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International audience; AT-752 is a guanosine analogue prodrug active against dengue virus (DENV). In infected cells, it is metabolized into 2 '-methyl-2 '-fluoro guanosine 5 '-triphosphate (AT-9010) which inhibits RNA synthesis in acting as a RNA chain terminator. Here we show that AT-9010 has several modes of action on DENV full-length NS5. AT-9010 does not inhibit the primer pppApG synthesis step significantly. However, AT-9010 targets two NS5-associated enzyme activities, the RNA 2 '-O-MTase and the RNA-dependent RNA polymerase (RdRp) at its RNA elongation step. Crystal structure and RNA methyltransferase (MTase) activities of the DENV 2 MTase domain in complex with AT-9010 at 1.97 angstrom resolution shows the latter bound to the GTP/RNA-cap binding site, accounting for the observed inhibition of 2 '-O but not N7-methylation activity. AT-9010 is discriminated-10 to 14-fold against GTP at the NS5 active site of all four DENV1-4 NS5 RdRps, arguing for significant inhibi-tion through viral RNA synthesis termination. In Huh-7 cells, DENV1-4 are equally sensitive to AT-281, the free base of AT-752 (EC50 approximate to 0.50 mu M), suggesting broad spectrum antiviral properties of AT-752 against flaviviruses.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global public health crisis. The reduced efficacy of therapeutic monoclonal antibodies against emerging SARS-CoV-2 variants of concern (VOCs), such as omicron BA.5 subvariants, has underlined the need to explore a novel spectrum of antivirals that are effective against existing and evolving SARS-CoV-2 VOCs. To address the need for novel therapeutic options, we applied cell-based high-content screening to a library of natural products (NPs) obtained from plants, fungi, bacteria, and marine sponges, which represent a considerable diversity of chemical scaffolds. The antiviral effect of 373 NPs was evaluated using the mNeonGreen (mNG) reporter SARS-CoV-2 virus in a lung epithelial cell line (Calu-3). The screening identified 26 NPs with half-maximal effective concentrations (EC

Infections with mosquito-borne flaviviruses, such as Dengue virus, ZIKV virus, and West Nile virus, pose significant threats to public health. Flaviviruses cause up to 400 million infections each year, leading to many forms of diseases, including fatal hemorrhagic, encephalitis, congenital abnormalities, and deaths. Currently, there are no clinically approved antiviral drugs for the treatment of flavivirus infections. The non-structural protein NS4B is an emerging target for drug discovery due to its multiple roles in the flaviviral life cycle. In this review, we summarize the latest knowledge on the structure and function of flavivirus NS4B, as well as the progress on antiviral compounds that target NS4B.

SARS-CoV-2 has raised the alarm to search for effective therapy for this virus. To date several vaccines have been approved but few available drugs reported recently still need approval from FDA. Remdesivir was approved for emergency use only. In this report, the SARS-CoV-2 3CLpro was expressed and purified. By using a FRET-based enzymatic assay, we have screened a library consisting of more than 300 different niclosamide derivatives and identified three molecules JMX0286, JMX0301, and JMX0941 as potent allosteric inhibitors against SARS-CoV-2 3CLpro, with IC

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, has infected over 260 million people over the past 2 years. Remdesivir (RDV, VEKLURY®) is currently the only antiviral therapy fully approved by the FDA for the treatment of COVID-19. The parent nucleoside of RDV, GS-441524, exhibits antiviral activity against numerous respiratory viruses including SARS-CoV-2, although at reduced in vitro potency compared to RDV in most assays. Here we find in both human alveolar and bronchial primary cells, GS-441524 is metabolized to the pharmacologically active GS-441524 triphosphate (TP) less efficiently than RDV, which correlates with a lower in vitro SARS-CoV-2 antiviral activity. In vivo, African green monkeys (AGM) orally dosed with GS-441524 yielded low plasma levels due to limited oral bioavailability of10%. When GS-441524 was delivered via intravenous (IV) administration, although plasma concentrations of GS-441524 were significantly higher, lung TP levels were lower than observed from IV RDV. To determine the required systemic exposure of GS-441524 associated with in vivo antiviral efficacy, SARS-CoV-2 infected AGMs were treated with a once-daily IV dose of either 7.5 or 20 mg/kg GS-441524 or IV RDV for 5 days and compared to vehicle control. Despite the reduced lung TP formation compared to IV dosing of RDV, daily treatment with IV GS-441524 resulted in dose-dependent efficacy, with the 20 mg/kg GS-441524 treatment resulting in significant reductions of SARS-CoV-2 replication in the lower respiratory tract of infected animals. These findings demonstrate the in vivo SARS-CoV-2 antiviral efficacy of GS-441524 and support evaluation of its orally bioavailable prodrugs as potential therapies for COVID-19.

