: Infection, Activation Assay, Membrane, Generated, Western Blot, Staining, Comparison

Journal: IET Nanobiotechnology

Article Title: Synthesis of Zeolitic imidazolate frameworks‐8@ layered double hydroxide polyhedral nanocomposite with designed porous voids as an effective carrier for anti‐cancer drug‐controlled delivery

doi: 10.1049/nbt2.12125

Figure Lengend Snippet: The antimicrobial effect of concentrated bacterial cell‐free culture medium on the growth of (a) Klebsiella pneumonia strain Peterborough Town Cricket Club 10031 T and (b) Streptococcus aureus strain American Type Culture Collection 35668 T .

Article Snippet: In the current study, an inhibition zone was observed for two pathogenic bacteria including Klebsiella pneumonia strain Peterborough Town Cricket Club 10031 T and Staphylococcus aureus strain American Type Culture Collection 35668 T (Figure ), while others were observed without any inhibition zone.

Techniques:

Journal: Journal of Virology

Article Title: Harnessing the Noncanonical Keap1-Nrf2 Pathway for Human Cytomegalovirus Control

doi: 10.1128/jvi.00160-23

Figure Lengend Snippet: HCMV inhibition by ARP101. (A) A luciferase-based reporter assay was performed at 72 hpi on HFFs infected with the pp28-luciferase recombinant HCMV-Towne (MOI of 1 PFU/cell), followed by treatment with the indicated concentrations of ARP101. (B) HCMV-TB40 (200 PFU/well) was used to infect HFFs (1 × 10 6 cells in a 24-well plate), followed by treatment with the indicated concentrations of ARP101. On day 8 postinfection, plaques were enumerated. (C) HFFs were treated with the indicated concentrations of ARP101 for 72 h and 8 days, and a luminescence-based cell viability assay was performed to determine the 50% cellular viability (CC 50 ). (D) The activity of ARP101 (10 μM) against pp28-luciferase ganciclovir (GCV)-resistant HCMV-Towne (GCV R HCMV) was measured by luciferase assay at 72 hpi. (E) HFFs were infected with HCMV-Towne (MOI of 0.1) for 24, 48, and 72 h and treated with ARP101 (10 μM) or GCV (5 μM). Infected cells were lysed, DNA was isolated, and viral load was measured by a US17 quantitative real-time PCR. (F) HFFs were infected with HCMV-Towne (MOI of 1) and treated with ARP101 (10 μM) or GCV (5 μM). Infected cells were lysed at 24, 48, and 72 hpi, lysates were run on 12% SDS-PAGE gels, and immunoblot assays were performed to detect IE1/2 (immediate early), UL84 (early late), and pp65 (late) antigens. Numbers presented below each panel of immunoblots represent the relative band intensities of each blot. Relative luciferase activity was calculated by dividing the luciferase units obtained at a given drug concentration by the luciferase units obtained following treatment with the vehicle control, dimethyl sulfoxide. All experiments were performed in triplicates, and represented values are mean ± SD.

Article Snippet: HCMV-Towne (ATCC VR-977) was used for immunofluorescence, immunoblotting, and immunoprecipitation assays.

Techniques: Inhibition, Luciferase, Reporter Assay, Infection, Recombinant, Viability Assay, Activity Assay, Isolation, Real-time Polymerase Chain Reaction, SDS Page, Western Blot, Concentration Assay

Journal: Journal of Virology

Article Title: Harnessing the Noncanonical Keap1-Nrf2 Pathway for Human Cytomegalovirus Control

doi: 10.1128/jvi.00160-23

Figure Lengend Snippet: Timing of activity of ARP101. (A) An immunofluorescence assay was performed to test for HCMV entry. HFFs were pretreated with dimethyl sulfoxide (mock), ARP101, or GCV for 24 h and then infected with HCMV-Towne (MOI of 1). (B) Graph depicting percentage of pp65-positive cells in the microscopic data presented in panel A. (C and D) A luciferase-based reporter assay was performed to determine the timing of activity of ARP101. HFFs were infected with pp28-luciferase recombinant Towne, and ARP101 (10 μM) was either added to or removed from infected HFFs at 6, 24, 48, and 72 h postinfection. GCV (5 μM) was used as a control for timing of activity. Relative luciferase activity was calculated by dividing the luciferase units obtained at a given drug concentration by the luciferase units obtained following treatment with the vehicle control, dimethyl sulfoxide. All experiments were performed in triplicates, and represented values are mean ± SD.

