Live vaccine infection burden elicits adaptive humoral and cellular immunity required to prevent Zika virus infection

10.1016/j.ebiom.2020.103028EBioMedicine6110302

The B-Cell Specific Transcription Factor, Oct-2, Promotes Epstein-Barr Virus Latency by Inhibiting the Viral Immediate-Early Protein, BZLF1

The Epstein-Barr virus (EBV) latent-lytic switch is mediated by the BZLF1 immediate-early protein. EBV is normally latent in memory B cells, but cellular factors which promote viral latency specifically in B cells have not been identified. In this report, we demonstrate that the B-cell specific transcription factor, Oct-2, inhibits the function of the viral immediate-early protein, BZLF1, and prevents lytic viral reactivation. Co-transfected Oct-2 reduces the ability of BZLF1 to activate lytic gene expression in two different latently infected nasopharyngeal carcinoma cell lines. Furthermore, Oct-2 inhibits BZLF1 activation of lytic EBV promoters in reporter gene assays, and attenuates BZLF1 binding to lytic viral promoters in vivo. Oct-2 interacts directly with BZLF1, and this interaction requires the DNA-binding/dimerization domain of BZLF1 and the POU domain of Oct-2. An Oct-2 mutant (Δ262–302) deficient for interaction with BZLF1 is unable to inhibit BZLF1-mediated lytic reactivation. However, an Oct-2 mutant defective for DNA-binding (Q221A) retains the ability to inhibit BZLF1 transcriptional effects and DNA-binding. Importantly, shRNA-mediated knockdown of endogenous Oct-2 expression in several EBV-positive Burkitt lymphoma and lymphoblastoid cell lines increases the level of lytic EBV gene expression, while decreasing EBNA1 expression. Moreover, treatments which induce EBV lytic reactivation, such as anti-IgG cross-linking and chemical inducers, also decrease the level of Oct-2 protein expression at the transcriptional level. We conclude that Oct-2 potentiates establishment of EBV latency in B cells

AN INVESTIGATION INTO ZIKA LIVE ATTENUATED VACCINE DEVELOPMENT

Ph.DDOCTOR OF PHILOSOPH

Hypothesized model for Oct-1 and Oct-2 regulation of EBV lytic reactivation.

<p>(A) Oct-1 enhances lytic reactivation through a direct protein-protein interaction between Oct-1 and the IE protein BRLF1, which promotes BRLF1 DNA-binding and tethers Oct-1 to viral DNA. (RRE; BRLF1 responsive element) (B) Oct-2 promotes latency by interacting with the BZLF1 IE protein and inhibiting its DNA-binding. (ZRE; BZLF1 responsive element).</p

Loss of endogenous Oct-2 increases constitutive and induced lytic gene expression in EBV-infected B cells.

<p>(A) MutuI cells were infected with a pool of five different lentivirus vectors directed against Oct-2, individual lentivirus vectors directed against Oct-2, or control shRNAs. The cells were selected for 7 days using puromycin. Immunoblot analysis was performed to compare the levels of endogenous Oct-2, BRLF1, BZLF1, BMRF1, and β-actin (loading control) in each condition. The level of Oct-2 in each condition was quantitated relative to the Control #2 (set at 100) and is shown as a numerical value below the Oct-2 immunoblot. (B–D) KemI, Raji, and LCL cells were infected with a pool of five different lentivirus vectors directed against Oct-2 or control shRNAs and selected for 7 days using puromycin. The level of Oct-2 in each condition was quantitated relative to the untreated shControl (set at 100) and is shown as a numerical value below the Oct-2 immunoblot. (B) The type I BL cell line, KemI, was then subjected to immunoblot analysis to compare the levels of endogenous Oct-2, BZLF1, BMRF1, and β-actin (loading control) in both shOct-2 knockdown and control conditions. (C) Raji, a type III BL cell line, was treated with increasing amounts of TPA alone (4 or 20 ng/mL) or TPA (4 or 20 ng/mL) and sodium butyrate (0.6 or 3 mM) as indicated. Immunoblot analysis was performed after 24 hours to compare the levels of endogenous Oct-2, BZLF1, BMRF1, and β-actin (loading control) in each condition. (D) The type III lymphoblastoid cell line (LCL) was treated with 20 ng/mL TPA and 3 mM sodium butyrate for 48 hours to induce lytic reactivation, followed by immunoblot analysis for endogenous Oct-2, BZLF1, BMRF1, and β-actin (loading control). (E) RNA was isolated from control and shOct-2 infected MutuI cells, reverse-transcribed into cDNA, and analyzed for the level of viral gene expression using qRT-PCR as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002516#s4" target="_blank">Materials and Methods</a>. Shown is a heatmap depicting the fold-activation of gene expression in the shOct-2 MutuI cells relative to the control vector infected cells. The EBV genes are grouped according to their gene expression profile (IE lytic, early lytic, late lytic, and latent). The fold gene activation is indicated by the color shade.</p

