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Synthetic bone graft has gained considerable attention because of an increase in the aged population. In the present study, a biphasic bone graft composed of hydroxyapatite (HAp) and calcium sulfate (CS) is prepared using a gel-foaming technique. The gel is prepared at a temperature as low as 35 °C; many pores are introduced into the gel through an agitation process. The foamed gel can be cut into any shape and size. After sintering, the porosity of the biphasic bone graft is approximately 70%. Depending on the HAp/CS ratio, the amount of macropores and micropores can be tailored. The macropores of approximately 130 μm diameter are interconnected with each other through openings of 40 μm size. The biphasic bone graft exhibits no cytotoxicity; preosteoblast cells can adhere and proliferate on the surface of the fabricated bone graft. However, migration of these cells into the bone graft is considerably limited in the in vitro study. When the biphasic bone graft is implanted into the distal femur of rats, with the combined effect of osteoblast and osteoclast cells, more than 90% of the bone graft is degraded after 3 months. New trabeculae bone and bone marrow are observed within the bone defect. The HAp/CS composite can thus be used as degradable bone grafts.

Rta, a key transcription factor expressed by Epstein-Barr virus (EBV), primarily acts to induce activation of the EBV lytic cycle. Interestingly, we observed from an immunogold assay that Rta is also present on the EBV capsid in the host cell nucleus, and a centrifugation study further revealed that Rta cofractionates with EBV virions. Importantly, cofractionated Rta showed similar properties as the EBV tegument protein, BGLF4. Glutathione S-transferase (GST)-pulldown and coimmunoprecipitation assays subsequently demonstrated that Rta directly interacts with the EBV capsid protein, BORF1. Rta was observed to colocalize with BORF1 in the nucleus during EBV lytic induction, and this interaction appears to influence BORF1 stability. Moreover, we found that BORF1 is modified by ubiquitin, and Rta reduces this ubiquitination. These results indicate that Rta may act as an inner tegument protein to improve EBV capsid stability and critical to viral infection.

Rta of Epstein-Barr virus (EBV) is thought to be expressed only during the lytic cycle to promote the transcription of lytic genes. However, we found that Rta is expressed in EBV-infected B cells during viral latency, at levels detectable by immunoblot analysis. Latent Rta expression cannot be attributed to spontaneous lytic activation, as we observed that more than 90% of Akata, P3HR1, and 721 cells latently infected by EBV express Rta. We further found that Rta is sequestered in the nucleolus during EBV latency through its interaction with MCRS2, a nucleolar protein. When Rta is sequestered in the nucleolus, it no longer activates RNA polymerase II-driven transcription, thus explaining why Rta expression during latency does not transactivate EBV lytic genes. Additional experiments showed that Rta can bind to 18S rRNA and become incorporated into ribosomes, and a transient transfection experiment showed that Rta promotes translation from an mRNA reporter. These findings reveal that Rta has novel functions beyond transcriptional activation during EBV latency and may have interesting implications for the concept of EBV latency.

Protoflavones, a rare group of natural flavonoids with a non-aromatic B-ring, are best known for their antitumor properties. The protoflavone B-ring is a versatile moiety that might be explored for various pharmacological purposes, but the common cytotoxicity of these compounds is a limitation to such efforts. Protoapigenone was previously found to be active against the lytic cycle of Epstein&ndash, Barr virus (EBV). Further, the 5-hydroxyflavone moiety is a known pharmacophore against HIV-integrase. The aim of this work was to prepare a series of less cytotoxic protoflavone analogs and study their antiviral activity against HIV and EBV. Twenty-seven compounds, including 18 new derivatives, were prepared from apigenin through oxidative de-aromatization and subsequent continuous-flow hydrogenation, deuteration, and/or 4&prime, oxime formation. One compound was active against HIV at the micromolar range, and three compounds showed significant activity against the EBV lytic cycle at the medium-low nanomolar range. Among these derivatives, protoapigenone 1&prime, O-isopropyl ether (6) was identified as a promising lead that had a 73-times selectivity of antiviral over cytotoxic activity, which exceeds the selectivity of protoapigenone by 2.4-times. Our results open new opportunities for designing novel potent and safe anti-EBV agents that are based on the natural protoflavone moiety.

Protoflavones, a rare group of natural flavonoids with a non-aromatic B-ring, are best known for their antitumor properties. The protoflavone B-ring is a versatile moiety that might be explored for various pharmacological purposes, but the common cytotoxicity of these compounds is a limitation to such efforts. Protoapigenone was previously found to be active against the lytic cycle of Epstein–Barr virus (EBV). Further, the 5-hydroxyflavone moiety is a known pharmacophore against HIV-integrase. The aim of this work was to prepare a series of less cytotoxic protoflavone analogs and study their antiviral activity against HIV and EBV. Twenty-seven compounds, including 18 new derivatives, were prepared from apigenin through oxidative de-aromatization and subsequent continuous-flow hydrogenation, deuteration, and/or 4′-oxime formation. One compound was active against HIV at the micromolar range, and three compounds showed significant activity against the EBV lytic cycle at the medium-low nanomolar range. Among these derivatives, protoapigenone 1′-O-isopropyl ether (6) was identified as a promising lead that had a 73-times selectivity of antiviral over cytotoxic activity, which exceeds the selectivity of protoapigenone by 2.4-times. Our results open new opportunities for designing novel potent and safe anti-EBV agents that are based on the natural protoflavone moiety.

