Journal: Journal of Virology

Article Title: A single polymorphic residue in humans underlies species-specific restriction of HSV-1 by the antiviral protein MxB

doi: 10.1128/jvi.00830-23

Figure Lengend Snippet: The restriction of HSV-1 by MxB is species-specific. (A) Immunoblot analysis of cell lysates from IMR90s expressing the MxB orthologs (human, chimpanzee, African green monkey, or owl monkey) that were untreated or treated with dox (1 µg/mL) for 24 h to determine MxB expression levels. The ortholog-expressing IMR90s treated as in A were infected with HSV-1 strain KOS (B) or HSV-1 strain 17 (C) for 48 h (MOI = 0.1). The virus present in the medium was then titered, and the titers were normalized to those of the untreated controls. (D) The cells were treated as in A-C and then infected with HCMV (strain AD169; MOI = 0.1) for 6 days following which the virus in the medium was titered and the titers were normalized as above. (E) Immunoblot analysis of IMR90 cells expressing a non-MxB control protein (IRS1263) that were either untreated (control) or treated with doxycycline. (F) IRS263-expressing IMR90 cells were treated or untreated with dox for 24 h and then infected with HCMV (AD169; MOI = 0.1) for 6 days, following which the virus in the medium was titered and the titers were normalized to control without dox. B, C, D, and F were done in duplicate three independent times. (ns, P > 0.05; ****, P < 0.0001).

Article Snippet: HCMV (strain AD169; ATCC VR-538) was propagated in HFs.

Techniques: Western Blot, Expressing, Infection, Virus

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