Article Snippet: HCMV-Towne (ATCC VR-977) was used for immunofluorescence, immunoblotting, and immunoprecipitation assays.

Techniques: Activity Assay, Immunofluorescence, Infection, Luciferase, Reporter Assay, Recombinant, Concentration Assay

Journal: Journal of Virology

Article Title: Harnessing the Noncanonical Keap1-Nrf2 Pathway for Human Cytomegalovirus Control

doi: 10.1128/jvi.00160-23

Figure Lengend Snippet: ARP101 induces both p62 and LC3 II in HCMV-infected HFFs. (A) HFFs were infected with HCMV-Towne (MOI of 1) and treated with ARP101 (10 μM) for 24 and 72 h. Bafilomycin A1 (Baf A1, 50 nM) was added to the respective conditions 3 h before harvesting. Following Baf A1 treatment, cells were lysed, and an immunoblot assay was performed for LC3 I/II and p62. Red arrows indicate HCMV-infected HFF samples, and black arrows indicate ARP101-treated HCMV-infected HFF samples. HCMV pp65 and β-actin were probed as infection and loading controls, respectively. (B) Structure of active ARP101 (having the hydroxamic acid functional group) and inactive ARP101 (carboxylic acid replaces the hydroxamic acid functional group). (C) HCMV inhibition by ARP101 hydroxamic acid and the carboxylic acid (inactive) was measured at 72 hpi with pp28-luciferase recombinant Towne (MOI of 1). (D) Whole-cell lysates from infected HFFs were analyzed by immunoblotting for p62, LC3 I/II, and viral pp65 following treatment with the active and inactive ARP101 (10 μM) at the indicated time points. Red arrows indicate HCMV-infected HFF samples, and black arrows indicate ARP101-treated HCMV-infected HFF samples at 72 h postinfection. The experiments were performed in triplicates, and the best representative data are presented. INF, infected with HCMV-Towne.

Article Snippet: HCMV-Towne (ATCC VR-977) was used for immunofluorescence, immunoblotting, and immunoprecipitation assays.

Techniques: Infection, Western Blot, Functional Assay, Inhibition, Luciferase, Recombinant

Journal: Journal of Virology

Article Title: Harnessing the Noncanonical Keap1-Nrf2 Pathway for Human Cytomegalovirus Control

doi: 10.1128/jvi.00160-23

Figure Lengend Snippet: ARP101 induces phosphorylation of p62 at the KIR and UBA domains and increases expression of Nrf2 and heme oxygenase-1 (HO-1). (A) ATG5 knockdown (KD) by lentiviral transduction in HFFs. Cells expressing pLKO.1 (vector control) and plasmids containing ATG5 shRNA were grown to confluence following puromycin selection and lysed in cell lysis buffer. ATG5 expression in pLKO.1 control and ATG5 KD HFFs (#1, #2, and #3) was analyzed by immunoblotting. (B) A luciferase-based reporter assay was performed with ATG5 KD (#2 and #3) and pLKO.1 control HFFs infected with HCMV-Towne (MOI of 1) and treated with the indicated concentrations of ARP101 and GCV. The values are represented as the mean ± SD from two independent experiments. (C) HFFs were infected with HCMV-Towne (MOI of 1) and treated with ARP101 (10 μM) for 24, 48, and 72 h. Cells were lysed posttreatment, and equal amounts of proteins were analyzed on 12% SDS-PAGE gels. Immunoblot assays were performed for HO-1, Keap1, Nrf2, p62, and LC3 I/II. Viral pp65 and β-actin were probed for infection and loading controls, respectively. The red arrowhead indicates HCMV-infected HFF samples, and the black arrowhead indicates ARP101-treated HCMV-infected HFF samples at 72 hpi. (D) HCMV-infected cells (MOI of 1) were lysed posttreatment with active and inactive ARP101 (10 μM). Equal amounts of lysates were analyzed on 12% SDS-PAGE gels, and an immunoblot assay was performed for p-p62 (S349), p-p62 (S403), p62, viral UL84, and β-actin. The experiment was repeated in triplicate, and representative images are presented. (E) HFFs were treated with ARP101 (10 μM) and harvested at 24, 48, and 72 h posttreatment for analysis of levels of HO-1, Nrf2, Keap1, p-p62 (S349), p62, and LC3 I/II by Western blotting. β-Actin was probed as an internal control. NI, noninfected; INF, infected with HCMV-Towne.

