Subgenomic Replicon Research Articles

Epoxide based inhibitors of the hepatitis C virus non-structural 2 autoprotease

Hepatitis C virus (HCV) non-structural 2 (NS2) encodes an essential protease activity responsible for processing at the NS2–NS3 junction which represents an attractive antiviral target. Attempts to inhibit the NS2 autoprotease with mechanism-based protease inhibitors and substrate peptides have had limited success. We report a series of epoxide-containing small molecules capable of blocking NS2–NS3 proteolysis in vitro and demonstrate the potential for selectivity towards the NS2 autoprotease. A compound within this series was able to perturb HCV genome replication in a subgenomic replicon system only when polyprotein processing was dependent on NS2 autoprotease activity, in addition it inhibited replication of full length HCV. These findings suggest blocking HCV polyprotein processing through inhibition of the NS2 autoprotease represents a viable route to exert an antiviral effect.

Soraphen A: A Probe for Investigating the Role of de Novo Lipogenesis during Viral Infection.

Many viruses including the hepatitis C virus (HCV) induce changes to the infected host cell metabolism that include the up-regulation of lipogenesis to create a favorable environment for the virus to propagate. The enzyme acetyl-CoA carboxylase (ACC) polymerizes to form a supramolecular complex that catalyzes the rate-limiting step of de novo lipogenesis. The small molecule natural product Soraphen A (SorA) acts as a nanomolar inhibitor of acetyl-CoA carboxylase activity through disruption of the formation of long highly active ACC polymers from less active ACC dimers. We have shown that SorA inhibits HCV replication in HCV cell culture models expressing subgenomic and full-length replicons (IC50 = 5 nM) as well as a cell culture adapted virus. Using coherent anti-Stokes Raman scattering (CARS) microscopy, we have shown that SorA lowers the total cellular lipid volume in hepatoma cells, consistent with a reduction in de novo lipogenesis. Furthermore, SorA treatment was found to depolymerize the ACC complexes into less active dimers. Taken together, our results suggest that SorA treatment reverses HCV-induced lipid accumulation and demonstrate that SorA is a valuable probe to study the roles of ACC polymerization and enzymatic activity in viral pathogenesis.

Benzothiazole and Pyrrolone Flavivirus Inhibitors Targeting the Viral Helicase.

The flavivirus nonstructural protein 3 (NS3) is a protease and helicase, and on the basis of its similarity to its homologue encoded by the hepatitis C virus (HCV), the flavivirus NS3 might be a promising drug target. Few flavivirus helicase inhibitors have been reported, in part, because few specific inhibitors have been identified when nucleic acid unwinding assays have been used to screen for helicase inhibitors. To explore the possibility that compounds inhibiting NS3-catalyzed ATP hydrolysis might function as antivirals even if they do not inhibit RNA unwinding in vitro, we designed a robust dengue virus (DENV) NS3 ATPase assay suitable for high-throughput screening. Members of two classes of inhibitory compounds were further tested in DENV helicase-catalyzed RNA unwinding assays, assays monitoring HCV helicase action, subgenomic DENV replicon assays, and cell viability assays and for their ability to inhibit West Nile virus (Kunjin subtype) replication in cells. The first class contained analogues of NIH molecular probe ML283, a benzothiazole oligomer derived from the dye primuline, and they also inhibited HCV helicase and DENV NS3-catalyzed RNA unwinding. The most intriguing ML283 analogue inhibited DENV NS3 with an IC50 value of 500 nM and was active against the DENV replicon. The second class contained specific DENV ATPase inhibitors that did not inhibit DENV RNA unwinding or reactions catalyzed by HCV helicase. Members of this class contained a 4-hydroxy-3-(5-methylfuran-2-carbonyl)-2H-pyrrol-5-one scaffold, and about 20 μM of the most potent pyrrolone inhibited both DENV replicons and West Nile virus replication in cells by 50%.

Scanning mutagenesis studies reveal a potential intramolecular interaction within the C-terminal half of dengue virus NS2A involved in viral RNA replication and virus assembly and secretion.

