Selective monoaminergic and histaminergic circuit dysregulation following long-term HIV-1 protein exposure.

Between 30 and 60% of HIV-seropositive individuals develop symptoms of clinical depression and/or apathy. Dopamine and serotonin are associated with motivational alterations; however, histamine is less well studied. In the present study, we used fast-scan cyclic voltammetry in HIV-1 transgenic (Tg) rats to simultaneously analyze the kinetics of nucleus accumbens dopamine (DA), prefrontal cortical serotonin (5-HT), and hypothalamic histamine (HA). For voltammetry, subjects were 15 HIV-1 Tg (7 male, 8 female) and 20 F344/N (11 male, 9 female) adult rats. Both serotonergic and dopaminergic release and reuptake kinetics were decreased in HIV-1 Tg animals relative to controls. In contrast, rates of histamine release and reuptake increased in HIV-1 Tg rats. Additionally, we used immunohistochemical (IHC) methods to identify histaminergic neurons in the tuberomammillary nucleus (TMN) of the hypothalamus. For IHC, subjects were 9 HIV-1 Tg (5 male, 4 female) and 9 F344/N (5 male, 4 female) adult rats. Although the total number of TMN histaminergic cells did not differ between HIV-1 Tg rats and F344/N controls, a significant sex effect was found, with females having an increased number of histaminergic neurons, relative to males. Collectively, these findings illustrate neurochemical alterations that potentially underlie or exacerbate the pathogenesis of clinical depression and/or apathy in HIV-1.

Hsp27 Responds to and Facilitates Enterovirus A71 Replication by Enhancing Viral Internal Ribosome Entry Site-Mediated Translation.

Enterovirus 71 (EV-A71) is a human pathogen that causes hand, foot, and mouth disease (HFMD) and fatal neurological diseases, and no effective treatment is available. Characterization of key host factors is important for understanding its pathogenesis and developing antiviral drugs. Here we report that Hsp27 is one of the most upregulated proteins in response to EV-A71 infection, as revealed by two-dimensional gel electrophoresis-based proteomics studies. Depletion of Hsp27 by small interfering RNA or CRISPR/Cas9-mediated knockout significantly inhibited viral replication, protein expression, and reproduction, while restoration of Hsp27 restored such virus activities. Furthermore, we show that Hsp27 plays a crucial role in regulating viral internal ribosome entry site (IRES) activities by two different mechanisms. Hsp27 markedly promoted 2Apro-mediated eukaryotic initiation factor 4G cleavage, an important process for selecting and initiating IRES-mediated translation. hnRNP A1 is a key IRES trans-acting factor (ITAF) for enhancing IRES-mediated translation. Surprisingly, knockout of Hsp27 differentially blocked hnRNP A1 but not FBP1 translocation from the nucleus to the cytoplasm and therefore abolished the hnRNP A1 interaction with IRES. Most importantly, the Hsp27 inhibitor 1,3,5-trihydroxy-13,13-dimethyl-2H-pyran [7,6-b] xanthone (TDP), a compound isolated from a traditional Chinese herb, significantly protected against cytopathic effects and inhibited EV-A71 infection. Collectively, our results demonstrate new functions of Hsp27 in facilitating virus infection and provide novel options for combating EV-A71 infection by targeting Hsp27.IMPORTANCE Outbreaks of infections with EV-A71, which causes hand, foot, and mouth disease, severe neurological disorders, and even death, have been repeatedly reported worldwide in recent decades and are a great public health problem for which no approved treatments are available. We show that Hsp27, a heat shock protein, supports EV-A71 infection in two distinct ways to promote viral IRES-dependent translation. A small-molecule Hsp27 inhibitor isolated from a traditional Chinese medicinal herb effectively reduces virus yields. Together, our findings demonstrate that Hsp27 plays an important role in EV-A71 infection and may serve as an antiviral target.