Ethnopharmacological relevance Lindernia crustacea (L.) F.Muell. (Scrophulariaceae) was selected for phytochemical investigation owing to its traditional use against human herpes virus infection and its anti-Epstein–Barr virus (EBV) effect. Aims of the study The present study focused on the phytochemical investigation of L. crustacea including the isolation and structure determination of its biologically active compounds. Compounds with anti-EBV effects were also investigated. Materials and methods The EtOH extract of L. crustacea was subsequently partitioned using different solvents. The EtOAc fraction was subjected to several chromatographic methods to obtain pure compounds. The structures of all isolates were established by spectroscopic analysis and compared with previously reported physical data. The anti-EBV effect was evaluated in an EBV-containing Burkitt's lymphoma cell line (P3HR1) to study the expression of EBV lytic proteins. Results Thirty-three compounds, including one diterpene (1), four anthraquinones (2–5), two ionones (6 and 7), fourteen phenylpropanoid glycosides (8–21), five flavonoids (22–26), one lignan glycoside (27), one phenethyl alcohol glycoside (28), one phenylpropene glycoside (29), one glucosyl glycerol derivative (30), one furanone (31), and two cinnamic acid derivatives (32 and 33), were isolated from the ethanolic extract of the plant. All isolated compounds were obtained for the first time from Lindernia sp. The evaluation of the anti-EBV activity of L. crustacea crude extract, partitioned fractions, and constituents was performed for the first time. Phytol (1), aloe-emodin (2), byzantionoside B (7), a mixture of trans-martynoside (8) and cis-martynoside (9), a mixture of trans-isomartynoside (10) and cis-isomartynoside (11), luteolin-7-O-β-D-glucopyranoside (24), and apigenin-7-O-[β-D-apiofuranosyl (1→6)-β-D-glucopyranoside] (25) exhibited significant inhibitory effects on the EBV lytic cycle at 20 μg/mL in the immunoblot analysis. On the other hand, (6R,7E,9R)-3-oxo-α-ionol-β-D-glucopyranoside (6) and a mixture of trans-dolichandroside A (12) and cis-dolichandroside A (13) showed moderate anti-EBV activity at 20 μg/mL. Conclusions L. crustacea and its active isolates could be developed as potential candidates against EBV. Our findings provide scientific evidence for the traditional use of L. crustacea for its antiviral effects.

The Epstein-Barr virus (EBV) capsid contains a major capsid protein, VCA; two minor capsid proteins, BDLF1 and BORF1; and a small capsid protein, BFRF3. During the lytic cycle, these capsid proteins are synthesized and imported into the host nucleus for capsid assembly. This study finds that EBV capsid proteins colocalize with promyelocytic leukemia (PML) nuclear bodies (NBs) in P3HR1 cells during the viral lytic cycle, appearing as nuclear speckles under a confocal laser scanning microscope. In a glutathione S -transferase pulldown study, we show that BORF1 interacts with PML-NBs in vitro . BORF1 also colocalizes with PML-NBs in EBV-negative Akata cells after transfection and is responsible for bringing VCA and the VCA-BFRF3 complex from the cytoplasm to PML-NBs in the nucleus. Furthermore, BDLF1 is dispersed throughout the cell when expressed alone but colocalizes with PML-NBs when BORF1 is also present in the cell. In addition, this study finds that knockdown of PML expression by short hairpin RNA does not influence the intracellular levels of capsid proteins but reduces the number of viral particles produced by P3HR1 cells. Together, these results demonstrate that BORF1 plays a critical role in bringing capsid proteins to PML-NBs, which may likely be the assembly sites of EBV capsids. The mechanisms elucidated in this study are critical to understanding the process of EBV capsid assembly. IMPORTANCE Capsid assembly is an important event during the Epstein-Barr virus (EBV) lytic cycle, as this process is required for the production of virions. In this study, confocal microscopy revealed that the EBV capsid protein BORF1 interacts with promyelocytic leukemia (PML) nuclear bodies (NBs) in the host nucleus and is responsible for transporting the other EBV capsid proteins, including VCA, BDLF1, and BFRF3, to these subnuclear locations prior to initiation of capsid assembly. This study also found that knockdown of PML expression by short hairpin RNA significantly reduces EBV capsid assembly capabilities. This enhanced understanding of capsid assembly offers potential for the development of novel antiviral strategies and therapies that can prevent the propagation and spread of EBV.

During its lytic cycle, Epstein-Barr virus (EBV) expresses Rta, a factor encoded by BRLF1 that activates the transcription of viral lytic genes. We found that upstream stimulating factor (USF) binds to E1, one of the five E boxes located at - 79 in the BRLF1 promoter (Rp), to activate BRLF1 transcription. Furthermore, Rta was shown to interact with USF1 in coimmunoprecipitation and glutathione S-transferase (GST)-pulldown assays, and confocal laser-scanning microscopy further confirmed that these two proteins colocalize in the nucleus. Rta was also found to bind with the E1 sequence in a biotin-labelled E1 probe, but only in the presence of USF1, suggesting that these two proteins likely form a complex on E1. We subsequently constructed p188mSZ, a reporter plasmid that contained the sequence from - 188 to +5 in Rp, within which the Sp1 site and Zta response element were mutated. In EBV-negative Akata cells cotransfected with p188mSZ and plasmids expressing USF1 and Rta, synergistic activation of Rp transcription was observed. However, after mutating the E1 sequence in p188mSZ, USF1 and Rta were no longer able to transactivate Rp, indicating that Rta autoregulates BRLF1 transcription via its interaction with USF1 on E1. This study showed that pUSF1 transfection after EBV lytic induction in P3HR1 cells increases Rta expression, indicating that USF1 activates Rta expression after the virus enters the lytic cycle. Together, these results reveal a novel mechanism by which USF interacts with Rta to promote viral lytic development, and provide additional insight into the viral-host interactions of EBV.

Epstein-Barr virus (EBV) expresses two immediate-early proteins, Rta and Zta, which are key transcription factors that can form a complex with MCAF1 at Zta-responsive elements (ZREs) to synergistically activate several viral lytic genes. Our previous research indicated that RanBPM interacts with Rta and enhances Rta sumoylation. Here we showed that RanBPM binds to Zta in vitro and in vivo, and acts as an intermediary protein in Rta-Zta complex formation. The Rta-RanBPM-Zta complex was observed to bind with ZREs in the transcriptional activation of key viral genes, such as BHLF1 and BHRF1, while the introduction of RanBPM short hairpin RNA (shRNA) subsequently reduced the synergistic activity of Zta and Rta. RanBPM was found to enhance Zta-dependent transcriptional activity via the inhibition of Zta sumoylation. Interestingly, Z-K12R, a sumoylation-defective mutant of Zta, demonstrated transcriptional activation capabilities that were stronger than those of Zta and apparently unaffected by RanBPM modulation. Finally, RanBPM silencing inhibited the expression of lytic proteins. Taken together, these results shed light on the mechanisms by which RanBPM regulates Zta-mediated transcriptional activation, and point to an important role for RanBPM in EBV lytic progression.

