Search

Flaviviruses are important human pathogens and include dengue (DENV), West Nile (WNV), Yellow fever virus (YFV), Japanese encephalitis (JEV) and Zika virus (ZIKV). DENV, transmitted by mosquitoes, causes diseases ranging in severity from mild dengue fever with non-specific flu-like symptoms to fatal dengue hemorrhagic fever and dengue shock syndrome (Clyde et al., 2006). DENV infections are caused by four serotypes, DENV1-4, which interact differently with antibodies in blood serum. The incidence of DENV infection has increased dramatically in recent decades and the CDC estimates 400 million dengue infections occur each year, resulting in ∼25,000 deaths mostly among children and elderly people. Similarly, ZIKV infections are caused by infected mosquito bites to humans, can be transmitted sexually and through blood transfusions. If a pregnant woman is infected, the virus can cross the placental barrier and can spread to her fetus, causing severe brain malformations in the child including microcephaly and other birth defects. It is noteworthy that the neurological manifestations of ZIKV were also observed in DENV endemic regions, suggesting that pre-existing antibody response to DENV could augment ZIKV infection. WNV, previously unknown in the US (and known to cause only mild disease in Middle East), first arrived in New York city in 1999 (NY99) and spread throughout the US and Canada by Culex mosquitoes and birds. WNV is now endemic in North America. Thus, emerging and re-emerging flaviviruses are significant threat to human health. However, vaccines are available for only a limited number of flaviviruses, and antiviral therapies are not available for any flavivirus. Hence, there is an urgent need to develop therapeutics that interfere with essential enzymatic steps, such as protease in the flavivirus lifecycle as these viruses possess significant threat to future pandemics. In this review, we focus on our E. coli expression of NS2B hydrophilic domain (NS2BH) covalently linked to NS3 protease domain (NS3Pro) in their natural context which is processed by the combined action of both subunits of the NS2B-NS3Pro precursor. Biochemical activities of the viral protease such as solubility and autoproteolysis of NS2BH-NS3Pro linkage depended on the C-terminal portion of NS2BH linked to the NS3Pro domain. Since 2008, we also focus on the use of the recombinant protease in high throughput screens and characterization of small molecular compounds identified in these screens.

Flaviviruses use a ~70 nucleotide stem-loop structure called stem-loop A (SLA) at the 5′ end of the RNA genome as a promoter for RNA synthesis. Flaviviral polymerase NS5 specifically recognizes SLA to initiate RNA synthesis and methylate the 5′ guanosine cap. We report the crystal structures of dengue (DENV) and Zika virus (ZIKV) SLAs. DENV and ZIKV SLAs differ in the relative orientations of their top stem-loop helices to bottom stems, but both form an intermolecular three-way junction with a neighboring SLA molecule. To understand how NS5 engages SLA, we determined the SLA-binding site on NS5 and modeled the NS5-SLA complex of DENV and ZIKV. Our results show that the gross conformational differences seen in DENV and ZIKV SLAs can be compensated by the differences in the domain arrangements in DENV and ZIKV NS5s. We describe two binding modes of SLA and NS5 and propose an SLA-mediated RNA synthesis mechanism., Flaviviruses use a ~70 nucleotide stem-loop structure called stem-loop A (SLA) at the 5’ end of the RNA genome as a promoter for RNA synthesis by the viral polymerase NS5. Here the authors describe the structures of dengue and Zika virus SLAs, identify the SLA-binding site on NS5, and propose models for how NS5 recognizes the RNA promoter.

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

The HEK-293 cell line was created in 1977 by transformation of primary human embryonic kidney cells with sheared adenovirus type 5 DNA. A previous study determined that the HEK-293 cells have neuronal markers rather than kidney markers. In this study, we tested the hypothesis whether Zika virus (ZIKV), a neurotropic virus, is able to infect and replicate in the HEK-293 cells. We show that the HEK-293 cells infected with ZIKV support viral replication as shown by indirect immunofluorescence (IFA) and quantitative reverse transcriptase-PCR (qRT-PCR). We performed RNA-seq analysis on the ZIKV-infected and the control uninfected HEK-293 cells and find 659 genes that are differentially transcribed in ZIKV-infected HEK-293 cells as compared to uninfected cells. The results show that the top 10 differentially transcribed and upregulated genes are involved in antiviral and inflammatory responses. Seven upregulated genes, IFNL1, DDX58 , CXCL10, ISG15, KCNJ15, IFNIH1, and IFIT2, were validated by qRT-PCR. Altogether, our findings show that ZIKV infection alters host gene expression by affecting their antiviral and inflammatory responses.

Four serotypes of mosquito-borne dengue virus (DENV), evolved from a common ancestor, are human pathogens of global significance for which there is no vaccine or antiviral drug available. The N-terminal domain of DENV NS5 has guanylyltransferase and methyltransferase (MTase), and the C-terminal region has the polymerase (POL), all of which are important for 5'-capping and RNA replication. The crystal structure of NS5 shows it as a dimer, but the functional evidence for NS5 dimer is lacking. Our studies showed that the substitution of DENV2 NS5 MTase or POL for DENV4 NS5 within DENV2 RNA resulted in a severe attenuation of replication in the transfected BHK-21 cells. A replication-competent species was evolved with the acquired mutations in the DENV2 and DENV4 NS5 MTase or POL domain or in the DENV2 NS3 helicase domain in the DENV2 chimera RNAs by repeated passaging of infected BHK-21 or mosquito cells. The linker region of seven residues in NS5, rich in serotype-specific residues, is important for the recovery of replication fitness in the chimera RNA. Our results, taken together, provide genetic evidence for a serotype-specific interaction between NS3 and NS5 as well as specific interdomain interaction within NS5 required for RNA replication. Genome-wide RNAseq analysis revealed the distribution of adaptive mutations in RNA quasispecies. Those within NS3 and NS5 are located at the surface and/or within the NS5 dimer interface, providing a functional significance to the crystal structure NS5 dimer.

