Hyosun Cho

CTCF Regulates Kaposi's Sarcoma-Associated Herpesvirus Latency Transcription by Nucleosome Displacement and RNA Polymerase Programming

ABSTRACT CCCTC-binding factor (CTCF) has been implicated in various aspects of viral and host chromatin organization and transcriptional control. We showed previously that CTCF binds to a cluster of three sites in the first intron of the Kaposis sarcoma-associated herpesvirus (KSHV) multicistronic latency-associated transcript that encodes latency-associated nuclear antigen (LANA), viral cyclin (vCyclin), vFLIP, viral microRNAs, and kaposin. We show here that these CTCF binding sites regulate mRNA production, RNA polymerase II (RNAPII) programming, and nucleosome organization of the KSHV latency transcript control region. We also show that KSHV bacmids lacking these CTCF binding sites have elevated and altered ratios of spliced latency transcripts. CTCF binding site mutations altered RNAPII and RNAPII-accessory factor interactions with the latency control region. CTCF binding sites were required for the in vitro recruitment of RNAPII to the latency control region, suggesting that direct interactions between CTCF and RNAPII contribute to transcription regulation. Histone modifications in the latency control region were also altered by mutations in the CTCF binding sites. Finally, we show that CTCF binding alters the regular phasing of nucleosomes in the latency gene transcript and intron, suggesting that nucleosome positioning can be an underlying biochemical mechanism of CTCF function. We propose that RNAPII interactions and nucleosome displacement serve as a biochemical basis for programming RNAPII in the KSHV transcriptional control region.

Genipin as a novel chemical activator of EBV lytic cycle

Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus that causes acute infection and establishes life-long latency. EBV causes several human cancers, including Burkitt’s lymphoma, nasopharyngeal and gastric carcinoma. Antiviral agents can be categorized as virucides, antiviral chemotherapeutic agents, and immunomodulators. Most antiviral agents affect actively replicating viruses, but not their latent forms. Novel antiviral agents must be active on both the replicating and the latent forms of the virus. Gardenia jasminoides is an evergreen flowering plant belonging to the Rubiaceae family and is most commonly found growing wild in Vietnam, Southern China, Taiwan, Japan, Myanmar, and India. Genipin is an aglycone derived from an iridoid glycoside called geniposide, which is present in large quantities in the fruit of G. jasminoides. In this study, genipin was evaluated for its role as an antitumor and antiviral agent that produces inhibitory effects against EBV and EBV associated gastric carcinoma (EBVaGC). In SNU719 cells, one of EBVaGCs, genipin caused significant cytotoxicity (70 μM), induced methylation on EBV C promoter and tumor suppressor gene BCL7A, arrested cell-cycle progress (S phases), upregulated EBV latent/lytic genes in a dose-dependent manner, stimulated EBV progeny production, activated EBV F promoter for EBV lytic activation, and suppressed EBV infection. These results indicated that genipin could be a promising candidate for antiviral and antitumor agents against EBV and EBVaGC.

The PseEF efflux system is a virulence factor of Pseudomonas syringae pv. syringae

An ATP-binding cassette (ABC) transporter, called the PseEF efflux system, was identified at the left border of the syr-syp genomic island of Pseudomonas syringae pv. syringae strain B301D. The PseEF efflux system was located within a 3.3-kb operon that encodes a periplasmic membrane fusion protein (PseE), and an ABC-type cytoplasmic membrane protein (PseF). The PseEF efflux system exhibited amino acid homology to a putative ABC efflux system (MacAB) of E. coli W3104 with identities of 47.2% (i.e., PseE to MacA) and 57.6% (i.e., PseF to MacB). A nonpolar mutation within the pseF gene was generated by nptII insertional mutagenesis. The resultant mutant strain showed significant reduction in secretion of syringomycin (74%) and syringopeptin (71%), as compared to parental strain B301D. Quantitative real-time RT-PCR was used to determine transcript levels of the syringomycin (syrB1) and syringopeptin (sypA) synthetase genes in strain B301D-HK7 (a pseF mutant). Expression of the sypA gene by mutant strain B301D-HK7 was approximately 6.9% as compared to that of parental strain B301D, while the syrB1 gene expression by mutant strain B301D-HK7 was nearly 14.6%. In addition, mutant strain B301D-HK7 was less virulent by approximately 67% than parental strain B301D in immature cherry fruits. Mutant strain B301D-HK7 was not reduced in resistance to any antibiotics used in this study as compared to parental strain B301D. Expression (transcript levels) of the pseF gene was induced approximately six times by strain B301D grown on syringomycin minimum medium (SRM) supplemented with the plant signal molecules arbutin and D-fructose (SRMAF), as compared to that of strain B301D grown on SRM (in the absence of plant signal molecules). In addition, during infection of bean plants by P. syringae pv. syringae strain B728a, expression of the pseF gene increased at 3 days after inoculation (dai). More than 180-fold induction was observed in transcript levels of the pseF gene by parental strain B728a as compared to strain B728a-SL7 (a salA mutant). Thus, the PseEF efflux system, an ABC-type efflux system, has an important role in secretion of syringomycin and syringopeptin, and is required for full virulence in P. syringae pv. syringae.