Yin Yang 1 (YY1) is a multifunctional zinc finger transcription factor that regulates many key cellular processes. In this study, we report the cloning of YY1 from Litopenaeus vannamei shrimp (LvYY1). This study shows that LvYY1 is ubiquitously expressed in shrimp tissues, and knockdown of LvYY1 expression by double-stranded RNA (dsRNA) injection in white spot syndrome virus (WSSV)-infected shrimp reduced both mRNA levels of the WSSV immediate early gene ie1 as well as overall copy numbers of the WSSV genome. The cumulative mortality rate of infected shrimp also declined with LvYY1 dsRNA injection. Using an insect cell model, we observed that LvYY1 activates ie1 expression, and a mutation introduced into the ie1 promoter subsequently repressed this capability. Moreover, reporter assay results suggested that LvYY1 is involved in basal transcriptional regulation via an interaction with L. vannamei TATA-binding protein (LvTBP). Electrophoretic mobility shift assay (EMSA) results further indicated that LvYY1 binds to a YY1-binding site in the region between positions −119 and −126 in the ie1 promoter. Chromatin immunoprecipitation analysis also confirmed that LvYY1 binds to the ie1 promoter in WSSV-infected shrimp. Taken together, these results indicate that WSSV uses host LvYY1 to enhance ie1 expression via a YY1-binding site and the TATA box in the ie1 promoter, thereby facilitating lytic activation and viral replication. IMPORTANCE WSSV has long been a scourge of the shrimp industry and remains a serious global threat. Thus, there is a pressing need to understand how the interactions between WSSV and its host drive infection, lytic development, pathogenesis, and mortality. Our successful cloning of L. vannamei YY1 (LvYY1) led to the elucidation of a critical virus-host interaction between LvYY1 and the WSSV immediate early gene ie1 . We observed that LvYY1 regulates ie1 expression via a consensus YY1-binding site and TATA box. LvYY1 was also found to interact with L. vannamei TATA-binding protein (LvTBP), which may have an effect on basal transcription. Knockdown of LvYY1 expression inhibited ie1 transcription and subsequently reduced viral DNA replication and decreased cumulative mortality rates of WSSV-infected shrimp. These findings are expected to contribute to future studies involving WSSV-host interactions.

Replication and transcription activator (Rta), a key protein expressed by Epstein-Barr virus (EBV) during the immediate-early stage of the lytic cycle, is responsible for the activation of viral lytic genes. In this study, GST-pulldown and coimmunoprecipitation assays showed that Rta interacts in vitro and in vivo with TRIM5α, a host factor known to be involved in the restriction of retroviral infections. Confocal microscopy results revealed that Rta colocalizes with TRIM5α in the nucleus during lytic progression. The interaction involves 190 amino acids in the N-terminal of Rta and the RING domain in TRIM5α, and it was further found that TRIM5α acts as an E3 ubiquitin ligase to promote Rta ubiquitination. Overexpression of TRIM5α reduced the transactivating capabilities of Rta, while reducing TRIM5α expression enhanced EBV lytic protein expression and DNA replication. Taken together, these results point to a critical role for TRIM5α in attenuating EBV lytic progression through the targeting of Rta for ubiquitination, and suggest that the restrictive capabilities of TRIM5α may go beyond retroviral infections.

The switch between latency and the lytic cycle of Kaposi’s sarcoma-associated herpesvirus (KSHV) is controlled by the expression of virally encoded ORF50 protein. Thus far, the regulatory mechanism underlying the protein stability of ORF50 is unknown. Our earlier studies have demonstrated that a protein abundance regulatory signal (PARS) at the ORF50 C-terminal region modulates its protein abundance. The PARS region consists of PARS-I (aa 490–535) and PARS-II (aa 590–650), and mutations in either component result in abundant expression of ORF50. Here, we show that ORF50 protein is polyubiquitinated and its abundance is controlled through the proteasomal degradation pathway. The PARS-I motif mainly functions as a nuclear localization signal in the control of ORF50 abundance, whereas the PARS-II motif is required for the binding of ubiquitin enzymes in the nucleus. We find that human oncoprotein MDM2, an ubiquitin E3 ligase, is capable of interacting with ORF50 and promoting ORF50 degradation in cells. The interaction domains between both proteins are mapped to the PARS region of ORF50 and the N-terminal 220-aa region of MDM2. Additionally, we identify lysine residues at positions 152 and 154 in the N-terminal domain of ORF50 critically involved in MDM2-mediated downregulation of ORF50 levels. Within KSHV-infected cells, the levels of MDM2 were greatly reduced during viral lytic cycle and genetic knockdown of MDM2 in these cells favored the enhancement of ORF50 expression, supporting that MDM2 is a negative regulator of ORF50 expression. Collectively, the study elucidates the regulatory mechanism of ORF50 stability and implicates that MDM2 may have a significant role in the maintenance of viral latency by lowering basal level of ORF50., Author Summary The switch of Kaposi’s sarcoma-associated herpesvirus (KSHV) from latency to lytic replication is an essential process for controlling viral dissemination and the pathogenesis of KSHV-associated diseases. The virally encoded ORF50 protein is known as the master controller of viral reactivation. We previously reported that a two-component PARS region, which contains PARS-I and PARS-II, in ORF50 influences its protein abundance. In this study, we elucidate the regulatory mechanism of the ORF50 protein stability. We find that degradation of ORF50 involves at least two critical steps, including nuclear translocation and the subsequent degradation in the nucleus. The PARS-I motif is responsible for the nuclear translocation of ORF50, whereas the PARS-II motif is required for ORF50 degradation in the nucleus. Furthermore, we show that the PARS region is the primary docking site for cellular oncoprotein MDM2, an E3 ubiquitin ligase. Direct interaction of MDM2 with ORF50 promotes ORF50 degradation. The putative ubiquitin acceptor sites targeted by MDM2 are mapped to K152 and K154 in the N-terminal region of ORF50. In the context of KSHV-infected cells, we also provide evidence that MDM2 is involved in the regulation of the ORF50 expression.

