Epstein-Barr Virus BGLF4 Kinase Retards Cellular S-Phase Progression and Induces Chromosomal Abnormality

Epstein-Barr virus (EBV) induces an uncoordinated S-phase-like cellular environment coupled with multiple prophase-like events in cells replicating the virus. The EBV encoded Ser/Thr kinase BGLF4 has been shown to induce premature chromosome condensation through activation of condensin and topoisomerase II and reorganization of the nuclear lamina to facilitate the nuclear egress of nucleocapsids in a pathway mimicking Cdk1. However, the observation that RB is hyperphosphorylated in the presence of BGLF4 raised the possibility that BGLF4 may have a Cdk2-like activity to promote S-phase progression. Here, we investigated the regulatory effects of BGLF4 on cell cycle progression and found that S-phase progression and DNA synthesis were interrupted by BGLF4 in mammalian cells. Expression of BGLF4 did not compensate Cdk1 defects for DNA replication in S. cerevisiae. Using time-lapse microscopy, we found the fate of individual HeLa cells was determined by the expression level of BGLF4. In addition to slight cell growth retardation, BGLF4 elicits abnormal chromosomal structure and micronucleus formation in 293 and NCP-TW01 cells. In Saos-2 cells, BGLF4 induced the hyperphosphorylation of co-transfected RB, while E2F1 was not released from RB-E2F1 complexes. The E2F1 regulated activities of the cyclin D1 and ZBRK1 promoters were suppressed by BGLF4 in a dose dependent manner. Detection with phosphoamino acid specific antibodies revealed that, in addition to Ser780, phosphorylation of the DNA damage-responsive Ser612 on RB was enhanced by BGLF4. Taken together, our study indicates that BGLF4 may directly or indirectly induce a DNA damage signal that eventually interferes with host DNA synthesis and delays S-phase progression

Epstein-Barr virus immediate-early protein Rta activates 4-3-3 σ expression to regulate cell cycle progression

Epstein-Barr virus (EBV)為人類傳染性單核球增多症及口腔髮狀白斑瘤的病原，亦被發現與許多人類癌症，例如巴氏淋巴瘤(Burkitt''s lymphoma)、何杰金氏淋巴瘤(Hodgkin''s lymphoma)及鼻咽癌(nasopharyngeal carcinoma, NPC)等有高度相關性。病毒通常會利用本身所產生的蛋白質，影響細胞週期運行所需的相關分子，達到有利於病毒複製的環境。在實驗室先前的研究中，已知EB病毒的特早期蛋白質Rta，具有轉活化p21啟動子之能力，使p21蛋白質表現量上升，p21蛋白質屬於cyclin-dependent kinase inhibitor (CKIs)的一員，Rta藉由轉活化p21啟動子使細胞週期停在G1時期，造成G1-arrest的現象。此外，從Rta cDNA microarray資料顯示，Rta的存在亦活化具有調控細胞週期能力的14-3-3 σ蛋白質。在本研究當中，進一步研究Rta是否會藉由調控14-3-3 σ蛋白質的表現而對細胞週期有所影響。首先，以即時同步偵測定量聚合酶連鎖反應及西方墨點法，確認Rta可在轉錄、轉譯的層面上調控14-3-3 σ蛋白質的表現。接著，欲探討Rta調控14-3-3 σ蛋白質之機制，以螢光酵素-報導基因檢測方法，觀察到Rta具有轉活化14-3-3 σ啟動子的能力，但此轉活化能力並不是透過14-3-3 σ啟動子上的一個putative Rta-responsive element (RRE)。欲觀察Rta調控14-3-3 σ蛋白質是否對細胞週期運行而有所影響，以免疫螢光染色法觀察到Rta可能透過14-3-3 σ蛋白質而影響CDK1、CDK2的入核，使其堆積在細胞質當中，干擾細胞週期的正常運作。此外，Rta亦會造成細胞週期當中G2/M期的遲緩，然而是否是透過14-3-3 σ而造成此現象，還需進一步之探討。在本研究當中，我們發現Rta除了可藉由調控p21之外，亦會藉由影響14-3-3 σ的表現量而共同影響細胞週期的運行。Epstein-Barr virus (EBV) is the etiologic agent responsible for infectious mononucleosis and oral hairy leukoplakia. It is also highly associated with several human malignancies, such as Burkitt’s lymphoma, Hodgkin’s lymphoma and nasopharyngeal carcinoma (NPC). Many researches have suggested that regulation of the cell cycle is one strategy frequently used by viruses to create a more favorable environment for viral replication. From the previous studies of our lab, we have demonstrated that EBV Rta had the ability to block cell cycle at the G1 phase through regulating the expression of p21 protein. Furthermore, data from a cDNA microarray indicated that Rta could upregulate 14-3-3 σ in transcriptional level. The 14-3-3 σ protein is reported to interact with many cell cycle-related molecules. In this study, we intend to elucidate whether Rta activates 14-3-3 σ expression to affect cell cycle progression. First, we confirmed that Rta could upregulate 14-3-3 σ mRNA and protein expression by using RT-Q-PCR and western blotting assay. Moreover, we found that Rta could transactivate the 14-3-3 σ promoter in a manner independent of its putative Rta-responsive element. In addition, we demonstrated that Rta affected the subcellular localization of CDK1 and CDK2 in 293-TREx-Rta cells via the upregulation of 14-3-3σ expression. Finally, we discovered that Rta also was able to interfere with the G2-M phase progression of cell cycle. Taken together, our results clearly suggest that EBV protein Rta acts through the transcriptional induction of 14-3-3 σ, in addition to p21, to influence cell cycle progression.論文口試委員審定書…………………………………………i文摘要……………………………………………………… ii文摘要……………………………………………………… iii論……………………………………………………………1驗材料………………………………………………………9驗方法……………………………………………………… 14果…………………………………………………………… 22論……………………………………………………………27表…………………………………………………………… 30錄…………………………………………………………… 42amp;#63851;考文獻……………………………………………………… 4