International audience; In response to the outbreak of Zika virus (ZIKV) infection in the Western Hemisphere and the recognition of a causal association with fetal malformations, the Global Virus Network (GVN) assembled an international taskforce of virologists to promote basic research, recommend public health measures and encourage the rapid development of vaccines, antiviral therapies and new diagnostic tests. In this article, taskforce members and other experts review what has been learned about ZIKV-induced disease in humans, its modes of transmission and the cause and nature of associated congenital manifestations. After describing the make-up of the taskforce, we summarize the emergence of ZIKV in the Americas, Africa and Asia, its spread by mosquitoes, and current control measures. We then review the spectrum of primary ZIKV-induced disease in adults and children, sites of persistent infection and sexual transmission, then examine what has been learned about maternal-fetal transmission and the congenital Zika syndrome, including knowledge obtained from studies in laboratory animals. Subsequent sections focus on vaccine development, antiviral therapeutics and new diagnostic tests. After reviewing current understanding of the mechanisms of emergence of Zika virus, we consider the likely future of the pandemic.

Nucleoside analogs represent the largest class of antiviral agents and have been actively pursued for potential therapy of dengue virus (DENV) infection. Early success in the treatment of human immunodeficiency virus (HIV) infection and the recent approval of sofosbuvir for chronic hepatitis C have provided proof of concept for this class of compounds in clinics. Here we review (i) nucleoside analogs with known anti-DENV activity; (ii) challenges of the nucleoside antiviral approach for dengue; and (iii) potential strategies to overcome these challenges. This article forms part of a symposium in Antiviral Research on flavivirus drug discovery.

Zika virus (ZIKV) has drawn global attention as the etiologic agent of Zika Congenital Syndrome in babies born to infected pregnant women. To prevent future ZIKV outbreaks and protect persons at risk for severe disease, we developed two live-attenuated vaccine (LAV) candidates containing 10- or 20-nucleotide deletions in the 3′UTR of the viral genome (Δ10 and Δ20). After a single-dose immunization, both Δ10 and Δ20 LAVs protected mice and non-human primates against ZIKV infection. Here, we characterized the stability, safety, and efficacy of the LAVs after continuously culturing them on manufacture Vero cells for ten rounds. Whole genome sequencing showed that passage 10 (P10) LAVs retained the engineered Δ10 and Δ20 deletions; one to four additional mutations emerged at different regions of the genome. In A129 mice, the P10 LAVs exhibited viremia higher than the un-passaged LAVs, but lower than wild-type ZIKV; unlike wild-type ZIKV-infected mice, none of the P10 LAV-infected mice developed disease or death, demonstrating that the P10 LAVs remained attenuated. Mice immunized with a single dose of the P10 LAVs developed robust neutralizing antibody titers (1/1,000 to 1/10,000) and were protected against epidemic ZIKV challenge. The P10 LAVs did not exhibit increased neurovirulence. Intracranial inoculation of one-day-old CD1 pups with 103 focus-forming units of the P10 Δ10 and Δ20 LAVs resulted in 100% and ≥80% survival, respectively. Furthermore, the P10 LAVs remained incompetent in infecting Aedes aegypti mosquitoes after intrathoracic microinjection. Our results support the phenotypic stability and further development of these promising LAVs for ZIKV.

