Regulation of autophagic activation by Rta of Epstein-Barr virus via the extracellular signal-regulated kinase pathway - PubMed (original) (raw)

Regulation of autophagic activation by Rta of Epstein-Barr virus via the extracellular signal-regulated kinase pathway

Chien-Hui Hung et al. J Virol. 2014 Oct.

Abstract

Autophagy is an intracellular degradation pathway that provides a host defense mechanism against intracellular pathogens. However, many viruses exploit this mechanism to promote their replication. This study shows that lytic induction of Epstein-Barr virus (EBV) increases the membrane-bound form of LC3 (LC3-II) and LC3-containing punctate structures in EBV-positive cells. Transfecting 293T cells with a plasmid that expresses Rta also induces autophagy, revealing that Rta is responsible for autophagic activation. The activation involves Atg5, a key component of autophagy, but not the mTOR pathway. The expression of Rta also activates the transcription of the genes that participate in the formation of autophagosomes, including LC3A, LC3B, and ATG9B genes, as well as those that are involved in the regulation of autophagy, including the genes TNF, IRGM, and TRAIL. Additionally, treatment with U0126 inhibits the Rta-induced autophagy and the expression of autophagy genes, indicating that the autophagic activation is caused by the activation of extracellular signal-regulated kinase (ERK) signaling by Rta. Finally, the inhibition of autophagic activity by an autophagy inhibitor, 3-methyladenine, or Atg5 small interfering RNA, reduces the expression of EBV lytic proteins and the production of viral particles, revealing that autophagy is critical to EBV lytic progression. This investigation reveals how an EBV-encoded transcription factor promotes autophagy to affect viral lytic development.

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Figures

FIG 1

EBV lytic cycle and autophagy activation. (A) P3HR1 cells were transfected with control plasmid pCMV3 (Crt) or pCMV-Zta (Zta) to activate the lytic cycle. Cell lysates were prepared 24 h (lane 2) and 48 h (lane 1 and lane 3) after transfection and analyzed by immunoblotting using antibodies against the indicated proteins. (B) P3HR1 cells that were treated with TPA and SB for 48 h were incubated with anti-gp350/220 antibody and then labeled with fluorescence-conjugated secondary antibody. The cells that expressed gp350/220 (lane 2) were separated from those did not (lane 1) by using a cell sorter. Cell lysates were prepared and analyzed by immunoblotting. (C) Akata cells were treated with anti-IgG for 24 and 48 h. Cell lysates were prepared and analyzed by immunoblotting using antibodies against the indicated proteins. (D) 293EBV(2089) cells were transfected with control plasmid pCMV3 (Crt; lanes 1 and 3) or pCMV-Zta (Z; lanes 2 and 4). Cell lysates were prepared at 48 and 72 h after transfection and analyzed by immunoblotting. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (E) P3HR1 cells were cotransfected with pGFP-LC3 and pCMV3 or pCMV-Zta to induce the lytic cycle. Cells were fixed and adhered to poly-

-lysine-coated coverslips and then stained using anti-Rta or anti-gp350/220 antibodies. 4′,6-Diamidino-2-phenyldole staining revealed the nucleus. The images of fluorescence were captured under a Leica SP5 confocal laser scanning microscope. _z_-stacks with 20 planes were acquired in each experiment. Compressed images of multiple z-stacks are shown. Bar, 5 μm.

FIG 2

Expression of Rta and autophagic activation. (A) 293T cells were transfected with an empty vector pCMV3 (Ctr), pCMV-Zta (Z), and pCMV-Rta (R). Cells were treated with 100 nM bafilomycin A1 for 2 h, and lysates were prepared at 48 h after transfection. Proteins were separated by SDS-PAGE and analyzed by immunoblot analysis using antibodies against the indicated proteins. (B) The intensity of the LC3-II band from the cells that were transfected with pCMV-Zta or pCMV-Rta was compared to that from cells that were transfected with an empty vector. The intensity of LC3-II was measured using ImageJ software and normalized to the band intensity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) based on three blots. (C) 293T cells were cotransfected with both pLC3-GFP and pCMV-Zta or pCMV-Rta. (D) 293T cells were transfected with pCMV-Rta and cultured in various media as indicated. Cells were fixed at 48 h after transfection. Indirect immunofluorescence staining was performed using anti-Zta or anti-Rta (C) and anti-LC3B and anti-Rta (D). 4′,6-Diamidino-2-phenylindole staining revealed the nucleus. _z_-stacks of 20 planes were acquired in each experiment. Compressed images of multiple z-stacks are presented. Bar, 10 μm; white arrow, Rta-expressing cell. (E) Electron micrographs of 293T cells transfected with pCMV3 and pCMV-Rta. Two boxed regions in the image were enlarged (a and b). The black arrow indicates an autophagosome; the blue arrow indicates an autolysosome. (F) Lysates were prepared from P3HR1 cells that had been treated with TPA and sodium butyrate and from 293T cells that had been transfected with pCMV-R. Rta and tubulin (Tub) in the lysates were analyzed by immunoblotting.

FIG 3

Involvement of Atg5 in Rta-induced autophagy activation. (A) 293T cells were cotransfected with control siRNA (scr) or Atg5 siRNA (100 pmol) and pCMV3 (Crt) or pCMV-Rta (R), as indicated. The lysates were prepared at 48 h following transfection and analyzed by immunoblotting using antibodies against the indicated proteins. (B) 293T cells were cotransfected with control siRNA (scr) or Atg5 siRNA (100 pmol), and pCMV-Rta, and examined by indirect immunofluorescence using anti-Rta and anti-LC3B antibodies. 4′,6-Diamidino-2-phenylindole staining revealed the nucleus. Bar, 10 μm. (C) The percentages of Rta expressing cells (red) containing autophagic punctate vesicles (green dots) was determined by counting the numbers of cells that exhibited autophagic vesicles (green dots) in approximately 400 Rta-expressing cells (red).

