Identification of natural compounds extracted from crude drugs as novel inhibitors of hepatitis C virus.

Natural product-derived crude drugs are expected to yield an abundance of new drugs to treat infectious diseases. Hepatitis C virus (HCV) is an oncogenic virus that significantly impacts public health. In this study, we sought to identify anti-HCV compounds in extracts of natural products. A total of 110 natural compounds extracted from several herbal medicine plants were examined for antiviral activity against HCV. Using a Huh7-mCherry-NLS-IPS reporter system for HCV infection, we first performed a rapid screening for anti-HCV compounds extracted from crude drugs. The compounds threo-2,3-bis(4-hydroxy-3-methoxyphenyl)-3-butoxypropan-1-ol (#106) and medioresinol (#110), which were extracted from Crataegus cuneate, exhibited anti-HCV activity and significantly inhibited HCV production in a dose-dependent manner. Analyses using HCV pseudoparticle and subgenomic replicon systems indicated that compounds #106 and #110 specifically inhibit HCV RNA replication but not viral entry or translation. Interestingly, compound #106 also inhibited the replication and production of hepatitis A virus. Our findings suggest that C. cuneate is a new source for novel anti-hepatitis virus drug development.

N-Terminal PreS1 Sequence Regulates Efficient Infection of Cell-Culture-Generated Hepatitis B Virus.

BACKGROUND AND AIMS: An efficient cell-culture system for hepatitis B virus (HBV) is indispensable for research on viral characteristics and antiviral reagents. Currently, for the HBV infection assay in cell culture, viruses derived from HBV genome-integrated cell lines of HepG2.2.15 or HepAD-38 are commonly used. However, these viruses are not suitable for the evaluation of polymorphism-dependent viral characteristics or resistant mutations against antiviral reagents. HBV obtained by the transient transfection of the ordinary HBV molecular clone has limited infection efficiencies in cell culture. APPROACH AND RESULTS: We found that an 11-amino-acid deletion (d11) in the preS1 region enhances the infectivity of cell-culture-generated HBV (HBVcc) to sodium taurocholate cotransporting polypeptide-transduced HepG2 (HepG2/NTCP) cells. Infection of HBVcc derived from a d11-introduced genotype C strain (GTC-d11) was ~10-fold more efficient than infection of wild-type GTC (GTC-wt), and the number of infected cells was comparable between GTC-d11- and HepG2.2.15-derived viruses when inoculated with the same genome equivalents. A time-dependent increase in pregenomic RNA and efficient synthesis of covalently closed circular DNA were detected after infection with the GTC-d11 virus. The involvement of d11 in the HBV large surface protein in the enhanced infectivity was confirmed by an HBV reporter virus and hepatitis D virus infection system. The binding step of the GTC-d11 virus onto the cell surface was responsible for this efficient infection. CONCLUSIONS: This system provides a powerful tool for studying the infection and propagation of HBV in cell culture and also for developing the antiviral strategy against HBV infection.

Non-nucleoside hepatitis B virus polymerase inhibitors identified by an in vitro polymerase elongation assay.

BACKGROUND: Hepatitis B virus (HBV) polymerase is the only virus-encoded enzyme essential for producing the HBV genome and is regarded as an attractive drug target. However, the difficulty of synthesizing and purifying recombinant HBV polymerase protein has hampered the development of new drugs targeting this enzyme, especially compounds unrelated to the nucleoside structure. We recently have developed a technique for the synthesis and purification of recombinant HBV polymerase containing the reverse transcriptase (RT) domain that carried DNA elongation activity in vitro. METHODS: We used the overproduced protein to establish an in vitro high-throughput screening system to identify compounds that inhibit the elongation activity of HBV polymerase. RESULTS: We screened 1120 compounds and identified a stilbene derivative, piceatannol, as a potential anti-HBV agent. Derivative analysis identified another stilbene derivative, PDM2, that was able to inhibit HBV replication with an IC50 of 14.4 ± 7.7 µM. An infection experiment suggested that the compounds inhibit the replication of HBV rather than the entry process, as expected. Surface plasmon resonance analysis demonstrated a specific interaction between PDM2 and the RT domain. Importantly, PDM2 showed similar inhibitory activity against the replication of both wild-type HBV and a lamivudine/entecavir-resistant HBV variant. Furthermore, PDM2 showed an additive effect in combination with clinically used nucleos(t)ide analogs. CONCLUSIONS: We report the development of a screening system that is useful for identifying non-nucleos(t)ide RT inhibitors.