The non-structural protein 5 (NS5) of flaviviruses is the most conserved amongst the viral proteins. It is about 900 kDa and bears enzymatic activities that play vital roles in virus replication. Its N-terminal domain encodes dual N7 and 2'-O methyltransferase activities (MTase), and possibly guanylyltransferase (GTase) involved in RNA cap formation. The C-terminal region comprises a RNA-dependent RNA polymerase (RdRp) required for viral RNA synthesis. Both MTase and RdRp activities of dengue virus NS5 are well characterized, structurally and functionally. Numerous crystal structures of the flavivirus MTase and RdRp domains have been solved. Inhibitors of both functions have been identified through screening activities using biochemical and cell-based assays, as well as via rational design approaches. This review summaries the current knowledge as well as prospective views on these aspects. This article forms part of a symposium on flavivirus drug discovery in Antiviral Research.

The flavivirus nonstructural 4B protein (NS4B) has recently emerged as a valid antiviral target for drug discovery. Here we review (i) the current understanding of the structure and function of DENV NS4B, (ii) the approaches that have been taken to identify NS4B inhibitors, and (iii) the known inhibitors of flavivirus NS4B protein. This article forms part of a symposium in Antiviral Research on flavivirus drug discovery.

Highlights • A method to produce the dengue methyltransferase without a bound SAM is presented. • The enzyme retains enzymatic activity. • The high-resolution crystal structure of the free MTase shows there is no conformational change on SAM binding. • This allows rational drug discovery of compounds that compete with SAM binding. • This is demonstrated by solving the crystal structure of DENV MTase bound to Sinefungin., Flavivirus methyltransferase is a genetically-validated antiviral target. Crystal structures of almost all available flavivirus methyltransferases contain S-adenosyl-L-methionine (SAM), the methyl donor molecule that co-purifies with the enzymes. This raises a possibility that SAM is an integral structural component required for the folding of dengue virus (DENV) methyltransferase. Here we exclude this possibility by solving the crystal structure of DENV methyltransferase without SAM. The SAM ligand was removed from the enzyme through a urea-mediated denaturation-and-renaturation protocol. The crystal structure of the SAM-depleted enzyme exhibits a vacant SAM-binding pocket, with a conformation identical to that of the SAM-enzyme co-crystal structure. Functionally, equivalent enzymatic activities (N-7 methylation, 2′-O methylation, and GMP-enzyme complex formation) were detected for the SAM-depleted and SAM-containing recombinant proteins. These results clearly indicate that the SAM molecule is not an essential component for the correct folding of DENV methyltransferase. Furthermore, the results imply a potential antiviral approach to search for inhibitors that can bind to the SAM-binding pocket and compete against SAM binding. To demonstrate this potential, we have soaked crystals of DENV methyltransferase without a bound SAM with the natural product Sinefungin and show that preformed crystals are capable of binding ligands in this pocket.

West Nile virus (WNV), a mosquito-borne flavivirus, is an important neurotropic human pathogen. As a biosafety level-3 (BSL-3) agent, WNV is strictly to BSL-3 laboratories for experimentations, thus greatly hindering the development of vaccine and antiviral drug. Here, we developed a novel pseudo-infectious WNV reporter virus expressing the Gaussia luciferase (Gluc). A stable 293TNS1 cell line expressing NS1 was selected for trans-supplying NS1 protein to support the replication of WNV-ΔNS1 virus and WNV-ΔNS1-Gluc reporter virus with large-fragment deletion of NS1. WNV-ΔNS1 virus and WNV-Gluc-ΔNS1 reporter virus were confined to complete their replication cycle in this 293TNS1 cell line, displaying nearly identical growth kinetics to WT WNV although the viral titers were lower than those of WT WNV. The reporter gene was stably maintained in virus genome at least within three rounds of passage in 293TNS1 cell line. Using a known flaviviruses inhibitor, NITD008, we demonstrated that the pseudo-infectious WNV-Gluc-ΔNS1 could be used for antiviral screening. Furthermore, a high-throughput screening (HTS) assay in a 96-well format was optimized and validated using several known WNV inhibitors, indicating that the optimized HTS assay was suitable for high-throughput screening WNV inhibitors. Our work provides a stable and safe tool to handle WNV outside of a BSL-3 facility and facilitates high throughput screening for anti-WNV drugs.