The NS2A protein of dengue virus (DENV) has eight predicted transmembrane segments (pTMSs; pTMS1 to pTMS8). NS2A has been shown to participate in RNA replication, virion assembly, and the host antiviral response. However, the role of the amino acid residues within the pTMS regions of NS2A during the virus life cycle is poorly understood. In the study described here, we explored the function of DENV NS2A by introducing a series of double or triple alanine substitutions into the C-terminal half (pTMS4 to pTMS8) of NS2A in the context of a DENV infectious clone or subgenomic replicon. Fourteen (8 within pTMS8) of 35 NS2A mutants displayed a lethal phenotype due to impairment of RNA replication by a replicon assay. Three NS2A mutants with mutations within pTMS7, the CM20, CM25, and CM27 mutants, displayed similar phenotypes, low virus yields (>100-fold reduction), wild-type-like replicon activity, and low infectious virus-like particle yields by transient trans-packaging experiments, suggesting a defect in virus assembly and secretion. The sequencing of revertant viruses derived from CM20, CM25, and CM27 mutant viruses revealed a consensus reversion mutation, leucine (L) to phenylalanine (F), at codon 181 within pTMS7. The introduction of an L181F mutation into a full-length NS2A mutant, i.e., the CM20, CM25, and CM27 constructs, completely restored wild-type infectivity. Notably, L181F also substantially rescued the other severely RNA replication-defective mutants with mutations within pTMS4, pTMS6, and pTMS8, i.e., the CM2, CM3, CM13, CM31, and CM32 mutants. In conclusion, the results revealed the essential roles of pTMS4 to pTMS8 of NS2A in RNA replication and/or virus assembly and secretion. The intramolecular interaction between pTMS7 and pTMS4, pTMS6, or pTMS8 of the NS2A protein was also implicated. The reported characterization of the C-terminal half of dengue virus NS2A is the first comprehensive mutagenesis study to investigate the function of flavivirus NS2A involved in the steps of the virus life cycle. In particular, detailed mapping of the amino acid residues within the predicted transmembrane segments (pTMSs) of NS2A involved in RNA replication and/or virus assembly and secretion was performed. A revertant genetics study also revealed that L181F within pTMS7 is a consensus reversion mutation that rescues both RNA replication-defective and virus assembly- and secretion-defective mutants with mutations within the other three pTMSs of NS2A. Collectively, these findings elucidate the role played by NS2A during the virus life cycle, possibly through the intricate intramolecular interaction between pTMS7 and other pTMSs within the NS2A protein.

Synthesis of Novel 4′α-Trifluoromethyl-2′β-C-Methyl-Carbodine Analogs as Anti-Hepatitis C Virus Agents

Novel 4 ′α-trifluoromethyl-2 ′β-methyl carbocyclic nucleoside analogs have been prepared and evaluated for inhibition of hepatitis C virus (HCV) RNA replication in cell cultures. Construction of cyclopentene intermediate 10a was achieved via sequential Johnson–Claisen orthoester rearrangement and ring-closing metathesis starting from the α-trifluoromethyl-α,β-unsaturated ester 5. Stereoselective dihydroxylation and desilylation yielded the target carbodine analogs. The synthesized nucleoside analogs mentioned above (18 and 19) were assayed for their ability to inhibit HCV RNA replication in a subgenomic replicon Huh7 cell line (LucNeo#2). However, the synthesized nucleosides showed neither significant antiviral activity nor toxicity up to 50 μM.

Characterization of the activity of 2′-C-methylcytidine against dengue virus replication

Dengue virus (DENV) is a severe mosquito-borne viral pathogen. Neither vaccines nor antiviral therapy is currently available to treat DENV infection. Nucleoside inhibitors targeting viral polymerase have proved promising for the development of drugs against viruses. In this study, we report a nucleoside analog, 2′-C-methylcytidine (2CMC), which exerts potent anti-DENV activity in DENV subgenomic RNA replicon and infectious systems, with an IC50 value of 11.2±0.3μM. This study utilized both cell-based and cell-free reporter assay systems to reveal the specific anti-DENV RNA polymerase activity of 2CMC. In addition, both xenograft bioluminescence-based DENV replicon and DENV-infected Institute of Cancer Research (ICR) suckling mice models evaluated the anti-DENV replication activity of 2CMC in vivo. Collectively, these findings provide a promising compound for the development of direct-acting antivirals against DENV infection.