Article Snippet: HCMV-Towne (ATCC VR-977) was used for immunofluorescence, immunoblotting, and immunoprecipitation assays.

Techniques: Expressing, Transduction, Plasmid Preparation, shRNA, Selection, Lysis, Western Blot, Luciferase, Reporter Assay, Infection, SDS Page

Journal: Journal of Virology

Article Title: Harnessing the Noncanonical Keap1-Nrf2 Pathway for Human Cytomegalovirus Control

doi: 10.1128/jvi.00160-23

Figure Lengend Snippet: ARP101 releases Nrf2 from the p62-Keap1-Nrf2 complex, resulting in nuclear translocation. (A) Immunoprecipitation (IP) of p62 was performed in HCMV-infected (MOI of 1) ARP101 (10 μM)-treated cells at 48 and 72 hpi. Keap1, Nrf2, p-p62 (S349), and p-p62 (S403) were probed to measure the affinity of p62 for Keap1 and its effect on Nrf2 release from Keap1. Viral pp65 and pp28 were probed for analysis of the interaction of p62 with viral proteins. The right panel shows the profile of different proteins in the soluble fraction of the lysates (input), which was used for IP. IB, immunoblot. (B) Reverse IP of Nrf2 was performed in HCMV-infected ARP101-treated cells at 48 and 72 hpi. Keap1 and p62 were probed to measure the status of the p62-Keap1-Nrf2 complex. The right panel shows the input used for the IP. (C) HFFs were infected with HCMV-Towne (MOI of 1) and treated with ARP101 (10 μM) for 48 and 72 h. Cells were fractionated into cytoplasmic (C) and nuclear (N) fractions, concentrated by acetone precipitation, and quantified. Equal amounts of proteins were analyzed on 12% SDS-PAGE gels, and immunoblot assays were performed to measure the level of p62, p-p62 (Ser349), p-p62 (S403), Keap1, Nrf2, and HO-1. Histone H3 and GAPDH were probed as nuclear (N) and cytoplasmic (C) controls, respectively. Viral pp65 was probed as an infection control. The experiments were performed thrice, and representative images are presented. NI, noninfected; INF, infected with HCMV-Towne. IgG control experiments were conducted with INF cell lysates.

Article Snippet: HCMV-Towne (ATCC VR-977) was used for immunofluorescence, immunoblotting, and immunoprecipitation assays.

Techniques: Translocation Assay, Immunoprecipitation, Infection, Western Blot, SDS Page

Journal: Journal of Virology

Article Title: Harnessing the Noncanonical Keap1-Nrf2 Pathway for Human Cytomegalovirus Control

doi: 10.1128/jvi.00160-23

Figure Lengend Snippet: ARP101 induces binding of Nrf2 to specific promoters and activation of the antioxidant response element (ARE). (A to D) Binding of Nrf2 to several ARE promoters was analyzed by ChIP assay. HFFs were infected with HCMV-Towne (MOI of1) and treated with ARP101 or inactive ARP101 (10 μM) for 48 and 72 h. (E to H) Quantitative PCRs were performed for SQSTM1/p62 (E), HMOX1 (F), NQO1 (G), and GCLC (H) transcribed upon ARE activation in infected HFFs treated/untreated with ARP101 at 48 and 72 hpi. (I) Viral IE1 and IE2 expression was analyzed by quantitative PCR for infection control in the same samples from panels E to H. The experiments were performed thrice, and data from a single experiment with three technical replicates are presented. NI, noninfected; INF, infected with HCMV-Towne.

Article Snippet: HCMV-Towne (ATCC VR-977) was used for immunofluorescence, immunoblotting, and immunoprecipitation assays.