To combat neglected diseases, the Novartis Institute of Tropical Diseases (NITD) was founded in 2002 through private-public funding from Novartis and the Singapore Economic Development Board. One of NITD's missions is to develop antivirals for dengue virus (DENV), the most prevalent mosquito-borne viral pathogen. Neither vaccine nor antiviral is currently available for DENV. Here we review the progress in dengue drug discovery made at NITD as well as the major discoveries made by academia and other companies. Four strategies have been pursued to identify inhibitors of DENV through targeting both viral and host proteins: (i) HTS (high-throughput screening) using virus replication assays; (ii) HTS using viral enzyme assays; (iii) structure-based in silico docking and rational design; (iv) repurposing hepatitis C virus inhibitors for DENV. Along the developmental process from hit finding to clinical candidate, many inhibitors did not advance beyond the stage of hit-to-lead optimization, due to their poor selectivity, physiochemical or pharmacokinetic properties. Only a few compounds showed efficacy in the AG129 DENV mouse model. Two nucleoside analogs, NITD-008 and Balapiravir, entered preclinical animal safety study and clinic trial, but both were terminated due to toxicity and lack of potency, respectively. Celgosivir, a host alpha-glucosidase inhibitor, is currently under clinical trial; its clinical efficacy remains to be determined. The knowledge accumulated during the past decade has provided a better rationale for ongoing dengue drug discovery. Though challenging, we are optimistic that this continuous, concerted effort will lead to an effective dengue therapy.

Development of anti-dengue therapy represents an urgent un-met medical need. Towards antiviral therapy, recent advances in crystal structures of DENV enzymes have led to the possibility of structure-based rational design of inhibitors for anti-dengue therapy. These include (i) the structure of the 'active' form of the DENV protease in complex with a peptide substrate; (ii) the structure of DENV methyltransferase bound to an inhibitor that selectively suppresses viral methyltransferase, but not human methyltransferases; (iii) the structure of DENV RNA-dependent RNA polymerase in complex with a small-molecule compound. This review summarizes the structural biology of these three key enzymes (protease, methyltransferase, and polymerase) that are essential for DENV replication. The new structural information has provided new avenues for development of anti-dengue therapy.

Zika virus (ZIKV), a previously obscure flavivirus closely related to dengue, West Nile, Japanese encephalitis and yellow fever viruses, has emerged explosively since 2007 to cause a series of epidemics in Micronesia, the South Pacific, and most recently the Americas. After its putative evolution in sub-Saharan Africa, ZIKV spread in the distant past to Asia and has probably emerged on multiple occasions into urban transmission cycles involving Aedes (Stegomyia) spp. mosquitoes and human amplification hosts, accompanied by a relatively mild dengue-like illness. The unprecedented numbers of people infected during recent outbreaks in the South Pacific and the Americas may have resulted in enough ZIKV infections to notice relatively rare congenital microcephaly and Guillain-Barre syndromes. Another hypothesis is that phenotypic changes in Asian lineage ZIKV strains led to these disease outcomes. Here, we review potential strategies to control the ongoing outbreak through vector-centric approaches as well as the prospects for the development of vaccines and therapeutics.

The four serotypes of dengue virus (DENV) are the causative agents of the most prevalent mosquito-borne viral disease in human. No clinically approved antiviral therapy is currently available. Therapeutic antibodies represent a viable approach for potential treatment of DENV infection. We recently isolated a human monoclonal antibody (HM14c10) that selectively neutralizes DENV serotype 1 (DENV-1), but not serotypes 2, 3, and 4. Here we report the resistance profile of DENV-1 against HM14c10 in cell culture. Escape mutant viruses readily emerged by culturing wild-type DENV-1 in the presence of the HM14c10 antibody. Sequencing of resistant viruses revealed a single T51K substitution in the domain I/II hinge region of the viral envelope protein. Residue T51 is located within the HM14c10 epitope and is highly conserved among various DENV-1 isolates. Recombinant DENV-1 containing the T51K mutation could not be neutralized by HM14c10 in vitro or in vivo. Biochemical assay revealed that the T51K mutation completely abolished the antibody binding to the DENV-1 virion. Collectively, the results demonstrate that a single amino acid change in DENV envelope protein can confer resistance to a potent antibody through abolishing the antibody-virus interaction.