FIG 4

Rta-induced autophagy is independent of the mTOR signaling pathway. 293T cells were cultured for 24 h, and lysates were prepared after 1 h treatment with dimethyl sulfoxide (lane 1) or rapamycin (Rap; 100 mM) (lane 2). 293T cells were transfected with control plasmid (C, lane 3) or pCMV-Rta (R, lane 4) and lysates were prepared at 48 h after transfection. Lysates from cells treated with rapamycin or transfected with plasmids were analyzed by immunoblotting using antibodies against the indicated proteins.

FIG 5

Mutations in Rta and autophagy activation. (A) Schematic diagram of the structure of Rta and its mutant derivatives. Dimerization, DNA binding, transactivation domain, and mutations in Rta are indicated. (B) The reporter plasmid pZpluc was cotransfected with pCMV3 (Crt), pCMV-Rta (Rta), pCMV-R550, or pCMV-Rta F600/605. Luciferase activity was measured at 24 h after transfection. (C) 293T cells were transfected with pCMV3, a control plasmid (Crt), pCMV-Rta (Rta), or plasmids that expressed Rta mutants, as indicated. The lysates were prepared 48 h after transfection and analyzed by immunoblotting using antibodies against the indicated proteins. (D) Quantification of LC3-II levels shown in panel C. The amount of LC3-II was measured using ImageJ software and normalized using that of the internal control, tubulin. (E) 293T cells were transfected with pCMV-Rta (Rta) or plasmids that expressed Rta mutants. At 48 h after transfection, cells were fixed and stained with anti-Rta and anti-LC3B antibodies. 4′,6-Diamidino-2-phenylindole reveals the nucleus. Bar, 20 μm. (F) The percentage of the cells in the population that had autophagic punctate vesicles in panel E was determined by counting the numbers of cells that exhibited autophagic vesicles (red dot) in approximately total 400 Rta-expressing cells (green).

FIG 6

Involvement of ERK activity in Rta-induced autophagy activation. (A) 293T cells that had been transfected with pCMV-Rta (R) and cells were treated with U0126 (10 μM) after 6 h transfection. The lysates were harvested after 48 h of transfection and analyzed by immunoblotting using antibodies against the indicated proteins. (B) Cells were fixed and stained with anti-Rta and anti-LC3B antibodies. 4′,6-Diamidino-2-phenylindole stain reveals the nucleus. Bar, 10 μm. (C) The number of cells that had autophagic punctate vesicles (green) in a total population of about 400 Rta-expressing cells (red) was counted and the percentages were calculated.

FIG 7

Rta activates transcription of autophagy-related genes. The expression levels of LC3A (A), LC3B (B), ATG9B (C), TNF (D), IRGM (E) and TNFSF10 (F) in control (C) and Rta- (R) and R550-expressing cells that were treated (+) or were not treated (−) with U0126 were determined by RT-qPCR. The control (C) or Rta-expressing cells were treated with U0126 (10 μM) at 6 h after transfection. The relative expression of each gene was normalized to the amount of actin and expressed as the fold change relative to that in control cells. *, P < 0.05; **, P < 0.01 (determined with Student's t test).

FIG 8

Relationship between autophagy and EBV lytic development. (A) P3HR1 cells were pretreated with 10 mM 3-MA for 1 h and then treated with TPA and SB to activate the EBV lytic cycle. Lysates were prepared at 48 or 24 h (for LC3) after lytic induction and analyzed by immunoblotting using antibodies to the indicated proteins. (B) Viral particles in the cell (intracellular) or in the culture medium (extracellular) were analyzed by qPCR after 4 days of lytic induction. (C) The numbers for P3HR1 cells that had been treated with 3-MA or/and TPA and SB were determined at different times. (D) P3HR1 cells that were infected with lentiviruses expressing β-galactosidase (lacZ) or Atg5 shRNA and were treated with TPA and SB. The lysates were prepared 48 h after infection and analyzed by immunoblotting using antibodies against the indicated proteins. (E) The production of viral particles after 4 days of lytic induction was analyzed by qPCR. (F) The numbers of latent P3HR1 cells that were infected with lentiviruses that expressed lacZ or Atg5 shRNA were determined at different times. (G) 293EBV(2089) cells were first transfected with control siRNA (scr) (lanes 1 and 2) or Atg5 siRNA (lanes 3 and 4) and then transfected with control plasmid (Crt) or pCMV-Zta (Z) a day later. Proteins in cell lysates were harvested 48 h after transfection and were analyzed by immunblotting using antibodies against the indicated proteins. (H) The production of viral particles after 4 days of lytic induction was analyzed by qPCR. The numbers of virus particles as percentages of those detected from cells treated with control siRNA are presented. (I) Raji cells were infected with virus particles that were harvested from cells treated with control siRNA or Atg5 siRNA following lytic induction. After 3 days, the percentage of Raji cells that were GFP positive was determined by FACS analysis. The relative infectivity of EBV virions is presented as the fold change of virion infectivity from 293EBV(2089) treated with control siRNA after normalization for virus particles.

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Regulation of autophagic activation by Rta of Epstein-Barr virus via the extracellular signal-regulated kinase pathway - PubMed (original) (raw)