Techniques: Binding Assay, Activation Assay, Infection, Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of Virology

Article Title: Harnessing the Noncanonical Keap1-Nrf2 Pathway for Human Cytomegalovirus Control

doi: 10.1128/jvi.00160-23

Figure Lengend Snippet: ARP101 fails to activate the ARE in Nrf2 KD cells and does not inhibit HCMV replication. (A) Nrf2 knockdown efficiency was analyzed by immunoblotting in the lysates of lentivirus-transduced HFF cells following puromycin selection. GAPDH was probed as a loading control. (B) A luciferase-based reporter assay was performed with infected (MOI of 1) Nrf2 KD and pLKO.1 control cells treated with the indicated concentrations of ARP101 and GCV. (C) Lysates from panel B were analyzed by Western blotting for Nrf2, viral pp65, and β-actin. (D) A plaque reduction assay was performed using Nrf2 KD and pLKO.1 control HFFs by infecting cells with HCMV-TB40 (200 PFU/well, used to infect 1 × 10 6 cells in a 24-well plate) and treating them with ARP101 (5 and 10 μM) and GCV (5 μM). (E) Nrf2 KD and pLKO.1 cells were infected with HCMV-TB40 (MOI of 1 PFU/cell), and supernatants were harvested after 120 h followed by titration using plaque assay. (F) Nrf2 KD and pLKO.1 control cells were infected with HCMV-Towne (MOI of 1) and treated with ARP101 (10 μM), and lysates were prepared after 72 hpi. Levels of Nrf2, Keap1, p-p62 (S349), p62, HO-1, and LC3 I/II were measured by Western blotting. Viral pp65 and GAPDH were probed as infection and internal controls, respectively. Experiments were performed thrice, and the best representative images are depicted. NI, noninfected; INF, infected with HCMV-Towne.

Article Snippet: HCMV-Towne (ATCC VR-977) was used for immunofluorescence, immunoblotting, and immunoprecipitation assays.

Techniques: Western Blot, Selection, Luciferase, Reporter Assay, Infection, Titration, Plaque Assay

Journal: Journal of Virology

Article Title: Harnessing the Noncanonical Keap1-Nrf2 Pathway for Human Cytomegalovirus Control

doi: 10.1128/jvi.00160-23

Figure Lengend Snippet: Keap1 is degraded through a p62-mediated process induced by ARP101 in HCMV-infected cells. (A) A cycloheximide chase assay was performed at 72 hpi, and expression of Keap1 was analyzed by immunoblotting. (B) Graphical representation of Keap1 levels measured at 72 hpi in panel A. The level of Keap1 was normalized to β-actin at the indicated time points after the addition of cycloheximide. (C) The levels of Keap1, along with several other proteins, at 72 hpi following ARP101 (10 μM) treatment were analyzed by Western blotting. Bafilomycin A1 (Baf A1; 50 nM) was added 3 h before harvesting at 72 hpi, whereas MG132 (10 μM) was added 8 h before harvesting at 72 hpi. β-Actin was probed as an internal control. (D) HFFs were treated with MG132 (10 μM), K67 (25 μM), and their combination for 18 h, and lysates were prepared for analysis of Keap1, Nrf2, p-p62 (S349), and p62 by immunoblotting. (E) HFFs were infected with HCMV-Towne (MOI of 1 PFU/cell) for 72 h and treated with ARP101 (10 μM), K67 (25 μM), and their combination. Lysates were analyzed by immunoblotting for Keap1, Nrf2, p-p62 (S349), viral pp65, and β-actin, respectively. (F) Immunoprecipitation of p-p62 (S349) followed by detection of Keap1 and LC3 II using noninfected, infected, and ARP101-treated cellular lysates harvested at 72 hpi. (G) IP of p-p62 (S349) followed by detection of Keap1 and LC3 II using HCMV-infected HFFs (MOI of 1 PFU/cell) treated with ARP101 or ARP101 plus K67 and harvested at 72 h postinfection. Experiments were performed thrice, and representative data are presented.

Article Snippet: HCMV-Towne (ATCC VR-977) was used for immunofluorescence, immunoblotting, and immunoprecipitation assays.