Sp1-like proteins and Kruppel-like factors (KLFs) are highly related zinc-finger proteins that have crucial roles in transcription. One expressed sequence tag (EST, HPA-N-S01-EST0038) from shrimps is homologous to Sp1. This study reports the cloning and characteristics of a KLF from shrimp, Penaeus monodon (PmKLF). The full-length PmKLF cDNA is 1702 bp, encoding a polypeptide of 360 amino acids. Sequence analysis revealed that the sequence of PmKLF is similar to that of KLF11 in humans, mice and zebrafish. RT-PCR analysis indicated that PmKLF mRNA is expressed in all examined tissues. Additionally, immunofluorescence analysis revealed that GFP-KLF fusion protein is located in the nucleus as dots in an insect cell line, Sf9. Localization of PmKLF in the nucleus is also observed in the hemolymph from white spot syndrome virus (WSSV)-infected and WSSV-uninfected Litopenaeus vannamei. Knockdown of the expression of PmKLF transcript in WSSV-infected shrimp resulted in delayed cumulative mortalities, suggesting that PmKLF is important to WSSV infection. Moreover, inhibition of PmKLF expression reduced the copy number of WSSV and ie1 expression, revealing that PmKLF affects WSSV infection via interfering with ie1 expression.

The capsids of herpesviruses, which comprise major and minor capsid proteins, have a common icosahedral structure with 162 capsomers. An electron microscopic study shows that Epstein-Barr virus (EBV) capsids in the nucleus are immunolabeled by anti-BDLF1 and anti-BORF1 antibodies, indicating that BDLF1 and BORF1 are the minor capsid proteins of EBV. Cross-linking and electrophoresis studies of purified BDLF1 and BORF1 revealed that these two proteins form a triplex that is similar to that formed by the minor capsid proteins, VP19C and VP23, of herpes simplex virus type 1 (HSV-1). Although the interaction between VP23, a homolog of BDLF1, and the major capsid protein VP5 could not be verified biochemically in earlier studies, the interaction between BDLF1 and the EBV major capsid protein, viral capsid antigen (VCA), can be confirmed by glutathione S -transferase (GST) pulldown assay and coimmunoprecipitation. Additionally, in HSV-1, VP5 interacts with only the middle region of VP19C; in EBV, VCA interacts with both the N-terminal and middle regions of BORF1, a homolog of VP19C, revealing that the proteins in the EBV triplex interact with the major capsid protein differently from those in HSV-1. A GST pulldown study also identifies the oligomerization domains in VCA and the dimerization domain in BDLF1. The results presented herein reveal how the EBV capsid proteins interact and thereby improve our understanding of the capsid structure of the virus.

Zta, encoded by the BZLF1 gene of Epstein-Barr virus (EBV), is a transcription factor that is expressed during the immediate-early stage of the lytic cycle. The expression of Zta is crucial to viral lytic development. Earlier studies showed that Ku80 is a binding partner of Zta in ZKO-293 cells and is co-purified with Zta. This study verifies the interaction between Ku80 and Zta by using glutathione S-transferase-pull-down and co-immunoprecipitation assays, and also by indirect immunofluorescence analysis. This investigation also reveals that Ku80 binds to Zta on Zta-response elements in the BHLF1 promoter, enhancing the promoter activity. This study also reveals that the interaction between Zta and Ku80 involves the C-terminal region of Zta and the 425 aa N-terminal region of Ku80. The interaction between these two proteins and the enhancement of transcription that is activated by Zta suggest that Ku80 is important to EBV lytic development.

Dynamic modification of target proteins by small ubiquitin-like modifier (SUMO) is known to modulate many important cellular processes and is required for cell viability and development in all eukaryotes. However, understanding of SUMO systems in plants, especially in unicellular green algae, remains elusive. In this study, Chlamydomonas reinhardtii CrSUMO96, CrSUMO97 and CrSUMO148 were characterized. We show that the formation of polymeric CrSUMO96 and CrSUMO97 chains can be catalyzed either by the human SAE1/SAE2 and Ubc9 SUMOylation system in vitro or by an Escherichia coli chimeric SUMOylation system in vivo. An exposed C-terminal di-glycine motif of CrSUMO96 or CrSUMO97 is essential for functional SUMOylation. The human SUMO-specific protease, SENP1, demonstrates more processing activity for CrSUMO97 than for CrSUMO96. The CrSUMO148 precursor notably has four repeated di-glycine motifs at the C-terminus. This unique feature is not found in other known SUMO proteins. Interestingly, only 83-residual CrSUMO148(1-83) with the first di-glycine motif can form SAE1/SAE2-SUMO complex and further form polymeric chains with the help of Ubc9. More surprisingly, CrSUMO148 precursor is digested by SENP1, solely at the peptide bond after the first di-glycine motif although there are four theoretically identical processing sites in the primary sequence. This process directly generates 83-residual CrSUMO148(1-83) mature protein, which is exactly the form suitable for activation and conjugation. We also show that SENP1 displays similar isopeptidase activity in the deconjugation of polymeric CrSUMO96, CrSUMO97 or CrSUMO148 chains, revealing that the catalytic mechanisms of processing and deconjugation of CrSUMOs by SENP1 may differ.

Epstein–Barr virus (EBV) expresses two transcription factors, Rta and Zta, during the immediate-early stage of the lytic cycle. The two proteins often collaborate to activate the transcription of EBV lytic genes synergistically. This study demonstrates that Rta and Zta form a complex via an intermediary protein, MCAF1, on Zta response element (ZRE) in vitro. The interaction among these three proteins in P3HR1 cells is also verified via coimmunoprecipitation, CHIP analysis and confocal microscopy. The interaction between Rta and Zta in vitro depends on the region between amino acid 562 and 816 in MCAF1. In addition, overexpressing MCAF1 enhances and introducing MCAF1 siRNA into the cells markedly reduces the level of the synergistic activation in 293T cells. Moreover, the fact that the synergistic activation depends on ZRE but not on Rta response element (RRE) originates from the fact that Rta and Zta are capable of activating the BMRF1 promoter synergistically after an RRE but not ZREs in the promoter are mutated. The binding of Rta–MCAF1–Zta complex to ZRE but not RRE also explains why Rta and Zta do not use RRE to activate transcription synergistically. Importantly, this study elucidates the mechanism underlying synergistic activation, which is important to the lytic development of EBV.