Linear and branched alkyl-esters and amides of gallic acid and other (mono-, di- and tri-) hydroxy benzoyl derivatives as promising anti-HCV inhibitors

Linear and branched compounds that contain two, three or five units of galloyl (3,4,5-trihydroxybenzoyl) or its isomer 2,3,4-trihydroxybenzoyl, as well as other mono- or dihydroxybenzoyl moieties have been synthesized. These molecules have been evaluated for their in vitro inhibitory effects against a wide panel of viruses showing preferential activity against HIV and HCV.Our structure-activity relationship studies demonstrated that the 2,3,4-trihydroxybenzoyl moiety provides better antiviral activities than the galloyl (3,4,5-trihydroxybenzoyl) moiety that is present in natural green tea catechins. This observation can be of interest for the further rational exploration of compounds with anti-HCV/HIV properties.The most notable finding with respect to HIV is that the tripodal compounds 43 and 45, with three 2,3,4-trihydroxybenzoyl moieties, showed higher activities than linear compounds with only one or two. With respect to HCV, the linear compounds, 52 and 41, containing a 12 polymethylene chain and two 2,3 di- or 2,3,4 tri-hydroxybenzoyl groups respectively at the ends of the molecule showed good antiviral efficiency. Furthermore, the anti-HCV activity of both compounds was observed at concentrations well below the cytotoxicity threshold. A representative member of these compounds, 41, showed that the anti-HCV activity was largely independent of the genetic make-up of the HCV subgenomic replicon and cell lines used.

The interaction of the Hepatitis C Virus with intra-cellular signaling converts EGF into a potent inducer of the production of CXCR2-ligands by the infected host cell

Introduction: To establish persistent infection the hepatitis C virus (HCV) must have evolved strategies to escape antiviral and innate immunity and to influence the inflammatory response. Migration of the different cell types of adaptive and innate immunity is largely controlled by chemokines. Hence chemokines are decisive for the local composition of the different types of immune- or inflammatory cells and therefore play a major role for the formation of the local inflammatory- or immune-milieu. It is therefore not surprising that HCV has evolved strategies to gain influence on the expression of chemokines. The molecular mechanisms, which are employed by HCV to modify the expression of these factors, are incompletely understood. Likewise the influence of HCV on the regulation of chemokine expression in response to different stimuli is not clear. The present study investigates the impact of HCV on the production of the major chemo-attractants of neutrophils, the CXCR2 ligands CXCL1, 2, 3 and 8, in its host cell upon stimulation with the growth factor EGF. Methods: Huh-7 cells either infected with the HCV cc strain JC1 or harbouring the HCV subgenomic replicon were used and the impact of HCV on cellular signalling and gene expression was analysed employing targeted gene knockdown by siRNA, inhibitor-studies, rtPCR and Immunoblot-analysis. Results: Expression of the CXCR2 ligands in the hepatoma cell lines Huh-7 or Huh-7.5 is not or only weakly influenced upon stimulation with EGF. Therefore, the observation was surprising, that in the presence of either the sub-genomic or the full-length HCV replicon or upon infection of Huh-7.5 cells with the HCVcc strain JC1 EGF is converted in to a potent inducer of the different CXCR2 ligands. The further analysis of the molecular mechanism involved in this phenomenon revealed that with respect to the regulation of CXCL8 the NS3/4A mediated down-regulation of the endogenous feedback inhibitor of the EGF receptor (EGFR), the ubiquitously expressed T cell protein-tyrosine phosphatase (TC-PTP) and subsequent sensitization of the EGFR is important. Furthermore inhibitor studies and studies using targeted gene knockdown by specific siRNA suggest that downstream of the EGFR activation of the mitogen activated protein kinase (MAPK) activating protein kinase (MEK)1 and of its down-stream effector kinases the extracellular signal stimulated kinase (Erk)1 and 2 play a role for EGF inducible expression of CXCL8 in the presence of HCV. Interestingly this mechanism is not responsible for the EGFR-dependent up-regulation of the other CXCR2 ligands CXCL-1, 2 and 3 in HCV infected cells upon stimulation with EGF. Conclusion: The study provide evidence that HCV utilizes EGFR and EGFR-dependent signalling not only as cofactors for viral entry and replication but also interferes with EGF-dependent signalling thereby modifying the inter-cellular communication of the infected host cell.

