Journal:

Article Title: Antiapoptotic and Oncogenic Potentials of Hepatitis C Virus Are Linked to Interferon Resistance by Viral Repression of the PKR Protein Kinase

doi:

Figure Lengend Snippet: Inducible expression of NS5A reduces IFN sensitivity of viral mRNA translation. (A) Inducible expression of NS5A 1B in HeLa 1B cells. Extracts (50 μg) prepared from Tet− (lane 1) and Tet+ (lane 2) cultures of HeLa 1B cells were analyzed by immunoblotting using a MAb specific to NS5A. The arrowhead denotes the position of NS5A 1B. We confirmed that PKR was efficiently expressed in cells from Tet− and Tet+ cultures (not shown). Positions of molecular mass standards are indicated in kilodaltons. (B) Expression of NS5A supports viral mRNA translation in IFN-treated HeLa 1B cells infected with VSV. Viral protein synthesis in cells treated with increasing concentrations of IFN was determined by biosynthetic labeling and autoradiography as described in Materials and Methods. The level of each viral protein was quantitated from autoradiograms by using a Bio-Rad GS700 imaging densitometer and computer software supplied by the manufacturer. Panels show the biosynthesis of the 29-kDa VSV matrix protein (denoted by arrowheads) in the presence (NS5A 1B+; lower panel) and absence (NS5A 1B−; upper panel) of NS5A 1B expression. The far-left lane of each panel shows an extract prepared from uninfected, untreated control cultures. Lanes represent IFN concentrations of 0 (lanes 1 and 2), 50 (lane 3), 100 (lane 4), 150 (lane 5), 200 (lane 6), 300 (lane 7), 400 (lane 8), 500 (lane 9), 600 (lane 10), 750 (lane 11), and 1,000 (lane 12) U/ml. (C) The relative level of matrix protein translation for each sample was determined by first subtracting the optical density (within the region indicated for mock-infected extracts by brackets in panel B) from that obtained for each subsequent lane. Data are presented as a ratio of VSV matrix protein translation in cells expressing NS5A to that observed in cells not expressing NS5A, for each concentration of IFN. The value of each ratio is shown above the corresponding bar.

Article Snippet: HeLa S3 Tet-Off cells (Clontech) were transfected with pTRE-NS5A 1B or pTRE-NS5A 1B-5 and selected in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), l -glutamine (200 μg/ml), G418 (100 μg/ml), hygomycin B (100 μg/ml), and Tet (2 μg/ml).

Techniques: Expressing, Western Blot, Infection, Labeling, Autoradiography, Imaging, Software, Concentration Assay

Journal:

Article Title: Antiapoptotic and Oncogenic Potentials of Hepatitis C Virus Are Linked to Interferon Resistance by Viral Repression of the PKR Protein Kinase

doi:

Figure Lengend Snippet: ISDR mutations within the PKR-binding domain of NS5A confer IFN sensitivity to viral mRNA translation. (A) Inducible expression of NS5A 1B and NS5A 1B-5 in HeLa S3 cells. Extracts (50 μg) prepared from Tet− (lanes 1 and 3) and Tet+ (lane 2 and 4) cultures of HeLa 1B (lanes 1 and 2) and HeLa 1B-5 (lanes 3 and 4) cell lines were analyzed by immunoblotting using a MAb specific to NS5A. Arrowheads denote the positions NS5A, which migrates on SDS-PAGE as hypo- and hyperphosphorylated isoforms (60). We confirmed that PKR was efficiently expressed in both cell lines in the presence and absence of Tet (not shown). Positions of molecular mass standards are indicated in kilodaltons. (B) Expression of NS5A 1B, but not NS5A 1B-5, supports viral mRNA translation in IFN-treated HeLa cell lines infected with VSV. Viral protein synthesis in HeLa 1B (upper panel) and HeLa 1B-5 (lower panel) cell lines, cultured in the absence of Tet and treated with increasing concentrations of IFN, was determined by biosynthetic labeling and autoradiography as described in Materials and Methods. The level of each viral protein was quantitated as described for Fig. ​Fig.2.2. Panels show the biosynthesis of the 29-kDa VSV matrix protein (denoted by arrowheads) in the presence NS5A 1B and NS5A 1B-5 (upper and lower panels, respectively). The far-left lane of each panel shows an extract prepared from uninfected, untreated control cultures. Lanes represent IFN concentrations of 0 (lanes 1 and 2), 50 (lane 3), 100 (lane 4), 150 (lane 5), 200 (lane 6), 300 (lane 7), 400 (lane 8), 500 (lane 9), 600 (lane 10), 750 (lane 11), and 1,000 (lane 12) U/ml. (C) Translation ratios. The relative level of matrix protein translation for each sample was determined as for Fig. ​Fig.2.2. Data are presented as a ratio of VSV matrix protein translation in cells expressing NS5A 1B to that observed in cells expressing NS5A 1B-5, for each concentration of IFN. The value of each ratio is shown above the corresponding bar.