The functions of centrosomal protein ninein may be involved in microtubule minus end capping, centriole positioning, protein anchoring and microtubule nucleation, but the true physiological function of various human hNinein isoforms remains to be determined. Here we describe the identification of four diverse CCII-termini of human hNinein isoforms, including a novel isoform 6, by differential expression in a tissue-specific manner. These hNinein isoforms exhibit centrosomal (concentrated) and noncentrosomal (aggregated) localization when GFP-tagged fusion proteins are expressed transiently in mammalian cells. In a kinase assay, we show that the CCII region of hNinein provides a differential phosphorylation site by GSK3beta. In addition, our data indicate that either N-terminal or CCIIZ domain disruption may cause hNinein conformational change which recruits gamma-tubulin to centrosomal or noncentrosomal hNinein-containing sites, implying that the gamma-tubulin localization may be hNinein-dependent. Further, our RNA interference experiment against all hNinein isoforms caused a significant decrease in the gamma-tubulin signal in the centrosome. In domain swapping, we clearly show that the CCIIX-CCIIY region provides docking sites for gamma-tubulin. Moreover, our data also show that nucleation of microtubules from the centrosome is significantly affected by the presence of either the full -length hNinein or CCIIX-CCIIY region overexpression. Taken together, these results show that the centrosomal targeting signals of hNinein have a role not only in regulating hNinein conformation, resulting in localization change, but also provide docking sites to recruit gamma-tubulin at centrosomal and noncentrosomal sites.

To explore more hNinein interacting proteins, the yeast two-hybrid screening using ninein C-terminal domain as bait protein was performed. One novel gene, CGI-99, was demonstrated to associate with hNinein in the yeast two-hybrid method and in vitro GST pull-down assay. Molecular characterization also showed that CGI-99 possessed a transcriptional activity at the N-terminal. In addition, CGI-99 formed a dimer with the C-terminal, which overlapped with hNinein binding site. In kinase assay, CGI-99 binds to hNinein and completely blocks the phosphorylation of hNinein by GSK3β. Moreover, CGI-99 was highly expressed in all brain tumors which is in agreement with the Northern blot analysis. Taken together, we have isolated a novel protein CGI-99, which may be involved in the functional regulation of human ninein in the centrosome structure and may also be important in brain development and tumorigenesis.

A series of deletion and mutation assays of the white spot syndrome virus (WSSV) immediate-early gene WSSV108 promoter showed that a Kruppel-like factor (KLF) binding site located from -504 to -495 (relative to the transcription start site) is important for the overall level of WSSV108 promoter activity. Electrophoretic mobility shift assays further showed that overexpressed recombinant Penaeus monodon KLF (rPmKLF) formed a specific protein-DNA complex with the (32)P-labeled KLF binding site of the WSSV108 promoter, and that higher levels of Litopenaeus vannamei KLF (LvKLF) were expressed in WSSV-infected shrimp. A transactivation assay indicated that the WSSV108 promoter was strongly activated by rPmKLF in a dose-dependent manner. Lastly, we found that specific silencing of LvKLF expression in vivo by dsRNA injection dramatically reduced both WSSV108 expression and WSSV replication. We conclude that shrimp KLF is important for WSSV genome replication and gene expression, and that it binds to the WSSV108 promoter to enhance the expression of this immediate-early gene.

Kruppel-like factors (KLFs) belong to a subclass of Cys2/His2 zinc-finger DNA-binding proteins, and act as important regulators with diverse roles in cell growth, proliferation, differentiation, apoptosis and tumorigenesis. Our previous research showed that PmKLF from Penaeus monodon is crucial for white spot syndrome virus (WSSV) infection, yet the mechanisms by which PmKLF influences WSSV infection remain unclear. This study cloned KLF from Litopenaeus vannamei (LvKLF), which had 93% similarity with PmKLF. LvKLF formed a dimer via the C-terminal zinc-finger motif. Knockdown of LvKLF expression by dsRNA injection in WSSV-challenged shrimps was found to significantly inhibit the transcription of two important immediate-early (IE) genes, IE1 and WSSV304, and also reduced WSSV copy numbers. Moreover, reporter assays revealed that the promoter activities of these two WSSV IE genes were substantially enhanced by LvKLF. Mutations introduced in the promoter sequences of IE1 and WSSV304 were shown to abolish LvKLF activation of promoter activities; and an electrophoretic mobility shift assay demonstrated that LvKLF binds to putative KLF-response elements (KRE) in the promoters. Taken together, these results indicate that LvKLF transcriptional regulation of key IE genes is critical to WSSV replication.

Epstein-Barr virus (EBV) expresses two transcription factors, Rta and Zta, which are involved in the transcriptional activation of EBV lytic genes. This study sought to elucidate the mechanism by which Rta activates transcription of the Zta-encoding gene, BZLF1, through the ZII element in the gene promoter. In a DNA affinity precipitation assay, ATF2 was found to associate with an Rta-interacting protein, MCAF1, at the ZII element. The interaction between Rta, MCAF1, and ATF2 at the same site in the ZII region was further verified in vivo by chromatin immunoprecipitation assay. The complex appears to be crucial for the activation of BZLF1 transcription, as the overexpression of two ATF2-dominant negative mutants, or the introduction of MCAF1 siRNA into 293T cells, were both found to substantially reduce Rta-mediated transcription levels of BZLF1. Moreover, this study also found that the Rta-MCAF1-ATF2 complex binds to a typical AP-1 binding sequence on the promoter of BMRF2, a key viral gene for EBV infection. Mutation of this sequence decreased Rta-mediated promoter activity significantly. Taken together, these results indicate a critical role for MCAF1 in AP-1-dependent Rta activation of BZLF1 transcription.

Rta is a transcription factor encoded by BRLF1 of the Epstein-Barr virus (EBV). This factor is expressed during the immediate-early stage of the lytic cycle to activate the genes required for EBV lytic develop- ment. Although transcription activation by Rta is 15 frequently associated with the binding of Rta to the Rta-response element (RRE) in promoters, Rta some- times activates promoters without an RRE. Here we show that Rta interacts with an Sp1-interacting pro- tein, MBD1-containing chromatin-associated factor 1 20 (MCAF1). This interaction is critical to the forma- tion of an Sp1-MCAF1-Rta complex at Sp1 sites. Therefore, following lytic induction and the expres- sion of Rta, Rta increases Sp1-mediated transcription. The genes that are thus activated include p16, p21, 25 SNRPN and BRLF1. However, the binding of Rta to RRE prevents the interaction between Rta and MCAF1; therefore, transcription activation by RRE depends only on Rta, and not on MCAF1 or Sp1. Furthermore, this study finds that MCAF1 promotes 30 the expression of Rta and Zta from EBV, indicating that MCAF1 participates EBV lytic activation. Our study documents the critical role of Rta in regulat- ing the transcription of the genes that are mediated by Sp1.