Conserved motifs in a tombusvirus polymerase modulate genome replication, subgenomic transcription, and amplification of defective interfering RNAs.

The replication of plus-strand RNA virus genomes is mediated by virally encoded RNA-dependent RNA polymerases (RdRps). We have investigated the role of the C-proximal region in the RdRp of tomato bushy stunt virus (TBSV) in mediating viral RNA synthesis. TBSV is the prototype species in the genus Tombusvirus, family Tombusviridae, and its RdRp is responsible for replicating the viral genome, transcribing two subgenomic mRNAs, and supporting replication of defective interfering RNAs. Comparative sequence analysis of the RdRps of tombusvirids identified three highly conserved motifs in their C-proximal regions, and these sequences were subsequently targeted for mutational analysis in TBSV. The results revealed that these motifs are important for (i) synthesizing viral genomic RNA and subgenomic mRNAs, (ii) facilitating plus- and/or minus-strand synthesis, and (iii) modulating trans-replication of a defective interfering RNA. These motifs were also found to be conserved in other plant viruses as well as in a fungal and insect virus. The collective findings are discussed in relation to viral RNA synthesis and taxonomy. Little is currently known about the structure and function of the viral polymerases that replicate the genomes of RNA plant viruses. Tombusviruses, the prototype of the tombusvirids, have been used as model plus-strand RNA plant viruses for understanding many of the steps in the infectious process; however, their polymerases remain poorly characterized. To help address this issue, the function of the C-terminal region of the polymerase of a tombusvirus was investigated. Three conserved motifs were identified and targeted for mutational analysis. The results revealed that these polymerase motifs are important for determining what type of viral RNA is produced, facilitating different steps in viral RNA production, and amplifying subgenomic RNA replicons. Accordingly, the C-terminal region of the tombusvirus polymerase is needed for a variety of fundamental activities. Furthermore, as these motifs are also present in distantly related viruses, the significance of these results extends beyond tombusvirids.

A zebrafish model for subgenomic hepatitis C virus replication

Persistent infection with hepatitis C virus (HCV) is a major risk factor in the development of hepatocellular carcinoma. The elucidation of the pathogenesis of HCV-associated liver disease is hampered by the absence of an appropriate small animal model. Zebrafish exhibits high genetic homology to mammals, and is easily manipulated experimentally. In this study, we describe the use of a zebrafish model for the analysis of HCV replication mechanisms. As the 5' untranslated region (UTR), the core protein, the non-structural protein 5B (NS5B) and the 3'UTR are essential for HCV replication, we constructed a HCV sub-replicon gene construct including the 4 gene sequences and the enhanced green fluorescent protein (EGFP) reporter gene; these genes were transcribed through the mouse hepatocyte nuclear factor 4 (mHNF4) promoter. By microinjection of the subgenomic replicon vector into zebrafish larvae, the virus was easily detected by observing EGFP fluorescence in the liver. The positive core and NS5B signals showed positive expression of the HCV gene construct in zebrafish by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. Importantly, the negative strand sequence of the HCV subgenomic RNA was detected by RT-PCR and hybridization in situ, demonstrating that the HCV sub-replicon has positive replication activity. Furthermore, the hybridization signal mainly appeared in the liver region of larvae, as detected by the sense probe of the core protein or NS5B, which confirmed that the sub-replicon amplification occurred in the zebrafish liver. The amplification of the sub-replicon caused alterations in the expression of certain genes, which is similar to HCV infection in human liver cells. To verify the use of this zebrafish model in drug evaluation, two drugs against HCV used in clinical practice, ribavirin and oxymatrine, were tested and these drugs showed significant inhibition of replication of the HCV sub-replicon in the larvae. In conclusion, this zebrafish model of HCV may prove to be a novel and simple in vivo model for the study of the mechanisms of HCV replication and may also prove useful in the disovery of new anti-HCV drugs.

