Short Intracellular HIV-1 Transcripts as Biomarkers of Residual Immune Activation in Patients on Antiretroviral Therapy.

UNLABELLED: HIV-1 patients continue to remain at an abnormal immune status despite prolonged combination antiretroviral therapy (cART), which results in an increased risk of non-AIDS-related diseases. Given the growing recognition of the importance of understanding and controlling the residual virus in patients, additional virological markers to monitor infected cells are required. However, viral replication in circulating cells is much poorer than that in lymph nodes, which results in the absence of markers to distinguish these cells from uninfected cells in the blood. In this study, we identified prematurely terminated short HIV-1 transcripts (STs) in peripheral blood mononuclear cells (PBMCs) as an efficient intracellular biomarker to monitor viral activation and immune status in patients with cART-mediated full viral suppression in plasma. STs were detected in PBMCs obtained from both treated and untreated patients. ST levels in untreated patients generally increased with disease progression and decreased after treatment initiation. However, some patients exhibited sustained high levels of ST and low CD4(+) cell counts despite full viral suppression by treatment. The levels of STs strongly reflected chronic immune activation defined by coexpression of HLA-DR and CD38 on CD8(+) T cells, rather than circulating proviral load. These observations represent evidence for a relationship between viral persistence and host immune activation, which in turn results in the suboptimal increase in CD4(+) cells despite suppressive antiretroviral therapy. This cell-based measurement of viral persistence contributes to an improved understanding of the dynamics of viral persistence in cART patients and will guide therapeutic approaches targeting viral reservoirs. IMPORTANCE: Combination antiretroviral therapy (cART) suppresses HIV-1 load to below the detectable limit in plasma. However, the virus persists, and patients remain at an abnormal immune status, which results in an increased risk of non-AIDS-related complications. To achieve a functional cure for HIV-1 infection, activities of viral reservoirs must be quantified and monitored. However, latently infected cells are difficult to be monitored. Here, we identified prematurely terminated short HIV-1 transcripts (STs) as an efficient biomarker for monitoring viral activation and immune status in patients with cART-mediated full viral suppression in plasma. This cell-based measurement of viral persistence will contribute to our understanding of the impact of residual virus on chronic immune activation in HIV-1 patients during cART.

Translation initiation from the ribosomal A site or the P site, dependent on the conformation of RNA pseudoknot I in dicistrovirus RNAs.

Translation initiation of the second ORF of insect dicistrovirus RNA depends on an internal ribosomal entry site (IRES) in its intergenic region (IGR) and is exceptional in using a codon other than AUG and in not using the canonical initiator methionine tRNA. Studies in vitro suggest that pseudoknot I (PKI) immediately preceding the initiation codon occupies the ribosomal P site and that an elongator tRNA initiates translation from the ribosomal A site. Using dicistronic reporters carrying mutations in the initiation codon of the second ORF and mutant elongator or initiator tRNAs capable of reading these codons, we provide direct evidence for initiation from the A site in mammalian cells and, under certain conditions, also from the P site. Initiation from the A but not the P site requires PKI. Thus, PKI structure may be dynamic, and optimal IGR IRES-mediated translation of dicistroviral RNAs may require trans-acting factors to stabilize PKI.

Actinocrispum wychmicini gen. nov., sp. nov., a novel member of the family Pseudonocardiaceae, isolated from soil.

A novel actinomycete, designated MI503-A4T, was isolated from soil. Comparative analysis of 16S rRNA gene sequences indicated that MI503-A4T was phylogenetically related to members of the family Pseudonocardiaceae. The most closely related genus was Kibdelosporangium (95.7-96.2 % sequence similarity). Substrate mycelia were branched and pale yellow to pale yellowish-brown. Straight- to zigzag-shaped aerial mycelia were observed, but Sporangium-like structures were absent. The whole-cell hydrolysate contained meso-diaminopimelic acid. The muramic acid residues in the peptidoglycan were N-acetylated. Whole-cell sugars were rhamnose, ribose, arabinose and galactose (cell wall chemotype IV). The predominant menaquinone was MK-9(H4). A small amount of MK-8(H4) was also detected. The DNA G+C content was 70.3-71.1 mol%. Polar lipids contained diphosphatidylglycerol, phosphatidylethanolamine and hydroxyl-phosphatidylethanolamine. Cellular fatty acid analysis of MI503-A4T detected predominantly iso-C14 : 0 (11.5 %), iso-C15 : 0 (13.3 %) and iso-C16 : 0 (35.7 %). Phenotypic and phylogenetic characteristics differentiated MI503-A4T from members of all genera within the family Pseudonocardiaceae with validly published names. Therefore, MI503-A4T is proposed to be a representative of a novel species in a novel genus, Actinocrispum wychmicini gen. nov., sp. nov. The type strain of the type species is MI503-A4T (=NBRC 109632T=DSM 45934T).