Techniques: Infection, Expressing, Western Blot, Immunoprecipitation

Journal: Journal of Virology

Article Title: Harnessing the Noncanonical Keap1-Nrf2 Pathway for Human Cytomegalovirus Control

doi: 10.1128/jvi.00160-23

Figure Lengend Snippet: ARP101-induced p62 phosphorylation at S349/S403 is not modulated by the mTOR kinase or casein kinases 1 and 2 in HCMV-infected HFFs. HFFs were infected with HCMV-Towne (MOI of 1 PFU/cell) and treated with ARP101 (10 μM) for 24, 48, and 72 h. Torin-1 (250 nM) was added at 8 h (in panel B) or 24 h (in panel C) before harvesting the cells at 48 or 72 hpi, respectively. TBCA (30 μM), an inhibitor of casein kinase 2, and CK1-7 (50 μM), an inhibitor of casein kinase 1, were also added 8 h before harvesting at 72 hpi. (A) p-mTOR (S2448) and mTOR were analyzed by immunoblotting at the indicated time points. (B) Immunoblot analysis of indicated proteins at 72 hpi under various experimental conditions where kinase inhibitors including Torin-1 were added 8 h before harvesting and ARP101 was added immediately after infection. (C) p-p62 (S349), p-p62 (S403), p62, and p-4EBP1 were analyzed at 48 and 72 hpi following Torin-1 and ARP101 treatments, as mentioned above. Torin-1 was added 24 h before harvesting at indicated time points, and ARP101 was added immediately after infection. Viral pp65/UL84 and β-actin were probed for infection and loading controls, respectively. The experiments were repeated twice, and representative images are presented. NI, noninfected; INF, infected with HCMV-Towne.

Article Snippet: HCMV-Towne (ATCC VR-977) was used for immunofluorescence, immunoblotting, and immunoprecipitation assays.

Techniques: Infection, Western Blot

Journal: PLoS ONE

Article Title: Exploitation of Herpesviral Transactivation Allows Quantitative Reporter Gene-Based Assessment of Virus Entry and Neutralization

doi: 10.1371/journal.pone.0014532

Figure Lengend Snippet: ( A ) Human MRC-5 cells, transiently transfected with the pTA-Control plasmid (left panel) or pTA-GAS (right panel), were split into aliquots and seeded. Cells were infected with 1 PFU/cell HCMV-AD169, HCMV-Towne, HCMV-TB40/E (black bars) or left uninfected (white bars). After 24 h the cells were treated for additional 5 h with 500 U/ml human IFN-γ. Cells were lysed and luciferase activity was measured. The experiment was performed in triplicate and the arithmetic mean with the standard deviation is shown. The significance was tested with the Student's t-test: * p<0.05 and ** p<0.01. ( B ) ARPE-19, CV-1, Vero and Jurkat cells were transfected with 2.5 µg pTA-Control plasmid using the Lonza nucleotransfection system. Cells were split so that parallel measurements and infections originated from the same transfection. 20 h post transfection cells were left uninfected (mock; open bars) infected with 3 PFU/cell HCMV-AD169, HSV-1 F strain (black bars) or UV-inactivated virus (hatched bar) for additional 20 h. Cells were lysed and luciferase activity was measured. ( C ) MRC-5 cells were infected with 3 PFU/cell HCMV-AD169 or UV-inactivated HCMV-AD169. After the indicated time cells were lysed and total RNA or protein was prepared. Protein lysates were subjected to native sodium-deoxycholate-PAGE for the analysis of IRF-3 dimerization or to SDS-PAGE for the analysis of pp72-IE1 and β-actin protein amount. Proteins were transferred to filters and probed with the indicated antibodies. IFN-β and GAPDH mRNA was assessed by semi-quantitative RT-PCR from total RNA as. For GAPDH two log 10 dilutions were used as template to confirm measurement in the linear amplification range.

Article Snippet: Purified stocks of HCMV strains AD169 , Towne (ATCC VR-977), the BAC-derived HB5 and the BAC-derived endotheliotropic strain TB40/E were used.

Techniques: Transfection, Plasmid Preparation, Infection, Luciferase, Activity Assay, Standard Deviation, SDS Page, Quantitative RT-PCR, Amplification