Glycyrrhizin inhibits human parainfluenza virus type 2 replication by the inhibition of genome RNA, mRNA and protein syntheses.

The effect of glycyrrhizin on the replication of human parainfluenza virus type 2 (hPIV-2) was examined. Cell fusion induced by hPIV-2 was inhibited by glycyrrhizin, and glycyrrhizin reduced the number of viruses released from the cells. Glycyrrhizin did not change cell morphology at 1 day of culture, but caused some damage at 4 days, as determined by the effect on actin microfilaments. However, it affected the cell viability at 1 day: about 20% of the cells were not alive by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 1 day of culture. Real-time polymerase chain reaction (PCR) and PCR showed that virus genome synthesis was largely inhibited. mRNA synthesis was also inhibited by glycyrrhizin. Viral protein synthesis was largely inhibited as observed by an indirect immunofluorescence study. Multinucleated giant cell formation was studied using a recombinant green fluorescence protein (GFP)-expressing hPIV-2 without matrix protein (rhPIV-2ΔMGFP). A few single cells with fluorescence were observed, but the formation of giant cells was completely blocked. Taken together, it was shown that viral genome, mRNA and protein syntheses, including F and HN proteins, were inhibited by glycyrrhizin, and consequently multinucleated giant cell formation was not observed and the infectious virus was not detected in the culture medium.

Tyrosine kinase/phosphatase inhibitors decrease dengue virus production in HepG2 cells.

Dengue virus is the causative agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. High rates of dengue virus replication and virion production are related to disease severity. To identify anti-DENV compounds, we performed cell-based ELISA testing to detect the level of DENV E protein expression. Among a total of 83 inhibitors, eight were identified as inhibitors with antiviral activity. Epidermal growth factor receptor inhibitor II (EGFR/ErbB-2/ErbB-4 inhibitor II) and protein tyrosine phosphatase inhibitor IV (PTP inhibitor IV) significantly inhibited dengue virus production and demonstrated low toxicity in hepatocyte cell lines. Our results suggest the efficacy of tyrosine kinase/phosphatase inhibitors in decreasing dengue virus production in HepG2 cells.

Phage M13 for the treatment of Alzheimer and Parkinson disease.

The studies of microbes have been instrumental in combatting infectious diseases, but they have also led to great insights into basic biological mechanism like DNA replication, transcription, and translation of mRNA. In particular, the studies of bacterial viruses, also called bacteriophage, have been quite useful to study specific cellular processes because of the ease to isolate their DNA, mRNA, and proteins. Here, I review the recent discovery of how properties of the filamentous phage M13 emerge as a novel approach to combat neurodegenerative diseases.

JC virus-iLOV fluorescent strains enable the detection of early and late viral protein expression.

JC virus (JCV) is highly prevalent in humans, and may cause progressive multifocal leukoencephalopathy (PML), JCV granule cell neuronopathy (JCV GCN), JCV encephalopathy (JCVE) and JCV meningitis (JCVM) in immunocompromised individuals. There is no treatment for JCV, and a growing number of multiple sclerosis patients treated with immunomodulatory medications have developed PML. Antiviral agents against JCV are therefore highly desirable but remain elusive, due to the difficulty of determining their effect in vitro. A JCV strain carrying a fluorescent protein gene would greatly simplify and accelerate the drug screening process. To achieve this goal, we selected the 366bp improved Light, Oxygen or Voltage-sensing domain (iLOV) of plant phototropin gene and created two full-length JCV-iLOV constructs on the prototype JCV Mad1 backbone. The iLOV gene was inserted either before the early regulatory T gene (iLOV-T), or after the late Agno gene (iLOV-Agno). Both JCV iLOV strains were replication-competent in vitro and emitted a fluorescent signal detectable by confocal microscope, but JCV iLOV-T exhibited higher cellular and supernatant viral loads compared to JCV iLOV-Agno. JCV iLOV-T could also produce infectious pseudovirions. These data suggest that JCV iLOV constructs may become valuable tools for anti-JCV drug screening.