New metastatic model of human small-cell lung cancer by orthotopic transplantation in mice.

Small-cell lung cancer (SCLC) is an aggressive cancer with high metastatic ability and novel strategies against the metastasis are urgently needed to improve SCLC treatment. However, the mechanism of metastasis of SCLC remains largely to be elucidated. For further studies of SCLC metastasis, we developed a new orthotopic transplantation model in mice. We established a GFP-labeled subline from the human SCLC cell line DMS273 and transplanted them orthotopically into the lung of nude mice with Matrigel. The GFP-labeled cells showed significant metastatic activity and formed metastatic foci in distant tissues such as bone, kidney, and brain, as observed in SCLC patients. From a bone metastasis focus of the mouse, we isolated another subline, termed G3H, with enhanced metastatic potential and higher hepatocyte growth factor (HGF) expression than the parental line. Further studies indicated that the HGF/MET signaling pathway was involved in in vitro motility and invasion activities of the G3H cells and treatments with MET inhibitors decreased formation of distant metastases in our orthotopic model using G3H cells. These data indicated that our model mimics the clinical aspect of SCLC such as metastatic tropism and autocrine of HGF/MET signaling. Compared with other orthotopic SCLC models, our model has a superior ability to form distant metastases. Therefore, our model will provide a valuable tool for the study of SCLC metastasis.

Identification of self-growth-inhibiting compounds lauric acid and 7-(Z)-tetradecenoic acid from Helicobacter pylori.

Helicobacter pylori growth medium is usually supplemented with horse serum (HS) or FCS. However, cyclodextrin derivatives or activated charcoal can replace serum. In this study, we purified self-growth-inhibiting (SGI) compounds from H. pylori growth medium. The compounds were recovered from porous resin, Diaion HP-20, which was added to the H. pylori growth medium instead of known supplements. These SGI compounds were also identified from 2,6-di-O-methyl-ß-cyclodextrin, which was supplemented in a pleuropneumonia-like organisms broth. The growth-inhibiting compounds were identified as lauric acid (LA) and 7-(Z)-tetradecenoic acid [7-(Z)-TDA]. Although several fatty acids had been identified in H. pylori, these specific compounds were not previously found in this species. However, we confirmed that these fatty acids were universally present in the cultivation medium of the H. pylori strains examined in this study. A live/dead assay carried out without HS indicated that these compounds were bacteriostatic; however, no significant growth-inhibiting effect was observed against other tested bacterial species that constituted the indigenous bacterial flora. These findings suggested that LA and 7-(Z)-TDA might play important roles in the survival of H. pylori in human stomach epithelial cells.

Asteltoxins from the Entomopathogenic Fungus Pochonia bulbillosa 8-H-28.

New asteltoxins C (3) and D (4) were found in the extract of the entomopathogenic fungus Pochonia bulbillosa 8-H-28. Compound 2, which was spectroscopically identical with the known asteltoxin B, was isolated, and structural analysis led to a revision of the structure of asteltoxin B. Compounds 2 and 4 have a novel tricyclic ring system connected to a dienyl α-pyrone structure. Compound 3 has a 2,8-dioxabicyclo[3.3.0]octane ring similar to that of asteltoxin (1). Compound 3 showed potent antiproliferative activity against NIAS-SL64 cells derived from the fat body of Spodoptera litura larvae, while 2 and 4 were inactive.

Quinofuracins A-E, produced by the fungus Staphylotrichum boninense PF1444, show p53-dependent growth suppression.

Quinofuracins A-E, novel anthraquinone derivatives containing ß-D-galactofuranose that were isolated from the fungus Staphylotrichum boninense PF1444, induced p53-dependent cell death in human tumor cells. The structures of quinofuracins A-E, including absolute configurations, were elucidated by extensive spectroscopic analysis and chemical transformation studies. Quinofuracins were classified into three groups according to the aglycone moieties. 5'-Oxoaverantin was present in quinofuracins A-C, whereas averantin and versicolorin B were identified in quinofuracins D and E, respectively. These quinofuracins induced p53-dependent growth suppression in human glioblastoma LNZTA3 cells.

