Rie Yamada

SUMO-1 modification of the major immediate-early (IE) 1 and 2 proteins of human cytomegalovirus is regulated by different mechanisms and modulates the intracellular localization of the IE1, but not IE2, protein

Summary.We have previously shown that two proteins with apparent molecular masses of 91- and 102-kDa (p91 and p102, respectively) in human cytomegalovirus (HCMV)-infected cells are antigenically and structurally related to the major immediate-early (IE) 1 and 2 proteins (IE1p68 and IE2p80, respectively) of HCMV, respectively. In this study, we extended the characterization of p91 and p102 and our results were as follows; (i) Lysine at amino acid position 450 in IE1p68 and at amino acid position 175 or 180 in IE2p80, to which SUMO-1 has been shown to be covalently linked, are required for production of p91 and p102, respectively. (ii) A reversal of cycloheximide (CH) block in the presence of actinomycin D imposed at the time of infection inhibited production of p91, but not p102. (iii) The steady-state levels of p91, but not p102, were remarkably decreased by treatment with proteasome inhibitor MG132, but coincubation with CH inhibited this decrease of p91. (iv) Cell fractionation by differential detergent extraction demonstrated that p91 is preferentially found in detergent-insoluble fraction, although p102 as well as IE1p68 and IE2p80 distributes into all fractions. These results demonstrate that p91 and p102 correspond to SUMO-1-modified IE1p68 and IE2p80, respectively, that the production and degradation of SUMO-1-modified IE1p68 is regulated by mechanisms different from those of SUMO-1-modified IE2p80, and that SUMO-1 modification modulates the intracellular localization of IE1p68, but not IE2p80.

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.

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.

The major immediate-early genes of human cytomegalovirus induce two novel proteins with molecular weights of 91 and 102 kilodaltons

Summary. Mouse monoclonal antibody MAB810 is known to recognize the major immediate-early (IE) proteins, 68 kDa IE1 (IE1p68) and 80 kDa IE2 (IE2p80), of human cytomegalovirus (HCMV). Using this antibody we found that two additional proteins with higher molecular weights of approximately 91 (p91) and 102 kDa (p102) are also synthesized in HCMV-infected cells. p91 and p102 were produced in cells stably transfected with plasmid expressing IE1p68 and IE2p80, respectively, and shown to be related with IE1p68 and IE2p80, respectively, in primary amino acid sequence. Taken together, these results indicate that p91 and p102 are expressed from the IE1 and IE2 genes, respectively.

The effect of cyclic AMP on expression of the major immediate-early genes and replication of human cytomegalovirus in human central nervous system cell lines

SummaryThe effect of dibutyryl cyclic AMP (dbcAMP) on interaction of human cytomegalovirus (HCMV) with human central nervous system cell lines was examined. Activation of the major immediate-early (IE) promoter (MIEP) of HCMV by dbcAMP was observed in human neuroblastoma IMR-32 cells, but not in glioma 118MGC and astrocytoma U373-MG cells. The 19 bp repeat element in the enhancer of the MIEP was most likely to be the cis-element that responded to dbcAMP in IMR-32 cells as we expected. In IMR-32 cells activation of the MIEP led to enhanced synthesis of the major IE proteins and infectious HCMV.

Human cytomegalovirus replication supported by virus-induced activation of CCL2-CCR2 interactions.

We previously revealed that human cytomegalovirus (HCMV) infection can cause aberrant expression of the chemokine IL-8/CXCL8. We first examined the effects of HCMV infection on the expression of another chemokine, CCL2. HCMV infection induced CCL2 expression at the mRNA and protein levels in human embryonic lung fibroblasts cells (HEL). Moreover, HCMV induced the mRNA expression of CCR2, a specific receptor for CCL2. CCL2 siRNA treatment reduced HCMV virion production, and this reduction was reversed by the addition of CCL2. We further observed that CCL2 siRNA, but not control siRNA, reduced the expression of HCMV immediate early gene (IE1) and HCMV UL54 gene (DNA polymerase) in a dose-dependent manner. Thus, HCMV infection is able to activate the CCL2-CCR2 interactions to further enhance HCMV infection and/or replication.