Pyrimidotriazine derivatives as selective inhibitors of HBV capsid assembly.

Chronic hepatitis B virus (HBV) infection is currently treated with nucleoside/nucleotides analogs. They are potent inhibitors of HBV DNA polymerase, which also functions as reverse transcriptase. Although nucleoside/nucleotide analogs efficiently suppress HBV replication in liver cells, they cannot eradicate HBV DNA from liver cells and cure the disease. Therefore, it is still mandatory to identify and develop effective inhibitors that target a step other than reverse transcription in the viral replication cycle. HBV capsid assembly is a critical step for viral replication and an attractive target for inhibition of HBV replication. We conducted in silico screening of compounds expected to bind to the HBV capsid dimer-dimer interaction site. The selected compounds were further examined for their anti-HBV activity in vitro. Among the test compounds, novel pyrimidotriazine derivatives were found to be selective inhibitors of HBV replication in HepG2.2.15.7 cells. Among the compounds, 2-[(2,3-dichlorophenyl)amino]-4-(4-tert-butylphenyl)-8-methyl-4H,9H-pyrimido[1,2-a][1,3,5]triazin-6-one was the most active against HBV replication. Studies on its mechanism of action revealed that the compound interfered with HBV capsid assembly determined by a cell-free capsid assembly system. Thus, the pyrimidotriazine derivatives are considered to be potential leads for novel HBV capsid assembly inhibitors.

Screening for inhibitor of episomal DNA identified dicumarol as a hepatitis B virus inhibitor.

Currently, there is no available therapy to eradicate hepatitis B virus (HBV) in chronically infected individuals. This is due to the difficulty in eliminating viral covalently closed circular (ccc) DNA, which is central to the gene expression and replication of HBV. We developed an assay system for nuclear circular DNA using an integration-deficient lentiviral vector. This vector produced non-integrated circular DNA in nuclei of infected cells. We engineered this vector to encode firefly luciferase to monitor the lentiviral episome DNA. We screened 3,840 chemicals by this assay for luciferase-reducing activity and identified dicumarol, which is known to have anticoagulation activity. We confirmed that dicumarol reduced lentiviral episome DNA. Furthermore, dicumarol inhibited HBV replication in cell culture using NTCP-expressing HepG2 and primary human hepatocytes. Dicumarol reduced intracellular HBV RNA, DNA, supernatant HBV antigens and DNA. We also found that dicumarol reduced the cccDNA level in HBV infected cells, but did not affect HBV adsorption/entry. This is a novel assay system for screening inhibitors targeting nuclear cccDNA and is useful for finding new antiviral substances for HBV.

Vitamin D derivatives inhibit hepatitis C virus production through the suppression of apolipoprotein.

Supplementation with vitamin D (VD) has been reported to improve the efficacy of interferon-based therapy for chronic hepatitis C. We found that 25-hydroxyvitamin D3 (25-(OH)D3), one of the metabolites of VD, has antiviral effects by inhibiting the infectious virus production of the hepatitis C virus (HCV). In this study, to clarify the underlying mechanisms of the anti-HCV effects, we searched VD derivatives that have anti-HCV effects and identified the common target molecule in the HCV life cycle by using an HCV cell culture system. After infection of Huh-7.5.1 cells with cell culture-generated HCV, VD derivatives were added to culture media, and the propagation of HCV was assessed by measuring the HCV core antigen levels in culture media and cell lysates. To determine the step in the HCV life cycle affected by these compounds, the single-cycle virus production assay was used with a CD81-negative cell line. Of the 14 structural derivatives of VD, an anti-HCV effect was detected in 9 compounds. Cell viability was not affected by these effective compounds. The 2 representative VD derivatives inhibited the infectious virus production in the single-cycle virus production assay. Treatment with these compounds and 25-(OH)D3 suppressed the expression of apolipoprotein A1 and C3, which are known to be involved in infectious virus production of HCV, and the knockdown of these apolipoproteins reduced infectious virus production. In conclusion, we identified several compounds with anti-HCV activity by screening VD derivatives. These compounds reduce the infectious virus production of HCV by suppressing the expression of apolipoproteins in host cells.

Novel stable HBV producing cell line systems for expression and screening antiviral inhibitor of hepatitis B virus in human hepatoma cell line.