In a previous study, twelve antimalarial compounds, amodiaquine (AQ) and derivatives, were shown to have potent anti-dengue viral (DENV) activity by using the stable DENV2 Renilla luciferase reporter replicon expressing BHK-21 cells, infectivity (plaque), and the qRT-PCR assays. In this study, we performed molecular modeling on these compounds to determine their stereo-electronic properties required for optimal antiviral activity. Based on the similarity of calculated stereo-electronic profiles, specifically the electrostatic potential profiles of the compounds, and in silico screening of related compounds from literature, we identified three additional compounds, Quinacrine (QC), Mefloquine (MQ), and GSK369796. Analysis of their antiviral activities indicated that all three compounds have high anti-DENV activity in the DENV2 replicon expressing cells with EC50 values of 5.30 ± 1.31 μM (QC), 3.22 ± 0.37 μM (MQ), and 5.06 ± 0.86 μM (GSK369796). The infectivity assays revealed the EC50 values of 7.09 ± 1.67 μM (QC), 4.36 ± 0.31 μM (MQ) and 3.03 ± 0.35 μM (GSK369796). The mode of action of these compounds is through inhibition of autophagy, thereby affecting DENV2 replication. Moreover, these compounds also showed antiviral activity against the rapidly emerging Zika virus (ZIKV) with EC50 values of 2.27 ± 0.14 μM (QC), 3.95 ± 0.21 μM (MQ), and 2.57 ± 0.09 μM (GSK369796).

Dengue virus (DENV) causes frequent epidemics infecting ∼390 million people annually in over 100 countries. There are no approved vaccines or antiviral drugs for treatment of infected patients. However, there is a novel approach to control DENV transmission by the mosquito vectors, Aedes aegypti and Aedes albopictus, using the Wolbachia symbiont. The wMelPop strain of Wolbachia suppresses DENV transmission and shortens the mosquito life span. However, the underlying mechanism is poorly understood. To clarify this mechanism, either naive A. albopictus (C6/36) or wMelPop-C6/36 cells were infected with DENV serotype 2 (DENV2). Analysis of host transcript profiles by transcriptome sequencing (RNAseq) revealed that the presence of wMelPop dramatically altered the mosquito host cell transcription in response to DENV2 infection. The viral RNA evolved from wMelPop-C6/36 cells contained low-frequency mutations (∼25%) within the coding region of transmembrane domain 1 (TMD1) of E protein. Mutations with >97% frequencies were distributed within other regions of E, the NS5 RNA-dependent RNA polymerase (NS5POL) domain, and the TMDs of NS2A, NS2B, and NS4B. Moreover, while DENV2-infected naive C6/36 cells showed syncytium formation, DENV2-infected wMelPop-C6/36 cells did not. The Wolbachia-induced mutant DENV2 can readily infect and replicate in naive C6/36 cells, whereas in mutant DENV2-infected BHK-21 or Vero cells, virus replication was delayed. In LLC-MK2 cells, the mutant failed to produce plaques. Additionally, in BHK-21 cells, many mutations in the viral genome reverted to the wild type (WT) and compensatory mutations in NS3 gene appeared. Our results indicate that wMelPop impacts significantly the interactions of DENV2 with mosquito and mammalian host cells.IMPORTANCE Mosquito-borne diseases are of global significance causing considerable morbidity and mortality throughout the world. Dengue virus (DENV; serotypes 1 to 4), a member of the Flavivirus genus of the Flaviviridae family, causes millions of infections annually. Development of a safe vaccine is hampered due to absence of cross-protection and increased risk in secondary infections due to antibody-mediated immune enhancement. Infection of vector mosquitoes with Wolbachia bacteria offers a novel countermeasure to suppress DENV transmission, but the mechanisms are poorly understood. In this study, the host transcription profiles and viral RNA sequences were analyzed in naive A. albopictus (C6/36) and wMelPop-C6/36 cells by RNAseq. Our results showed that the wMelPop symbiont caused profound changes in host transcription profiles and morphology of DENV2-infected C6/36 cells. Accumulation of several mutations throughout DENV2 RNA resulted in loss of infectivity of progeny virions. Our findings offer new insights into the mechanism of Wolbachia-mediated suppression of DENV transmission.

Dengue virus (DENV) causes frequent epidemics infecting ∼390 million people annually in over 100 countries. There are no approved vaccines or antiviral drugs for treatment of infected patients. However, there is a novel approach to control DENV transmission by the mosquito vectors, Aedes aegypti and Aedes albopictus, using the Wolbachia symbiont. The wMelPop strain of Wolbachia suppresses DENV transmission and shortens the mosquito life span. However, the underlying mechanism is poorly understood. To clarify this mechanism, either naive A. albopictus (C6/36) or wMelPop-C6/36 cells were infected with DENV serotype 2 (DENV2). Analysis of host transcript profiles by transcriptome sequencing (RNAseq) revealed that the presence of wMelPop dramatically altered the mosquito host cell transcription in response to DENV2 infection. The viral RNA evolved from wMelPop-C6/36 cells contained low-frequency mutations (∼25%) within the coding region of transmembrane domain 1 (TMD1) of E protein. Mutations with >97% frequencies were distributed within other regions of E, the NS5 RNA-dependent RNA polymerase (NS5POL) domain, and the TMDs of NS2A, NS2B, and NS4B. Moreover, while DENV2-infected naive C6/36 cells showed syncytium formation, DENV2-infected wMelPop-C6/36 cells did not. The Wolbachia-induced mutant DENV2 can readily infect and replicate in naive C6/36 cells, whereas in mutant DENV2-infected BHK-21 or Vero cells, virus replication was delayed. In LLC-MK2 cells, the mutant failed to produce plaques. Additionally, in BHK-21 cells, many mutations in the viral genome reverted to the wild type (WT) and compensatory mutations in NS3 gene appeared. Our results indicate that wMelPop impacts significantly the interactions of DENV2 with mosquito and mammalian host cells. IMPORTANCE Mosquito-borne diseases are of global significance causing considerable morbidity and mortality throughout the world. Dengue virus (DENV; serotypes 1 to 4), a member of the Flavivirus genus of the Flaviviridae family, causes millions of infections annually. Development of a safe vaccine is hampered due to absence of cross-protection and increased risk in secondary infections due to antibody-mediated immune enhancement. Infection of vector mosquitoes with Wolbachia bacteria offers a novel countermeasure to suppress DENV transmission, but the mechanisms are poorly understood. In this study, the host transcription profiles and viral RNA sequences were analyzed in naive A. albopictus (C6/36) and wMelPop-C6/36 cells by RNAseq. Our results showed that the wMelPop symbiont caused profound changes in host transcription profiles and morphology of DENV2-infected C6/36 cells. Accumulation of several mutations throughout DENV2 RNA resulted in loss of infectivity of progeny virions. Our findings offer new insights into the mechanism of Wolbachia-mediated suppression of DENV transmission.