Oct-2 inhibits BZLF1 activation of multiple lytic EBV promoters in EBV-negative cells.

<p>EBV-negative HONE-1 cells were transfected with (A) BRLF1p-luciferase construct, (B) BMRF1p-luciferase construct, (C) BXLF1p-luciferase construct, or (D) BALF2p-luciferase construct in the presence or absence of co-transfected BZLF1 (5 ng), Oct-2 (100 ng), or control vectors as indicated. The fold luciferase activity for each condition is shown relative to control vector; the value for the activity of the promoter construct plus the vector control is set at 1. Values are given as means ± standard deviations of results from two replicates. EBV-negative HONE-1 cells were transfected with the (E) BMRF1p-luciferase construct or (F) SMp-luciferase construct in the presence or absence of co-transfected BZLF1 (5 ng), BRLF1 (5 ng), Oct-2 (100 ng), or control vectors as indicted.</p

Oct-2 knockdown decreases EBNA1 protein expression in cells with type I and type III latency.

<p>(A) Type I BL cell lines, MutuI and KemI, and the type III lymphoblastoid cell line (LCL) were infected with a pool of five different lentivirus vectors directed against Oct-2, or control shRNAs. The cells were selected for 7 days using puromycin. The expression levels of the latency proteins EBNA1, EBNA2, EBNA-LP, and LMP1 were examined using immunoblot analysis. β-actin expression was used as a loading control. (B) The level of the EBNA1 transcript was examined by RT-PCR in type III LCL and Raji cells which were infected with a pool of five different lentivirus vectors directed against Oct-2, or control shRNAs. The cells were selected for 7 days using puromycin prior to RT-PCR analysis. The β₂-microglobulin gene was used as a control.</p

Oct-2 inhibits lytic EBV replication.

<p>(A) CNE-2 Akata cells were transfected with 5 ng BZLF1, 500 ng Oct-2, or control expression vectors as indicated. Immunoblot analysis was performed to compare the levels of transfected Oct-2 and BZLF1, as well as the lytic viral proteins BMRF1 and BRLF1 derived from the endogenous viral genome. β-actin expression was used as a loading control. (B) The amount of infectious virus produced by each condition in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002516#ppat-1002516-g002" target="_blank">Figure 2A</a> was determined using the green Raji cell assay as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002516#s4" target="_blank">Materials and Methods</a>.</p

Oct-2 interacts with BZLF1 DNA-binding/dimerization domain, and BZLF1 interacts with Oct-2 POU domain.