Natural compounds isolated from Brazilian plants are potent inhibitors of hepatitis C virus replication in vitro

Compounds extracted from plants can provide an alternative approach to new therapies. They present characteristics such as high chemical diversity, lower cost of production and milder or inexistent side effects compared with conventional treatment. The Brazilian flora represents a vast, largely untapped, resource of potential antiviral compounds. In this study, we investigate the antiviral effects of a panel of natural compounds isolated from Brazilian plants species on hepatitis C virus (HCV) genome replication. To do this we used firefly luciferase-based HCV sub-genomic replicons of genotypes 2a (JFH-1), 1b and 3a and the compounds were assessed for their effects on both HCV replication and cellular toxicity. Initial screening of compounds was performed using the maximum non-toxic concentration and 4 compounds that exhibited a useful therapeutic index (favourable ratio of cytotoxicity to antiviral potency) were selected for extra analysis. The compounds APS (EC50=2.3μM), a natural alkaloid isolated from Maytrenus ilicifolia, and the lignans 3∗43 (EC50=4.0μM), 3∗20 (EC50=8.2μM) and 5∗362 (EC50=38.9μM) from Peperomia blanda dramatically inhibited HCV replication as judged by reductions in luciferase activity and HCV protein expression in both the subgenomic and infectious systems. We further show that these compounds are active against a daclatasvir resistance mutant subgenomic replicon. Consistent with inhibition of genome replication, production of infectious JFH-1 virus was significantly reduced by all 4 compounds. These data are the first description of Brazilian natural compounds possessing anti-HCV activity and further analyses are being performed in order to investigate the mode of action of those compounds.

Serine Phosphorylation of the Hepatitis C Virus NS5A Protein Controls the Establishment of Replication Complexes

ABSTRACTThe hepatitis C virus (HCV) nonstructural 5A (NS5A) protein is highly phosphorylated and involved in both virus genome replication and virion assembly. We and others have identified serine 225 in NS5A to be a phosphorylation site, but the function of this posttranslational modification in the virus life cycle remains obscure. Here we describe the phenotype of mutants with mutations at serine 225; this residue was mutated to either alanine (S225A; phosphoablatant) or aspartic acid (S225D; phosphomimetic) in the context of both the JFH-1 cell culture infectious virus and a corresponding subgenomic replicon. The S225A mutant exhibited a 10-fold reduction in genome replication, whereas the S225D mutant replicated like the wild type. By confocal microscopy, we show that, in the case of the S225A mutant, the replication phenotype correlated with an altered subcellular distribution of NS5A. This phenotype was shared by viruses with other mutations in the low-complexity sequence I (LCS I), namely, S229D, S232A, and S235D, but not by viruses with mutations that caused a comparable replication defect that mapped to domain II of NS5A (P315A, L321A). Together with other components of the genome replication complex (NS3, double-stranded RNA, and cellular lipids, including phosphatidylinositol 4-phosphate), the mutation in NS5A was restricted to a perinuclear region. This phenotype was not due to cell confluence or another environmental factor and could be partially transcomplemented by wild-type NS5A. We propose that serine phosphorylation within LCS I may regulate the assembly of an active genome replication complex.IMPORTANCE The mechanisms by which hepatitis C virus replicates its RNA genome remain poorly characterized. We show here that phosphorylation of the viral nonstructural protein NS5A at serine residues is important for the efficient assembly of a complex that is able to replicate the viral genome. This research implicates cellular protein kinases in the control of virus replication and highlights the need to further understand the interplay between the virus and the host cell in order to develop potential avenues for future antiviral therapy.

In vitro activity and resistance profile of dasabuvir, a nonnucleoside hepatitis C virus polymerase inhibitor.

