Journal: bioRxiv

Article Title: The Host DHX29 RNA Helicase Regulates HCMV Immediate Early Protein Synthesis

doi: 10.1101/2025.01.27.635168

Figure Lengend Snippet: A) Human fibroblasts were transduced with lentivirus encoding scrambled (control) or DHX29 specific shRNAs. Seventy -two hours later cells were infected with HCMV (MOI of 3). Cell free virus was harvested at the indicated times after infection and quantified by the TCID50 assay. B) Cells were transduced and infected as in A. The amount of HCMV DNA in infected cells was measured at six hours post infection by qPCR. The mean data from three independent experiments is shown. C) Cells were transduced and infected as in A, and viral protein expression was measured by Western blot at the indicated times after infection. The data are representative of at least three independent experiments.

Article Snippet: Membranes were probed with antibodies to DHX29 (Santa Cruz Biotechnology), eIF4E (Cell Signaling Technology), β-Actin (Santa Cruz Biotechnology), rpS6 (Cell Signaling Technology), rpL13a (Cell Signaling Technology), HCMV IE1, IE2, UL44, UL99 (pp28), eIF4G (Cell Signaling Technology), 4E-BP (Cell Signaling Technology), 4E-BP-phospho (Cell Signaling Technology), rpS6-phospho (Cell Signaling Technology) and HSP90 (Stressgen).

Techniques: Transduction, Control, Infection, Virus, TCID50 Assay, Expressing, Western Blot

Journal: bioRxiv

Article Title: The Host DHX29 RNA Helicase Regulates HCMV Immediate Early Protein Synthesis

doi: 10.1101/2025.01.27.635168

Figure Lengend Snippet: Human fibroblasts transduced with lentivirus encoding scrambled control or DHX29 specific shRNAs were infected with HCMV (MOI of 3). Six hours post infection HCMV immediate early mRNA (A) and protein levels (B) were analyzed by qRT-PCR and Western blot, respectively (B). C) Polysome formation in control or DHX29-depleted cells was measured by sucrose gradient centrifugation at 6 hours after HCMV infection. D) The percent of cytosolic RNA found in each gradient fraction was measured by qRT-PCR for the IE1, IE2, and β-actin mRNA. E) Graph of data in D, comparing the amount of each mRNA found in gradient fractions containing polysomes in control or DHX29 -depleted cells. The amount of each mRNA in control cells was set to 100%. The mean data from 3 independent experiments is shown. (* p<0.05; ** p<0.005; *** p<0.001)

Article Snippet: Membranes were probed with antibodies to DHX29 (Santa Cruz Biotechnology), eIF4E (Cell Signaling Technology), β-Actin (Santa Cruz Biotechnology), rpS6 (Cell Signaling Technology), rpL13a (Cell Signaling Technology), HCMV IE1, IE2, UL44, UL99 (pp28), eIF4G (Cell Signaling Technology), 4E-BP (Cell Signaling Technology), 4E-BP-phospho (Cell Signaling Technology), rpS6-phospho (Cell Signaling Technology) and HSP90 (Stressgen).

Techniques: Transduction, Control, Infection, Quantitative RT-PCR, Western Blot, Gradient Centrifugation

Journal: bioRxiv

Article Title: The Host DHX29 RNA Helicase Regulates HCMV Immediate Early Protein Synthesis

doi: 10.1101/2025.01.27.635168

Figure Lengend Snippet: A) Human fibroblasts transduced with lentivirus expressing scrambled control or DHX29 specific shRNAs were infected with HCMV (MOI of 3). Six hours post infection m 7 G cap binding complexes were precipitated from cell lysates and analyzed by Western blot. B) Cells were treated as in A, and cytoplasmic extracts were fractionated by sucrose gradient centrifugation. The percent of the eIF4G, HSP90, or Actin in each gradient fraction was measured by qRT-PCR. C) Graph comparing the amount of each RNA in gradient fractions containing polysomes in control or shDHX29 expressing cells. The amount of each mRNA in control cells was set to 100%. D) Western blot showing the phoshorylation state of the mTOR substrates 4E-BP1 and rpS6 in control or DHX29 depleted cells. The mean data from 3 independent experiments is shown. (* p<0.05; ** p<0.005; *** p<0.001)

Article Snippet: Membranes were probed with antibodies to DHX29 (Santa Cruz Biotechnology), eIF4E (Cell Signaling Technology), β-Actin (Santa Cruz Biotechnology), rpS6 (Cell Signaling Technology), rpL13a (Cell Signaling Technology), HCMV IE1, IE2, UL44, UL99 (pp28), eIF4G (Cell Signaling Technology), 4E-BP (Cell Signaling Technology), 4E-BP-phospho (Cell Signaling Technology), rpS6-phospho (Cell Signaling Technology) and HSP90 (Stressgen).

Techniques: Transduction, Expressing, Control, Infection, Binding Assay, Western Blot, Gradient Centrifugation, Quantitative RT-PCR