Chronic hepatitis B virus (HBV) infection is currently a major public health burden. Therefore, there is an urgent need for the development of novel antiviral inhibitors. The stable HBV-producing cell lines of genotype D are widely used to investigate the HBV life cycle and to evaluate antiviral agents. However, stable HBV-producing cell lines of different genotypes do not exist. To construct more convenient and efficient novel cell systems, stable cell lines of genotypes A, B, and C were established using a full-length HBV genome sequence isolated from chronic HBV patients in human hepatoma HepG2 cells. Novel HBV clones were identified and stable HBV-producing cell lines derived from these clones were constructed. HBV replication activities demonstrated time-dependent expression, and the novel cell lines were susceptible to several antiviral inhibitors with no cytotoxicity. Furthermore, infectious viruses were produced from these cell lines. In conclusion, we have established novel stable HBV-producing cell line systems of genotypes A, B, and C. These systems can provide valuable tools for screening antiviral agents and analyzing viral phenotypes in vitro.

Quantifying antiviral activity optimizes drug combinations against hepatitis C virus infection.

With the introduction of direct-acting antivirals (DAAs), treatment against hepatitis C virus (HCV) has significantly improved. To manage and control this worldwide infectious disease better, the "best" multidrug treatment is demanded based on scientific evidence. However, there is no method available that systematically quantifies and compares the antiviral efficacy and drug-resistance profiles of drug combinations. Based on experimental anti-HCV profiles in a cell culture system, we quantified the instantaneous inhibitory potential (IIP), which is the logarithm of the reduction in viral replication events, for both single drugs and multiple-drug combinations. From the calculated IIP of 15 anti-HCV drugs from different classes [telaprevir, danoprevir, asunaprevir, simeprevir, sofosbuvir (SOF), VX-222, dasabuvir, nesbuvir, tegobuvir, daclatasvir, ledipasvir, IFN-α, IFN-λ1, cyclosporin A, and SCY-635], we found that the nucleoside polymerase inhibitor SOF had one of the largest potentials to inhibit viral replication events. We also compared intrinsic antiviral activities of a panel of drug combinations. Our quantification analysis clearly indicated an advantage of triple-DAA treatments over double-DAA treatments, with triple-DAA treatments showing enhanced antiviral activity and a significantly lower probability for drug resistance to emerge at clinically relevant drug concentrations. Our framework provides quantitative information to consider in designing multidrug strategies before costly clinical trials.

Fungus-Derived Neoechinulin B as a Novel Antagonist of Liver X Receptor, Identified by Chemical Genetics Using a Hepatitis C Virus Cell Culture System.

UNLABELLED: Cell culture systems reproducing virus replication can serve as unique models for the discovery of novel bioactive molecules. Here, using a hepatitis C virus (HCV) cell culture system, we identified neoechinulin B (NeoB), a fungus-derived compound, as an inhibitor of the liver X receptor (LXR). NeoB was initially identified by chemical screening as a compound that impeded the production of infectious HCV. Genome-wide transcriptome analysis and reporter assays revealed that NeoB specifically inhibits LXR-mediated transcription. NeoB was also shown to interact directly with LXRs. Analysis of structural analogs suggested that the molecular interaction of NeoB with LXR correlated with the capacity to inactivate LXR-mediated transcription and to modulate lipid metabolism in hepatocytes. Our data strongly suggested that NeoB is a novel LXR antagonist. Analysis using NeoB as a bioprobe revealed that LXRs support HCV replication: LXR inactivation resulted in dispersion of double-membrane vesicles, putative viral replication sites. Indeed, cells treated with NeoB showed decreased replicative permissiveness for poliovirus, which also replicates in double-membrane vesicles, but not for dengue virus, which replicates via a distinct membrane compartment. Together, our data suggest that LXR-mediated transcription regulates the formation of virus-associated membrane compartments. Significantly, inhibition of LXRs by NeoB enhanced the activity of all known classes of anti-HCV agents, and NeoB showed especially strong synergy when combined with interferon or an HCV NS5A inhibitor. Thus, our chemical genetics analysis demonstrates the utility of the HCV cell culture system for identifying novel bioactive molecules and characterizing the virus-host interaction machinery. IMPORTANCE: Hepatitis C virus (HCV) is highly dependent on host factors for efficient replication. In the present study, we used an HCV cell culture system to screen an uncharacterized chemical library. Our results identified neoechinulin B (NeoB) as a novel inhibitor of the liver X receptor (LXR). NeoB inhibited the induction of LXR-regulated genes and altered lipid metabolism. Intriguingly, our results indicated that LXRs are critical to the process of HCV replication: LXR inactivation by NeoB disrupted double-membrane vesicles, putative sites of viral replication. Moreover, NeoB augmented the antiviral activity of all known classes of currently approved anti-HCV agents without increasing cytotoxicity. Thus, our strategy directly links the identification of novel bioactive compounds to basic virology and the development of new antiviral agents.