Dasabuvir (ABT-333) is a nonnucleoside inhibitor of the RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) NS5B gene. Dasabuvir inhibited recombinant NS5B polymerases derived from HCV genotype 1a and 1b clinical isolates, with 50% inhibitory concentration (IC50) values between 2.2 and 10.7 nM, and was at least 7,000-fold selective for the inhibition of HCV genotype 1 polymerases over human/mammalian polymerases. In the HCV subgenomic replicon system, dasabuvir inhibited genotype 1a (strain H77) and 1b (strain Con1) replicons with 50% effective concentration (EC50) values of 7.7 and 1.8 nM, respectively, with a 13-fold decrease in inhibitory activity in the presence of 40% human plasma. This level of activity was retained against a panel of chimeric subgenomic replicons that contained HCV NS5B genes from 22 genotype 1 clinical isolates from treatment-naive patients, with EC50s ranging between 0.15 and 8.57 nM. Maintenance of replicon-containing cells in medium containing dasabuvir at concentrations 10-fold or 100-fold greater than the EC50 resulted in selection of resistant replicon clones. Sequencing of the NS5B coding regions from these clones revealed the presence of variants, including C316Y, M414T, Y448C, Y448H, and S556G, that are consistent with binding to the palm I site of HCV polymerase. Consequently, dasabuvir retained full activity against replicons known to confer resistance to other polymerase inhibitors, including the S282T variant in the nucleoside binding site and the M423T, P495A, P495S, and V499A single variants in the thumb domain. The use of dasabuvir in combination with inhibitors targeting HCV NS3/NS4A protease (ABT-450 with ritonavir) and NS5A (ombitasvir) is in development for the treatment of HCV genotype 1 infections.

Hydroxamic acids block replication of hepatitis C virus.

Intrigued by the role of protein acetylation in hepatitis C virus (HCV) replication, we tested known histone deacetylase (HDAC) inhibitors and a focused library of structurally simple hydroxamic acids for inhibition of a HCV subgenomic replicon. While known HDAC inhibitors with varied inhibitory profiles proved to be either relatively toxic or ineffective, structure-activity relationship (SAR) studies on cinnamic hydroxamic acid and benzo[b]thiophen-2-hydroxamic acid gave rise to compounds 22 and 53, which showed potent and selective anti-HCV activity and therefore are promising starting points for further structural optimization and mechanistic studies.

In Vitro Assessment of Re-treatment Options for Patients with Hepatitis C Virus Genotype 1b Infection Resistant to Daclatasvir Plus Asunaprevir.

IntroductionDaclatasvir is a non-structural protein 5A (NS5A) inhibitor with activity against hepatitis C virus (HCV) genotypes 1–6 in vitro, and asunaprevir is a non-structural protein 3 (NS3) protease inhibitor with activity against genotypes 1, 4, 5, and 6. This study evaluates potential options for the re-treatment of HCV genotype 1b-infected patients who have failed combination therapy with daclatasvir plus asunaprevir.MethodsThe antiviral activity of drug combination regimens in HCV subgenomic replicon cell lines representing genotype 1b (Con1 strain) wild-type or a variant with specific NS5A and NS3 amino acid substitutions conferring resistance to daclatasvir and asunaprevir were compared using replicon elimination assays. Drug concentrations representing multiple 50% effective concentrations (EC50) derived in vitro and trough plasma concentrations observed in a clinical setting were utilized.ResultsAt multiple EC50 values of each drug (3×, 10×, and 30× EC50), combinations of daclatasvir plus sofosbuvir, sofosbuvir plus ledipasvir, sofosbuvir plus simeprevir, and sofosbuvir plus either a next-generation NS3 or NS5A inhibitor demonstrated comparable activity in wild-type and daclatasvir/asunaprevir-resistant cell lines. At clinically relevant drug trough concentrations, combination regimens of daclatasvir plus asunaprevir plus beclabuvir (±ribavirin), and daclatasvir plus asunaprevir plus beclabuvir plus sofosbuvir efficiently cleared daclatasvir + asunaprevir-resistant replicons from cells within 5 days of treatment.ConclusionOur in vitro results highlight a number of potential all-oral treatment options for patients who do not achieve a sustained virologic response following therapy with daclatasvir plus asunaprevir. These results require further evaluation in clinical studies.Electronic supplementary materialThe online version of this article (doi:10.1007/s40121-014-0052-8) contains supplementary material, which is available to authorized users.