Inhibitory effects of Pycnogenol® on hepatitis C virus replication.

Chronic hepatitis C virus (HCV) infection increases the risk of liver cirrhosis and hepatocellular carcinoma. In the last decade, the current standard HCV treatment, pegylated interferon and ribavirin, have limited efficacy and significant side effects. Novel direct acting antivirals show promise, but escape mutants are expected, along with potential side effects. Pycnogenol®, a French maritime pine extract, has been reported to have antioxidant and antiviral effects. Here, we evaluated the effect of Pycnogenol® on HCV replication. Wild-type and protease inhibitor (VX-950; telaprevir)-resistant HCV replicon cells were treated with Pycnogenol®, Pycnogenol® and interferon-alpha, and ribavirin and telaprevir. Pycnogenol® effects on replication were also evaluated in HCV-infected chimeric mice. Pycnogenol® treatment showed antiviral effects without cytotoxicity at doses up to 50 µg/mL. Pycnogenol® in combination with interferon-alpha or ribavirin showed synergistic effects. Moreover, Pycnogenol® inhibited HCV replication in telaprevir-resistant replicon cells; telaprevir and Pycnogenol® acted additively to reduce HCV RNA levels in wild-type HCV replicon cells without significantly increasing cytotoxicity. Pycnogenol® antiviral activity was higher than its components procyanidin and taxifolin. Further, treatment of infected chimeric mice with Pycnogenol® suppressed HCV replication and showed a synergistic effect with interferon-alpha. In addition, Pycnogenol® treatment resulted in dose-dependent reduction of reactive oxygen species in HCV replicon cell lines. Pycnogenol® is a natural product that may be used to improve the efficacy of the current standard antiviral agents and even to eliminate resistant HCV mutants.

Bacterial lipid modification of proteins requires appropriate secretory signals even for expression - implications for biogenesis and protein engineering.

Sec- and Tat-mediated bacterial lipid modification of proteins are important posttranslational processes owing to their vital roles in cellular functions, membrane targeting and biotechnological applications like ELISA, biosensor, adjuvant-free vaccines, liposomal drug delivery etc. However a better understanding of the tight coupling of secretory and lipid modification machineries and the processes associated will help unravel this essential biological event and utilize it for engineering applications. Further, there is a need for a systematic and convincing investigation into membrane targeting, solubilization and ease-of-purification of engineered lipoproteins to facilitate scientists in readily applying this new protein engineering tool. Therefore, in this study, we have investigated systematically recombinant expression, translocation, solubilization and purification of three White Spot Syndrome Viral (WSSV) proteins, ICP11, VP28 and VP281. Our study shows that the lipid modification and secretion processes are tightly coupled to the extent that mismatch between folding kinetics and signal sequence of target proteins could lead to transcriptional-translational uncoupling or aborted translation. The proteins expressed as lipoproteins through Tat-pathway were targeted to the inner membrane achieving considerable enrichment. These His-tagged proteins were then purified to apparent homogeneity in detergent-free form using single-step Immobilized Metal Affinity Chromatography. This study has interesting findings in lipoprotein biogenesis enhancing the scope of this unique post-translational protein engineering tool for obtaining pure detergent-free, membrane or hydrophobic surface-associating diagnostic targets and vaccine candidates for WSSV.

Aqueous extract of the edible Gracilaria tenuistipitata inhibits hepatitis C viral replication via cyclooxygenase-2 suppression and reduces virus-induced inflammation.

