). In this study, we show that BGLF4 interacts with lamin A/C and phosphorylates lamin A protein in vitro. Using a green fluorescent protein (GFP)-lamin A system, we found that Ser-22, Ser-390, and Ser-392 of lamin A are important for the BGLF4-induced disassembly of the nuclear lamina and the EBV reactivation-mediated redistribution of nuclear lamin. Virion production and protein levels of two EBV primary envelope proteins, BFRF1 and BFLF2, were reduced significantly by the expression of GFP-lamin A(5A), which has five Ser residues replaced by Ala at amino acids 22, 390, 392, 652, and 657 of lamin A. Our data indicate that BGLF4 kinase phosphorylates lamin A/C to promote the reorganization of the nuclear lamina, which then may facilitate the interaction of BFRF1 and BFLF2s and subsequent virion maturation. UL kinases of alpha-and betaherpesviruses also induce the disassembly of the nuclear lamina through similar sites on lamin A/C, suggesting a conserved mechanism for the nuclear egress of herpesviruses.Most DNA viruses replicate and assemble their genomes into nucleocapsids in the nuclei of infected cells. To facilitate efficient replication, viruses regulate the nuclear environment by affecting cellular chromatin and nuclear lamina (30,35,40). The nuclear lamina is a thin electron-dense meshwork lining the nucleoplasmic face of the inner nuclear membrane (INM) (14, 20) and provides structural support for the major components of the nuclear envelope (36, 39). The lamina also functions as a transverse scaffold for INM proteins (e.g., emerin and lamin B receptor), chromatin proteins (histone H2A/H2B dimers), and cytoskeleton-interacting proteins (nesprin1/2) (7, 52).The nuclear lamina comprises a series of type V intermediate filaments composed of lamin types A, B1, B2, and C. Types A and C are products of RNA splicing variants of the lmnA transcripts, whereas types B1 and B2 are derived from two other genes, lmnB1 and B2 (15, 22). The INM-associated lamin B layer provides the fundamental structure of the lamina and is essential for the nuclear shape, whereas the lamin A/C layer adjacent to the nucleoplasm has more specialized functions and contributes to nuclear stiffness (7, 23, 52). Similarly to other intermediate filaments, lamins contain globular head and tail domains flanked by a central rod domain (15). The rod domains of two lamin molecules can intertwine to form dimers, whereas regions flanking the head/rod and rod/tail domains potentially interact with other lamin dimers to form longer filaments (45). Physiologically, the nuclear lamina is reorganized dynamically throughout the cell cycle via a mechanism regulated by phosphorylation. Phosphorylation by mitotic Cdc2 kinase at Ser-22, Ser-390, and Ser-392 residues on lamin A/C, or by protein kinase C (PKC) during apoptosis, leads to the depolymerization of lamin (disassembly of the nuclear lamina), which may lead to their release from the INM (11,21,44).The intact meshwork of the nuclear lamina also presents a barrier to most DNA viruses. Upon infection,...

Intratumoral cytotoxic T lymphocytes are critical for controlling tumor recurrence, and programmed death-1 (PD-1) is a recognized marker of T-cell dysfunction. We analyzed this marker and its binding ligands in nasopharyngeal tumor tissue and non-cancerous nasopharyngeal control tissue to retrospectively evaluate the correlation between its expression and the post-treatment outcome of nasopharyngeal carcinoma patients. Using double immunofluorescence staining, we found that the expression of PD-1 in CD8 T cells in tumor tissue was significantly higher than in control tissue (mean: 28.4 vs 3.9%, Po0.0001). Although the expression rate of PD-1 in intratumoral CD8 cells was not associated with the other clinicopathological parameters examined, the higher expression rate in this subset of T cells significantly correlated with a poorer prognosis of overall survival, disease-free survival, and locoregional recurrence-free survival of the cancer patients (P ¼ 0.05, 0.007, and 0.004, respectively). Multivariate analysis confirmed it as an independent risk factor for death, treatment failure, and local recurrence of nasopharyngeal carcinoma. On the other hand, the expression of PD-1 in CD4 T cells and of its ligands in epithelial and stromal cells was not significantly different between tumor and control tissue, and its expression was not associated with clinical outcome of the cancer patients. We propose that PD-1 expression in CD8 cells reflects the selective suppression of cytotoxic lymphocytes in the tumor microenvironment and predicts recurrence of nasopharyngeal carcinoma after conventional therapies.