Histone acetylation alters the chromatin structure and activates the genes that are repressed by histone deacetylation. This investigation demonstrates that treating P3HR1 cells with trichostatin A (TSA) activates the Epstein-Barr virus (EBV) lytic cycle, allowing the virus to synthesize three viral lytic proteins-Rta, Zta and EA-D. Experimental results indicate that TSA and 12-O:-tetradecanoylphorbol-13-acetate synergistically activate the transcription of BRLF1, an immediate-early gene of EBV. Chromatin immunoprecipitation assay reveals that histone H4 at the BRLF1 promoter is acetylated after P3HR1 cells are treated with TSA, suggesting that histone acetylation activates BRLF1 transcription. Furthermore, results in this study demonstrate that mutation of a YY1-binding site in the BRLF1 promoter activates BRLF1 transcription 1.6- and 2.3-fold in P3HR1 cells and C33A cells, respectively. Real time PCR analysis reveals that the mutation also increases the histone acetylation level of the nucleosomes at the BRLF1 promoter 1. 64- and 3.08-fold in P3HR1 and C33A cells, respectively. Results presented herein suggest that histone deacetylation plays an important role in maintaining the viral latency and histone acetylation at the BRLF1 promoter allows the virus to express Rta and to activate the viral lytic cycle.

A 37-kb DNA fragment containing five fengycin synthetase genes, includingfenC,fenD,fenE,fenA, andfenB, was cloned and sequenced. Among these genes,fenCencodes a fengycin synthetase 2,560 amino acids long with an estimated molecular mass of 287 kDa. This protein contains two amino acid activation modules, FenC1 and FenC2, which activatel-glutamic acid andl-ornithine, respectively. Primer extension, using mRNA isolated from the log-phase cells, identified a transcription start site located 86 nucleotides upstream from the initiation codon offenC, implying that a promoter is located upstream from the start site. Primer extension using total RNA isolated from stationary-phase cells also identified a transcription start site located 61 nucleotides upstream from the initiation codon offenC. Gene fusion studies demonstrated that in nHA medium, the cells transcribe the fengycin synthetase genes at two different stages of cell growth. The promoter is active during the log phase, and the activity reaches the highest level during the late log phase. The activity decreases sharply but is maintained at a low level for approximately 24 h after cells enter the early stationary phase. The results of this investigation also suggest that the transcription offenCis positively regulated during the late log phase. Results presented herein provide further insight into fengycin synthesis byB. subtilisF29-3.

Epstein-Barr virus (EBV) encodes a transcription factor, Rta, which is required to activate the transcription of EBV lytic genes. This study demonstrates that treating P3HR1 cells with a proteasome inhibitor, MG132, causes the accumulation of SUMO-Rta and promotes the expression of EA-D. GST pulldown and coimmunoprecipitation studies reveal that RNF4, a RING-domain-containing ubiquitin E3 ligase, interacts with Rta. RNF4 also targets SUMO-2-conjugated Rta and promotes its ubiquitination in vitro. Additionally, SUMO interaction motifs in RNF4 are important to the ubiquitination of Rta because the RNF4 mutant with a mutation at the motifs eliminates ubiquitination. The mutation of four lysine residues on Rta that abrogated SUMO-3 conjugation to Rta also decreases the enhancement of the ubiquitination of Rta by RNF4. This finding demonstrates that RNF4 is a SUMO-targeted ubiquitin E3 ligase of Rta. Finally, knockdown of RNF4 enhances the expression of Rta and EA-D, subsequently promoting EBV lytic replication and virions production. Results of this study significantly contribute to efforts to elucidate a SUMO-targeted ubiquitin E3 ligase that regulates Rta ubiquitination to influence the lytic development of EBV.

A total of 20 Bacillus subtilis F29-3 mutants defective in fengycin biosynthesis was obtained by Tn917 mutagenesis. Cloning and mapping results showed that the transposon in these mutants was inserted in eleven different locations on the chromosome. We were able to use the chromosomal sequence adjacent to the transposon as a probe to screen for cosmid clones containing the fengycin biosynthesis genes. One of the clones obtained, pFC660, was 46 kb long. Eight transposon insertion sites were mapped within this plasmid. Among the eleven different mutants analyzed, four mutants had Tn917 inserted in regions which encoded peptide sequences similar to part of gramicidin S synthetase, surfactin synthetase, and tyrocidine synthetase. Our results suggest that fengycin is synthesized nonribosomally by the multienzyme thiotemplate mechanism.

Reactivation of Epstein-Barr virus (EBV) from latency to the lytic cycle is required for the production of viral particles. Here, we examine the capacity of resveratrol to inhibit the EBV lytic cycle. Our results show that resveratrol inhibits the transcription of EBV immediate early genes, the expression of EBV lytic proteins, including Rta, Zta, and EA-D and reduces viron production, suggesting that this compound may be useful for preventing the proliferation of the virus.

The hedgehog (Hh) transcription factor Gli induces transformation of epithelial cells via induction of Snail, a repressor of E-cadherin. Epithelial-mesenchymal transition is also a determinant of the progression of tumorigenesis, following down-regulation of E-cadherin. However, the role of Hh signaling components and Snail/E-cadherin in brain tumors is not yet fully understood. We analyzed the expression of Hh signaling components and Snail/E-cadherin in 69 brain tumors by reverse transcription-polymerase chain reaction (RT-PCR). The data showed that overexpression of Smo (35/69), Ptch (50/69), Gli1 (56/69), Gli2 (29/69) and N-myc (39/69) might contribute to brain tumorigenesis. Our results also indicated that Snail and E-cadherin showed opposing expression in malignant tumors (high grade astrocytoma and metastasis). Snail and E-cadherin showed less correlation in benign brain tumors. We further investigated mutations of Gli2 and Snail by RT-PCR and direct sequencing. No mutation was observed on Gli2 but several sporadic mutations on Snail were found, including S96G, S111L, S111L/ S119Y and one nonsense mutation at codon 158 (Y158*) . An in vitro E-cadherin promoter assay showed that S96G, S111L, S111L/S119Y Snail mutants were decreased by 15, 25 and 50%, respectively, whereas Y158 * was increased by 40% compared to wild-type. Furthermore, our data showed that wild-type Snail and S96G, S 111L, S111L/S119Y translocated to the nucleus, while the Y158 * mutant failed to translocate to the nucleus. Taken together, our results demonstrate that Hh signaling components, the expression and mutations of Snail and the expression of E-cadherin may play an important role in human brain tumorigenesis.