Hepatitis C virus (HCV) is an important human pathogen leading to hepatocellular carcinoma. Using an in vitro cell-based HCV replicon and JFH-1 infection system, we demonstrated that an aqueous extract of the seaweed Gracilaria tenuistipitata (AEGT) concentration-dependently inhibited HCV replication at nontoxic concentrations. AEGT synergistically enhanced interferon-α (IFN-α) anti-HCV activity in a combination treatment. We found that AEGT also significantly suppressed virus-induced cyclooxygenase-2 (COX-2) expression at promoter transactivation and protein levels. Notably, addition of exogenous COX-2 expression in AEGT-treated HCV replicon cells gradually abolished AEGT anti-HCV activity, suggesting that COX-2 down-regulation was responsible for AEGT antiviral effects. Furthermore, we highlighted the inhibitory effect of AEGT in HCV-induced pro-inflammatory gene expression such as the expression of tumour necrosis factor-α, interleukin-1ß, inducible nitrite oxide synthase and COX-2 in a concentration-dependent manner to evaluate the potential therapeutic supplement in the management of patients with chronic HCV infections.

Allitridin inhibits human cytomegalovirus replication in vitro.

Human cytomegalovirus (HCMV) has been associated with a wide spectrum of diseases. There is currently no effective treatment for eliminating the virus. Garlic bulb extract has been reported to possess anti-viral efficacy. This study aimed to investigate the expression of the immediate­early (IE; ul122 and ul123), early (E; ul54) and late (L; ul83) genes of HCMV as well as the inhibitory effect of allitridin on the transcription levels of these genes. The results indicated that a HCMV gene expression cascade occurred, and that the deletion of IE72 had no influence on the transcription of the ul122 gene, while it led to significant reductions of ul54 and ul83 mRNA expression levels. Additionally, allitridin effectively suppressed the transcription of the HCMV IE, E and L genes; the inhibition rates of the transcription of the ul122 and ul123 genes were higher compared with those of ul54 and ul83 mRNA expression, while the expression of the IE genes was not significantly reduced by ganciclovir (GCV). Our results indicate that the HCMV IE72 deletion mutant strain affects the transcription of the virus downstream gene, allitridin inhibits HCMV infection in vitro, and that the IE genes may be the key target of allitridin in its action against HCMV.

Mechanism by which ma-xing-shi-gan-tang inhibits the entry of influenza virus.

ETHNOPHARMACOLOGICAL RELEVANCE: Ma-xing-shi-gan-tang (MXSGT, aka maxing shigan powder), a Chinese herbal decoction, has been used for the treatment of the common cold, fever, and influenza virus infections. However, the underlying mechanisms of its activity against the influenza virus are not fully understood. In this study, we examined the antiviral effects of MXSGT in influenza-virus-infected MDCK cells and their underlying mechanisms, including the damage of the viral surface ultrastructure and the consequent inhibition of viral entry. MATERIALS AND METHODS: The antiviral activity of nontoxic concentrations of MXSGT against influenza virus A/WSN/33 was examined by assaying (neutralization assay) its inhibition of the virus-induced cytopathic effects. The mode of MXSGT action was first examined with a time-of-addition assay of synchronized infections, followed by viral attachment and penetration assays. Viral endocytosis was evaluated with attachment and penetration assays. We also performed assays related to the inhibition of viral entry, such as neuraminidase activity, hemagglutinin activity, and phosphoinositide-3-kinase (PI3K)/AKT phosphorylation assays. The inhibition of viral replication was demonstrated by quantitative real-time PCR, immunoblotting, and immunofluorescence microscopy. The surface ultrastructure of the MXSGT-treated virus was revealed by atomic force microscopy. RESULTS: MXSGT exhibited an EC(50) of 0.83±0.41mg/ml against influenza virus A/WSN/33 (H1N1), with broad-spectrum inhibitory activity against different strains of human influenza A viruses, including clinical oseltamivir-resistant isolates and an H1N1pdm strain. The synthesis of both viral RNA and protein was profoundly inhibited when the cells were treated with MXSGT. The time-of-addition assay demonstrated that MXSGT blocks the virus entry phase. This was confirmed with attachment and penetration assays, in which MXSGT showed similar inhibitory potencies (IC(50) of 0.58±0.07 and 0.47±0.08mg/ml). High-resolution images and quantitative measurements made with atomic force microscopy confirmed that the viral surface structure was disrupted by MXSGT. We also established that viral entry, regulated by the PI3K/AKT signaling pathway, was abolished by MXSGT. CONCLUSIONS: Our results give scientific support to the use of MXSGT in the treatment of influenza virus infections. MXSGT has potential utility in the management of seasonal pandemics of influenza virus infections, like other clinically available drugs.

