Tsugiya Murayama

Potential involvement of IL-8 in the pathogenesis of human cytomegalovirus infection.

The observations that several types of viruses induced interleukin (IL)‐8 production prompted us to investigate the interrelationship between IL‐8 and cytomegalovirus (CMV) infection. CMV infection caused IL‐8 production in a human monocytic cell line, THP‐1, in dose‐ and time‐dependent manners. Moreover, CMV induced IL‐8 gene expression by concurrently activating transcription factors, NF‐κB and AP‐1. Furthermore, CMV infection of human fibroblast cell lines increased gene expression of a specific receptor for IL‐8, CXCR1. IL‐8 in turn enhanced CMV replication in a human embryonic fibroblast, MRC‐5, in dose‐ and time‐dependent manners. Augmented replication eventually culminated in the increased production of infectious CMV virions. Moreover, IL‐8 can attenuate the antiviral activity of interferon (IFN), particularly that of α‐type against picornaviruses such as encephalomyocarditis virus and poliovirus. The inhibitory effects were associated with reduced 2′,5′‐A oligoadenylate synthetase activity. These results would imply that CMV can induce IL‐8, which can augment CMV replication directly and indirectly by counteracting antiviral activity of IFN. J. Leukoc. Biol. 64: 62–67; 1998.

Herpes Simplex Virus Requires Poly(ADP-Ribose) Polymerase Activity for Efficient Replication and Induces Extracellular Signal-Related Kinase-Dependent Phosphorylation and ICP0-Dependent Nuclear Localization of Tankyrase 1

ABSTRACT Tankyrase 1 is a poly(ADP-ribose) polymerase (PARP) which localizes to multiple subcellular sites, including telomeres and mitotic centrosomes. Poly(ADP-ribosyl)ation of the nuclear mitotic apparatus (NuMA) protein by tankyrase 1 during mitosis is essential for sister telomere resolution and mitotic spindle pole formation. In interphase cells, tankyrase 1 resides in the cytoplasm, and its role therein is not well understood. In this study, we found that herpes simplex virus (HSV) infection induced extensive modification of tankyrase 1 but not tankyrase 2. This modification was dependent on extracellular signal-regulated kinase (ERK) activity triggered by HSV infection. Following HSV-1 infection, tankyrase 1 was recruited to the nucleus. In the early phase of infection, tankyrase 1 colocalized with ICP0 and thereafter localized within the HSV replication compartment, which was blocked in cells infected with the HSV-1 ICP0-null mutant R7910. In the absence of infection, ICP0 interacted with tankyrase 1 and efficiently promoted its nuclear localization. HSV did not replicate efficiently in cells depleted of both tankyrases 1 and 2. Moreover, XAV939, an inhibitor of tankyrase PARP activity, decreased viral titers to 2 to 5% of control values. We concluded that HSV targets tankyrase 1 in an ICP0- and ERK-dependent manner to facilitate its replication.

Transgenic rice seed synthesizing diverse flavonoids at high levels: a new platform for flavonoid production with associated health benefits

Flavonoids possess diverse health-promoting benefits but are nearly absent from rice, because most of the genes encoding enzymes for flavonoid biosynthesis are not expressed in rice seeds. In the present study, a transgenic rice plant producing several classes of flavonoids in seeds was developed by introducing multiple genes encoding enzymes involved in flavonoid synthesis, from phenylalanine to the target flavonoids, into rice. Rice accumulating naringenin was developed by introducing phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS) genes. Rice producing other classes of flavonoids, kaempferol, genistein, and apigenin, was developed by introducing, together with PAL and CHS, genes encoding flavonol synthase/flavanone-3-hydroxylase, isoflavone synthase, and flavone synthases, respectively. The endosperm-specific GluB-1 promoter or embryo- and aleurone-specific 18-kDa oleosin promoters were used to express these biosynthetic genes in seed. The target flavonoids of naringenin, kaempferol, genistein, and apigenin were highly accumulated in each transgenic rice, respectively. Furthermore, tricin was accumulated by introducing hydroxylase and methyltransferase, demonstrating that modification to flavonoid backbones can be also well manipulated in rice seeds. The flavonoids accumulated as both aglycones and several types of glycosides, and flavonoids in the endosperm were deposited into PB-II-type protein bodies. Therefore, these rice seeds provide an ideal platform for the production of particular flavonoids due to efficient glycosylation, the presence of appropriate organelles for flavonoid accumulation, and the small effect of endogenous enzymes on the production of flavonoids by exogenous enzymes.