Article Snippet: HeLa S3 Tet-Off cells (Clontech) were transfected with pTRE-NS5A 1B or pTRE-NS5A 1B-5 and selected in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), l -glutamine (200 μg/ml), G418 (100 μg/ml), hygomycin B (100 μg/ml), and Tet (2 μg/ml).

Techniques: Binding Assay, Expressing, Western Blot, SDS Page, Infection, Cell Culture, Labeling, Autoradiography, Concentration Assay

Journal:

Article Title: U2AF modulates poly(A) length control by the poly(A)-limiting element

doi: 10.1093/nar/gkg823

Figure Lengend Snippet: Impact of U2AF65 and PAP-interacting domain deletions on PLE regulation of poly(A) tail length. (A) Plasmids expressing wild-type U2AF65 or U2AF65 deleted for amino acids 17–27 (Δ17–27) or 17–47 (Δ17–47) were transfected into CHO cells and expression was analyzed by western blot using a polyclonal antibody to human U2AF65. In lane 1 cells were transfected with pcDNA3 alone. (B) HeLa S3 (Tet-Off) cells were transfected in tetracycline-containing medium with the indicated U2AF65 constructs plus tetracycline-regulated plasmids bearing human β-globin reporter genes that lack (SPA) or contain a PLE (PLE). Poly(A) tail length was analyzed by RT–PCR on RNA isolated 30 h after transfection and 6 h after removing tetracycline from the medium. The center lane (lane 5) contains a marker of Hinf φX174 DNA fragments. (C) The graphing function of the ImageQuant™ program was used to determine the distribution of radioactivity in each of the lanes in (B). Note that the scales for control and PLE-containing mRNAs are different.

Article Snippet: Tet-Off HeLa S3 cells and CHO cells were purchased from Clontech and maintained in DMEM with 10% FBS, 4 mM glutamine and 100 µg/ml G418 (Invitrogen).

Techniques: Expressing, Transfection, Western Blot, Construct, Reverse Transcription Polymerase Chain Reaction, Isolation, Marker, Radioactivity

Journal:

Article Title: U2AF modulates poly(A) length control by the poly(A)-limiting element

doi: 10.1093/nar/gkg823

Figure Lengend Snippet: Impact of overexpressing U2AF65 on polyadenylation of mRNA with the C14G mutation. HeLa S3 (Tet-Off™) cells were transfected as in Figure ​Figure66 with tetracycline-regulated plasmids bearing the PLE or C14G element in the last exon of the β-globin reporter gene plus empty vector (pcDNA) and CMV-driven plasmids expressing full-length U2AF65 or the Δ17–47 mutant form of U2AF lacking the PAP-interacting domain. (A) Poly(A) tail length was determined by RT–PCR as in Figure ​Figure66 and equal amounts of radiolabeled products were applied to the gel. Lane 1 (M) contains a marker of Hinf φX174 DNA fragments. (B) The graphing function of the ImageQuant™ program was used to determine the distribution of radioactivity in each of the lanes for PLE-containing mRNA. The dashed line corresponds to cells transfected with the Δ17–47 form of U2AF65.

Article Snippet: Tet-Off HeLa S3 cells and CHO cells were purchased from Clontech and maintained in DMEM with 10% FBS, 4 mM glutamine and 100 µg/ml G418 (Invitrogen).

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction, Marker, Radioactivity