The mosquito-borne dengue virus serotypes 1–4 (DENV1-4) and West Nile virus (WNV) cause serious illnesses worldwide associated with considerable morbidity and mortality. According to the World Health Organization (WHO) estimates, there are about 390 million infections every year leading to ∼500,000 dengue haemorrhagic fever (DHF) cases and ∼25,000 deaths, mostly among children. Antiviral therapies could reduce the morbidity and mortality associated with flaviviral infections, but currently there are no drugs available for treatment. In this study, a high-throughput screening assay for the Dengue protease was employed to screen ∼120,000 small molecule compounds for identification of inhibitors. Eight of these inhibitors have been extensively analyzed for inhibition of the viral protease in vitro and cell-based viral replication using Renilla luciferase reporter replicon, infectivity (plaque) and cytotoxicity assays. Three of these compounds were identified as potent inhibitors of DENV and WNV proteases, and viral replication of DENV2 replicon and infectious RNA. Fluorescence quenching, kinetic analysis and molecular modeling of these inhibitors into the structure of NS2B-NS3 protease suggest a mode of inhibition for three compounds that they bind to the substrate binding pocket.

The dengue virus is considered to be a globally important human pathogen prevalent in tropical and subtropical regions of the world. According to a recent estimate, the disease burden due to DENV infections is ∼390 million infections per year globally in ∼100 countries including the southern US, Puerto Rico and Hawaii, resulting in nearly ∼25,000 deaths mostly among children. Despite the significant morbidity and mortality that results from DENV infections, there is currently no effective chemotherapeutic treatment for DENV infections. We identified curcumin as an inhibitor of DENV2 NS2B/NS3protease in a previous high-throughput screening (HTS) campaign. We synthesized four analogues of curcumin (curcuminoids) and tested the in vitro protease inhibition activity and inhibition of replication by cell-based assays. The results revealed that curcumin is a weak inhibitor of the viral protease. However, the analogues exhibited more potent inhibition of DENV infectivity in plaque assays suggesting that the cellular pathway(s) required for viral replication and/or assembly are targeted by these compounds. Further analysis shows that inhibition of genes involved in lipid biosynthesis, and of actin polymerization by curcuminoids, are likely to be involved as their mode of action in DENV2-infected cells. Three of the curcumin derivatives possess good selectivity indices (SI) (>10) when compared to the parent curcumin.

Establishment of in vitro systems to study mechanisms of RNA synthesis for positive strand RNA viruses have been very useful in the past and have shed light on the composition of protein and RNA components, optimum conditions, the nature of the products formed, cis-acting RNA elements and trans-acting protein factors required for efficient synthesis. In this review, we summarize our current understanding regarding the requirements for flavivirus RNA synthesis in vitro. We describe details of reaction conditions, the specificity of template used by either the multi-component membrane-bound viral replicase complex or by purified, recombinant RNA-dependent RNA polymerase. We also discuss future perspectives to extend the boundaries of our knowledge.

1,2-Benzisothiazol-3(2H)-ones and 1,3,4-oxadiazoles individually have recently attracted considerable interest in drug discovery, including as antibacterial and antifungal agents. In this study, a series of functionalized 1,2-benzisothiazol-3(2H)-one - 1,3,4-oxadiazole hybrid derivatives were synthesized and subsequently screened against Dengue and West Nile virus proteases. Ten out of twenty-four compounds showed greater than 50% inhibition against DENV2 and WNV proteases ([I] = 10 μM). The IC50 values of compound 7n against DENV2 and WNV NS2B/NS3 were found to be 3.75 ± 0.06 and 4.22 ± 0.07 μM, respectively. The kinetics data support a competitive mode of inhibition by compound 7n. Molecular modeling studies were performed to delineate the putative binding mode of this series of compounds. This study reveals that the hybrid series arising from the linking of the two scaffolds provides a suitable platform for conducting a hit-to-lead optimization campaign via iterative structure-activity relationship studies, in vitro screening and X-ray crystallography.

Flavivirus nonstructural protein 5 (NS5) consists of methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, which catalyze 5’-RNA capping/methylation and RNA synthesis, respectively, during viral genome replication. Although the crystal structure of flavivirus NS5 is known, no data about the quaternary organization of the functional enzyme are available. We report the crystal structure of dengue virus full-length NS5, where eight molecules of NS5 are arranged as four independent dimers in the crystallographic asymmetric unit. The relative orientation of each monomer within the dimer, as well as the orientations of the MTase and RdRp domains within each monomer, is conserved, suggesting that these structural arrangements represent the biologically relevant conformation and assembly of this multi-functional enzyme. Essential interactions between MTase and RdRp domains are maintained in the NS5 dimer via inter-molecular interactions, providing evidence that flavivirus NS5 can adopt multiple conformations while preserving necessary interactions between the MTase and RdRp domains. Furthermore, many NS5 residues that reduce viral replication are located at either the inter-domain interface within a monomer or at the inter-molecular interface within the dimer. Hence the X-ray structure of NS5 presented here suggests that MTase and RdRp activities could be coordinated as a dimer during viral genome replication., Author Summary Many plus-strand RNA viruses encode a viral RNA polymerase and capping enzymes to synthesize a 5’-capped RNA genome. However, how these two activities are coordinated during viral replication is not understood. In flaviviruses, polymerase and capping enzymes are encoded in a single multifunctional protein, where separate domains within the polypeptide are responsible for these activities; flavivirus NS5, composed of the polymerase and methyltransferase domains, carries out viral RNA synthesis, 5’-RNA capping, and RNA cap methylations. Previous NS5 monomer structures were unable to provide mechanistic insight into how the two domains communicate or the quaternary organization of the functional enzyme. We have determined the crystal structure of dengue virus NS5 and show that the NS5 dimer is likely the biological assembly of NS5, and RNA synthesis and RNA capping may be coordinated by the dimer. We found that essential interactions between the two NS5 domains can be maintained either within a monomer or via inter-molecular interactions within a dimer, and thus NS5 can adopt multiple conformations while preserving necessary interactions between the methyltransferase and polymerase domains. Using dengue virus, we additionally determined that such specific interaction between the two NS5 domains is the major determinant of viral replication.