Aurora-A is involved in chromosome alignment, centrosome maturation, mitotic spindle assembly and regards to an oncogene. Aurora-A is also known to bind to several other proteins affecting its up-regulation or down-regulation and localization. However, how these different binding signals work together to regulate Aurora-A is not properly known. To explore more Aurora-A interacting proteins, the low-copy yeast two-hybrid screening using Aurora-A as bait protein was performed. One novel gene, AIBp, was demonstrated to associate with Aurora-A by the yeast two-hybrid method and in vitro GST pull-down assay. Molecular characterization showed that AIBp possessed a binding site at the C-terminal with Aurora-A (kinase domain). Interestingly, AIBp also interacts with hNinein at the N-terminal, which overlaps with a previously reported hNinein and GSK3beta binding site. Using a kinase assay, AIBp interacts with the Aurora-A kinase domain functions as a positive regulator, whereas AIBp binding to hNinein appears to block the phosphorylation of hNinein by both Aurora-A and GSK3beta. siRNA-mediated elimination of AIBp from HeLa cells, results in a doughnut-like shape, asymmetrical spindle pole and multiple spindle pole formation. We also demonstrated that both AIBp and Aurora-A are co-overexpressed in various brain tumors. These studies demonstrate that AIBp may not only be required for the dynamic movement of Aurora-A at the centrosomes and spindle apparatus during the cell cycle, but may also be important during brain tumorigenesis.

Epstein–Barr virus (EBV) expresses two transcription factors, Rta and Zta, during the immediate-early stage of the lytic cycle to activate the transcription of viral lytic genes. Our immunoblotting and flow cytometry analyses find that moronic acid, found in galls of Rhus chinensis and Brazilian propolis, at 10 μM inhibits the expression of Rta, Zta, and an EBV early protein, EA-D, after lytic induction with sodium butyrate. This study also finds that moronic acids inhibits the capacity of Rta to activate a promoter that contains an Rta-response element, indicating that moronic acid interferes with the function of Rta. On the other hand, moronic acid does not appear to influence with the transactivation function of Zta. Therefore, the lack of expression of Zta and EA-D after moronic acid treatment is attributable to the inhibition of the transactivation functions of Rta. Because the expression of Zta, EA-D and many EBV lytic genes depends on Rta, the treatment of P3HR1 cells with moronic acid substantially reduces the numbers of EBV particles produced by the cells after lytic induction. This study suggests that moronic acid is a new structural lead for anti-EBV drug development.

BRCA1-associated protein 2 (BRAP2) is known to interact with the kinase suppressor of Ras 1 (KSR1), inhibiting the ERK signal transduction cascade. This study found that an Epstein–Barr virus (EBV) immediate-early protein, Rta, is a binding partner of BRAP2 in yeast and confirmed the bindingin vitroby a glutathioneS-transferase pull-down assay andin vivoby co-immunoprecipitation in 293(maxi-EBV) cells. Binding studies also showed that Rta and KSR1 interacted with the C-terminal 202 aa region in BRAP2. Additionally, Rta appeared to prevent the binding of KSR1 to BRAP2, activating the ERK signal transduction pathway and the transcription of an EBV immediate-early gene,BZLF1. Activation of the ERK signal transduction pathway by Rta may be critical for the maintenance of the lytic state of EBV.

Epstein-Barr virus (EBV) expresses the immediate-early protein Rta to activate the transcription of EBV lytic genes and the lytic cycle. We show that RanBPM acts as a binding partner of Rta in yeast two-hybrid analysis. The binding was confirmed by glutathione-S-transferase pull-down assay. A coimmunoprecipitation experiment and confocal microscopy revealed that RanBPM and Rta interact in vivo and colocalize in the nucleus. The interaction appears to involve the SPRY domain in RanBPM and the region between amino acid residues 416 to 476 in Rta. The interaction promotes the transactivation activity of Rta in activating the transcription of BMLF1 and p21 in transient transfection assays. Additionally, RanBPM interacts with SUMO-E2 (Ubc9) to promote sumoylation of Rta by SUMO-1. This fact explains why the expression of RanBPM enhances the transactivation activity of Rta. Taken together, the present results indicate a new role of RanBPM in regulating a viral protein that is critical to EBV lytic activation.

Human cIAP1 protein is a member of the inhibitor of apoptosis proteins (IAPs) that are involved in apoptosis regulation and an increasing number of other functions, including cell cycle and intracellular signal transduction. In order to identify novel proteins involved in cIAP1 regulation, we performed a yeast two-hybrid screen and identified an F-box protein Fbxo7 as a cIAP1 interacting protein. Co-immunoprecipitation assay showed that cIAP1 can interact with Fbxo7 in human cells. When co-expressed in cells, cIAP1 and Fbxo7 co-localized remarkably both in the cytoplasm and nucleus, and considerable amounts of these often co-localized at one or few distinct Golgi-like structures close to the nucleus. Furthermore, we showed that overexpression of Fbxo7 promotes the ubiquitination of cIAP1. Since F-box proteins are specificity determining subunits of SCF ubiquitin protein ligases, our results suggest that Fbxo7 can mediate the ubiquitination of cIAP1 by SCF ubiquitin protein ligase and thus have important implication in the regulation of cIAP1 function.

Epstein-Barr virus (EBV) expresses an immediate-early protein, Rta, to activate the viral lytic cycle. This study identifies PIASxalpha and PIASxbeta as binding partners of Rta in a yeast two-hybrid screen and demonstrates the binding of Rta to PIASxalpha and PIASxbeta in vitro by GST pull-down analysis. Coimmunoprecipitation experiments and indirect immunofluorescence analysis show that Rta interacts and colocalizes with PIASxalpha and PIASxbeta in the nucleus. These interactions seem to enhance Rta sumoylation as transfecting plasmids expressing PIASxalpha, PIASxbeta, Ubc9, or SUMO-1 increase the capacity of Rta to transactivate a promoter that contains an Rta-response element and the promoters of p21 and BNLF1 in transient transfection assay. This study also finds that Rta sumoylation is preferentially enhanced by PIASxbeta, which could be attributed to the fact that PIASxbeta, compared to PIASxalpha, has a strong affinity to Rta, suggesting that affinity of a SUMO E3 ligase to its target protein influences the function of protein sumoylation.