<p>(A) Schematic of the BZLF1 protein transcriptional activation (TA), basic DNA-binding (DNA), dimerization (DIM), and C-terminal tail (Tail) domains. Numbers represent amino acid positions. Full-length (FL), as well as various BZLF1 truncation proteins used in subsequent GST pull-down assays are also depicted. (B) GST pull-down assays were performed using GST, GST-Oct-2, or various GST-BZLF1 truncation fusion proteins incubated with ³⁵S-labeled, <i>in vitro</i>-translated Oct-2 protein. Twenty percent of the direct load was used for autoradiography. The amount of Oct-2 binding in each condition, quantified using ImageJ software, is depicted in a bar graph at the bottom of the gel; the level of binding obtained with the full-length (1–245) BZLF1 protein is set at 100 percent. (C) GST pull-down assays were performed using GST, GST-BZLF1, or GST-Oct-2 fusion proteins incubated with ³⁵S-labeled, <i>in vitro</i>-translated wild-type BZLF1 or mutant BZLF1 (Y200E/L225E) protein. Twenty percent of the direct load was used for autoradiography. (D) Schematic of the Oct-2 protein POU specific domain (POUs), linker region (L), and POU homeodomain (POUh). Numbers represent amino acid positions. Various Oct-2 truncation proteins used in subsequent GST pull-down assays are also depicted. (E) GST pull-down assays were performed using GST, GST-BZLF1, or various GST-Oct-2 truncation fusion proteins incubated with ³⁵S-labeled, <i>in vitro</i>-translated BZLF1 protein. Twenty percent of the direct load was used for autoradiography. The amount of BZLF1 binding in each condition, quantified using ImageJ software, is depicted in a bar graph at the bottom of the gel; the level of binding obtained with Oct-2 (179–343) protein is set at 100 percent. (F) GST pull-down assays were performed using GST or GST-BZLF1 fusion protein incubated with ³⁵S-labeled, <i>in vitro</i>-translated (full-length) wild-type Oct-2 or mutant Oct-2 (Δ262–302) protein. Twenty percent of the direct load was used for autoradiography. (G) HONE-Akata cells were transfected with 5 ng BZLF1, 50 ng wild-type Oct-2, 50 ng mutant Oct-2 (Δ262–302), or control vectors as indicated. Immunoblot analysis was performed two days after transfection to compare levels of transfected BZLF1 and Oct-2, as well as the level of BMRF1 derived from the endogenous viral genome. β-actin served as a loading control.</p

Oct-2 DNA-binding activity is not required for Oct-2 inhibition of BZLF1 function.

<p>(A) (Left panel) An EMSA was performed using <i>in vitro</i>-translated wild-type or mutant Oct-2 (Q221A) protein and a ³²P-labelled oligonucleotide probe containing the consensus Oct-2 binding site. Protein-DNA complexes are indicated by arrows. (Right panel) Immunoblot analysis was performed to compare the levels of wild-type and mutant Oct-2 (Q221A) in the <i>in vitro</i>-translated samples. (B) HONE-Akata cells were transfected with 5 ng BZLF1, 500 ng wild-type Oct-2, 500 ng mutant Oct-2 (Q221A), or control vectors as indicated. Immunoblot analysis was performed two days after transfection to compare levels of transfected BZLF1 and Oct-2, as well as the levels of BMRF1 and BRLF1 protein derived from the endogenous viral genome. β-actin served as a loading control. (C) EBV-negative BJAB cells were transfected with a BMRF1p-luciferase construct in the presence or absence of co-transfected BZLF1 (30 ng), wild-type Oct-2 (1470 ng), two different mutant Oct-2 proteins (Oct-2 Q221A or Oct-2 Δ262–302) (1470 ng), or control vectors as indicated. The fold luciferase activity for each condition is shown relative to control vector; the value for the activity of the promoter construct plus the vector control is set at 1. Values are given as means ± standard deviations of results from two replicates. (D) A ChIP assay was performed using HONE-Akata cells transfected with Flag-tagged-BZLF1, wild-type Oct-2, mutant Oct-2 (Q221A), or control vectors as indicated. Cross-linked DNA-protein complexes were immunoprecipitated using antibodies against Flag (BZLF1) or an IgG control. Antibody-bound DNA sequences were then PCR-amplified using primers spanning the EBV BRLF1, BMRF1, or SM promoters, the viral FR repeats, cellular Gadd45a promoter, or the GAPDH gene (negative control). Binding bands were quantified using ImageJ software and represented as numerical values in bar diagrams in the lower panel. The amount of BZLF1 binding to each promoter in the presence or absence of wild-type or mutant Oct-2 (Q221A) was compared to input. (E) A ChIP assay was performed using HONE-Akata cells transfected with 3 µg wild-type Oct-2 or mutant Oct-2 (Q221A), with or without 3 µg co-transfected BZLF1, or control vectors as indicated. Cross-linked DNA-protein complexes were immunoprecipitated using antibodies against Oct-2 or an IgG control. Antibody-bound DNA sequences were then PCR-amplified using primers spanning the cellular Gadd45a promoter, viral FR repeats, viral latency Qp, or the GAPDH gene (negative control). Binding bands were quantified using ImageJ software and represented as numerical values in bar diagrams in the lower panel. The amount of Oct-2 binding to each DNA region in the presence or absence of BZLF1 was compared to input.</p