alkbh1 specific  (Revvity Signals)

Journal: bioRxiv

Article Title: Dengue virus exploits the host tRNA epitranscriptome to promote viral replication

doi: 10.1101/2023.11.05.565734

Figure Lengend Snippet: ( a ) ALKBH1 and DENV2 NS5 immunoblots of DENV2 NGC-and mock-infected cells (left). Densitometric quantitation of NS5 protein (right). ( b ) siRNA knockdown of ALKBH1 assessed by immunoblotting. ( c ) LC-MS/MS profiling of tRNA modifications in ALKBH1 knockdown versus control knockdown cells. ( d ) DENV2 NS3 immunoblot analysis in ALKBH1 knockdown versus control knockdown cells (left). Densitometry quantitation of NS3 protein (right). ( e ) New virus particle production in ALKBH1 knockdown versus control knockdown cells determined by plaque assay. ( f ) DENV2 NS3 immunoblot analysis in ALKBH1-overexpressing versus control cells (left). Densitometry quantitation of NS3 protein (right). ( g ) LC-MS/MS profiling of tRNA modifications in ALKBH1-overexpressing versus control cells. ( h ) New virus particle production at 24 hpi in ALKBH1-overexpressing versus control cells determined by plaque assay. Graphs in all panels show mean ±} SD for 3 biological replicates. All statistical significance was determined using Student’s t test, *p<0.05; **p<0.01.

Article Snippet: Transfections were performed using 25 nM ALKBH1-specific (8846, Dharmacon) or non-targeting control (D-001810-10-05, Dharmacon) siRNA, 500 ng plasmid DNA and lipofectamine 3000 (Invitrogen) as indicated, according to the manufacturer’s instructions.

Techniques: Western Blot, Infection, Quantitation Assay, Liquid Chromatography with Mass Spectroscopy, Virus, Plaque Assay

Journal: bioRxiv

Article Title: Dengue virus exploits the host tRNA epitranscriptome to promote viral replication

doi: 10.1101/2023.11.05.565734

Figure Lengend Snippet: Cell viability determined by MTT assay of Huh-7 cells treated with (a) non-targeting and ALKBH1-specific siRNA, and (b) control empty vector and ALKBH1 plasmid, at 24h and 48h post-transfection.

Article Snippet: Transfections were performed using 25 nM ALKBH1-specific (8846, Dharmacon) or non-targeting control (D-001810-10-05, Dharmacon) siRNA, 500 ng plasmid DNA and lipofectamine 3000 (Invitrogen) as indicated, according to the manufacturer’s instructions.

Techniques: MTT Assay, Plasmid Preparation, Transfection

Journal: bioRxiv

Article Title: Dengue virus exploits the host tRNA epitranscriptome to promote viral replication

doi: 10.1101/2023.11.05.565734

Figure Lengend Snippet: ( a ) Illustration of ALKBH1 transcript with indicated siRNA-targeting sites. ( b ) Manipulation of ALKBH1 levels by siRNA knockdown, plasmid overexpression, and complementation using a siRNA-resistant ALKBH1 plasmid as assessed by immunoblotting. ( c ) LC-MS/MS profiling of modifications in f 5 Cm biogenesis of cells with indicated treatments versus cells treated with control siRNA and control plasmid. ( d ) Table of ALKBH1-dependent proteins obtained from proteomics analysis of treated cells from panel c . ( e ) Plot of p values derived from analysis of codon Z-scores of ALKBH1 negatively-regulated versus positively-regulated transcripts. ( f,g ) Z-score analysis (number of standard deviations above/below the mean) of leucine ( f ) and arginine ( g ) codons in ALKBH1 negatively-regulated (□) versus ALKBH1 positively-regulated (▪) transcripts. ( h ) Table of GO term and KEGG analysis of proteins deregulated by ALKBH1 depletion assessed by DAVID. Statistical significance was determined using Student’s t test, *p<0.05; **p<0.01.

Article Snippet: Transfections were performed using 25 nM ALKBH1-specific (8846, Dharmacon) or non-targeting control (D-001810-10-05, Dharmacon) siRNA, 500 ng plasmid DNA and lipofectamine 3000 (Invitrogen) as indicated, according to the manufacturer’s instructions.

Techniques: Plasmid Preparation, Over Expression, Western Blot, Liquid Chromatography with Mass Spectroscopy, Derivative Assay

Journal: bioRxiv

Article Title: Dengue virus exploits the host tRNA epitranscriptome to promote viral replication

doi: 10.1101/2023.11.05.565734

Figure Lengend Snippet: (a) Densitometric quantitation of NS3 protein levels at indicated times after infection of Huh-7 cells with DENV strain EDEN2 infection. Huh-7 cells were pre-treated with control non-targeting siRNA (blue circles) and ALKBH1-specific siRNA (orange circles). (b) Differences in codon usage frequencies in DENV2 NGC strain versus human host genome. Leucine UUA codon (red arrow) is nearly equally represented in both human and DENV2 genomes, while other leucine codons (black arrows) are not shared equally.

Article Snippet: Transfections were performed using 25 nM ALKBH1-specific (8846, Dharmacon) or non-targeting control (D-001810-10-05, Dharmacon) siRNA, 500 ng plasmid DNA and lipofectamine 3000 (Invitrogen) as indicated, according to the manufacturer’s instructions.

Techniques: Quantitation Assay, Infection

Journal: bioRxiv

Article Title: Dengue virus exploits the host tRNA epitranscriptome to promote viral replication

doi: 10.1101/2023.11.05.565734

Figure Lengend Snippet: In early stages of DENV infection, host cellular ALKBH1 levels decrease resulting in reduced f 5 Cm-modified ct-tRNA Leu(CAA) reduced decoding of non-cognate UUA codon. Transcripts lacking UUA are consequently preferentially translated and many of which have virus-associated functions, resulting in pro-viral translational remodeling of the host proteome to facilitate viral replication. As the virus replicates and translates its own mRNA, accumulating the MTase NS5 in the cell at later stages of infection, NS5 contributes to f 5 Cm-modification of tRNA, increasing UUA decoding and further cellular translational remodeling that may contribute to viral exocytosis.

Article Snippet: Transfections were performed using 25 nM ALKBH1-specific (8846, Dharmacon) or non-targeting control (D-001810-10-05, Dharmacon) siRNA, 500 ng plasmid DNA and lipofectamine 3000 (Invitrogen) as indicated, according to the manufacturer’s instructions.

Techniques: Infection, Modification, Virus