A centrosomal-associated protein, ninein is a microtubules minus end capping, centrosome position, and anchoring protein, but the underlying structure and physiological functions are still unknown. To identify the molecules that regulate the function of human ninein in centrosome, we performed yeast two-hybrid screen and isolated the SUMO-conjugating E2 enzyme, Ubc9, and SUMOylation enhancing enzymes, including PIAS1 and PIASxalpha, as binding partners of hNinein. These interactions as well as the interaction between hNinein and SUMO-1 are also confirmed by a glutathione S-transferase (GST) pull-down experiment. Furthermore, the C-terminal region of hNinein can be SUMOylated in vitro and in HeLa cells transfected with a plasmid expressing GFP-hNinein. Our findings firstly place SUMOylation target on the centrosome structure protein, hNinein, which results in the switch localization from centrosome to nucleus, suggesting the importance of the SUMOylation of hNinein and probably other centrosomal proteins may also be involved in the centrosome activity.

To explore more possible roles for GSK3beta function, the yeast two-hybrid screening using GSK3beta as a bait protein was performed. In this study, we demonstrated that two variants of CABYR (281 and 379) interacted with GSK3beta in the yeast two-hybrid and GST pull down assay. Molecular characterization showed that CABYR variants formed a dimer with a proline-rich extensin-like domain, which slightly overlapped with GSK3beta-binding site. In kinase assay, we also showed that CABYR variants act as an ideal substrate for GSK3beta within the extensin-like domain and phosphorylation sites on CABYR were mapped. Interestingly, Northern blot showed that CABYR transcripts were expressed more distinctly in the fetal brain than in the adult brain, suggesting that this protein may play a role during brain development. Moreover, differential expression of CABYR variants may exhibit aberrant expression in brain tumors and cancer cell lines.

Fengycin is a cyclic lipopeptidic antibiotic produced nonribosomally by Bacillus subtilis. A fengycin synthetase mutant of B. subtilis F29-3 was generated with Tn917lux, which contains a transposon inserted in a 7716-bp gene, fenD. The mutation can be genetically complemented by transforming a plasmid carrying a wild-type fenD, confirming the participation of the gene in fengycin synthesis. Sequencing and biochemical analysis reveal that this gene encodes an enzyme that includes two amino acid-activating modules, FenD1 and FenD2, which activate l-Tyr and l-Thr, the third and the fourth amino acids in fengycin, respectively.

Epstein-Barr virus (EBV) expresses an immediate-early protein, Rta, to activate the transcription of EBV lytic genes and the lytic cycle. This work identifies Ubc9 and PIAS1 as binding partners of Rta in a yeast two-hybrid screen. These bindings are verified by glutathione S-transferase pull-down assay, coimmunoprecipitation, and confocal microscopy. The interactions appear to cause Rta sumoylation, because not only can Rta be sumoylated in vitro but also sumoylated Rta can be detected in P3HR1 cells following lytic induction and in 293T cells after transfecting plasmids that express Rta and SUMO-1. Moreover, PIAS1 stimulates conjugation of SUMO-1 to Rta, thus acting as an E3 ligase. Furthermore, transfecting plasmids that express Ubc9, PIAS1, and SUMO-1 increases the capacity of Rta to transactivate the promoter that includes an Rta response element, indicating that the modification by SUMO-1 increases the transactivation activity of Rta. This study reveals that Rta is sumoylated at the Lys-19, Lys-213, and Lys-517 residues and that SUMO-1 conjugation at the Lys-19 residue is crucial for enhancing the transactivation activity of Rta. These results indicate that sumoylation of Rta may be important in EBV lytic activation.

(-)-Epigallocatechin gallate (EGCG), abundant in green tea, is a potent anti-microbial and anti-tumor compound. This investigation used immunoblot, flow cytometry, microarray, and indirect immunofluorescence analyses to show that at concentrations exceeding 50 microM, EGCG inhibits the expression of Epstein-Barr virus (EBV) lytic proteins, including Rta, Zta, and EA-D, but does not affect the expression of EBNA-1. Moreover, DNA microarray and transient transfection analyses demonstrated that EGCG blocks EBV lytic cycle by inhibiting the transcription of immediate-early genes, thus inhibiting the initiation of EBV lytic cascade.

Epstein-Barr virus (EBV) expresses an immediate-early protein, Rta, to activate the transcription of EBV lytic genes. This protein usually binds to Rta-response elements or interacts with Sp1 or Zta via a mediator protein, MCAF1, to activate transcription. Rta is also known to interact with TBP and TFIIB to activate transcription. This study finds that Rta interacts with TAF4, a component of TFIID complex, in vitro and in vivo, and on the TATA sequence in the BcLF1 promoter. Rta also interacts with TAF4 and Sp1 on Sp1-binding sequences on TATA-less promoters, including those of BNLF1, BALF5, and the human androgen receptor. These interactions are important to the transcriptional activation of these genes by Rta since introducing TAF4 shRNA substantially reduces the ability of Rta to activate these promoters. This investigation reveals how Rta interacts with TFIID to stimulate transcription.

A Tn977 derivative was constructed for the purposes of mutagenesis and cloning of Bacillus subtilis genes. This transposon, Tn 917ac1 (4.6 kb), consisted of terminal inverted repeats of Tn 977 , the res sequence, a ColE1 origin of replication ( ori ) and two drug-resistance genes. The plasmid carrying this transposon, named pD917, contained the ermtnpR-tnpA gene cluster of Tn977 and a temperature-sensitive ori of pE194. For the purpose of mutagenesis, transposition of Tn917ac1 was induced by culturing strains harboring pD917 in a medium containing a low concentration of erythromycin. Cells with a Tn 917ac1 insertion in the chromosome were selected on agar containing chloramphenicol after heat treatment to eliminate the plasmidic form of pD917. DNA fragments adjacent to Tn917ac1 could be cloned by restriction digestion of the chromosomal DNA and by transforming the self-ligated restriction fragments into Escherichia coli . Sequence analysis revealed that Tn 917ac1 was integrated into the chromosome of B. subtilis by transposition in a recE strain and by transposition or integration of pD917 in a wild-type strain. Tn917ac1 has been demonstrated to be useful for mutating and cloning of the genes involved in the biosynthesis of fengycin in B. subtilis F29-3.