Vegetation Pattern and Woody Species Composition of a Broad-Leaved Forest at the Upstream Basin of Nantzuhsienhsi in Mid-southern Taiwan

A 8.37-ha plot of the broad-leaved forest at an elevation of about 2000 m in the upstream basin of the Nantzuhsienhsi River in mid-southern Taiwan was set up for long-term monitoring of forest dynamics. All stems with diameter at breast height (d.b.h.) ≧ 1 cm were identified, measured, tagged, and mapped to analyze the forest composition, structure and species diversity of the plot. A total of 18,790 woody plant individuals, belonging to 64 species in 27 families, were recorded. The dominant families were Lauraceae, Fagaceae, and Theaceae, accounting for 78.8% of total individuals. The dominant species were Castanopsis carlesii, Machilus japonica, Listea acuminate, and Cyclobalanopsis stenophylloides. The most abundant species in the canopy layer was Castanopsis carlesii, in the subcanopy layer was Listea acuminate, and in the shrub layer were Machilus japonica and Listea acuminata. Four plant communities were identified based on Two-way Indicator Species Analysis (TWINSPAN) classification, including three evergreen forest types and one deciduous forest type. The three evergreen types are Machilus japonica type, locating on the east and west valleys and partial lower slopes, Machilus japonica-Castanopsis carlesii type, locating on middle to lower slopes and the central dry valley, and Schima superba-Castanopsis carlesii type, locating on eastern ridge and upper slopes. The deciduous type is Alnus formosana forest which is distributed on mid-west and southwestern ridges. The means of species number, density and basal area for different forest types declined gradually from ridge to valley habitats. These results reveal that topography is an important factor which is closely related to the distribution of evergreen broad-leaved forest types in the plot

The BGLF4 expression level is the key factor that determines cell fates.

<p>(A) Slide-cultured GFP-H2B-HeLa cells were transfected with DsRed-BGLF4, DsRed-K102I and DsRed-monomer. At 24 h post transfection, cells were fixed with 4% paraformaldehyde, stained for DNA using Hoechst 33258 and examined using fluorescence microscopy. (B)(C)(D) GFP-H2B-HeLa cells were seeded and transfected with (B) DsRed-monomer vector or (C)(D) Ds-Red-BGLF4. At 6 h post transfection, fields of view were selected randomly and filmed using an Axiovert 200 M inverted fluorescence microscope every 10 min.</p

A hypothetical model of BGLF4-induced RB phosphorylation and DNA damage response (DDR).