Dengue and West Nile viruses (WNV) are mosquito-borne members of flaviviruses that cause significant morbidity and mortality. There is no approved vaccine or antiviral drugs for human use to date. In this study, a series of functionalized meta and para aminobenzamide derivatives were synthesized and subsequently screened in vitro against Dengue virus and West Nile virus proteases. Four active compounds were identified which showed comparable activity toward the two proteases and shared in common a meta or para(phenoxy)phenyl group. The inhibition constants (K(i)) for the most potent compound 7n against Dengue and West Nile virus proteases were 8.77 and 5.55 μM, respectively. The kinetics data support a competitive mode of inhibition of both proteases by compound 7n. This conclusion is further supported by molecular modeling. This study reveals a new chemical scaffold which is amenable to further optimization to yield potent inhibitors of the viral proteases via the combined utilization of iterative medicinal chemistry/structure-activity relationship studies and in vitro screening.

Two click chemistry-derived focused libraries based on the benz[d]isothiazol-3(2H)-one scaffold were synthesized and screened against Dengue virus and West Nile virus NS2B-NS3 proteases. Several compounds (4l, 7j–n) displayed noteworthy inhibitory activity toward Dengue virus NS2B-NS3 protease in the absence and presence of added detergent. These compounds could potentially serve as a launching pad for a hit-to-lead optimization campaign.

Detergents such as Triton X-100 are often used in drug discovery research to weed out small molecule promiscuous and non-specific inhibitors which act by aggregation in solution and undesirable precipitation in aqueous assay buffers. We evaluated the effects of commonly used detergents, Triton X-100, Tween-20, Nonidet-40 (NP-40), Brij-35, and CHAPS, on the enzymatic activity of West Nile virus (WNV) protease. Unexpectedly, Triton X-100, Tween-20, and NP-40 showed an enhancement of in vitro WNV protease activity from 2 to 2.5-fold depending on the detergent and its concentration. On the other hand, Brij-35, at ⩾0.001% enhanced the protease activity by 1.5-fold and CHAPS had the least enhancing effect. The kinetic analysis showed that the increase in protease activity by Triton X-100 was dose-dependent. Furthermore, at Triton X-100 and Tween-20 concentrations higher than 0.001%, the inhibition of compound B, one of the lead compounds against WNV protease identified in a high throughput screen (IC50 value of 5.7 ± 2.5 μM), was reversed. However, in the presence of CHAPS, compound B still showed good inhibition of WNV protease. Our results, taken together, indicate that nonionic detergents, Triton X-100, Tween, and NP-40 are unsuitable for the purpose of discrimination of true versus promiscuous inhibitors of WNV protease in high throughput assays.

Genomes of RNA viruses encounter a continual threat from host cellular ribonucleases. Therefore, viruses have evolved mechanisms to protect the integrity of their genomes. To study the mechanism of 3′-end repair in dengue virus-2 in mammalian cells, a series of 3′-end deletions in the genome were evaluated for virus replication by detection of viral antigen NS1 and by sequence analysis. Limited deletions did not cause any delay in the detection of NS1 within 5 d. However, deletions of 7–10 nucleotides caused a delay of 9 d in the detection of NS1. Sequence analysis of RNAs from recovered viruses showed that at early times, virus progenies evolved through RNA molecules of heterogeneous lengths and nucleotide sequences at the 3′ end, suggesting a possible role for terminal nucleotidyl transferase activity of the viral polymerase (NS5). However, this diversity gradually diminished and consensus sequences emerged. Template activities of 3′-end mutants in the synthesis of negative-strand RNA in vitro by purified NS5 correlate well with the abilities of mutant RNAs to repair and produce virus progenies. Using the Mfold program for RNA structure prediction, we show that if the 3′ stem–loop (3′ SL) structure was abrogated by mutations, viruses eventually restored the 3′ SL structure. Taken together, these results favor a two-step repair process: non-template-based nucleotide addition followed by evolutionary selection of 3′-end sequences based on the best-fit RNA structure that can support viral replication.

Background and Aim: Oval cells, liver stem cell-derived cells, are generated from the liver periportal region and spread into the parenchyma by an autocrine signaling pathway. The mechanism behind how oval cells take their place among packed silent hepatocytes, however, is not well understood. We hypothesized that apoptosis involves a decrease in hepatocytes surrounding oval cells. Methods: Male Fisher rats were treated using the AAF/PH protocol to induce oval cells in the liver. Apoptosis was assessed by measuring the activity of caspase-3, -8 and -9, and apoptosis-related molecules such as caspase-3, Fas, Fas-L and Bax were also assessed by immunohistochemical analysis and reverse transcriptase-polymerase chain reaction (RT–PCR). Apoptosis was confirmed by TUNEL staining. Regarding antiapoptotic factors, nuclear factor-κB (NF-κB) DNA binding activity and proliferating cell nuclear antigen (PCNA) expression were examined. Results: NF-κB elevated at the early stage of oval cell proliferation. Conversely, caspase activity increased after NF-κB elevation. The mRNA of caspase-3, Fas, Fas-L and Bax was induced during and after AAF/PH treatment. Immunohistochemically, oval cells lacked the expression of these proteins, whereas the hepatocytes, particularly those surrounding oval cells, expressed strongly. Conclusions: The present study suggests that the apoptosis in hepatocytes through both extrinsic and intrinsic pathways mediates oval cell proliferation.