Synthesis and Structure-Activity Relationship Study of NBRI16716B, an Antitumor Natural Product.

The total synthesis of NBRI16716B (2), a naturally occurring modulator of tumor-stroma interactions, was successfully achieved. Using this synthetic route, a dehydroxy analogue (21) and a derivative lacking the 5-hydroxy-3-methylpentenoyl side chain (22) became accessible. A preliminary structure-activity relationship study to unveil the structural requirements for selective inhibition of tumor cells cocultured with stromal cells revealed that both of the hydroxamate structures of 2 are indispensable, whereas the 5-hydroxy-3-methylpentenoyl side chain is not essential.

Inhibition of the first step in synthesis of the mycobacterial cell wall core, catalyzed by the GlcNAc-1-phosphate transferase WecA, by the novel caprazamycin derivative CPZEN-45.

Because tuberculosis is one of the most prevalent and serious infections, countermeasures against it are urgently required. We isolated the antitubercular agents caprazamycins from the culture of an actinomycete strain and created CPZEN-45 as the most promising derivative of the caprazamycins. Herein, we describe the mode of action of CPZEN-45 first against Bacillus subtilis. Unlike the caprazamycins, CPZEN-45 strongly inhibited incorporation of radiolabeled glycerol into growing cultures and showed antibacterial activity against caprazamycin-resistant strains, including a strain overexpressing translocase-I (MraY, involved in the biosynthesis of peptidoglycan), the target of the caprazamycins. By contrast, CPZEN-45 was not effective against a strain overexpressing undecaprenyl-phosphate-GlcNAc-1-phosphate transferase (TagO, involved in the biosynthesis of teichoic acid), and a mutation was found in the tagO gene of the spontaneous CPZEN-45-resistant strain. This suggested that the primary target of CPZEN-45 in B. subtilis is TagO, which is a different target from that of the parent caprazamycins. This suggestion was confirmed by evaluation of the activities of these enzymes. Finally, we showed that CPZEN-45 was effective against WecA (Rv1302, also called Rfe) of Mycobacterium tuberculosis, the ortholog of TagO and involved in the biosynthesis of the mycolylarabinogalactan of the cell wall of M. tuberculosis. The outlook for WecA as a promising target for the development of antituberculous drugs as a countermeasure of drug resistant tuberculosis is discussed.

Antitumor effects of tyropeptin-boronic acid derivatives: New proteasome inhibitors.

The proteasome degrades numerous regulatory proteins that are critical for tumor growth. Thus, proteasome inhibitors are promising antitumor agents. New proteasome inhibitors, such as tyropeptins and tyropeptin-boronic acid derivatives, have a potent inhibitory activity. Here we report the antitumor effects of two new tyropeptin-boronic acid derivatives, AS-06 and AS-29. AS-06 and AS-29 significantly suppress the degradation of the proteasome-sensitive fluorescent proteins in HEK293PS cells, and induce the accumulation of ubiquitinated proteins in human multiple myeloma cells. We show that these derivatives also suppress the degradation of the NF-κB inhibitor IκB-α and the nuclear translocation of NF-κB p65 in multiple myeloma cells, resulting in the inhibition of NF-κB activation. Furthermore, we demonstrate that AS-06 and AS-29 induce apoptosis through the caspase-8 and caspase-9 cascades. In a xenograft mouse model, i.v. administration of tyropeptin-boronic acid derivatives inhibits proteasome in tumors and clearly suppresses tumor growth in mice bearing human multiple myeloma. Our results indicate that tyropeptin-boronic acid derivatives could be lead therapeutic agents against human multiple myeloma.

Poliomyelitis in transgenic mice expressing CD155 under the control of the Tage4 promoter after oral and parenteral poliovirus inoculation.

