cells human primary embryonic lung fibroblasts  (ATCC)

Journal: Journal of Virology

Article Title: Stabilization of the human cytomegalovirus UL136p33 reactivation determinant overcomes the requirement for UL135 for replication in hematopoietic cells

doi: 10.1128/jvi.00148-23

Figure Lengend Snippet: Stabilizing UL136p33 modestly alters viral gene expression in a productive infection. (A) MRC-5 lung fibroblast cells were infected with UL136 myc, UL136mycΔK→R, ΔUL135STOP/UL136myc, or ΔUL135STOP/UL136mycΔK→R recombinant viruses at MOI of 1. Lysates were collected over a time course of infection and immunoblotted using antibodies specific to the myc epitope tag (UL136 isoforms), UL135 (UL135 isoforms), UL44, IE1&2 (clone 3H4), pp28, and pp150. Tubulin was used as a loading control. Representative blots are shown. (B and C) Protein levels quantified over multiple independent experiments using Image Studio Lite quantification software. All protein bands are normalized to tubulin. Bars represent the averages of at least three independent experiments with standard deviation shown. Significance is calculated using a two-way ANOVA with Tukey’s multiple comparison tests. *P < 0.01; **P < 0.001; ***P < 0.0001.

Article Snippet: Cells Human primary embryonic lung fibroblasts (MRC-5, purchased from ATCC; Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-alanyl-glutamine, 0.1 mM nonessential amino acids, 100 U/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Gene Expression, Infection, Recombinant, Control, Software, Standard Deviation, Comparison

Journal: Journal of Virology

Article Title: Stabilization of the human cytomegalovirus UL136p33 reactivation determinant overcomes the requirement for UL135 for replication in hematopoietic cells

doi: 10.1128/jvi.00148-23

Figure Lengend Snippet: Stabilizing UL136p33 does not direct the middle isoforms of UL136 for proteasomal degradation. MRC-5 fibroblasts were infected with either UL136 myc or UL136mycΔK→R. 6 h prior to lysate collection, infected cells were treated with 20 µM MG132 to inhibit the proteasome or vehicle control. Lysates were collected at 24 hpi and immunoblotted for myc to detect protein isoforms and tubulin (as a loading control) with antibodies described in Table 2. Three independent biological replicates were used to calculate statistical significance. Statistical significance was calculated through a two-way ANOVA with Tukey’s multiple comparison tests for each isoform as follows. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Cells Human primary embryonic lung fibroblasts (MRC-5, purchased from ATCC; Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-alanyl-glutamine, 0.1 mM nonessential amino acids, 100 U/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Infection, Control, Comparison

Journal: Journal of Virology

Article Title: Stabilization of the human cytomegalovirus UL136p33 reactivation determinant overcomes the requirement for UL135 for replication in hematopoietic cells

doi: 10.1128/jvi.00148-23

Figure Lengend Snippet: Stabilizing UL136p33 rescues viral replication in the absence of UL135 in CD34+ HPCs. (A) CD34+ HPCs were infected with WT UL136myc, UL136mycΔ33kDa, UL136mycΔK→R, and ΔUL135STOP/UL136mycΔK→R at an MOI of 2. At 24 hpi, CD34+/GFP+ (infected cells) were sorted and seeded into long-term bone marrow culture. After 10 days in culture, parallel populations of either mechanically lysed cells or live cells were plated onto fibroblast monolayers in cytokine-rich media. 14 days later, GFP+ wells were scored, and the frequency of infectious centers was determined by extreme limiting dilution analysis. The mechanically lysed population defines the quantity of virus present prior to reactivation (pre-reactivation; white bar). The live-cell population defines the quantity of virus present after reactivation (reactivation; gray bar). The frequency was normalized to WT UL136myc pre-reactivation, and the average of three independent experiments is shown. Statistical significance was calculated using a two-way ANOVA with Tukey’s multiple comparison tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001). (B) Total DNA was isolated from CD34+ HPCs infected with WT UL136myc, UL136mycΔ33kDa, UL136mycΔK→R, and ΔUL135STOP/UL136mycΔK→R at an MOI of 2 at 10 dpi. The number of viral genomes relative to the level of RNaseP expression was quantified by qPCR using β2.7kb RNA gene- and RNaseP-specific primers. Two biological replicates from two independent cell donors are shown.

Article Snippet: Cells Human primary embryonic lung fibroblasts (MRC-5, purchased from ATCC; Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-alanyl-glutamine, 0.1 mM nonessential amino acids, 100 U/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Infection, Virus, Comparison, Isolation, Expressing

Journal: Journal of Virology

Article Title: Stabilization of the human cytomegalovirus UL136p33 reactivation determinant overcomes the requirement for UL135 for replication in hematopoietic cells

doi: 10.1128/jvi.00148-23

Figure Lengend Snippet: Stabilizing UL136p33 compensates for a loss of UL135 for viral replication in huNSG mice. Humanized NSG mice were injected with fibroblasts infected with either HCMV WT UL136myc, UL136mycΔ33kDa, UL136mycΔK→R, and ΔUL135STOP/UL136mycΔK→R (n = 10 per group). At 4 wk post-infection, half of the mice were treated with G-CSF and AMD-3100 to induce cellular mobilization and promote HCMV reactivation. Control mice were left untreated. At 1 wk post-mobilization, mice were euthanized, and tissues were collected. Total DNA was extracted using DNAzol, and HCMV viral load was determined by qPCR on 1 µg of total DNA prepared from spleen or liver tissue. Error bars represent standard error of the mean between average DNA copies from two or four tissue sections, respectively, for individual animals. All samples were compared by a two-way ANOVA with Tukey’s multiple comparison tests within experimental groups (nonmobilized [-G-CSF] versus mobilized [+G-CSF] for each virus and between all virus groups for both nonmobilized and mobilized conditions). Statistical significance where **, P < 0.01 and ***, P < 0.001.

Article Snippet: Cells Human primary embryonic lung fibroblasts (MRC-5, purchased from ATCC; Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-alanyl-glutamine, 0.1 mM nonessential amino acids, 100 U/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Injection, Infection, Control, Comparison, Virus