<p>BGLF4 induces premature chromosome condensation through activation of condensin and Topo II. The prophase-like chromosomes may cause abnormal DNA structure and micronucleus formation. In addition, BGLF4 elicits DNA damage signaling by phosphorylating TIP60. TIP60 is a histone acetyltransferase that regulates chromatin remodeling and the DNA damage response (DDR). Upon DNA double-stranded breakage, ATM kinase is acetylated by TIP60. The acetylation of ATM is essential for its autophosphorylation and activation. Therefore, BGLF4 is able to turn on DNA damage signaling in a TIP60/Chk2-dependent manner. Here we found that the exogenous expression of BGLF4 induces RB hyperphosphorylation at Ser780 and Ser612, which is an indicator of DNA damage signaling activation. Based on our observation that the RB-E2F1 complex does not dissociate and the E2F1-downstream cyclin D1 promoter and ZBRK1 activities are repressed upon BGLF4 expression, we propose that BGLF4-induced RB phosphorylation may be a downstream event of DDR. Because the DNA damage pathway is necessary for efficient EBV replication, BGLF4-induced RB phosphorylation is favorable for lytic cycle progression, while cellular DNA synthesis is blocked.</p

BGLF4 cannot compensate for the kinase activity of Cdk1 for DNA replication in <i>S. cerevisiae.</i>

<p>(A) Five-fold serial dilutions of <i>cdc28-4</i> mutant yeasts containing pVT-U101 vector, BGLF4, K102I, or HA-Cdc2 expressing plasmids were spotted onto YPD plates and incubated at 23°C, 30°C or 37°C for 2 days to observe colony growth. (B) The protein expression levels of BGLF4, K102I and Cdc2 in (A) were detected by immunoblotting. The blot was detected with anti-BGLF4 and anti-HA antibody. (C) Saturated <i>cdc28-13</i> cells carrying indicated plasmids were treated with 0.2 M hydroxyurea (HU) at 23°C for 3 h to synchronize transformed yeast cells at an early stage of S-phase. The synchronized cells were released into fresh YPD medium and incubated at the restrictive temperature for 4 h. The percentages of budded cells were determined at the time of temperature shift and 4 h later. The asterisk (*) indicates a statistically significant difference from vector control cells (P<0.01). (D) Total cell extracts of <i>cdc28-13</i> transformed with pVTU vector, HA-BGLF4, HA-UL97 or HA-Cdc2 were harvested before and after HU release and subjected to immunoblotting with HA antibody. (E) Asynchro, synchronized or released <i>cdc28-13</i> cells containing the plasmids indicated were fixed and stained with propidium iodide to analyze DNA contents by FACS.</p

Expression of BGLF4 elicits abnormal chromosomal structure and micronuclei formation in various cell types.

<p>(A) Slide-cultured HeLa cells with stable GFP-H2B expression were transfected with DsRed-BGLF4, DsRed-K102I or pDsRed-monomer. At 48 h or 72 h post transfection, cells were fixed, stained for DNA with Hoechst 33258 and examined using fluorescence microscopy. (B) Slide-cultured 293 T-REx B22, K9, and V4 cells were treated with 100 ng/ml doxycycline (Dox). At 72 h post induction (hpi), cells were fixed with 4% paraformaldehyde and stained for DNA with Hoechst 33258. Micronucleus (MN) formation was observed using fluorescence microscopy. Detection of MN formation (MN%) was calculated as the percentage of cells containing micronuclei, derived from the analysis of 1,000 cells. Bars represent mean values in MN% (duplicates±SD). One of the two independent experiments conducted is shown. (C) Slide-cultured NPC-TW01 T-REx KIT2 and VIT7 cells were incubated with 50 ng/ml Dox for 72 h. Cells were fixed with 4% paraformaldehyde and examined using fluorescence microscopy. Detection of MN formation (MN%) was calculated as the percentage of cells containing micronuclei, derived from the analysis of 1,200-1,600 cells. An asterisk (*) indicates a statistically significant difference from vector control cells (P<0.01). One of the two independent experiments conducted is shown.</p

RB is hyperphosphorylated but the RB-E2F1 complex is not disrupted in the presence of BGLF4.

