The Interferon-Inducible Human PLSCR1 Protein Is a Restriction Factor of Human Cytomegalovirus.

Human phospholipid scramblase 1 (PLSCR1) is strongly expressed in response to interferon (IFN) treatment and viral infection, and it has been suggested to play an important role in IFN-dependent antiviral responses. In this study, we showed that the levels of human cytomegalovirus (HCMV) plaque formation in OUMS-36T-3 (36T-3) cells with high basal expression of PLSCR1 were significantly lower than those in human embryonic lung (HEL) cells with low basal expression of PLSCR1. In addition, the levels of HCMV plaque formation and replication in PLSCR1-knockout (KO) 36T-3 cells were significantly higher than those in parental 36T-3 cells and were comparable to those in HEL cells. Furthermore, compared to that in PLSCR1-KO cells, the expression of HCMV major immediate early (MIE) proteins was repressed and/or delayed in parental 36T-3 cells after HCMV infection. We also showed that PLSCR1 expression decreased the levels of the cAMP-responsive element (CRE)-binding protein (CREB)•HCMV immediate early protein 2 (IE2) and CREB-binding protein (CBP)•IE2 complexes, which have been suggested to play important roles in the IE2-mediated transactivation of the viral early promoter through interactions with CREB, CBP, and IE2. Interestingly, PLSCR1 expression repressed CRE- and HCMV MIE promoter-regulated reporter gene activities. These observations reveal, for the first time, that PLSCR1 negatively regulates HCMV replication by repressing the transcription from viral MIE and early promoters, and that PLSCR1 expression may contribute to the IFN-mediated suppression of HCMV infection. IMPORTANCE Because several IFN-stimulated genes (ISGs) have been reported to suppress HCMV replication, HCMV replication is thought to be regulated by an IFN-mediated host defense mechanism, but the mechanism remains unclear. PLSCR1 expression is induced in response to viral infection and IFN treatment, and PLSCR1 has been reported to play an important role in IFN-dependent antiviral responses. Here, we demonstrate that HCMV plaque formation and major immediate early (MIE) gene expression are significantly increased in PLSCR1-KO human fibroblast cells. PLSCR1 reduces levels of the CREB•IE2 and CBP•IE2 complexes, which have been suggested to play important roles in HCMV replication through its interactions with CREB, CBP, and IE2. In addition, PLSCR1 expression represses transcription from the HCMV MIE promoter. Our results indicate that PLSCR1 plays important roles in the suppression of HCMV replication in the IFN-mediated host defense system.

The anti-human cytomegalovirus drug tricin inhibits cyclin-dependent kinase 9.

4',5,7-trihydroxy-3',5'-dimethoxyflavone (tricin), derived from Sasa albo-marginata, has been reported to suppress significantly human cytomegalovirus (HCMV) replication in human embryonic lung (HEL) fibroblast cells. However, the target protein of tricin remains unclear. This study focused on the anti-HCMV activity of tricin in terms of its binding affinity to cyclin-dependent kinase 9 (CDK9). A molecular docking study predicted that tricin binds well to the ATP-binding site of CDK9. Experimental measurements then revealed that tricin inhibits the kinase activity of CDK9 and affects the phosphorylation of the carboxy-terminal domain of RNA polymerase II. Based on these results, we conclude that CDK9 is one of the target proteins of tricin. We also found that tricin possesses anti-HCMV activity with no cytotoxicity against HEL cells.

Tricin inhibits the CCL5 induction required for efficient growth of human cytomegalovirus.

Treatment of human embryonic lung fibroblast (HEL) cells with tricin (4', 5, 7-trihydroxy-3', 5'-dimethoxyflavone) following infection with human cytomegalovirus (HCMV) reportedly significantly suppresses HCMV replication. In the present work, the mechanisms for the anti-HCMV effects of tricin in HEL cells were examined. It was found that exposure of HEL cells to tricin inhibited HCMV replication, with concomitant decreases in amounts of transcripts of the CC chemokine RANTES (CCL5)-encoding gene and in expression of the CCL5 protein. It was also found that transcripts of HCMV immediate early 1 (IE1), and HCMV UL54 (encoding DNA polymerase) and replication of HCMV was significantly lower in CCL5 gene-knockdown cells. These results suggest that the anti-HCMV activity of tricin differs from that of ganciclovir and that CCL5 is one of the chemokines involved in HCMV replication. In addition, it is possible that chemokine CCL5 is one of the targets of tricin.

An in silico-designed flavone derivative, 6-fluoro-4'-hydroxy-3',5'-dimetoxyflavone, has a greater anti-human cytomegalovirus effect than ganciclovir in infected cells.