Targeting cellular squalene synthase, an enzyme essential for cholesterol biosynthesis, is a potential antiviral strategy against hepatitis C virus.

Hepatitis C virus (HCV) exploits host membrane cholesterol and its metabolism for progeny virus production. Here, we examined the impact of targeting cellular squalene synthase (SQS), the first committed enzyme for cholesterol biosynthesis, on HCV production. By using the HCV JFH-1 strain and human hepatoma Huh-7.5.1-derived cells, we found that the SQS inhibitors YM-53601 and zaragozic acid A decreased viral RNA, protein, and progeny production in HCV-infected cells without affecting cell viability. Similarly, small interfering RNA (siRNA)-mediated knockdown of SQS led to significantly reduced HCV production, confirming the enzyme as an antiviral target. A metabolic labeling study demonstrated that YM-53601 suppressed the biosynthesis of cholesterol and cholesteryl esters at antiviral concentrations. Unlike YM-53601, the cholesterol esterification inhibitor Sandoz 58-035 did not exhibit an antiviral effect, suggesting that biosynthesis of cholesterol is more important than that of cholesteryl esters for HCV production. YM-53601 inhibited transient replication of a JFH-1 subgenomic replicon and entry of JFH-1 pseudoparticles, suggesting that at least suppression of viral RNA replication and entry contributes to the antiviral effect of the drug. Collectively, our findings highlight the importance of the cholesterol biosynthetic pathway in HCV production and implicate SQS as a potential target for antiviral strategies against HCV. Hepatitis C virus (HCV) is known to be closely associated with host cholesterol and its metabolism throughout the viral life cycle. However, the impact of targeting cholesterol biosynthetic enzymes on HCV production is not fully understood. We found that squalene synthase, the first committed enzyme for cholesterol biosynthesis, is important for HCV production, and we propose this enzyme as a potential anti-HCV target. We provide evidence that synthesis of free cholesterol is more important than that of esterified cholesterol for HCV production, highlighting a marked free cholesterol dependency of HCV production. Our findings also offer a new insight into a role of the intracellular cholesterol pool that is coupled to its biosynthesis in the HCV life cycle.

Mechanism of action for respiratory syncytial virus inhibitor RSV604.

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in young children and other high-risk populations. RSV nucleoprotein (N) is essential for virus assembly and replication as part of the viral ribonucleoprotein (RNP) complex. RSV604 was a putative N inhibitor in phase 2 clinical trials whose molecular mechanism of action (MoA) was not well understood. This study investigated the cell line-dependent potency of RSV604 and demonstrated its direct binding to the N protein in vitro, providing the first evidence of direct target engagement for this class of inhibitors reported to date. The affinity of RSV604 N binding was not affected by RSV604 resistance mutations in the N protein. RSV604 engaged in two different MoAs in HeLa cells, inhibiting both RSV RNA synthesis and the infectivity of released virus. The lack of inhibition of viral RNA synthesis in some cell lines explained the cell-type-dependent potency of the inhibitor. RSV604 did not inhibit viral RNA synthesis in the RSV subgenomic replicon cells or in the cell-free RNP assay, suggesting that it might act prior to viral replication complex formation. RSV604 did not alter N protein localization in the infected cells. Taken together, these results provide new insights leading to an understanding of the MoAs of RSV604 and other similar N inhibitors.

New 1-phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides inhibit hepatitis C virus replication via suppression of cyclooxygenase-2.