Anti-influenza effect of Cordyceps militaris through immunomodulation in a DBA/2 mouse model

The immune-modulatory as well as anti-influenza effects of Cordyceps extract were investigated using a DBA/2 mouse model. Three different concentrations of Cordyceps extract, red ginseng extract, or drinking water were orally administered to mice for seven days, and then the mice were intranasally infected with 2009 pandemic influenza H1N1 virus. Body weight changes and survival rate were measured daily post-infection. Plasma IL-12, TNF-α, and the frequency of natural killer (NK) cells were measured on day 4 post-infection. The DBA/2 strain was highly susceptible to H1N1 virus infection. We also found that Cordyceps extract had an antiinfluenza effect that was associated with stable body weight and reduced mortality. The anti-viral effect of Cordyceps extract on influenza infection was mediated presumably by increased IL-12 expression and greater number of NK cells. However, high TNF-α expression after infection of H1N1 virus in mice not receiving treatment with Cordyceps extract suggested a two-sided effect of the extract on host immune regulation.

Anti-tumor effect of Cordyceps militaris in HCV-infected human hepatocarcinoma 7.5 cells

Cordyceps extract has been reported to have various pharmacological activities including an anti-cancer effect. We investigated the inhibitory effect of Cordyceps militaris on hepatitis C virus-infected human hepatocarcinoma 7.5 cells (J6/JFH1-huh 7.5 cells). The huh7.5 cells with or without HCV infection were treated with various concentrations of ethanol extract of Cordyceps militaris (CME) for 48 h and the cytotoxicity was measured by CCK-8 assay. Both J6/JFH1-huh7.5 cells and huh7.5 cells were highly susceptible to CME. To examine the molecular mechanisms of the inhibitory effect on huh7.5 cells, the effect of CME on cell apoptosis was measured using flow cytometry and the expressions of p53, Bim, Bax, PARP, (cleaved) caspase-9, and (cleaved) caspase- 3 in huh 7.5 cells were detected by western blot assays. CME significantly increased early apoptosis and up-regulated the expression of Bim, Bax, cleaved PARP, cleaved caspase 9 and cleaved caspase-3. We also found the decrease of HCV Core or NS3 protein by CME in HCV-infected huh 7.5 cells.

Apoptosis-Inducing Activity of Marine Sponge Haliclona sp. Extracts Collected from Kosrae in Nonsmall Cell Lung Cancer A549 Cells.

Although various anticancer drugs have been developed for the treatment of nonsmall cell lung cancer, chemotherapeutic efficacy is still limited. Natural products such as phytochemicals have been screened as novel alternative materials, but alternative funds such as marine bioresources remain largely untapped. Of these resources, marine sponges have undergone the most scrutiny for their biological activities, including antiinflammatory, antiviral, and anticancer properties. However, the biological mechanisms of the activities of these marine sponges are still unclear. We investigated the anticancer activity of marine sponges collected from Kosrae in Micronesia and examined their mechanisms of action using nonsmall cell lung cancer A549 cells as a model system. Of 20 specimens, the Haliclona sp. (KO1304-328) showed both dose- and time-dependent cytotoxicity. Further, methanol extracts of Haliclona sp. significantly inhibited cell proliferation and cell viability. A549 cells treated with Haliclona sp. demonstrated induced expression of c-Jun N-terminal kinase (JNK), p53, p21, caspase-8, and caspase-3. The percentage of apoptotic cells significantly increased in A549 cultures treated with Haliclona sp. These results indicate that Haliclona sp. induces apoptosis via the JNK-p53 pathway and caspase-8, suggesting that this marine sponge is a good resource for the development of drugs for treatment of nonsmall cell lung cancer.