A novel type of antiviral agent for human cytomegalovirus (HCMV) is required, because the appearance of ganciclovir (GCV) resistant viruses has been reported. Tricin (4',5,7-trihydroxy-3',5'-dimethoxyflavone) has been shown to suppress significantly HCMV replication in human embryonic lung (HEL) fibroblast cells. Recently, we revealed that the action of tricin is different from that of GCV and cyclin-dependent kinase 9 (CDK9) is one of the target proteins of tricin. These results suggested that tricin is considered as a novel type of anti-HCMV agent. However, its anti-HCMV potency is not greater than that of GCV. This study tried to develop novel compounds with much greater anti-HCMV activity than GCV. We first made modifications to tricin by introducing fluorine atom, and then performed molecular docking simulations using the designed compounds and CDK9. The calculated binding energies showed that 6F-tricin (6-fluoro-4'-hydroxy-3',5'-dimetoxyflavone) binds to CDK9 much stronger than tricin. Based on these results, 6F-tricin was synthesized, and then its anti-HCMV effect was analyzed in HEL cell cultures. As a result, 6F-tricin strongly suppressed HCMV replication in a dose-dependent manner. The anti-HCMV activity with a 50% effective concentration (EC50) was 0.126 nM, corresponding to about 1/200 and 1/400 of EC50 of GCV (27.5 nM) and tricin (54.3 nM), respectively. Moreover, 6F-tricin had no cytotoxicity against HEL cells at concentrations up to 10 µM. We further performed detailed analysis on the amino acid contributions to the binding energies and found that the strong binding affinity for 6F-tricin to CDK9 is attributed to the specific binding orientation of 6F-tricin in the ATP-binding site. These results suggest that 6F-tricin is a promising candidate for anti-HCMV drug development.

Inhibition of human cytomegalovirus replication by tricin is associated with depressed CCL2 expression.

We previously reported that treatment with tricin (4',5,7-trihydroxy-3',5'-dimethoxyflavone) after human cytomegalovirus (HCMV) infection significantly suppressed both infectious virion production and HCMV replication in human embryonic lung fibroblast (HEL) cells. Moreover, we recently demonstrated that HCMV infection can increase the expression of CC-motif ligand 2 (CCL2/MCP-1) and of CCR2, a CCL2-specific receptor, effects that can in turn enhance HCMV infection and replication. Hence, we here examined whether the CCL2-CCR2 axis is involved in the anti-HCMV effects of tricin in HEL cells. Tricin exposure yielded dose-dependent decreases in the accumulation of transcripts for the HCMV immediate early gene and the DNA polymerase gene in HCMV-infected cells, along with decreased production of infectious HCMV. Concomitantly, tricin caused dose-dependent attenuation of HCMV infection-induced up-regulation of expression of CCL2 and CCR2 mRNAs and of CCL2 protein. Moreover, CCL2 reversed tricin-mediated inhibition of HCMV virion production in a dose-dependent manner. Thus, tricin appears to exert anti-HCMV activity by depressing CCL2 expression.

Regulation of Matrix Metalloproteinases-2 and -9 Gene Expression in Cultured Human Fetal Membrane Cells by Influenza Virus Infection.

In order to understand a possible etiology of adverse pregnancy outcomes associated with intrauterine influenza virus infection, we examined the effect of influenza virus infection on gene expression of matrix metalloproteinases (MMPs) in cultured amnion epithelial, amnion mesenchymal and chorion trophoblast cells prepared from human fetal membrane tissues by gelatin zymography, Western blotting and reverse transcriptase-PCR. The cells were infected with influenza A (H1N1) virus. The levels of pro-MMP-9 activity in culture supernatants of three types of cells were increased during the period of 24-48 h after the virus infection as compared to those of mock infection. Chorion trophoblast cells spontaneously released a much greater level of pro-MMP-2 activity than amnion epithelial and amnion mesenchymal cells. The cleavage of pro-MMP-2 into an active intermediate form was enhanced in chorion trophoblast cells by the virus infection. The activity levels of MMP-2 and MMP-9 in culture supernatants were consistent with their protein levels. The virus infection induced the mRNA expression of MMP-9, but not MMP-2, in three types of cells. These results suggest that influenza virus infection induces the gene expression of MMP-9 and the cleavage of pro-MMP-2 into an active intermediate form in human fetal membrane cells, resulting in weakening of the membranes through extracellular matrix degradation. Therefore, it is possible that the regulation of MMPs gene expression in fetal membrane cells by influenza virus infection is implicated in a part of the etiology of adverse pregnancy outcomes associated with intrauterine infection with the virus.

Synergistic effects by combination of ganciclovir and tricin on human cytomegalovirus replication in vitro.