To facilitate dengue virus (DENV) drug discovery, we developed a stable luciferase reporter DENV-2. A renilla luciferase gene was engineered into the capsid-coding region of an infectious cDNA clone of DENV-2. Transfection of BHK-21 cells with the cDNA clone-derived RNA generated high titers (>10(6)PFU/ml) of luciferase reporter DENV-2. The reporter virus was infectious to a variety of cells, producing robust luciferase signals. Compared with wild-type virus, the reporter virus replicated slower in both mammalian Vero and mosquito C6/36 cells. To examine the stability of the reporter virus, we continuously passaged the virus on Vero cells for five rounds. All passaged viruses stably maintained the luciferase gene, demonstrating the stability of the reporter virus. Furthermore, we found that the passaged virus accumulated a mutation (T108M) in viral NS4B gene that could enhance viral RNA replication in a cell-type specific manner. Using the reporter virus, we developed a HTS assay in a 384-well format. The HTS assay was validated with known DENV inhibitors and showed a robust Z' factor of 0.79. The Luc-DENV-2 HTS assay allows screening for inhibitors of all steps of the viral life cycle. The reporter virus will also be a useful tool for studying DENV replication and pathogenesis.

Dengue virus (DENV) is a mosquito-borne flavivirus responsible for 50-100 million human infections each year. The development of DENV chemotherapy requires high-throughput screening (HTS) assays. A dengue virus-like particle (VLP) has been constructed using viral structural proteins to package a Renilla luciferase reporter replicon. VLP could be produced by either the sequential electroporation of the replicon RNAs and the structural gene RNAs or by electroporating replicon RNA into a stable cell line expressing the structural proteins. In both approaches, the key to produce high titer VLP (3x10(6)foci-forming unit/ml) is to use low temperature (30 degrees C) in the packaging step. In addition, exogenous expression of host protease furin increased VLP infectivity. The infection could be blocked by antibodies against viral envelope protein and by an inhibitor of viral NS5 polymerase, but not by an inhibitor of host alpha-glucosidase (castanospermine). The VLP infection assay was optimized for HTS in a 384-well format with consistent and robust signal, providing a simple and rapid cell-based assay for screening inhibitors against DENV entry, translation, and replication in an HTS format.

The dengue virus envelope protein plays an essential role in viral entry by mediating fusion between the viral and host membranes. The crystal structure of the envelope protein shows a pocket (located at a "hinge" between Domains I and II) that can be occupied by ligand n-octyl-beta-D-glucoside (beta OG). Compounds blocking the beta OG pocket are thought to interfere with conformational changes in the envelope protein that are essential for fusion. Two fusion assays were developed to examine the anti-fusion activities of compounds. The first assay measures the cellular internalization of propidium iodide upon membrane fusion. The second assay measures the protease activity of trypsin upon fusion between dengue virions and trypsin-containing liposomes. We performed an in silico virtual screening for small molecules that can potentially bind to the beta OG pocket and tested these candidate molecules in the two fusion assays. We identified one compound that inhibits dengue fusion in both assays with an IC50 Of 6.8 mu M and reduces viral titers with an EC50 of 9.8 mu M. Time-of-addition experiments showed that the compound was only active when present during viral infection but not when added 1 h later, in agreement with a mechanism of action through fusion inhibition. (C) 2009 Elsevier B.V. All rights reserved.