Anti-influenza virus activity of tricin, 4',5,7-trihydroxy-3',5'-dimethoxyflavone.

Background: We examined the anti-influenza virus activity of tricin, 4′,5,7-trihydroxy-3′,5′-dimethoxyflavone, against five viruses: A/Solomon islands/3/2006 (H1N1), A/Hiroshima/52/2005 (H3N2), A/California/07/2009 (H1N1pdm), A/Narita/1/2009 (H1N1pdm) and B/Malaysia/2506/2004 strains in vitro and against A/PR/8/34 virus in vivo. Methods: The effect of tricin was studied by an infectious virus yield reduction assay. The anti-influenza virus mechanism of the tricin was examined by western blot analysis, real-time reverse transcriptase PCR analysis, haemagglutination inhibition (HI) assay and neuraminidase (NA) inhibition assay. The anti-influenza virus efficacy of tricin was further examined in a murine influenza virus infection model. Results: Tricin of 3.3 to 30 μM significantly reduced seasonal A (H1N1), (H3N2) viruses, novel A (H1N1pdm) virus, as well as B virus in a dose-dependent manner. The 50% effective concentrations of tricin were 3.4 μM for seasonal A (H3N2) virus, 4.9 μM for B virus and 8.2 μM for A/Narita (H1N1pdm) virus. Tricin decreased the expression of haemagglutinin (HA) protein and matrix (M) protein, and messenger RNA expression of HA and M of influenza virus in the infected cells. Tricin exhibited little or no effects on influenza virus HI and NA activities. In the mouse infection model, tricin was significantly effective in reducing body weight loss, and also effective in prolonging survival times of infected mice. Conclusions: Tricin was indicated to possess anti-influenza virus activity and to ameliorate body weight loss and survival rate of influenza-A-virus-infected mice. Tricin is a novel compound with potential anti-influenza virus activity in vitro and in vivo.

Proteasome inhibitor differentially regulates expression of the major immediate early genes of human cytomegalovirus in human central nervous system-derived cell lines

Proteasome inhibitor, which inhibits NF-kappaB activation, has been reported to activate c-Jun N-terminal kinase (JNK)-c-Jun pathway. In this study, we investigated the effects of proteasome inhibitor on the human cytomegalovirus (HCMV) major immediate early (MIE) gene expression in human central nervous system (CNS)-derived cell lines. Treatment of HCMV-infected 118MGC glioma and U373-MG astrocytoma cells with three proteasome inhibitors, MG132, clasto-lactacystin beta-lactone, and epoxomicin, suppressed MIE protein expression. In contrast, in HCMV-infected IMR-32 neuroblastoma cells, the proteasome inhibitors increased MIE protein expression, even in the presence of NF-kappaB inhibitor SN-50. A luciferase reporter assay demonstrated that MG132 markedly elevated the MIE promoter/enhancer (MIEP) activity in IMR-32 cells, but down-regulated it in 118MGC and U373-MG cells. Mutation in five cAMP response elements (CREs) within the MIEP resulted in a loss of the ability to respond to MG132 in IMR-32 cells. Moreover, Western blotting analysis revealed that MG132 induced c-Jun phosphorylation in all three CNS-derived cell lines, whereas a high level of activating transcription factor-2 (ATF-2) phosphorylation was observed only in IMR-32 cells. Finally, MG132-induced MIE protein expression was suppressed by JNK inhibitor that reduced the phosphorylation levels of both c-Jun and ATF-2. Taken together, these results suggest that the proteasome inhibitors activate CRE binding proteins consisting of c-Jun and ATF-2 through activating the JNK-c-Jun pathway, thereby inducing MIE protein synthesis in IMR-32 cells under the condition where NF-kappaB activity is inhibited.

Analysis of Human Cytomegalovirus UL144 Variability in Low-Passage Clinical Isolates in Japan

To explore a possible role for viral genes as determinants of virulence, portions of the UL144 tumor necrosis factor-like receptor gene and the UL55 envelope glycoprotein B gene from 42 patients with congenital human cytomegalovirus (HCMV) infection or other diseases were sequenced. Of the 42 patients, 16 (38%) had UL144 group 1 [group 1A, 15 of 16 (94%); group 1B, 1 of 16 (6%); group 1C, 0 of 16 (0%)], 5 patients (12%) had UL144 group 2, and 21 patients (50%) had UL144 group 3. Although group 1C was not found in Japan strains (0%), it was found in USA strains (22%). Other HCMV polymorphisms should be further evaluated for their potential relevance to neonatal infection, and acquired immunodeficiency syndrome-associated HCMV diseases.