Flavivirus NS3 and NS5 are required in viral replication and 5′-capping. NS3 has NS2B-dependent protease, RNA helicase, and 5′-RNA triphosphatase activities. NS5 has 5′-RNA methyltransferase (MT)/guanylyltransferase (GT) activities within the N-terminal 270 amino acids and the RNA-dependent RNA polymerase (POL) activity within amino acids 271–900. A chimeric NS5 containing the D4MT/D4GT and the D2POL domains in the context of wild-type (WT) D2 RNA was constructed. RNAs synthesized in vitro were transfected into baby hamster kidney cells. The viral replication was analyzed by an indirect immunofluorescence assay to monitor NS1 expression and by quantitative real-time PCR. WT D2 RNA-transfected cells were NS1- positive by day 5, whereas the chimeric RNA-transfected cells became NS1-positive ∼30 days post-transfection in three independent experiments. Sequence analysis covering the entire genome revealed the appearance of a single K74I mutation within the D4MT domain ∼16 days post-transfection in two experiments. In the third, D290N mutation in the conserved NS3 Walker B motif appeared ≥16 days post-transfection. A time course study of serial passages revealed that the 30-day supernatant had gradually evolved to gain replication fitness. Trans-complementation by co-expression of WT D2 NS5 accelerated viral replication of chimeric RNA without changing the K74I mutation. However, the MT and POL activities of NS5 WT D2 and the chimeric NS5 proteins with or without the K74I mutation are similar. Taken together, our results suggest that evolution of the functional interactions involving the chimeric NS5 protein encoded by the viral genome species is essential for gain of viral replication fitness.

Dengue virus serotypes 1-4 (DENV1-4) are mosquito-borne human pathogens of global significance causing ~390 million cases annually worldwide. The virus infections cause in general a self-limiting disease, known as dengue fever, but occasionally also more severe forms, especially during secondary infections, dengue hemorrhagic fever and dengue shock syndrome causing ~25,000 deaths annually. The DENV genome contains a single-strand positive sense RNA, approximately 11 kb in length. The 5'-end has a type I cap structure. The 3'-end has no poly(A) tail. The viral RNA has a single long open reading frame that is translated by the host translational machinery to yield a polyprotein precursor. Processing of the polyprotein precursor occurs co-translationally by cellular proteases and posttranslationally by the viral serine protease in the endoplasmic reticulum (ER) to yield three structural proteins (capsid (C), precursor membrane (prM), and envelope (E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The active viral protease consists of both NS2B, an integral membrane protein in the ER, and the N-terminal part of NS3 (180 amino acid residues) that contains the trypsin-like serine protease domain having a catalytic triad of H51, D75, and S135. The C-terminal part of NS3, ~170-618 amino acid residues, encodes an NTPase/RNA helicase and 5'-RNA triphosphatase activities; the latter enzyme is required for the first step in 5'-capping. The cleavage sites of the polyprotein by the viral protease consist of two basic amino acid residues such as KR, RR, or QR, followed by short chain amino acid residues, G, S, or T. Since the cleavage of the polyprotein by the viral protease is absolutely required for assembly of the viral replicase, blockage of NS2B/NS3pro activity provides an effective means for designing dengue virus (DENV) small-molecule therapeutics. Here we describe the screening of small-molecule inhibitors against DENV2 protease.

The relationship between transforming growth factor-beta1 (TGF-beta1) induced growth arrest and apoptosis in rat liver derived epithelial (RLE) cells was analyzed. TGF-beta1 treatment of RLE cells induced both cell cycle arrest and apoptosis. However, pretreatment of the cells with either dexamethasone or cyclohexamide suppressed TGF-beta1 induced apoptosis, without preventing the cell cycle arrest. Both p53 and Bax were subsequently shown to be overexpressed during the TGF-beta1 induced apoptosis. Furthermore, it was revealed that cycloheximide suppressed expression of both p53 and Bax. In contrast, dexamethasone treatment prevented Bax expression alone. Treatment of RLE cells with several growth factors either alone or in combination was ineffective in counteracting TGF-beta1 induced apoptosis. In additon, we show that TGF-alpha also induced both p53 and Bax expressions and augmented TGF-beta-induced apoptosis. Thus, p53 and Bax are likely to be key factors in TGF-beta1 induced apoptosis in RLE cells.

The tightly controlled homeostatic mechanisms between cell growth and apoptosis that exist in normal liver tissue are disrupted during hepatocarcinogenesis. The TGF (transforming growth factor)-beta signaling system is a central component of the mechanisms by which cell growth and apoptosis are controlled in the liver. The recent delineation of the TGF-beta signaling pathway has provided a unique framework for analysis of the impact that disruption of individual components of this signaling pathway can have on apoptosis during hepatocarcinogenesis. Here we review recent data on involvement of the TGF-beta signaling pathway in the dysregulation of apoptosis frequently observed in hepatocellular carcinomas. The data indicate that disruption of the TGF-beta pathway at the pre-receptor, receptor, and post-receptor levels occurs in hepatocellular carcinomas and can cause dysregulation of apoptosis. Also, substantial evidence now exists that phosphatidylinositol-3-kinase (PI3K) may function as an important negative regulator of the TGF-beta 1-induced apoptosis in hepatocellular carcinomas. Taken together, the available evidence indicates that disruption of the TGF-beta 1-induced apoptosis as well as growth inhibition is an important and integral part of the multistage process of liver carcinogenesis.

The Dengue virus (DENV) genome contains multiple cis-acting elements required for translation and replication. Previous studies indicated that a 719-nt subgenomic minigenome (DENV-MINI) is an efficient template for translation and (-) strand RNA synthesis in vitro. We performed a detailed structural analysis of DENV-MINI RNA, combining chemical acylation techniques, Pb(2+) ion-induced hydrolysis and site-directed mutagenesis. Our results highlight protein-independent 5'-3' terminal interactions involving hybridization between recognized cis-acting motifs. Probing analyses identified tandem dumbbell structures (DBs) within the 3' terminus spaced by single-stranded regions, internal loops and hairpins with embedded GNRA-like motifs. Analysis of conserved motifs and top loops (TLs) of these dumbbells, and their proposed interactions with downstream pseudoknot (PK) regions, predicted an H-type pseudoknot involving TL1 of the 5' DB and the complementary region, PK2. As disrupting the TL1/PK2 interaction, via 'flipping' mutations of PK2, previously attenuated DENV replication, this pseudoknot may participate in regulation of RNA synthesis. Computer modeling implied that this motif might function as autonomous structural/regulatory element. In addition, our studies targeting elements of the 3' DB and its complementary region PK1 indicated that communication between 5'-3' terminal regions strongly depends on structure and sequence composition of the 5' cyclization region.