Although flaviviruses cause significant human diseases, no antiviral therapy is currently available for clinical treatment of these pathogens. To identify flavivirus inhibitors, we performed a high-throughput screening of compound libraries using cells containing luciferase-reporting replicon of West Nile viruses (WNV). Five novel small molecular inhibitors of WNV were identified from libraries containing 96,958 compounds. The inhibitors suppress epidemic strain of WNV in cell culture, with EC(50) (50% effective concentration) values of10microM and TI (therapeutic index) values of10. Viral titer reduction assays, using various flaviviruses and nonflaviviruses, showed that the compounds have distinct antiviral spectra. Mode-of-action analysis showed that the inhibitors block distinct steps of WNV replication: four compounds inhibit viral RNA syntheses, while the other compound suppresses both viral translation and RNA syntheses. Biochemical enzyme assays showed that two compounds selectively inhibit viral RNA-dependent RNA polymerase (RdRp), while another compound specifically inhibits both RdRp and methyltransferase. The identified compounds could potentially be developed for treatment of flavivirus infections.

Dengue virus (DENV) NS5 protein comprises an N-terminal methyltransferase domain and a C-terminal RNA-dependent RNA polymerase domain (RdRp). DENV RdRp is responsible for viral RNA synthesis via a de novo initiation mechanism and represents an attractive target for anti-viral therapy. Herein we describe the characterization of its de novo initiation activities by PAGE analyses and the knowledge gained was used to develop a fluorescent-based assay. A highly processive and robust assay was achieved by addition of cysteine in the assay buffer. This stabilized the apo-enzyme, and rendered optimal de novo initiation activity while balancing its intrinsic terminal transferase activity. Steady-state kinetic parameters of the NTP and RNA substrates under these optimal conditions were determined for DENV1–4 FL NS5. Heavy metal ions such as Zn++ and Co++ as well as high levels of monovalent salts, suppressed DENV polymerase de novo initiation activities. This assay was validated with nucleotide chain terminators and used to screen two diverse small library sets. The screen data obtained was further compared with concurrent screens performed with a DENV polymerase elongation fluorescent assay utilizing pre-complexed enzyme-RNA. A higher hit-rate was obtained for the de novo initiation assay compared to the elongation assay (∼2% versus ∼0.1%). All the hits from the latter assay are also identified in the de novo initiation assay, indicating that the de novo initiation assay performed with the stabilized apo-enzyme has the advantage of providing additional chemical starting entities for inhibiting this enzyme.

International audience; The family Flaviviridae comprises several major human pathogens including hepatitis C virus (genus hepacivirus), yellow fever virus, West Nile virus and dengue virus (genus flavivirus). Flaviviridae genomes comprise a single-stranded RNA segment encoding a single polyprotein that is subsequently processed into 10 mature viral proteins. The nonstructural proteins are released from the C-terminus of the polyprotein and contribute to the infectious cycle by forming membrane-bound, multi-protein compartments within host cells, named the replication complexes, where synthesis of new viral genomes takes place. Two nonstructural proteins are endowed with multiple enzymatic activities and represent important targets against which specific antiviral inhibitors have been developed. X-ray crystal structures of these viral enzymes as well as in-depth understanding of the molecular basis of their activities have contributed tremendously to the development of antiviral compounds, currently approved or in advanced clinical trials for hepatitis C treatment. One of the prime targets is the RNA-dependent RNA polymerase (RdRp, NS5B for hepatitis C virus, NS5 for flaviviruses). Here we review current knowledge of the structural basis for viral RNA synthesis by NS5B and NS5. These data offer perspectives for further drug design and constitute major advances in our basic understanding of viral RdRp. They thus point to future research directions in the field.

Enterovirus 71 (EV71) is one of the major causative agents for hand, foot, and mouth disease. There is currently no clinically approved vaccine or antiviral treatment for EV71 infection. To facilitate antiviral drug discovery, we developed an infectious cDNA clone of an epidemic strain of EV71 and a stable eGFP reporter EV71. The reporter virus was generated by engineering the eGFP gene between the 5' untranslated region and VP4 gene of the EV71 genome. Vero cells transfected with the cDNA clone-derived RNA generated high titers (>10(6)PFU/ml) of the eGFP reporter virus. The reporter virus was infectious to Vero cells, producing robust eGFP fluorescence signals. Compared with the wild type virus, the reporter virus replicated slower in cell culture. To examine the stability of the reporter virus, we continuously passaged the virus on Vero cells for five rounds. The passaged viruses maintained the eGFP gene, demonstrating the stability of the reporter virus. Using a known EV71 inhibitor, we demonstrate that the reporter virus could be used for antiviral screening. The infectious cDNA clones of the wild type virus and the eGFP reporter viruses will be useful for antiviral research as well as for studying viral replication and pathogenesis of EV71.