Antimicrobial peptide LL-37 attenuates infection of hepatitis C virus.

AIM: Although recent studies indicate that supplementation with vitamin D (VD) potentiates a sustained viral response by interferon-based therapy to chronic hepatitis C, detailed mechanisms are not fully defined. The production of cathelicidin, an antimicrobial peptide, has been demonstrated to be part of the VD-dependent antimicrobial pathway in innate immunity. Cathelicidin is known to directly kill or inhibit the growth of microbial pathogens including mycobacteria and viruses. METHODS: We used a hepatitis C virus (HCV) cell culture system to clarify the anti-HCV effects of the human cathelicidin, LL-37. HuH-7 cells were administrated with LL-37 and infected with cell culture-generated HCV (HCVcc). HCV propagation was estimated by measuring the level of HCV core antigen (Ag). RESULTS: Treatment with LL-37 resulted in decreased intra- and extracellular levels of HCV core Ag, suggesting inhibition of HCV propagation. To assess the effects of LL-37 on HCV replication, JFH-1 subgenomic replicon RNA-transfected cells were treated with LL-37. However, inhibition of HCV replication was not detected by this assay. To clarify the effects on HCV infection, we treated HCVcc with LL-37 and removed the antimicrobial peptide prior to use of the virus in infection. This exposure of HCVcc to LL-37 diminished the infectivity titers in a dose-dependent fashion. Iodixanol density gradient analysis revealed that the peak fraction of infectivity titer was eliminated by LL-37 treatment. CONCLUSION: The VD-associated antimicrobial peptide LL-37 attenuated the infectivity of HCV. This anti-HCV effect of LL-37 may explain the contribution of VD to the improved efficacy of interferon-based therapy.

Inhibitory Effects of Caffeic Acid, a Coffee-Related Organic Acid, on the Propagation of Hepatitis C Virus.

Multipurpose cohort studies have demonstrated that coffee consumption reduces the risk of hepatocellular carcinoma (HCC). Given that one of the main causes of HCC is hepatitis C virus (HCV) infection, we examined the effect of caffeic acid, a major organic acid derived from coffee, on the propagation of HCV using an in vitro naïve HCV particle-infection and production system within human hepatoma-derived Huh-7.5.1-8 cells. When cells were treated with 1% coffee extract or 0.1% caffeic acid for 1-h post HCV infection, the amount of HCV particles released into the medium at 3 and 4 days post-infection considerably decreased. In addition, HCV-infected cells cultured with 0.001% caffeic acid for 4 days, also released less HCV particles into the medium. Caffeic acid treatment inhibited the initial stage of HCV infection (i.e., between virion entry and the translation of the RNA genome) in both HCV genotypes 1b and 2a. These results suggest that the treatment of cells with caffeic acid may inhibit HCV propagation.

New preclinical antimalarial drugs potently inhibit hepatitis C virus genotype 1b RNA replication.

BACKGROUND: Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. Although new triple therapy (pegylated-interferon, ribavirin, and telaprevir/boceprevir) has recently been started and is expected to achieve a sustained virologic response of more than 70% in HCV genotype 1 patients, there are several problems to be resolved, including skin rash/ageusia and advanced anemia. Thus a new type of anti-HCV drug is still needed. METHODOLOGY/PRINCIPAL FINDINGS: Recently developed HCV drug assay systems using HCV-RNA-replicating cells (e.g., HuH-7-derived OR6 and Li23-derived ORL8) were used to evaluate the anti-HCV activity of drug candidates. During the course of the evaluation of anti-HCV candidates, we unexpectedly found that two preclinical antimalarial drugs (N-89 and its derivative N-251) showed potent anti-HCV activities at tens of nanomolar concentrations irrespective of the cell lines and HCV strains of genotype 1b. We confirmed that replication of authentic HCV-RNA was inhibited by these drugs. Interestingly, however, this anti-HCV activity did not work for JFH-1 strain of genotype 2a. We demonstrated that HCV-RNA-replicating cells were cured by treatment with only N-89. A comparative time course assay using N-89 and interferon-α demonstrated that N-89-treated ORL8 cells had more rapid anti-HCV kinetics than did interferon-α-treated cells. This anti-HCV activity was largely canceled by vitamin E. In combination with interferon-α and/or ribavirin, N-89 or N-251 exhibited a synergistic inhibitory effect. CONCLUSIONS/SIGNIFICANCE: We found that the preclinical antimalarial drugs N-89 and N-251 exhibited very fast and potent anti-HCV activities using cell-based HCV-RNA-replication assay systems. N-89 and N-251 may be useful as a new type of anti-HCV reagents when used singly or in combination with interferon and/or ribavirin.