Natural killer cells and tumor metastasis

Natural killer (NK) cells are cytotoxic lymphocytes that recognize tumor cells or stressed cells through ‘missing-self’ signals, such as altered or absent expression of MHC class I molecules. The function of NK cells is regulated by the activation or inhibition of receptors present on their surface. The activation of NK cells results in cytotoxic activity on target cells through release of toxic granules and inflammatory cytokines. However, NK cells infiltrating tumors have been frequently shown to exhibit a skewed phenotype that includes decreased antitumor activity and enhanced protumor activities, such as angiogenesis and metastasis. In fact, many studies have reported that tumor microenvironments induce a protumor phenotype in NK cells. Here, we review the biological properties of NK cells in the context of tumorigenesis and tumor progression, with a specific focus on the interactions between NK cells and critical tumor microenvironments, such as epithelial-to-mesenchymal transition, matrix metalloproteinases, and tumor-associated chronic inflammation in tumor metastasis.

DNA hypermethylation induced by Epstein-Barr virus in the development of Epstein-Barr virus-associated gastric carcinoma.

Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) is a recently recognized disease entity defined by the presence of EBV in gastric carcinoma cells. EBV infection causes major epigenetic alterations in the EBV genome and its cellular host genome, suggesting that EBV acts as a direct epigenetic driver for EBVaGC. One of the major epigenetic events in the viral and cellular genomes to control transcription is DNA hypo- or hyper-methylation. Particularly, local and global hypermethylation have been reported in EBVaGC. It is therefore important to understand the molecular mechanisms of DNA hypermethylation during EBVaGC carcinogenesis. To understand the functional roles of DNA methylation and suggest therapeutic target candidates for EBVaGC, we reviewed recent literature reporting DNA hypermethylation in EBVaGC. We summarized the identified candidate genes that are markedly hypermethylated in EBVaGC, which can potentially be targets for chemotherapies with demethylating agents.

Phenotypic characteristics of natural killer cells in acute hepatitis

Natural killer (NK) cells are the principal effector cell population in innate immune defense against many types of infections. These cells are enriched in the liver, where they comprise approximately 40% to 60% of the intrahepatic lymphocyte pool compared to the peripheral blood compartment. In chronic HBV and HCV infection, NK cells were reported to be partially dysfunctional due to impaired IFN-Γ secretion. Few studies have examined phenotypic features of NK cells in acute hepatitis. We identified NK (CD56+CD3-) cell populations in patients with acute hepatitis by examining the expressions of phenotypic NK cell markers (CD16, NKG2A, and NKG2D). Peripheral blood mononuclear cells were isolated from patients with acute hepatitis A (7) and patients with non-viral acute toxic hepatitis (6) during the symptomatic and convalescent phases. Expressions of NK (CD56+CD3-) cell markers, CD16, NKG2A, and NKG2D, were measured by flow cytometry. Symptomatic acute hepatitis including non-viral hepatitis and HAV infection showed significant increases of NKG2A expression compared to healthy controls. Interestingly, there was a direct correlation between the proportion of NK cell populations and liver function parameters (AST, ALT) in HAV infection. The strong correlation was also observed between the expression of NKG2A+NK cells and ALT, which suggests that most of NK cells in severe phase of disease express high level of NKG2A on their surface. In addition, decreased number of NK cells (CD56+CD3-) in symptomatic phase began to increase in the convalescent phase of acute hepatitis A. However, the expression of NKG2A tended to be reduced, which indicates that NKG2A, the inhibitory receptor on NK cells, can be a severity parameter in acute hepatitis.

KSHV infection of B-cell lymphoma using a modified KSHV BAC36 and coculturing system.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of two B cell lymphoproliferative diseases, namely primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD). KSHV infection of B cell lymphoma in vitro has been a long-standing battle in advancing human KSHV biology. In this study, a modified form of KSHV BAC36 named BAC36A significantly increased the fidelity of gene-targeted site-directed mutagenesis in the KSHV genome. This modification eliminates tedious screening steps required to obtain mutant clones when a KSHV BAC36 reverse genetic system is used. Coculturing B-cell lymphoma BJAB cells with KSHV BAC36A stably transfected 293T cells enabled us to infect BJAB cells with a KSHV virion derived from the KSHV BAC36A. The coculture system produced substantial amounts of KSHV infection to BJAB, meaning that KSHV virions were released from 293T cells and then infected neighboring BJAB cells. Owing to our success with the KSHV BAC36A and coculture system, we propose a new genetic system for the study of KSHV gene expression and regulation in B-cell lymphoma.