It has been demonstrated as the first report that combination treatment with ganciclovir (GCV) and tricin (4',5,7-trihydroxy-3',5' -dimethoxyflavone), a derivative of Sasa albo-marginata, after human cytomegalovirus (HCMV) infection has synergistic effects on both infectious virus production and HCMV DNA synthesis in the human embryonic fibroblast cell line MRC-5. In this paper, we examined the anti-HCMV effects of GCV plus various concentrations of tricin, and tricin plus various concentrations of GCV in MRC-5 cells. We found that expression of the HCMV UL54 gene was significantly inhibited by combination of GCV with tricin when compared with GCV mono-treatment. These results suggest that tricin is a novel compound for combination therapy with GCV against HCMV replication. In addition, reduced-dose combination therapy may provide a direction for treatment in patients with HCMV infection while reducing drug toxicity.

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.

Anti-cytomegalovirus effects of tricin are dependent on CXCL11.

It has been reported that treatment with tricin (4',5,7-trihydroxy-3',5'-dimethoxyflavone), a derivative of Sasa albo-marginata, after human cytomegalovirus (HCMV) infection significantly suppressed both infectious virus production and HCMV replication in the human embryonic fibroblast cell line MRC-5. In this paper, we examined the mechanisms for the anti-HCMV effects of tricin in MRC-5 cells. Exposure of fibroblasts to tricin inhibited infectious HCMV production, with concomitant decreases in levels of transcripts of the CXC chemokine IFN-inducible T cell alpha chemoattractant (I-TAC or CXCL11) gene. We also found that the transcripts of the HCMV immediate early (IE) gene and replication of HCMV were lower in CXCL11 gene-knockdown cells. These results suggest that tricin is a novel compound with potential anti-HCMV activity and that CXCL11 is one of the chemokines involved in HCMV replication. In addition, it is possible that CXCL11 is the one of the targets of tricin.

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.

Inhibitory effects of tricin derivative from Sasa albo-marginata on replication of human cytomegalovirus.

The anti-human cytomegalovirus (HCMV) activity of tricin (4',5,7-trihydroxy-3',5'-dimethoxyflavone), a derivative from Sasa albo-marginata, was studied in the human embryonic fibroblast cell line MRC-5. In a plaque assay, tricin and ganciclovir (GCV) showed concentration-dependent inhibitory properties from 0.05 to 3.6 µM and 0.01 to 1.0 µM, respectively. Tricin had no virucidal effects on cell-free HCMV. Treatment with tricin 1h before, or 1h or 3h after viral infection significantly suppressed HCMV replication. Moreover, tricin inhibited the expression of immediate early (IE) 2 mRNA and DNA polymerase (UL54) mRNA in HCMV-infected cells. Western blot analysis also demonstrated that tricin decreased the expression of IE antigen (especially IE2) and cyclooxygenase 2 (COX-2) expression in HCMV-infected cells. In the presence of tricin, prostaglandin E2 (PGE2) accumulation by HCMV infection was completely inhibited. These results suggest that tricin is a novel compound with potential COX inhibitor-dependent anti-HCMV activity.

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.

Hexamethylene bisacetamide can convert nonpermissive human cells to a permissive state for expressing the major immediate-early genes of human cytomegalovirus by up-regulating NF-kappaB activity.

Expression of the major immediate-early (MIE) genes of human cytomegalovirus (HCMV) in the human thyroid papillary carcinoma cell line TPC-1 is repressed at the transcriptional level. However, treatment of these cells with hexamethylene bisacetamide (HMBA), a chemical inducer of differentiation, for 12 to 24 h before infection enabled the cells to support IE1 and IE2 gene expression and consequently HCMV replication. In HMBA-treated cells the transcription factor NF-kappaB was induced and the MIE promoter (MIEP) was activated. The presence of a NF-kappaB inhibitory peptide SN-50 or expression of a dominant negative IkappaBalpha protein during the HMBA pretreatment period efficiently prevented the HMBA-induced MIEP activation and MIE protein synthesis. Moreover, introduction of mutations into the NF-kappaB binding sites in the MIEP in a plasmid expressing the IE1 protein diminished its ability to express the protein in HMBA-treated cells. Therefore, the NF-kappaB activity previously induced in HMBA-treated cells and the NF-kappaB sites in the MIEP were shown to be essential for HCMV to respond to HMBA action and to express the MIE genes. Investigation of the mechanisms by which HMBA activates NF-kappaB revealed that degradation of IkappaBalpha and translocation of the phosphorylated NF-kappaB p65 subunit to the nucleus, both of which are known to be critical steps in NF-kappaB activation, are stimulated in the HMBA-treated cells. These results indicate that treatment of nonpermissive TPC-1 cells with HMBA induces MIE gene permissiveness by up-regulating NF-kappaB activity.