Nasopharyngeal carcinoma (NPC) is an endemic malignancy prevalent in South East Asia. Epidemiological studies have associated this disease closely with Epstein-Barr virus (EBV) infection. Previous studies also showed that EBV reactivation is implicated in the progression of NPC. Thus, we proposed that recurrent reactivations of EBV may be important for its pathogenic role. In this study, NPC cell lines latently infected with EBV, NA and HA, and the corresponding EBV-negative NPC cell lines, NPC-TW01 (TW01) and HONE-1, were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium n-butyrate (SB) for lytic cycle induction. A single treatment with TPA/SB revealed that DNA double-strand breaks and formation of micronuclei (a marker for genome instability) were associated with EBV reactivation in NA and HA cells. Examination of EBV early genes had identified several lytic proteins, particularly EBV DNase, as potent activators that induced DNA double-strand breaks and contribute to genome instability. Recurrent reactivations of EBV in NA and HA cells resulted in a marked increase of genome instability. In addition, the degree of chromosomal aberrations, as shown by chromosome structural variants and DNA copy-number alterations, is proportional to the frequency of TPA/SB-induced EBV reactivation. Whereas these DNA abnormalities were limited in EBV-negative TW01 cells with mock or TPA/SB treatment, and were few in mock-treated NA cells. The invasiveness and tumorigenesis assays also revealed a profound increase in both characteristics of the repeatedly reactivated NA cells. These results suggest that recurrent EBV reactivations may result in accumulation of genome instability and promote the tumor progression of NPC.

Epstein-Barr virus (EBV)-

Early growth response 1 (Egr-1) is a cellular transcription factor involved in diverse biologic functions. Egr-1 has been associated with Epstein-Barr virus (EBV) infection, but it is still unknown whether any EBV protein regulates Egr-1 expression. In this study, we first showed that EBV reactivation is involved in upregulation of Egr-1 and that Egr-1 can be induced by Zta, an EBV lytic transactivator. Zta not only binds to the Egr-1 promoter but also activates the ERK signaling pathway to trigger binding of Elk-1 to the Egr-1 promoter. In addition, knockdown of Egr-1 significantly reduces the spontaneous expression of Zta and Rta in EBV-infected 293 cells, suggesting that a positive-feedback network involving Egr-1 is required for EBV reactivation. This study also implies that Zta has the potential to affect expression of certain genes through Egr-1.Early growth response 1 (Egr-1), also designated zif268, NGFI-A, and Krox24, is a cellular transcription factor belonging to a family of zinc finger DNA-binding proteins (49). According to its diverse target genes, Egr-1 has been associated with a broad range of biologic functions such as cell proliferation (3, 4, 41), apoptosis (39,51,54), and differentiation (28, 50) in a cell-type-dependent manner. Encoded by an immediate-early gene, Egr-1 can be rapidly induced by many stimuli, including growth factors, cytokines, and various stresses (6,31,44,46). Most of the stimuli trigger cellular signaling converged to mitogen-activated protein kinase (MAPK) pathways, which lead to phosphorylation and activation of a ternary complex factor, Elk-1 (26, 31, 46). Activated Elk-1 interacts with the serum response factor (SRF) to form a ternary complex that binds to a DNA element composed of a core serum response element (SRE) and the adjacent Ets motif (25, 52). There are five such binding sites for the ternary complex in the promoter of the Egr-1-encoding gene, and activation of signal transduction from MAPKs to the ternary complex is essential for Egr-1 expression induced by various stimuli (26,31,45,46).Several clues indicate that Egr-1 is also linked to infection with Epstein-Barr virus (EBV), a human gammaherpesvirus closely associated with several lymphoid and epithelial malignancies (43). First, Egr-1 is upregulated when EBV interacts with B lymphocytes at the initial infection stage, and constitutive expression of Egr-1 correlates with certain types of EBV latency in B-lymphoid cell lines (5). Notably, Egr-1 has also been associated with the lytic state of EBV infection (56). EBV reactivation into the lytic cycle is initiated from activation of two immediate-early viral promoters, Zp and Rp, which are driven to express the BZLF1 (Zta) and BRLF1 (Rta) proteins, respectively (22). Both Zta and Rta are key lytic transactivators which autostimulate their own expression, reciprocally induce each other, and cooperatively direct the downstream expression cascade of EBV lytic genes (18,20,42,48). A previous study showed that Egr-1 can activate Rp and identified two Egr-1-bindi...

No abstract

No abstract

FOXP1 belongs to the P-subfamily of forkhead transcription factors and contains a conserved forkhead DNA-binding domain. According to size exclusion chromatography analysis, the forkhead domain of FOXP1 existed as a mixture of monomer and dimer. The dissociation constants of the forkhead domain of wild-type, C61S, and C61Y mutants of FOXP1 were 27.3, 28.8, and 332.0 lM, respectively. In contrast, FOXP1 A39P mutant formed only a monomer. NMR analysis also showed that FOXP1 C61S and C61Y mutants existed as a mixture. The solution structure of FOXP1 A39P/C61Y mutant was similar to the X-ray structure of the FOXP2 monomer. Comparison of backbone dynamics of FOXP1 A39P/C61Y and C61Y mutants showed that the residues preceding helix 3, the hinge region, exhibited the largest conformational exchange in FOXP1 monomer. The A39 residue of FOXP1 dimer has a lower order parameter with internal motion on the ps-ns timescale, suggesting that the dynamics of the hinge region of FOXP1 are important in the formation of the swapped dimer. The analysis also showed that the residues exhibiting the motions on the ps-ns and ls-ms timescales were located at the DNA-binding surface of FOXP1, suggesting the interactions between FOXP1 and DNA may be highly dynamic.