The estrogen receptor was studied in a total of 42 cases of gastric carcinoma which included 14 cases of the penetrating type of early gastric carcinoma (PEN), 14 cases of the common type of early gastric carcinomas (EA) and 14 cases of the advanced gastric carcinomas (AD). An immunohistochemical study using the anti-ER-D5 antibody revealed that the detection of estrogen receptor-positive tumor cells was significantly higher in the PEN group and in the AD group than in the EA group. Moreover, the incidence of estrogen receptor immunoreactivity in infiltrative and scirrhous PENs was significantly higher than that in non-infiltrative and non-scirrhous PENs. These results suggests that the estrogen receptor may play an important role in infiltrative proliferation and scirrhous growth in the PENs. Furthermore, the results indicated that ER immunostaining procedures applied to endoscopic biopsy specimens could be useful for the endoscopic diagnosis of PEN-type early gastric cancer.

We studied a 57-year-old man who was diagnosed as having giant rugae at a mass-screening for gastric cancer. He was examined endoscopically for check-up purposes. In addition to giant rugal folds which occurred throughout the entire fundus and body of the stomach, a type IIc lesion was noted in the posterior lower wall of the gastric body. A biopsy revealed signet ring cell carcinoma. A total gastrectomy was performed. A resected specimen was serially sectioned and underwent histopathological examination; hypertrophy and cystic elongation of the gastric glands indicative of so-called giant hypertrophic gastritis was seen. The histological type of the type IIc lesion was undifferentiated adenocarcinoma with the signet ring cells being confined to the mucosal layer. This patient had a rare case of Menetrier's disease complicated by intramucosal carcinoma.

We studied a 66-year-old female who had a dull epigastric pain. An upper gastrointestinal (GI) series revealed a cordlike protrusion in the duodenal region, and a gastroendoscopic examination was performed and revealed a mucosal tag (MT) about 3 cm in length in the duodenal bulb. The MT was resected endoscopically and was found to be lined by normal duodenal mucosa and to have developed hyperplasia of the Brunner's glands. This MT was considered to be a congenitally-developed, hamartomatous hyperplasia of the duodenal mucosa, and of the Brunner's glands in particular.

大腸腺腫,腺腫内癌,sm癌,進行癌,平坦陥凹型癌,計51例における癌遣伝子産物(ras, c-myc, erbB-2),および増殖因子(EGF, TGF-β)の発現をパラフィン包埋材料の連続切片を用いて免疫組織化学染色により検討した,rasを除く遺伝子産物は腺腫群では低値を示し,浸潤傾向とともに高値を示した.進行癌周囲正常粘膜ではras, erbB-2が高値を示した.癌周囲異型粘膜の染色濃度には癌の病理学的形態による特異的な所見はなかった.sm浸潤部では進行癌が他群に比較して高値を示した.腫瘍性病変の各群とも癌遺伝子産物は重複して陽性を示した.またいずれの群においても浸潤を伴うものではこの部で非浸潤部と比較して重複陽性例が多かった.以上より大腸癌における発癌には複数の癌遺伝子の活性化が関与していることが示唆された.

Specific types of early gastric cancer were investigated in accordance with the cancer surface area and the degree of penetration by means of quantitative measurements of the surface area of early gastric cancer using the interactive image analysis system. The results indicated a significant correlation between the surface area and the penetration depth in ordinary early gastric cancer. However these correlations were not observed in both well and poorly differentiated adenocarcinoma cases of the so-called PEN and SUPER types, which showed a significant specificity when compared with ordinary early gastric cancer. The PEN and SUPER types of early gastric cancer also exhibited various clinicopathological characteristics, and it was suggested that the poorly differentiated PEN type might be the initial lesion of a linitis plastica type gastric cancer. Examination of the conditions of the mucosa surrounding the cancer revealed a difference between the PEN and the SUPER types, and this suggested that the environment at the site of a cancer growth influences the type of growth and the spread of early gastric cancer.

Dengue viruses (DENV) have 5'-capped RNA genomes of (+) polarity and encode a single polyprotein precursor that is processed into mature viral proteins. NS2B, NS3 and NS5 proteins catalyse/activate enzyme activities that are required for key processes in the virus life cycle. The heterodimeric NS2B/NS3 is a serine protease required for processing. Using a high-throughput protease assay, we screened a small molecule chemical library and identified -200 compounds having > or = 50% inhibition. Moreover, NS3 exhibits RNA-stimulated NTPase, RNA helicase and the 5'-RNA triphosphatase activities. The NTPase and the 5'-RTPase activities of NS3 are stimulated by interaction with NS5. Moreover, the conserved, positively charged motif in DENV-2 NS3, 184RKRK, is required for RNA binding and modulates the RNA-dependent enzyme activities of NS3. To study viral replication, a variety of methods are used such as the in vitro RNA-dependent RNA polymerase assays that utilize lysates from DENV-2-infected mosquito or mammalian cells or the purified NS5 along with exogenous short subgenomic viral RNAs or the replicative intracellular membrane-bound viral RNAs as templates. In addition, a cell-based DENV-2 replicon RNA encoding a luciferase reporter is also used to examine the role of cis-acting elements within the 3' UTR and the RKRK motif in viral replication.