Anti-human cytomegalovirus activity of constituents from Sasa albo-marginata (Kumazasa in Japan).

BACKGROUND: Effective new anti-human cytomegalovirus (HCMV) agents and regimens need to be developed. We examined the anti-HCMV properties of crude extract (True World Extract of Bambuseae sasa [TWEBS]) and five compounds (p-coumaric acid, 3-hydroxy-4-methoxybenzaldehyde [vanillin], p-hydroxybenzaldehyde, 3-hydroxypyridine and 4',5,7-trihydroxy-3',5'-dimethoxyflavone [tricin]), isolated from Sasa albo-marginata, a bamboo known in Japan as Sasa. METHODS: Among TWEBS and five compounds screened in a plaque reduction assay, four showed anti-HCMV activity in the MRC-5 human embryonic fibroblast cell line. The anti-HCMV mechanisms of the TWEBS was examined by western blot analysis using primary antibody specific for an immediate early (IE) antigen of HCMV, for a structural late antigen of HCMV and for beta-actin. RESULTS: Treatment of cells with > or = 0.001% of TWEBS inhibited the observable cytopathic effects of HCMV on infected cells. Western blot analysis demonstrated that TWEBS decreased the expression of IE antigen and late antigen of HCMV in the infected cells. Next, we examined the anti-HCMV properties of five compounds isolated from TWEBS. In a viral plaque reduction assay, tricin showed dose-dependent inhibitory properties with a 50% effective concentration of 0.17 microg/ml (selective index = 1,205.8). CONCLUSIONS: The hot water extract (TWEBS) of Sasa albo-marginata, with tricin isolated from it, has anti-HCMV activity in MRC-5 cells. TWEBS and/or tricin are a novel compound with potential anti-HCMV activity. Future studies should evaluate these findings in vivo.

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-beta-D-glucopyranosyl (1 --> 6)-beta-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 microg/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.

Epidemiology of Epstein-Barr virus, cytomegalovirus, and Kaposi's sarcoma-associated herpesvirus infections in peripheral blood leukocytes revealed by a multiplex PCR assay.

A multiplex polymerase chain reaction (PCR) has been developed for the simultaneous detection of Epstein-Barr virus (EBV), cytomegalovirus (CMV), and Kaposi's sarcoma-associated herpesvirus (KSHV) in a clinical sample. Primers of multiplex PCR were designed to amplify specific regions of the EBV EBNA1, CMV IE2, and KSHV LANA genes. This multiplex PCR assay was found to have detection sensitivities of 1-10 copies of purified viral DNA cloned into the plasmid. To assess diagnostic and pre-clinical applications with this method, we utilized KSHV-positive primary effusion lymphoma (PEL) cells, EBV-positive Burkitt's lymphoma cells, CMV-infected fibroblast cells, and clinically prepared peripheral blood leukocytes (PBLs) that had been infected with viruses. We found that this multiplex PCR assay has high sensitivity and specificity for simultaneous detection of EBV, CMV, and KSHV genomes in a single amplification from a clinical material. Using this multiplex PCR assay, we investigated the prevalence of EBV, CMV, and KSHV in PBL samples from normal Japanese randomly selected. KSHV, EBV, and CMV genomes were detected in samples from 2 (0.2%), 377 (39.5%), and 27 (2.8%) of the 953 blood donors, respectively. Interestingly, both EBV and CMV genomes were detected in samples from all KSHV-positive donors.

Multiplex PCR-based DNA array for simultaneous detection of three human herpesviruses, EVB, CMV and KSHV.

Human lymphotropic herpesviruses, Epstein-Barr virus (EBV), cytomegalovirus (CMV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are responsible for a wide variety of human diseases. Due to an increase in diseased states associated with immunosuppression, more instances of co-morbid infections with these herpesviruses have resulted in viral reactivations that have caused numerous fatalities. Therefore, the development of rapid and accurate method to detect these viruses in immunocompromised patients is vital for immediate treatment with antiviral prophylactic drugs. In this study, we developed a new multiplex PCR method coupled to DNA array hybridization, which can simultaneously detect all three human herpesviruses in one single cell sample. Multiplex PCR primers were designed to amplify specific regions of the EBV (EBER1), CMV (IE) and KSHV (LANA) viral genomes. Pre-clinical application of this method revealed that this approach is capable of detecting as few as 1 copy of the viral genomes for KSHV and CMV and 100 copies of the genome for EBV. Furthermore, this highly sensitive test showed no cross-reactivity among the three viruses and is capable of detecting both KSHV and EBV viral genomes simultaneously in the lymphoblastoid cells that have been double infected with both viruses. Thus, this array-based approach serves as a rapid and reliable diagnostic tool for clinical applications.