Inhibition of ganciclovir-resistant human cytomegalovirus replication by Kampo (Japanese herbal medicine)

We examined the effect of Kampo on the replication of ganciclovir (GCV)-resistant human cytomegalovirus (HCMV) in the human embryonic fibroblast cell line MRC-5. Treatment of HCMV-infected cells with Sho-seiryu-to (SST; Xiao-Qing-Long-Tang in Chinese) resulted in the inhibition of viral replication without affecting the cell growth. SST treatment decreased the synthesis of viral DNA, but had no virucidal effect on cell-free HCMV. However, the inhibitory effect of SST on HCMV replication was ablated by anti-interferon-β (IFN-β) antibody suggesting that SST inhibits the replication of GCV-resistant HCMV through the induction of IFN-β. These results suggest that SST is a novel compund with potential as an anti-HCMV.

Anticytomegalovirus activity of pristimerin, a triterpenoid quinone methide isolated from Maytenus heterophylla (Eckl. & Zeyh.)

We examined the anticytomegalovirus properties of four compounds: pristimerin, the pristimerin analogue, lupeol and 2-acetylphenol-1-β-D-glucopyranosyl (1→6)-β-D-xylpyranoside (acetophenol glycoside), isolated from Maytenus heterophylla, a Kenyan medicinal plant. The effects were studied on human cytomegalovirus (HCMV) replication in the human embryonic fibroblast cell line, MRC-5. In a viral plaque-reduction assay, pristimerin showed dose-dependent inhibitory properties with a 50% inhibitory concentration of 0.53 µg/ml (selective index=27.9). The cells treated with pristimerin inhibited the cytopathic effects in HCMV-infected cells. Moreover, pristimerin suppressed viral replication without affecting the cell growth. Pristimerin inhibited the synthesis of viral DNA but had no virucidal effect on cell-free HCMV. Furthermore, Western blot analysis demonstrated that pristimerin decreased the amount of immediate early (IE) antigen (especially IE2) expression in the infected cells. These results suggest that pristimerin is a unique compound with potential anti-HCMV activity.

In Vitro Anti-Cytomegalovirus Activity of Kampo (Japanese Herbal) Medicine

We examined the effect of three types of Kampo medicines on human cytomegalovirus (CMV) replication in the human embryonic fibroblast cell line, MRC-5. Treatment of cells with at least 0.01 μg/ml of Kampo medicines inhibited the cytopathic effects of CMV-infected MRC-5 cells. Moreover, Kampo medicine decreased the replication of CMV without affecting the inhibition of host cells, with a concomitant decrease in CMV DNA levels. However, Kampo medicine demonstrated no virocidal effect on cell-free CMV. Furthermore, western blotting analysis demonstrated that the Kampo medicine decreased the amount of 65 kDa late antigen expression in the infected cells. These results suggest that Kampo medicine may be sufficient to inhibit viral DNA replication and late protein synthesis, resulting in anti-CMV effects. Therefore, these three Kampo medicines have the potential of being a source of new powerful anti-CMV compounds.

Recombinant human monoclonal antibodies to human cytomegalovirus glycoprotein B neutralize virus in a complement-dependent manner

Human antibodies specific for HCMV are currently considered as potential anti-HCMV therapeutic agents. In this study, we used a combinatorial human antibody library to isolate and characterize complete human monoclonal antibodies that effectively neutralize HCMV in a complement-dependent manner. One hundred and six clones were isolated in two independent screens using HCMV virions and recombinant glycoprotein B, gB654, as antigens. All of the clones recognized the same molecule gB and were classified into 14 groups based on the amino acid sequence of the V(H) region. Seven representative clones from these 14 groups had a strong gB654 binding affinity by surface plasmon resonance (SPR). A pairwise binding competition analysis suggested that there were three groups based on differences in the gB recognition sites. Although Fab fragments of the seven groups showed strong affinity for gB, none of the Fab fragments neutralized HCMV infectivity in vitro. In contrast, complete human IgG(1) antibodies of at least three groups neutralized HCMV in a complement-dependent manner. These data suggest that potent therapeutic antibodies can be obtained from a human antibody library, including most of the functional antibodies that mediate humoral immunity to the selected pathogen.