A cell-based, microplate colorimetric screen identifies 7,8-benzoflavone and green tea gallate catechins as inhibitors of the hepatitis C virus.

We describe a cell-based, microplate colorimetric screen for anti-hepatitis C virus (HCV) drugs that exploits the HCV-JFH1 viral culture system. Antiviral activity was assessed by measuring protection against the HCV-JFH1-induced cytopathic effect (CPE) in Huh7.5.1 cells using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) viability assay. The use of serum-free medium substantially sensitized Huh7.5.1 cells to HCV-induced CPE, causing sufficient cell death to perform colorimetric assays for anti-HCV activity in 96-well plates. As a proof of concept, we carried out a pilot screen of an inhibitor library and identified cyclosporin A and tamoxifen, two compounds with reported anti-HCV activity. Using the assay, we discovered the anti-HCV properties of the plant flavonoids epigallocatechin gallate (EGCG) and 7,8-benzoflavone (α-naphthoflavone). Other gallate-type catechins and flavones also displayed anti-HCV activity, but 5,6-benzoflavone (ß-naphthoflavone), flavanone, and non-gallate catechins were inactive. EGCG apparently acted mainly on HCV entry, although it may also block other steps. In contrast, 7,8-benzoflavone was presumed to inhibit later stages of the HCV life cycle. This assay is simple, reliable and cost-effective; does not require any specially engineered cell lines or viruses; and should be useful in the identification of compounds with anti-HCV activity.

25-Hydroxyvitamin D3 suppresses hepatitis C virus production.

UNLABELLED: Because the current interferon (IFN)-based treatment for hepatitis C virus (HCV) infection has a therapeutic limitation and side effects, a more efficient therapeutic strategy is desired. Recent studies show that supplementation of vitamin D significantly improves sustained viral response via IFN-based therapy. However, mechanisms and an active molecular form of vitamin D for its anti-HCV effects have not been fully clarified. To address these questions, we infected HuH-7 cells with cell culture-generated HCV in the presence or absence of vitamin D(3) or its metabolites. To our surprise, 25-hydroxyvitamin D(3) [25(OH)D(3) ], but not vitamin D(3) or 1,25-dihydroxyvitamin D(3) , reduced the extra- and intracellular levels of HCV core antigen in a concentration-dependent manner. Single-cycle virus production assay with a CD81-negative cell line reveals that the inhibitory effect of 25(OH)D(3) is at the level of infectious virus assembly but not entry or replication. Long-term 25(OH)D(3) treatment generates a HCV mutant with acquired resistance to 25(OH)D(3) , and this mutation resulting in a N1279Y substitution in the nonstructural region 3 helicase domain is responsible for the resistance. CONCLUSION: 25(OH)D(3) is a novel anti-HCV agent that targets an infectious viral particle assembly step. This finding provides insight into the improved efficacy of anti-HCV treatment via the combination of vitamin D(3) and IFN. Our results also suggest that 25(OH)D(3) , not vitamin D(3) , is a better therapeutic option in patients with hepatic dysfunction and reduced enzymatic activity for generation of 25(OH)D(3) .

Anti-ulcer agent teprenone inhibits hepatitis C virus replication: potential treatment for hepatitis C.

