Journal: PLoS ONE

Article Title: Poly(A) Binding Protein 1 Enhances Cap-Independent Translation Initiation of Neurovirulence Factor from Avian Herpesvirus

doi: 10.1371/journal.pone.0114466

Figure Lengend Snippet: (A) Partial sequence from the full length 5L IRES from the immediate-early 1.8-kb mRNA that encodes pp14b isoform from Marek’s disease virus serotype 1. The 5L IRES spans nucleotides 129339-129798 (acc: AF243348). The internal poly-pyrimidine sequences C 13 and U 11 are boxed as well as the internal poly(A) sequences A 11 and A 9 . (B) Schematic of the DNA constructs used for the luciferase reporter assay. In this vector the translation of R-Luc is controlled by the 5L IRES whereas the F-Luc is under the control of the intercistronic IRES (ICR). This configuration mimics the dual IRES bicistronic 1.8-kb mRNA from MDV1. DF-1 cells were transiently transfected with the indicated DNA vectors and after 24 h the cells were lysed and the luciferase activities were measured. The results are expressed as per cent change in luciferase activity relative to the control wild type sequence (5Lwt). (C) Northern blotting was performed on total RNA extracted from cells transfected with DNA constructs depicted in B. Hybridization was done with a random-primed 32P-labelled DNA fragment corresponding to the 5' end of the F-Luc open reading frame. Ethidium bromide-staining of the gel used for Northern blot is shown below the blot with 18S/28S rRNAs as size markers and loading control. (D) The mutated nucleotides within the internal poly(A) from the 5L IRES are underlined. The corresponding DNA vectors were used to transfect DF-1 cells as described in B. For simplicity, only the R-Luc values are shown as the F-Luc follows the same trend due to the coevolved synergistic functional relationship between the 5L IRES and the ICR IRES. The results are expressed as per cent change relative to the control wild type sequence (5Lwt). The experiment was repeated three times and the SEM is shown. (E) Purified recombinant human PABP1 (0.5 µM) was incubated with 32 P-end labelled 5L IRES RNAs from wild type or from the indicated mutants and separated on a native 6% polyacrylamide gel by Electrophoretic Mobility Shift Assay (EMSA). It should be noted that the RNA was obtained by in vitro transcription and that it has no 3' poly (A) tail. The complex between PABP1 and the 5L IRES RNA was visualized by autoradiography using phosphor screen. The complex 5L IRES/PABP1 is observed in all combinations except with the mutants 5Lmt2 and 5Lmt2&3.

Article Snippet: Additional TaqMan miRNA and gene expression assays used in this study were purchased from Applied Biosystems and are: mdv1-miR-M13 (assay ID 007739_mat), mdv1-miR-M10 (assay ID 007565_mat) and paip2 (assay ID Gg03370296_m1).

Techniques: Sequencing, Virus, Construct, Luciferase, Reporter Assay, Plasmid Preparation, Control, Transfection, Activity Assay, Northern Blot, Hybridization, Random Primed, Staining, Functional Assay, Purification, Recombinant, Incubation, Electrophoretic Mobility Shift Assay, In Vitro, Autoradiography

Journal: PLoS ONE

Article Title: Poly(A) Binding Protein 1 Enhances Cap-Independent Translation Initiation of Neurovirulence Factor from Avian Herpesvirus

doi: 10.1371/journal.pone.0114466

Figure Lengend Snippet: ( A ) The paip2 transcript showing paip2 open reading frame (ORF), the 3' untranslated region (UTR) and the miRNA response elements (MRE). ( B ) Schematic of the reporter construct containing individual or combined MREs sequences downstream of the simian virus 40 promoter-driven Renilla luciferase cassette from psiCHECK-2 vector. ( C ) The predicted duplexes between paip2 mRNA and MDV1 miRNAs. The mutated nucleotides within the seed regions of paip2 mRNA are underlined. ( D ) Luciferase-based miRNA reporter assay. The full length region from the paip2 mRNA that contains all the MREs or the individual MREs and their mutated versions were made as synthetic oligonucleotides and sub-cloned into the sensor plasmid downstream of the Renilla luciferase in psiCHECK-2 vector. The resulting constructs were used to transfect MSB1; an MDV1-transformed CD4+ T-cell line derived from a spleen lymphoma induced by BC-1 strain of MDV1 constitutively expressing viral miRNAs. As positive control for assay validation we have used MRE-M4 that was previously shown to be targeted by MDV1 miRNA-M4. The normalized Renilla luciferase activities from five experiments are shown with the error bars (SEM) relative to that seen for the empty vector psiCHECK-2 which value is set to 1.

Article Snippet: Additional TaqMan miRNA and gene expression assays used in this study were purchased from Applied Biosystems and are: mdv1-miR-M13 (assay ID 007739_mat), mdv1-miR-M10 (assay ID 007565_mat) and paip2 (assay ID Gg03370296_m1).

Techniques: Construct, Virus, Luciferase, Plasmid Preparation, Reporter Assay, Clone Assay, Transformation Assay, Derivative Assay, Expressing, Positive Control, Biomarker Discovery

Journal: PLoS ONE

Article Title: Poly(A) Binding Protein 1 Enhances Cap-Independent Translation Initiation of Neurovirulence Factor from Avian Herpesvirus

doi: 10.1371/journal.pone.0114466

Figure Lengend Snippet: ( A ) Chicken embryo fibroblasts (CEF) were transfected with oncogenic BAC clone pRB1B5 of MDV1 or mock-transfected for 72 h. Total proteins were harvested and analysed by immunoblotting with the indicated antibodies. Quantification of the immunoblots from panel A using ImageQuant software is shown to the right. The results are from two independent experiments each in duplicate. ( B ) Total proteins were extracted from control samples or from samples taken from chicken infected with the oncogenic BAC clone pRB1B5 derived from archive samples. Proteins were analysed by immunoblotting as in panel A . Quantification of the immunoblots from panel B using ImageQuant software is shown to the right. The results are repeats from two different archive samples derived from the same chicken challenge experiment. ( C ) Indirect immunofluorescence of pRB1B5-infected CEF 72 h posttransfection. A series of optical sections were taken sequentially for each channel along the z-axis using a step size of 0.290 µm. The resulting 3D confocal image was reconstructed using IMARIS software. DAPI-staining shows the nucleus in blue, PABP1 in red and pp14 in green, the scale bar: 10 µm.

Article Snippet: Additional TaqMan miRNA and gene expression assays used in this study were purchased from Applied Biosystems and are: mdv1-miR-M13 (assay ID 007739_mat), mdv1-miR-M10 (assay ID 007565_mat) and paip2 (assay ID Gg03370296_m1).

Techniques: Transfection, Western Blot, Software, Control, Infection, Derivative Assay, Immunofluorescence, Staining