Dengue virus type 2 (DEN2), a member of the Flaviviridae family, is a re-emerging human pathogen of global significance. DEN2 nonstructural protein 3 (NS3) has a serine protease domain (NS3-pro) and requires the hydrophilic domain of NS2B (NS2BH) for activation. NS3 is also an RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicase and a 5′-RNA triphosphatase (RTPase). In this study the first biochemical and kinetic properties of full-length NS3 (NS3FL)-associated NTPase, RTPase, and RNA helicase are presented. The NS3FL showed an enhanced RNA helicase activity compared with the NS3-pro-minus NS3, which was further enhanced by the presence of the NS2BH (NS2BH-NS3FL). An active protease catalytic triad is not required for the stimulatory effect, suggesting that the overall folding of the N-terminal protease domain contributes to this enhancement. In DEN2-infected mammalian cells, NS3 and NS5, the viral 5′-RNA methyltransferase/polymerase, exist as a complex. Therefore, the effect of NS5 on the NS3 NTPase activity was examined. The results show that NS5 stimulated the NS3 NTPase and RTPase activities. The NS5 stimulation of NS3 NTPase was dose-dependent until an equimolar ratio was reached. Moreover, the conserved motif, 184RKRK, of NS3 played a crucial role in binding to RNA substrate and modulating the NTPase/RNA helicase and RTPase activities of NS3.

PTEN (phosphatase and tensin homologue deleted on chromosome 10) and p53 alterations were expected to be diversely involved in endometrial carcinogenesis. Patients (n=92) with endometrial carcinoma (EC) were analyzed, and PTEN and p53 were immunostained in the tissue sections. Tumor histology, grade of differentiation, presence of endometrial hyperplasia, staining status of PTEN and p53 and clinical information were examined. There were 37 cases (40%) negative for PTEN staining, which suggests lost or reduced PTEN function. Loss of PTEN staining was significantly related to the advanced staging in the grade 1 (G1) and grade 2 (G2) endometrioid adenocarcinoma group (p=0.026). Also, 18 cases (20%) showed positive staining for p53. p53 staining was largely found in grade 3 (G3) endometrioid adenocarcinoma and other phenotypes of EC. In the G1 and G2 group, all 29 cases with reduced PTEN staining showed p53-negative staining (p=0.025). In the G3 and others group, 6 of 8 cases with reduced PTEN staining showed p53-positive staining. p53-positive staining was associated with a high probability of tumor recurrence in the G1 and G2 group (p=0.0234). In contrast, in the G3 and others group, p53-positive cases had a low probability of tumor recurrence (p=0.0473). Both PTEN and p53 staining may be good indicators of clinical stage and probability of tumor recurrence in EC. Reciprocal abnormality of p53 or PTEN occurred at an early phase of carcinogenesis, however simultaneous abnormality of p53 and PTEN often occurred at the a late phase of carcinogenesis. Thus, immunohistochemistry for PTEN and p53 in biopsy specimens of EC can provide supportive information for determining a treatment plan.

Ulcerative colitis (UC)-associated neoplasia is one of the complications seen in patients with long-standing UC. Based on many epidemiological studies, colitis is assumed to promote colon tumorigenesis. Tumorigenesis is known to be suppressed in rodents and humans by selective cyclooxygenase-2 inhibitors. However, whether these drugs would serve as protective agents against UC-associated neoplasia remains unclear. Therefore, using a colitis-induced tumorigenesis rat model, we investigated the effects of etodolac, a selective cyclooxygenase-2 inhibitor, on tumorigenesis. The following 4 groups were examined: group A, administered trinitrobenzene sulfonic acid and 1,2-dimethylhydrazine; group B, in addition to the treatment in group A, also received etodolac; group C, administered etodolac alone; and group D, did not receive any agent throughout the study and served as an untreated control. The rats were sacrificed 163 days after the start of experiment, and the number of aberrant crypt foci and tumors in the intestine were counted using a stereoscopic microscope following methylene blue staining. The mean number of aberrant crypt foci was 52.4 in group A, 18.9 in group B, 0 in group C and 0.5 in group D. A total of 9 tumors were observed in group A alone, with none in the remaining groups. The numbers of aberrant crypt foci and tumors in group B were significantly lower than in group A. Etodolac, a selective cyclooxygenase-2 inhibitor, suppresses the occurrence of aberrant crypt foci and tumors in colitis-induced tumorigenesis in rats.

RNA-dependent RNA polymerases (RdRPs) of the Flaviviridae family catalyze replication of positive (+)- strand viral RNA through synthesis of minus (-)-and progeny (+)-strand RNAs. West Nile virus (WNV), a mosquito-borne member, is a rapidly re-emerging human pathogen in the United States since its first outbreak in 1999. To study the replication of the WNV RNA in vitro, an assay is described here that utilizes the WNV RdRP and subgenomic (-)- and (+)-strand template RNAs containing 5'- and 3'-terminal regions (TR) with the conserved sequence elements. Our results show that both 5'- and 3'-TRs of the (+)-strand RNA template including the wild type cyclization (CYC) motifs are important for RNA synthesis. However, the 3'-TR of the (-)-strand RNA template alone is sufficient for RNA synthesis. Mutational analysis of the CYC motifs revealed that the (+)-strand 5'-CYC motif is critical for (-)-strand RNA synthesis but neither the (-)-strand 5'- nor 3'-CYC motif is important for the (+)-strand RNA synthesis. Moreover, the 5'-cap inhibits the (-)-strand RNA synthesis from the 3' fold-back structure of (+)-strand RNA template without affecting the de novo synthesis of RNA. These results support a model that "cyclization" of the viral RNA play a role for (-)-strand RNA synthesis but not for (+)-strand RNA synthesis.