An important step in poliovirus (PV) infection by the oral route in humans is replication of the virus in lymphatic tissues of the gastrointestinal (GI) tract, thought to be mainly in the Peyer's patches of the small intestine. No immunocompetent transgenic (tg) mice that express human PV receptor (CD155) under the control of different promoters can be infected orally. The mouse orthologue of human CD155 is Tage4, a protein expressed at the surface of enterocytes and in the Peyer's patches. We describe here the generation of a tg mouse model in which the Tage4 promoter was used to drive expression of the human PV receptor-coding region (Tage4-CD155tg mice). In this model, CD155 expression was observed by immunostaining in different regions in the Peyer's patches but not in their germinal centres. Although a similar pattern of staining was observed between 3- and 6-week-old Tage4-CD155tg mice, poliomyelitis was only seen in the younger mice after PV infection by the oral route. When compared with TgPVR21 mice that expressed CD155 driven by its human promoter, 3-week-old Tage4-CD155tg mice were more susceptible to gut infection and paralysis following feeding with PV. Also, Tage4-CD155tg mice exhibited higher susceptibility to poliomyelitis after parenteral inoculation of PV. Remarkably, the LD50 after intracerebral inoculation of PV was similar in both CD155 tg mouse strains. The CD155 tg mouse model reported here, although moderately susceptible to oral infection, may be suitable to study mechanisms of PV replication in the gastrointestinal tract and to dissect important aspects of PV neuroinvasiveness.

A novel evaluation method of survival motor neuron protein as a biomarker of spinal muscular atrophy by imaging flow cytometry.

Spinal muscular atrophy (SMA) is caused by mutations within the survival motor neuron 1 (SMN1) gene. These mutations result in the reduction of survival motor neuron (SMN) protein expression and SMN complex in spinal motor neurons and other tissues. SMN protein has been used as a therapeutic biomarker in recent SMA clinical studies using enzyme-linked immunosorbent assay (ELISA). Here, we investigated whether imaging flow cytometry can be a viable source of quantitative information on the SMN protein. Using a FlowSight imaging flow cytometer (Merck-Millipore, Germany), we demonstrated that imaging flow cytometry could successfully identify different expression patterns and subcellular localization of SMN protein in healthy human fibroblasts and SMA patient-derived fibroblasts. In addition, we could also evaluate the therapeutic effects of SMN protein expression by valproic acid treatment of SMA patient-derived cells in vitro. Therefore, we suggest that imaging flow cytometry technology has the potential for identifying SMN protein expression level and pattern as an evaluation tool of clinical studies.

Production of infectious chimeric hepatitis C virus genotype 2b harboring minimal regions of JFH-1.

To establish a cell culture system for chimeric hepatitis C virus (HCV) genotype 2b, we prepared a chimeric construct harboring the 5' untranslated region (UTR) to the E2 region of the MA strain (genotype 2b) and the region of p7 to the 3' UTR of the JFH-1 strain (genotype 2a). This chimeric RNA (MA/JFH-1.1) replicated and produced infectious virus in Huh7.5.1 cells. Replacement of the 5' UTR of this chimera with that from JFH-1 (MA/JFH-1.2) enhanced virus production, but infectivity remained low. In a long-term follow-up study, we identified a cell culture-adaptive mutation in the core region (R167G) and found that it enhanced virus assembly. We previously reported that the NS3 helicase (N3H) and the region of NS5B to 3' X (N5BX) of JFH-1 enabled replication of the J6CF strain (genotype 2a), which could not replicate in cells. To reduce JFH-1 content in MA/JFH-1.2, we produced a chimeric viral genome for MA harboring the N3H and N5BX regions of JFH-1, combined with a JFH-1 5' UTR replacement and the R167G mutation (MA/N3H+N5BX-JFH1/R167G). This chimeric RNA replicated efficiently, but virus production was low. After the introduction of four additional cell culture-adaptive mutations, MA/N3H+N5BX-JFH1/5am produced infectious virus efficiently. Using this chimeric virus harboring minimal regions of JFH-1, we analyzed interferon sensitivity and found that this chimeric virus was more sensitive to interferon than JFH-1 and another chimeric virus containing more regions from JFH-1 (MA/JFH-1.2/R167G). In conclusion, we established an HCV genotype 2b cell culture system using a chimeric genome harboring minimal regions of JFH-1. This cell culture system may be useful for characterizing genotype 2b viruses and developing antiviral strategies.

NAD(P)H quinone oxidoreductase 1 (NQO1)-bioactivated pronqodine A regulates prostaglandin release from human synovial sarcoma cells.

Natural products have contributed to the elucidation of biological mechanisms as well as drug discovery research. Even now, the expectation for natural products is undiminished. We screened prostaglandin release inhibitors that had no effect on in vitro cyclooxygenase activity derived from natural product sources and discovered pronqodine A. Using spectral analysis and total synthesis, the structure of pronqodine A was shown to be a benzo[d]isothiazole-4,7-dione analogue. Evaluation of the biological activity of pronqodine A revealed that the NAD(P)H dehydrogenase quinone 1 (NQO1) converted pronqodine A into a two-electron reductive form. The reductive form underwent autoxidation and reversed to its native form immediately with the generation of reactive oxygen species. Further investigations proved that pronqodine A inhibited cyclooxygenase enzyme activity only in the presence of NQO1. Pronqodine A acts as a potential bioreductive compound, inhibiting prostaglandin release in selectively activated NQO1-expressing cells.

