Journal: Journal of Virology

Article Title: Epstein-Barr Virus Induces Global Changes in Cellular mRNA Isoform Usage That Are Important for the Maintenance of Latency

doi: 10.1128/JVI.02464-13

Figure Lengend Snippet: XBP1 alternative isoform usage and lytic reactivation. (A) Exon schematics of the XBP1 gene. Red blocks, SplicerEX UP probe sets; blue blocks, DOWN probe sets; arrows above the schematics, the primers used in RT-PCRs; horizontal hashes between the isoform schematics, amplicons generated in qRT-PCRs. (B) Gels of RT-PCR specifically targeting the long (top; primer set 1) and short (middle; primer set 2) XBP1 isoforms as well as a downstream region common to both isoforms (bottom; primer set 3) from two donor-matched B-cell and LCL pairs and BL41 versus BL41/B95-8 cells using the primers shown in panel A. The quantification below the gel represents the frequency of the long XBP1 isoform relative to the total amount of XBP1 mRNA present. (C) Isoform-specific qRT-PCR depicting the frequency of long XBP1 relative to the total amount of XBP1 using the amplicons shown in panel A. (D) Exon schematics depicting the IRE1-dependent alternative splicing of XBP1. Arrows above the schematics, the primers used in RT-PCRs. (E) Gel of RT-PCR querying IRE1-mediated splicing. XBP1u contains a PstI restriction site, and this site is lost in XBP1s. Digestion of the PCR product with PstI results in selective cleavage of the XBP1u product. (F) Schematic of Ig antibody-induced transcriptional activation of Z via IRE1-dependent splicing of XBP1. (G) Gel of RT-PCR of Akata cells treated with 5 μg/ml anti-IgG antibody for the indicated times in the presence or absence of the IRE1 inhibitor STF. Arrows, XBP1u, the unspliced transcript, and XBP1h, the hybrid product. (H) Gel of RT-PCR of ES-1 cells treated with anti-Ig antibody in the presence or absence of STF, as described for panel G. (I) qRT-PCR of the induction of endogenous Z by Ig antibody and inhibition by STF in EBV-positive Akata cells. DMSO, dimethyl sulfoxide. (J) qRT-PCR of the induction of endogenous Z by anti-Ig antibody and inhibition by STF in ES-1 LCLs.

Article Snippet: GFP-positive cells were placed back in the R15 medium and treated for 0 or 8 h with 100 μg/ml anti-IgG antibody from Jackson ImmunoResearch before harvesting for RNA.

Techniques: Generated, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Activation Assay, Inhibition

Journal: Journal of Virology

Article Title: Epstein-Barr Virus Induces Global Changes in Cellular mRNA Isoform Usage That Are Important for the Maintenance of Latency

doi: 10.1128/JVI.02464-13

Figure Lengend Snippet: TCF4 alternative isoform usage and lytic reactivation. (A) Plot of expression levels of TCF4 meta-probe sets in the 5′ → 3′ direction from left to right in B cells and LCLs. (B) Exon schematics of the TCF4 gene. Red blocks, SplicerEX UP probe sets; blue blocks, DOWN probe sets, single-headed arrows above the schematics, the primers used in RT-PCRs; horizontal hashes between isoform schematics, amplicons generated in qRT-PCRs. Below the isoform schematics is a representation of the TCF4 protein organization. bHLH, basic helix-loop-helix. (C) Gel of isoform-specific RT-PCR of two donor-matched B-cell and LCL pairs and BL41 versus BL41/B95-8 cells using the primers depicted in panel B. Upper band (primers 2 and 3), total amount of the TCF4 transcript in the cells; lower band (primers 1 and 3), amount of TCF4-FL specifically. (D) Isoform-specific qRT-PCR depicting the frequency of TCF4-FL relative to the B-cell average using the amplicons shown in panel B. (E) Western blot of TCF4-FL in BL41 and BL41/B95-8 cells. Densitometry was performed, and the ratio of TCF4-FL/TCF4-S detected is shown. The results for EBNA3C as a control for EBV latent infection are shown. The results for MAGOH as a loading control are shown. (F) Endogenous BZLF1 expression in Akata cells in response to anti-IgG antibody treatment and/or TCF4-FL overexpression. GFP-only, samples transfected with GFP only; TCF4-FL, samples were cotransfected with GFP and a plasmid expressing TCF4-FL. Values are relative to the Z mRNA levels in B95-8 cells. (G) BMRF1 expression in Akata cells in response to anti-IgG antibody treatment and/or TCF4-FL overexpression. Values are relative to the BMRF1 levels in B95-8 cells.

Article Snippet: GFP-positive cells were placed back in the R15 medium and treated for 0 or 8 h with 100 μg/ml anti-IgG antibody from Jackson ImmunoResearch before harvesting for RNA.

Techniques: Expressing, Generated, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Infection, Over Expression, Transfection, Plasmid Preparation