Interferon (IFN)-alpha treatment is a common therapy for chronic viral hepatitis and contributes to preventing hepatocarcinogenesis. However, it is not clear whether IFN-alpha directly inhibits the clonal expansion of pre-neoplastic hepatocytes. To clarify the mechanism by which IFN-alpha prevents hepatocarcinogenesis, we examined the effect of IFN-alpha in a chemically induced hepatocarcinogenesis model initiated by diethylnitrosamine (DEN) and promoted by 2-acetylaminofluorene (2-AAF) and partial hepatectomy, in which hepatocellular carcinoma (HCC) arises through pre-neoplastic foci without inflammation or fibrosis. The protocols of IFN-alpha administration were started simultaneously with chemical initiation and lasted for either 4 or 40 weeks. The pre-neoplastic foci and neoplastic HCC were evaluated at 4 or 40 weeks after chemical initiation, respectively. The effects of IFN-alpha were assessed by the expression of tumor-related genes and cell cycle-related genes in the pre-neoplastic foci, using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). As a result of IFN-alpha treatment, the numbers and average volume of pre-neoplastic foci were reduced. The proliferating cell nuclear antigen index and the expression of G(1) cyclins were also reduced in the pre-neoplastic foci in the IFN-treated group. The expression of p21, which is an inhibitor of cyclin-kinase complexes was higher in the foci of the IFN-treated group, while p53 expression was not altered in this group, compared with the control group. IFN-alpha also suppressed the tumor development at 40 weeks after initiation. And in the long-term IFN-alpha-treated group, both the tumor numbers and average tumor size were markedly more reduced than those in the short-term-treated group. Therefore, it was demonstrated that longer treatment with IFN-alpha was more effective, compared with shorter treatment. In conclusion, it was shown that IFN-alpha directly prevented and delayed hepatocarcinogenesis through the suppression of pre-neoplastic cell proliferation and that it may partially depend on p21 induction through a p53-independent pathway.

The liver has an extremely effective regenerative capacity. When 70% of a rat liver is removed by surgery, the liver mass regrows in 7 to 10 days by the compensatory hyperplasia of the remnant part. In case of damage to the surviving hepatocytes, the facultative stem-cell compartment is activated and the liver regenerates by means of oval-cell proliferation/differentiation. In the present study, we demonstrate that when both hepatocyte proliferation and stem-cell activation were prevented by dexamethasone (Dex) administration, the liver mass was restored in the absence of DNA synthesis. The restoration of the liver was accomplished by the preferential enlargement/hypertrophy of the periportal hepatocytes. A similar response was observed when cell proliferation was arrested by 5-fluorouracil (FU) following partial hepatectomy. Therefore, the hepatocytic hypertrophy appears to provide an alternative mechanism of liver-mass restoration. This hypertrophic condition of the liver is not stable, because following the withdrawal of Dex, the enlarged hepatocytes entered in the cell cycle and the normal liver structure and DNA content was re-established.

Current studies have indicated both positive and negative roles for the hepatocyte growth factor (HGF)/c-met receptor signaling system in tumor development. Recently, we have shown that HGF has the capacity to induce both growth inhibition and programmed cell death in aflatoxin-transformed (AFLB8) rat liver epithelial cells. Using the same cell line, we have now investigated a potential mechanism for HGF-induced apoptosis. Immunoblot analysis of bcl-2 gene family member (bax, bcl-2, bclX-s/l) expression showed no correlation with HGF treatment, suggesting that HGF-mediated apoptosis is bax independent. Following HGF treatment retinoblastoma protein (pRB) was present in the hypophosphorylated state. HGF treatment increased cyclin A, cyclin G1 and nuclear transcriptional factor (NFkappaB) protein expression. However, electrophoretic mobility shift analysis showed that NFkappaB activity decreased with HGF treatment. Under these apoptotic conditions, c-Jun N-terminal kinase (JNK1) and extracellular signal-regulated kinase (ERK2) were activated with lower level activation of ERK2, while no involvement of phosphatidylinositol-3 kinase was observed. Epidermal growth factor (EGF) was not protective, and actually induced cells to undergo apoptosis to a level similar to that of HGF alone or EGF/HGF in combination. These results suggest the possibility of cross-talk between HGF/c-met and EGF/EGFR signaling pathways, and the involvement of JNK1 induction in HGF-mediated apoptotic cell death.

The sialomucin CD34 is expressed on human and mouse hematopoietic stem cells and is used as an important marker for isolating the hematopoietic stem/progenitor cells. The involvement of hepatic stem cells in liver regeneration under certain conditions in adult rats is now well supported. The objective of the present research was to explore the idea that CD34 might also be expressed on hepatic stem cell progeny. Polymerase chain reaction (PCR)-based cloning of rat CD34 was partially accomplished. During the hepatic stem cell activation (2-acetylaminofluorene/partial hepatectomy [AAF/PH] model), the CD34 transcripts were increased and reached the peak level between 9 and 12 days after partial hepatectomy when the progenitor cells (e.g., oval cells, early hepatocytes in basophilic foci, and intestinal type of cells) are most abundant. Both in situ hybridization and immunohistochemistry, using anti-mouse CD34 antibody, which recognizes the cytoplasmic domain, clearly showed the expression of CD34 on oval cells as well as on endothelial cells of large hepatic vessels. In addition, bile ductular epithelial (BDE) cells both in the AAF/PH model and in normal liver expressed CD34, suggesting a close relationship between BDE cells and the hepatic stem-cell compartment. Taken together, the data indicate that CD34 would, similar to its role in the hematopoietic system, be an important probe for characterizing the cellular biology of the hepatic stem-cell compartment.

A patient with pigmentary retinopathy, nephrotic syndrome, Menetrier's disease, and diabetes mellitus is presented. Other complications were congestive heart failure, hypothyroidism, hypertension, and hypertriglyceridemia. Hypogenitalism was also suspected. Pigmentary retinopathy is known to associate with many systemic diseases, which are classified into several syndromes. This case superficially resembles Alstrom's disease due to the common characteristics of pigmentary retinopathy, diabetes mellitus, renal disease, and hypogenitalism. But clinically and histologically, there are distinct differences. To our knowledge, this association has never been reported.

Leiomyoblastoma has been regarded as a neoplasm of smooth muscle origin. With recent progress in immunohistostaining techniques, many clinicopathological discrepancies have been pointed out about the origin of leiomyoblastoma. It has been claimed that gastrointestinal non-epithelial tumors should be regarded as stromal tumors in order to study their origin. In the present study, we performed various forms of immunohistostaining in seven cases of leiomyoblastoma to determine their origin. One case expressed desmine and muscle specific actin and was considered to be derived from smooth muscle. Four neoplasms expressed S-100 protein (two cases were also NSE positive) and were thought to be derived from the nerve. Two cases were of unknown derivation. These results suggest that the cells of leiomyoblastoma may arise from a primitive to totipotential cell of neural lineages that may anomalously express smooth muscle filaments.