Sacchathridine A, a prostaglandin release inhibitor from Saccharothrix sp.

Sacchathridine A (1) was isolated from the fermentation broth of strain Saccharothrix sp. MI559-46F5. The structure was determined as a new naphthoquinone derivative with an acetylhydrazino moiety by a combination of NMR, MS spectral analyses, and chemical degradation. Compound 1 showed inhibitory activity of prostaglandin E2 release in a concentration-dependent manner from human synovial sarcoma cells, SW982, with an IC50 value of 1.0 µM, but had no effect on cell growth up to 30 µM.

In vivo imaging of proteasome inhibition using a proteasome-sensitive fluorescent reporter.

A proteasome degrades numerous regulatory proteins that are critical for tumor growth and is therefore recognized as a promising anticancer target. Determining proteasome activity in the tumors of mice bearing xenografts is essential for the development of novel proteasome inhibitors. We developed a system for in vivo imaging of proteasome inhibition in the tumors of living mice, using a proteasome-sensitive fluorescent reporter, ZsProSensor-1. This reporter consists of a green fluorescent protein, ZsGreen, fused to mouse ornithine decarboxylase, which is degraded by the proteasome without being ubiquitinated. In stably transfected cells expressing ZsProSensor-1, the fluorescent reporter was rapidly degraded under steady-state conditions, whereas it was stabilized in the presence of proteasome inhibitors. Subcutaneous inoculation of the transfected cells into nude mice resulted in tumor formation. When the proteasome inhibitor bortezomib was intravenously administered to mice bearing these tumors, the ZsProSensor-1 protein accumulated in the tumors and emitted a fluorescent signal in a dose-dependent manner. Robust fluorescence was sustained for 3 days and then gradually decreased to baseline levels within 15 days. Intravenous administration of bortezomib also showed potent antitumor activity. In contrast, oral administration of bortezomib did not result in fluorescent protein accumulation in tumors or exhibit any antitumor activity. These results indicate that in vivo imaging using the ZsProSensor-1 fluorescent protein can be used as an indicator of antitumor activity and will be a powerful tool for the development of novel proteasome inhibitors.

Tripropeptin C blocks the lipid cycle of cell wall biosynthesis by complex formation with undecaprenyl pyrophosphate.

Tripropeptin C (TPPC) is a naturally occurring cyclic lipodepsipeptide antibiotic produced by a Lysobacter sp. TPPC exhibits potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and penicillin-resistant Streptococcus pneumoniae. This antibiotic also inhibits the incorporation of N-acetylglucosamine into the peptidoglycan of S. aureus at a 50% inhibitory concentration (IC(50)) of 0.7 µM, which is proportional to its MIC (0.87 µM; equivalent to 1.0 µg/ml). Treatment of exponential-phase S. aureus cells with TPPC resulted in accumulation of UDP-MurNAc-pentapeptide in the cytoplasm. The antimicrobial activity of TPPC was weakened by the addition of prenyl pyrophosphates but not by prenyl phosphates, UDP-linked sugars, or the pentapeptide of peptidoglycan. The direct interaction between TPPC and undecaprenyl pyrophosphate (C(55)-PP) was observed by mass spectrometry and thin-layer chromatography analysis, indicating that TPPC can potentially inhibit C(55)-PP phosphatase activity, which plays a crucial role in the lipid cycle of peptidoglycan synthesis. As expected, TPPC inhibits this enzymatic reaction at an IC(50) of 0.03 to 0.1 µM in vitro, as does bacitracin. From the analysis of accumulation of lipid carrier-related compounds, TPPC was found to cause the accumulation of C(55)-PP in situ, leading to the accumulation of a glycine-containing lipid intermediate. This suggested that the TPPC/C(55)-PP complex also inhibits the transglycosylation step or flippase activity, adding to the inhibition of C(55)-PP dephosphorylation. This mode of action is different from that of currently available drugs such as vancomycin, daptomycin, and bacitracin.

Development of recombinant hepatitis C virus with NS5A from strains of genotypes 1 and 2.

Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) plays multiple and diverse roles in the viral lifecycle, and is currently recognized as a novel target for anti-viral therapy. To establish an HCV cell culture system with NS5A of various strains, recombinant viruses were generated by replacing NS5A of strain JFH-1 with those of strains of genotypes 1 (H77; 1a and Con1; 1b) and 2 (J6CF; 2a and MA; 2b). All these recombinant viruses were capable of replication and infectious virus production. The replacement of JFH-1 NS5A with those of genotype 1 strains resulted in similar or slightly reduced virus production, whereas replacement with those of genotype 2 strains enhanced virus production as compared with JFH-1 wild-type. A single cycle virus production assay with a CD81-negative cell line revealed that the efficient virus production elicited by replacement with genotype 2 strains depended on enhanced viral assembly, and that substitutions in the C-terminus of NS5A were responsible for this phenotype. Pulse-chase assays revealed that these substitutions in the C-terminus of NS5A were possibly associated with accelerated cleavage kinetics at the NS5A-NS5B site. Using this cell culture system with NS5A-substituted recombinant viruses, the anti-viral effects of an NS5A inhibitor were then examined. A 300- to 1000-fold difference in susceptibility to the inhibitor was found between strains of genotypes 1 and 2. This system will facilitate not only a better understanding of strain-specific roles of NS5A in the HCV lifecycle, but also enable the evaluation of genotype and strain dependency of NS5A inhibitors.

2A protease is not a prerequisite for poliovirus replication.

Poliovirus (PV) 2A(pro) has been considered important for PV replication and is known to be toxic to host cells. A 2A(pro)-deficient PV would potentially be less toxic and ideal as a vector. To examine whether 2A(pro) is needed to form progeny virus, a full-length cDNA of dicistronic (dc) PV with (pOME) or without (pOMEDelta2A) 2A(pro) was constructed in the strain PV1(M)OM. RNAs of both pOME and pOMEDelta2A were capable of forming progeny viruses, called OME and OMEDelta2A, respectively. In their ability to induce a cytopathic effect (CPE), the strains ranked as OMEDelta2A < OME falling dots PV1(M)OM. These results suggest that 2A(pro) is not essential for full-length dc PV to form progeny virus and that it contributes to the efficient viral replication and/or induction of a CPE. To clarify whether 2A(pro) is essential for P1-null (lacking the entire coding sequence for capsid proteins) PV, the RNA replication activity of P1-null PV (pOMDeltaP1) or P1-null PV without 2A(pro) (pOMDeltaP1Delta2A) or without both 2A(pro) and 2B (pOMDeltaP1Delta2ADelta2B) was examined. The RNAs of pOMDeltaP1 and pOMDeltaP1Delta2A could replicate and form progeny viruses under a trans supply of P1 protein, whereas the RNA of pOMDeltaP1Delta2ADelta2B could not. These results suggest that 2A(pro) is not needed for the replication of P1-null PV, although it is important for PV RNA replication and inducing a CPE. To know whether a 2A(pro)-deficient PV can be used as a vector, a P1-null PV containing the enhanced green fluorescent protein (EGFP) coding sequence with or without 2A(pro) was examined. It expressed fluorescent protein. This result suggests that 2A(pro)-deficient PV can express foreign genes.

Acetophenone 4-nitrophenylhydrazone inhibits Hepatitis B virus replication by modulating capsid assembly.

Hepatitis B virus (HBV) is the causative agent of chronic liver disease and is correlated with the development of subsequent hepatic cirrhosis and hepatocellular carcinoma. Current antiviral therapy using nucleos(t)ide analogs is effective in suppressing viral replication and interrupting disease progression, but HBV is rarely cured completely. Thus, there remains an unmet need for the development of novel anti-HBV drugs. Here, we report the identification of N-(4-Nitrophenyl)-1-phenylethanone hydrazone (ANPH) as a novel structural class of selective inhibitors targeting the replication of the HBV genome using adenovirus vector-mediated HBV genome transduction. ANPH inhibited viral genome replication in HepG2.2.15 cells by inducing the formation of empty capsids devoid of pregenomic RNA without affecting its transcription and translation. Biochemical assays using a truncated core protein consisting of the assembly domain showed that ANPH accelerates the formation of morphologically intact capsids. Taken together, we propose that ANPH might provide a new structural scaffold to design a new anti-HBV drug in medicinal chemistry as well as chemical probes for HBV core protein functions in the future.