<p>(A) EBV-positive Akata cells were induced by 0.8% anti-human IgG and harvested at the time points indicated. The proteins were resolved by 7.5% SDS-PAGE (for total RB) or 10% SDS-PAGE (for RB specific phosphorylation, Zta, BGLF4 and GAPDH). Zta and BGLF4 were detected to confirm lytic cycle progression. GAPDH served as a loading control. (B) Saos-2 cells were transfected with the expressing plasmids indicated. Cells were harvested at 24 h post transfection. The protein expression levels were resolved by 7.5% SDS-PAGE (for RB) or 10% SDS-PAGE (for BGLF4, HCMV UL97, RB specific phosphorylation and GAPDH). GAPDH served as a loading control. (C) Saos-2 cells were transfected with the plasmids indicated. Cells were harvested at 24 h post transfection and cell extracts were subjected to immunoprecipitation assays using anti-RB antibody. The immunocomplexes were separated by 10% SDS-PAGE. The co-immunoprecipitated proteins RB and E2F1 were detected using specific antibodies. (D) Luciferase reporter assays were performed in Saos-2 cells by co-transfected with p-CCND1-luc (a 3.3-kb full length cyclin D1 promoter construct) as well as pWP1 (for normalization of transfection efficiency) and plasmids of effectors as indicated. Luciferase activities were measured at 48 h post transfection. Results are means ± SD from three separate transfections. Data are representative of two independent experiments. (E) Saos-2 cells were transfected with the expression plasmids as indicated. Cells were harvested at 48 h post transfection. The protein expression levels were resolved by 7.5% SDS-PAGE (for RB) or 10% SDS-PAGE (for BGLF4, Chk2, RB specific phosphorylation and β-actin). β-actin served as a loading control. (F) Luciferase reporter assays were performed in Saos-2 cells by co-transfection of p-ZBRK1-luc (containing ZBRK1 promoter fragment -624 to +47 bp) as well as pWP1 (for normalization of transfection efficiency), and plasmids of effectors as indicated in the figure. Luciferase activities were measured at 48 h post transfection. Results are means ± SD from two separate transfections. Data are representative of two independent experiments.</p

Expression of BGLF4 results in slight cell growth retardation.

<p>(A) 293 T-REx BGLF4 inducible B22, K102I kinase dead K9 and vector control V4 cells were seeded into 96-well plates in triplicate and incubated in 100 ng/ml Dox containing medium to induce the expression of BGLF4 or K102I. At 24, 48, 72, 96 and 120 h post induction (hpi), MTT assays were performed and the optical densities (OD) were determined by spectrophotometry at 550 nm. Three independent experiments were conducted and one representative result is shown. (B) NPC-TW01 T-REx BGLF4 inducible KIT2, KIT21 cells and vector control VIT7 cells were induced with 50 ng/ml Dox and subjected to an MTT assay as described in (A).</p

BGLF4 induces the accumulation of the S-phase cell population and represses [¹⁴C]-thymidine incorporation into HeLa cells.

<p>(A) Asynchronous HeLa cells were seeded and transfected with GFP-BGLF4, GFP-K102I expressing plasmids or control GFP vector. At 24 h post transfection, cells were harvested and stained with propidium iodide. The cell cycle profile was analyzed by FACS according to the GFP intensities (left panels). Cells with high GFP expression (middle panels) were further subgrouped from GFP low cells (right panels). The statistics of cell cycle profiles of GFP expressing cells were plotted. Cells with high GFP-BGLF4 expression showed significant accumulation in S-phase (middle panel). An asterisk (*) indicates a statistically significant difference from vector control cells (P<0.05). Two independent experiments were performed and one of the results is shown. (B) At 4 h post transfection, HeLa cells were labeled with [¹⁴C]-thymidine for newly synthesized DNA. At 20 h post transfection, [¹⁴C]-thymidine was measured using a liquid scintillation counter. An asterisk (*) indicates a statistically significant difference from vector control cells (P<0.05). Reproducible data were observed in at least three independent experiments. (C) The M-phase synchronous protocol for GFP-BGLF4 or GFP-K102I expressing cells is indicated. HeLa cells were seeded and transfected with GFP-BGLF4, GFP-K102I or GFP vector expressing plasmids. At 24 h post transfection, cells were treated with 200 ng/ml Nocodazole (Noc). After 20 h of Noc treatment, cells were harvested and stained with propidium iodide. Cell cycle profiles of whole cell populations were analyzed by FACS. Two independent experiments were performed.</p