BACKGROUND: Previously we reported that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, statins, inhibited hepatitis C virus (HCV) RNA replication. Furthermore, recent reports revealed that the statins are associated with a reduced risk of hepatocellular carcinoma and lower portal pressure in patients with cirrhosis. The statins exhibited anti-HCV activity by inhibiting geranylgeranylation of host proteins essential for HCV RNA replication. Geranylgeranyl pyrophosphate (GGPP) is a substrate for geranylgeranyltransferase. Therefore, we examined the potential of geranyl compounds with chemical structures similar to those of GGPP to inhibit HCV RNA replication. METHODS: We tested geranyl compounds [geranylgeraniol, geranylgeranoic acid, vitamin K(2) and teprenone (Selbex)] for their effects on HCV RNA replication using genome-length HCV RNA-replicating cells (the OR6 assay system) and a JFH-1 infection cell culture system. Teprenone is the major component of the anti-ulcer agent, Selbex. We also examined the anti-HCV activities of the geranyl compounds in combination with interferon (IFN)-α or statins. RESULTS: Among the geranyl compounds tested, only teprenone exhibited anti-HCV activity at a clinically achievable concentration. However, other anti-ulcer agents tested had no inhibitory effect on HCV RNA replication. The combination of teprenone and IFN-α exhibited a strong inhibitory effect on HCV RNA replication. Although teprenone alone did not inhibit geranylgeranylation, surprisingly, statins' inhibitory action against geranylgeranylation was enhanced by cotreatment with teprenone. CONCLUSIONS: The anti-ulcer agent teprenone inhibited HCV RNA replication and enhanced statins' inhibitory action against geranylgeranylation. This newly discovered function of teprenone may improve the treatment of HCV-associated liver diseases as an adjuvant to statins.

Identification of bisindolylmaleimides and indolocarbazoles as inhibitors of HCV replication by tube-capture-RT-PCR.

We devised a screening method for hepatitis C virus (HCV) inhibitors by exploiting the JFH1 viral culture system. The viral RNA released in the medium was adsorbed onto PCR plates, and real-time RT-PCR was performed by directly adding the one-step RT-PCR reaction mixture to the wells. The "tube-capture-RT-PCR" method obviates the need for labor-intensive RNA isolation and should allow high-throughput screening of HCV inhibitors. To substantiate the validity of the assay for drug screening, a pilot screen of an inhibitor library composed of 95 compounds was performed. In addition to the known inhibitors of HCV replication included in the library, the assay identified the PKC inhibitor bisindolylmaleimide I (BIM I) as an HCV replication inhibitor. BIM I was also effective in reducing the viral protein level in genotype 1b and 2a subgenomic replicon cells, indicating inhibition of HCV replication. Further assays revealed that a broad range of bisindolylmaleimides and indolocarbazoles inhibit HCV, but no correlation was found between the PKC inhibition pattern and anti-HCV activity. These series of compounds represent new classes of inhibitors that may warrant further development.

HCV research and anti-HCV drug discovery: toward the next generation.

Hepatitis C virus (HCV) causes persistent infection and induces chronic hepatitis, liver cirrhosis and finally hepatocellular carcinoma. Current therapies for HCV infection have not been satisfactory, and more effective anti-viral treatments are needed. In this regard, detailed analysis of HCV has been hampered by a lack of appropriate viral culture systems and small animal models of infection. However, rapid progress in HCV research has recently been achieved, such as a subgenomic replicon system, a viral culture system using JFH-1 clone and the Alb-uPA/SCID mouse transplanted with human liver cells. Such progress will propel HCV research and anti-HCV drug discovery toward the next generation.

Characterization of the E-138 (Glu/Lys) mutation in Japanese encephalitis virus by using a stable, full-length, infectious cDNA clone.

A stable plasmid DNA, pMWJEAT, was constructed by using full-length Japanese encephalitis virus (JEV) cDNA isolated from the wild-type strain JEV AT31. Recombinant JEV was obtained by synthetic RNA transfection into Vero cells and designated rAT virus. JEV rAT exhibited similar large-plaque morphology and antigenicity to the parental AT31 strain. Mutant clone pMWJEAT-E138K, containing a single Glu-to-Lys mutation at aa 138 of the envelope (E) protein, was also constructed to analyse the mechanisms of viral attenuation arising from this mutation. Recombinant JEV rAT-E138K was also recovered and displayed a smaller-plaque morphology and lower neurovirulence and neuroinvasiveness than either AT31 virus or rAT virus. JEV rAT-E138K exhibited greater plaque formation than rAT virus in virus-cell interactions under acidic conditions. Heparin or heparinase III treatment inhibited binding to Vero cells more efficiently for JEV rAT-E138K than for rAT virus. Inhibition of virus-cell interactions by using wheatgerm agglutinin was more effective for JEV rAT than for rAT-E138K on Vero cells. About 20 % of macropinoendocytosis of JEV rAT for Vero cells was inhibited by cytochalasin D treatment, but no such inhibition occurred for rAT-E138K virus. Furthermore, JEV rAT was predominantly secreted from infected cells, whereas rAT-E138K was more likely to be retained in infected cells. This study demonstrates clearly that a single Glu-to-Lys mutation at aa 138 of the envelope protein affects multiple steps of the viral life cycle. These multiple changes may induce substantial attenuation of JEV.