Multiple effects of Honokiol on the life cycle of hepatitis C virus.

BACKGROUND: Honokiol, a small active molecular compound extracted from magnolia, has recently been shown to inhibit hepatitis C virus (HCV) infection in vitro. AIMS: This study further characterized aspects of the HCV lifecycle affected by the antiviral functions of honokiol. METHODS: The influence of honokiol on HCV infection, entry, translation and replication was assessed in Huh-7.5.1 cells using cell culture-derived HCV (HCVcc), HCV pseudo-type (HCVpp) and sub-genomic replicons. RESULTS: Honokiol had strong antiviral effect against HCVcc infection at non-toxic concentrations. Combined with interferon-α, its inhibitory effect on HCVcc was more profound than that of ribavirin. Honokiol inhibited the cell entry of lentiviral particles pseudo-typed with glycoproteins from HCV genotypes 1a, 1b, and 2a, but not of the vesicular stomatitis virus. It had inefficient activity on HCV internal ribosome entry site (IRES)-translation at concentrations with significant anti-HCVcc effects. The expression levels of components of replication complex, NS3, NS5A and NS5B, were down-regulated by honokiol in a dose-dependent manner. It also inhibited HCV replication dose dependently in both genotypes 1b and 2a sub-genomic replicons. CONCLUSIONS: Honokiol inhibits HCV infection by targeting cell entry and replication and, only at a concentration >30 µM, IRES-mediated translation of HCV life cycle. Based on its high therapeutic index (LD(50) /EC(90)  = 5.4), honokiol may be a promising drug for the treatment of HCV infection.

[Matrix activity of mRNP of different cell localization from plants infected by minus-genome curly potato dwarfness virus in protein-synthesizing system in vitro].

Using structures of mRNP of different cell localization isolated from plants infected by minus-genomecurly potato dwarfness virus (CPDV), it was established that synthesis of virus proteins in vitro is mainly realized on membrane-related and free polysomal mRNP capable to direct synthesis of proteins programmed in RNA in cell-free protein-synthesizing systems. The obtained results prove that synthesis of virus proteins is actively realized on membrane-related nonpolysomal mRNP - 23520 imp/min. An analogous distribution of matrix activity is observed also in the case when mRNA isolated from mRNP of different cell localization was introduced in protein-synthesizing system. The least matrix activity was observed in cytoplasmic nonpolysomal mRNPs which are low-active in the translation system in vitro - only 1890 imp/min. The function of free cytoplasmic nonpolysomal mRNP is apparently mainly reduced to the transport and reserve of genetic information in a cell.

Inhibition of H1N1 influenza A virus growth and induction of inflammatory mediators by the isoquinoline alkaloid berberine and extracts of goldenseal (Hydrastis canadensis).

In this study we tested whether the isoquinoline alkaloid berberine can inhibit the growth of influenza A. Our experiments showed strong inhibition of the growth of H1N1 influenza A strains PR/8/34 or WS/33 in RAW 264.7 macrophage-like cells, A549 human lung epithelial-derived cells and murine bone marrow derived macrophages, but not MDCK canine kidney cells. Studies of the mechanism underlying this effect suggest that berberine acts post-translationally to inhibit virus protein trafficking/maturation which in turn inhibits virus growth. Berberine was also evaluated for its ability to inhibit production of TNF-α and PGE(2) from A/PR/8/34 infected-RAW 264.7 cells. Our studies revealed strong inhibition of production of both mediators and suggest that this effect is distinct from the anti-viral effect. Finally, we asked whether berberine-containing ethanol extracts of goldenseal also inhibit the growth of influenza A and production of inflammatory mediators. We found strong effectiveness at high concentrations, although upon dilution extracts were somewhat less effective than purified berberine. Taken together, our results suggest that berberine may indeed be useful for the treatment of infections with influenza A.