We report here the synthesis and mechanism of inhibition of pyrazolecarboxamide derivatives as a new class of HCV inhibitors. Compounds 6, 7, 8 and 16 inhibited the subgenomic HCV replicon 1b genotype at EC50 values between 5 and 8 μM and displayed an even higher potency against the infectious Jc1 HCV 2a genotype. Compound 6 exhibited an EC50 of 6.7 μM and selectivity index of 23 against HCV 1b, and reduced the RNA copies of the infectious Jc1 chimeric 2a clone by 82% at 7 μM. Evaluation of the mode of anti-HCV activity of 6 revealed that it suppressed HCV-induced COX-2 mRNA and protein expression, displaying an IC50 of 3.2 μM in COX-2 promoter-linked luciferase reporter assay. Conversely, the anti-HCV activity of 6 was abrogated upon over-expression of COX-2. These findings suggest that 6 as a representative of these pyrazolecarboxamides function as anti-HCV agents via targeting COX-2 at both the transcription and translation levels.

Hydrophobic and charged residues in the C-terminal arm of hepatitis C virus RNA-dependent RNA polymerase regulate initiation and elongation.

The RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV) is essential for viral genome replication. Crystal structures of the HCV RdRp reveal two C-terminal features, a β-loop and a C-terminal arm, suitably located for involvement in positioning components of the initiation complex. Here we show that these two elements intimately regulate template and nucleotide binding, initiation, and elongation. We constructed a series of β-loop and C-terminal arm mutants, which were used for in vitro analysis of RdRp de novo initiation and primer extension activities. All mutants showed a substantial decrease in initiation activities but a marked increase in primer extension activities, indicating an ability to form more stable elongation complexes with long primer-template RNAs. Structural studies of the mutants indicated that these enzyme properties might be attributed to an increased flexibility in the C-terminal features resulting in a more open polymerase cleft, which likely favors the elongation process but hampers the initiation steps. A UTP cocrystal structure of one mutant shows, in contrast to the wild-type protein, several alternate conformations of the substrate, confirming that even subtle changes in the C-terminal arm result in a more loosely organized active site and flexible binding modes of the nucleotide. We used a subgenomic replicon system to assess the effects of the same mutations on viral replication in cells. Even the subtlest mutations either severely impaired or completely abolished the ability of the replicon to replicate, further supporting the concept that the correct positioning of both the β-loop and C-terminal arm plays an essential role during initiation and in HCV replication in general. HCV RNA polymerase is a key target for the development of directly acting agents to cure HCV infections, which necessitates a thorough understanding of the functional roles of the various structural features of the RdRp. Here we show that even highly conservative changes, e.g., Tyr→Phe or Asp→Glu, in these seemingly peripheral structural features have profound effects on the initiation and elongation properties of the HCV polymerase.

Construction of an infectious molecular clone of Japanese encephalitis virus genotype V and its derivative subgenomic replicon capable of expressing a foreign gene

Japanese encephalitis virus (JEV) genotype V was originally isolated in Malaysia in 1952 and has long been restricted to the area. In 2009, sudden emergence of the genotype V in China and Korea was reported, suggesting expansion of its geographical distribution. Although studies on the genotype V are becoming more important, they have been limited partly due to lack of its infectious molecular clone. In this study, a plasmid carrying cDNA corresponding to the entire genome of JEV Muar strain, which belongs to genotype V, in the downstream of T7 promoter was constructed. Electroporation of viral RNA transcribed by T7 RNA polymerase (T7RNAP) in vitro from the plasmid led to production of progeny viruses both in mammalian and mosquito cells. Also, transfection of the infectious clone plasmid into mammalian cells expressing T7RNAP transiently or stably was demonstrated to generate infectious progenies. When the viral structural protein genes were partially deleted from the full-length cDNA, the subgenomic RNA transcribed in vitro from the modified plasmid was shown to replicate itself in mammalian cells as a replicon. The replicon carrying the firefly luciferase gene in place of the deleted structural protein genes was also shown to efficiently replicate itself and express luciferase in mammalian cells. Compared with the replicon derived from JEV genotype III (Nakayama strain), the genotype V-derived replicon appeared to be more tolerant to introduction of a foreign gene. The infectious clone and the replicons constructed in this study may serve as useful tools for characterizing JEV genotype V.