The level of cholesterol in host cells has been shown to affect viral infection. However, it is still not understood why this level of regulation is important for successful infection. We have shown in this study that dengue virus infection was affected when the cholesterol intake in infected cells was disrupted using a cholesterol transport inhibitor, U18666A. The antiviral effect was found to result from two events: retarded viral trafficking in the cholesterol-loaded late endosomes/lysosomes and suppressed de novo sterol biosynthesis in treated infected cells. We also observed an additive antiviral effect of U18666A with C75, a fatty acid synthase inhibitor, suggesting dengue virus relies on both the host cholesterol and fatty acid biosynthesis for successful replication.

Cellular α-glucosidases I and II are enzymes that sequentially trim the three terminal glucoses in the N-linked oligosaccharides of viral envelope glycoproteins. This process is essential for the proper folding of viral glycoproteins and subsequent assembly of many enveloped viruses, including dengue virus (DENV). Imino sugars are substrate mimics of α-glucosidases I and II. In this report, we show that two oxygenated alkyl imino sugar derivatives, CM-9-78 and CM-10-18, are potent inhibitors of both α-glucosidases I and II in vitro and in treated animals, and efficiently inhibit DENV infection of cultured human cells. Pharmacokinetic studies reveal that both compounds are well tolerated at doses up to 100mg/kg in rats and have favorable pharmacokinetic properties and bioavailability in mice. Moreover, we showed that oral administration of either CM-9-78 or CM-10-18 reduces the peak viremia of DENV in mice. Interestingly, while treatment of DENV infected mice with ribavirin alone did not reduce the viremia, combination therapy of ribavirin with sub-effective dose of CM-10-18 demonstrated a significantly enhanced antiviral activity, as indicated by a profound reduction of the viremia. Our findings thus suggest that combination therapy of two broad-spectrum antiviral agents may provide a practically useful approach for the treatment of DENV infection.

Many flaviviruses are significant human pathogens. No effective antiviral therapy is currently available for treatment of flavivirus infections. Development of antiviral treatment against these viruses is urgently needed. The flavivirus methyltransferase (MTase) responsible for N-7 and 2′-O methylation of the viral RNA cap has recently been mapped to the N-terminal region of nonstructural protein 5. Structural and functional studies suggest that the MTase represents a novel antiviral target. Here we review current understanding of flavivirus RNA cap methylation and its implications for development of antivirals. The 5′ end of the flavivirus plus-strand RNA genome contains a type 1 cap structure (m 7 GpppAmG). Flaviviruses encode a single MTase domain that catalyzes two sequential methylations of the viral RNA cap, GpppA-RNA → m 7 GpppA-RNA → m 7 GpppAm-RNA, using S -adenosyl- l -methionine (SAM) as the methyl donor. The two reactions require different viral RNA elements and distinct biochemical assay conditions. Despite exhibiting two distinct methylation activities, flavivirus MTase contains a single binding site for SAM in its crystal structure. Therefore, substrate GpppA-RNA must be re-positioned to accept the N-7 and 2′-O methyl groups from SAM during the two methylation reactions. Structure-guided mutagenesis studies indeed revealed two distinct sets of amino acids on the enzyme surface that are specifically required for N-7 and 2′-O methylation. In the context of virus, West Nile viruses (WNVs) defective in N-7 methylation are non-replicative; however, WNVs defective in 2′-O methylation are attenuated and can protect mice from subsequent wild-type WNV challenge. Collectively, the results demonstrate that the N-7 MTase represents a novel target for flavivirus therapy.