In vitro anti-herpes simplex virus activity of 1,2,4,6-tetra-O-galloyl-ß-D-glucose from Phyllanthus emblica L. (Euphorbiaceae).

In this study, 1,2,4,6-tetra-O-galloyl-ß-D-glucose (1246TGG), a polyphenolic compound isolated from traditional Chinese medicine Phyllanthus emblica L. (Euphorbiaceae), was found to inhibit herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infection at different magnitudes of activity in vitro. Further studies revealed that 1246TGG directly inactivated HSV-1 particles, leading to the failure of early infection, including viral attachment and penetration. 1246TGG also suppressed the intracellular growth of HSV-1 within a long period post-infection (from 0 h p.i. to 12 h p.i.), while it might exert an antiviral effect mainly before 3 h p.i. It inhibited HSV-1 E and L gene expressions as well as viral DNA replication but did not affect the RNA synthesis of IE gene in our study. Also, in the presence of 1246TGG, the synthesis of viral protein was reduced. Taken together, it was suggested that 1246TGG might exert anti-HSV activity both by inactivating extracellular viral particles and by inhibiting viral biosynthesis in host cells. These results warrant further studies on the antiviral mechanisms of 1246TGG and suggest that it might be a candidate for HSV therapy.

In vitro anti-viral activity of the total alkaloids from Tripterygium hypoglaucum against herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1) is a commonly occurring human pathogen worldwide. There is an urgent need to discover and develop new alternative agents for the management of HSV-1 infection. Tripterygium hypoglaucum (level) Hutch (Celastraceae) is a traditional Chinese medicine plant with many pharmacological activities such as anti-inflammation, anti-tumor and antifertility. The usual medicinal part is the roots which contain about a 1% yield of alkaloids. A crude total alkaloids extract was prepared from the roots of T. hypoglaucum amd its antiviral activity against HSV-1 in Vero cells was evaluated by cytopathic effect (CPE) assay, plaque reduction assay and by RT-PCR analysis. The alkaloids extract presented low cytotoxicity (CC(50) = 46.6 µg/mL) and potent CPE inhibition activity, the 50% inhibitory concentration (IC(50)) was 6.5 µg/mL, noticeably lower than that of Acyclovir (15.4 µg /mL). Plaque formation was significantly reduced by the alkaloids extract at concentrations of 6.25 µg/mL to 12.5 µg/mL, the plaque reduction ratio reached 55% to 75 which was 35% higher than that of Acyclovir at the same concentration. RT-PCR analysis showed that, the transcription of two important delayed early genes UL30 and UL39, and a late gene US6 of HSV-1 genome all were suppressed by the alkaloids extract, the expression inhibiting efficacy compared to the control was 74.6% (UL30), 70.9% (UL39) and 62.6% (US6) respectively at the working concentration of 12.5 µg/mL. The above results suggest a potent anti-HSV-1 activity of the alkaloids extract in vitro.

Identification and characterization of inhibitors of West Nile virus.

Although flaviviruses cause significant human diseases, no antiviral therapy is currently available for clinical treatment of these pathogens. To identify flavivirus inhibitors, we performed a high-throughput screening of compound libraries using cells containing luciferase-reporting replicon of West Nile viruses (WNV). Five novel small molecular inhibitors of WNV were identified from libraries containing 96,958 compounds. The inhibitors suppress epidemic strain of WNV in cell culture, with EC(50) (50% effective concentration) values of <10microM and TI (therapeutic index) values of >10. Viral titer reduction assays, using various flaviviruses and nonflaviviruses, showed that the compounds have distinct antiviral spectra. Mode-of-action analysis showed that the inhibitors block distinct steps of WNV replication: four compounds inhibit viral RNA syntheses, while the other compound suppresses both viral translation and RNA syntheses. Biochemical enzyme assays showed that two compounds selectively inhibit viral RNA-dependent RNA polymerase (RdRp), while another compound specifically inhibits both RdRp and methyltransferase. The identified compounds could potentially be developed for treatment of flavivirus infections.

Bioactivity-guided screening identifies pheophytin a as a potent anti-hepatitis C virus compound from Lonicera hypoglauca Miq.

Chronic hepatitis C virus (HCV) infection is a worldwide public issue. In this study, we performed bioactivity-guided screening of the Lonicera hypoglauca Miq. crude extracts to find for naturally chemical entities with anti-HCV activity. Pheophytin a was identified from the ethanol-soluble fraction of L. hypoglauca that elicited dose-dependent inhibition of HCV viral proteins and RNA expression in both replicon cells and cell culture infectious system. Computational modeling revealed that pheophytin a can bind to the active site of HCV-NS3, suggesting that NS3 is a potent molecular target of pheophytin a. Biochemical analysis further revealed that pheophytin a inhibited NS3 serine protease activity with IC(50)=0.89 microM. Notably, pheophytin a and IFNalpha-2a elicited synergistic anti-HCV activity in replicon cells with no significant cytotoxicity. This study thereby demonstrates for the first time that pheophytin a is a potent HCV-NS3 protease inhibitor and offers insight for development of novel anti-HCV regimens.

Inhibition of the epstein-barr virus lytic cycle by andrographolide.

Andrographis paniculata NEES is a medicinal plant that is commonly used in Asia. This work demonstrates that 25 microg/ml of ethanolic extract from A. paniculata (EEAP) and 5 microg/ml of andrographolide, a bioactive compound in EEAP, effectively inhibit the expression of Epstein-Barr virus (EBV) lytic proteins, Rta, Zta and EA-D, during the viral lytic cycle in P3HR1 cells. Transient transfection analysis revealed that the lack of expression of Rta, Zta and EA-D is caused by the inhibition of the transcription of BRLF1 and BZLF1, two EBV immediate-early genes that encode Rta and Zta, respectively. This study finds that the inhibition prevents the virus from producing mature viral particles. Meanwhile, andrographolide is not toxic to P3HR1 cells when the concentration is below 5 microg/ml, indicating that the compound is potentially useful as an anti-EBV drug.

Construction and characterization of a stable subgenomic dengue virus type 2 replicon system for antiviral compound and siRNA testing.

Self-replicating, non-infectious flavivirus subgenomic replicons have been broadly used in the studies of trans-complementation, adaptive mutation, viral assembly and packaging in Kunjin, yellow fever and West Nile viruses. We describe here the construction of subgenomic EGFP- or Renilla luciferase-reporter based dengue replicons of the type 2 New Guinea C (NGC) strain and the establishment of stable BHK21 cell lines harboring the replicons. In replicon cells, viral proteins and RNAs are stably expressed at levels similar to cells transfected with the full length NGC infectious RNA. Furthermore, the replicon can be packaged by separately transfected C (core)-prM (pre-membrane)-E (envelope) polyprotein construct. The replicon cells were subjected to treatment with several antiviral compounds and inhibition of the replicon was observed in treatment with known nucleoside analog inhibitors of NS5 such as 2'-C-methyladenosine (EC(50)=2.42 +/- 0.59 microM), or ribavirin (EC(50)=6.77 +/- 1.33 microM), mycophenolic acid (EC(50)=1.31 +/- 0.27 microM) and siRNA against NS3. The BHK-replicon cells have been stably maintained for about 10 passages without significant loss in reporter intensity and are sufficiently robust for both research and drug discovery.