An adenosine nucleoside inhibitor of dengue virus

Yin, Zheng; Chen, Yen-Liang; Schul, Wouter; Wang, Qing Yin; Gu, Feng; Duraiswamy, Jeyaraj; Kondreddi, Ravinder Reddy; Niyomrattanakit, Pornwaratt; Lakshminarayana, Suresh B.; Goh, Anne; Xu, Hao; Liu, Wei; Liu, Boping; Lim, Joanne Y.H.; Ng, Choon Ta; Qing, Min; Lim, Chin Chin; Yip, Andy M.; Wang, Gang; Chan, Wai Ling; Tan, Hui Pen; Lin, Kai; Zhang, Bo; Zou, Gang; Bernard, Kristen A.; Garrett, Christine; Beltz, Karen; Dong, Min; Weaver, M. Libby; He, Handan; Pichota, Arkadius; Dartois, Véronique; Keller, Thomas H.; Shi, Pei Yong

doi:10.1073/pnas.0907010106

Cited by 324 publications

(297 citation statements)

References 18 publications

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“…MPA and NITD008 inhibited DENVFluc with an IC 50 of 0.09 μg/mL and 1.5 μM, respectively (Fig. 1E), consistent with previously published studies on nonreporter DENV (20,21). DENV-Fluc replication was potently inhibited by human type I IFN (IFN-α2), and modestly suppressed by human type III IFNs (IL28B and IL29; Fig.…”

Section: Resultssupporting

confidence: 77%

Dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro

Schoggins

Dorner

Feulner

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

166

171

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Dengue virus (DENV) is a global disease threat for which there are no approved antivirals or vaccines. Establishing state-of-the-art screening systems that rely on fluorescent or luminescent reporters may accelerate the development of anti-DENV therapeutics. However, relatively few reporter DENV platforms exist. Here, we show that DENV can be genetically engineered to express a green fluorescent protein or firefly luciferase. Reporter viruses are infectious in vitro and in vivo and are sensitive to antiviral compounds, neutralizing antibodies, and interferons. Bioluminescence imaging was used to follow the dynamics of DENV infection in mice and revealed that the virus localized predominantly to lymphoid and gut-associated tissues. The high-throughput potential of reporter DENV was demonstrated by screening a library of more than 350 IFN-stimulated genes for antiviral activity. Several antiviral effectors were identified, and they targeted DENV at two distinct life cycle steps. These viruses provide a powerful platform for applications ranging from validation of vaccine candidates to antiviral discovery. Flaviviridae family that causes significant morbidity and mortality worldwide. Each year, over 50 million people are affected by dengue fever, and ∼500,000 are hospitalized with the more severe dengue hemorrhagic fever (1). The virus is endemic to tropical environments in Southeast Asia, the Pacific, and the Americas, and has recently reemerged as far north as southern Florida (2). Currently, no vaccines or antivirals have been approved for prevention or treatment of DENV infection.Four genetically and antigenically distinct DENV serotypes circulate, and each can be isolated from infected human sera and propagated in cell culture. The pathogenesis and immune response to patient-derived virus can be studied in vitro and in vivo by quantifying viral genomes or antigens. These detection methods are also used to study the molecular virology of DENV with reagents such as virus-like particles (VLPs) and subgenomic replicons. In some cases, VLPs and subgenomic replicons have been engineered to take advantage of reporter proteins, such as luciferase, which can be used in high-throughput screening platforms for discovery of inhibitors of viral entry or replication (3). A caveat to these tools is the inability to fully recapitulate the entire viral life cycle. This can be overcome by launching virus production from full-length infectious molecular clones, which have been generated for DENV serotypes 1, 2, and 4 (4-6).Full-length flavivirus infectious clones are often difficult to work with, largely due to instability in various bacterial cell lines and the inability to rescue sufficient quantities of DNA for downstream applications (7). Additionally, mutagenesis of viral sequences may result in disruption of the various long-range interactions required for establishment of a productive replication complex (8-10). Thus, strategies to generate infectious viruses expressing heterologous sequences have to contend with both sub...

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Section: Resultssupporting

confidence: 77%

Dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro

Schoggins

Dorner

Feulner

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

166

171

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“…Both nucleoside analogues and non-nucleoside inhibitors of DENV RdRp have recently been reported (25,(32)(33)(34). The crystal structures of WNV and DENV RdRps revealed two conserved cavities that could be used for development of allosteric inhibitors (18).…”

Section: Discussionmentioning

confidence: 99%

Functional Analysis of Two Cavities in Flavivirus NS5 Polymerase

Zou

Chen

Dong

et al. 2011

Journal of Biological Chemistry

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118

127

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Flavivirus NS5 protein encodes methyltransferase and RNAdependent RNA polymerase (RdRp) activities. Structural analysis of flavivirus RdRp domains uncovered two conserved cavities (A and B). Both cavities are located in the thumb subdomains and represent potential targets for development of allosteric inhibitors. In this study, we used dengue virus as a model to analyze the function of the two RdRp cavities. Amino acids from both cavities were subjected to mutagenesis analysis in the context of genome-length RNA and recombinant NS5 protein; residues critical for viral replication were subjected to revertant analysis. For cavity A, we found that only one (Lys-756) of the seven selected amino acids is critical for viral replication. Alanine substitution of Lys-756 did not affect the RdRp activity, suggesting that this residue functions through a nonenzymatic mechanism. For cavity B, all four selected amino acids (Leu-328, Lys-330, Trp-859, and Ile-863) are critical for viral replication. Biochemical and revertant analyses showed that three of the four mutated residues (Leu-328, Trp-859, and Ile-863) function at the step of initiation of RNA synthesis, whereas the fourth residue (Lys-330) functions by interacting with the viral NS3 helicase domain. Collectively, our results have provided direct evidence for the hypothesis that cavity B, but not cavity A, from dengue virus NS5 polymerase could be a target for rational drug design.The genus Flavivirus from the family Flaviviridae contains more than 70 viruses, many of which are important human pathogens causing major public health threats worldwide. Dengue virus (DENV) 2 is a mosquito-borne flavivirus responsible for 50 -100 million human infections and ϳ20,000 deaths each year (1). Besides DENV, West Nile virus (WNV), Japanese encephalitis virus, yellow fever virus, and tick-borne encephalitis virus also cause significant human diseases (1). No antiviral therapy is currently available for treatment of flavivirus infections. Therefore, development of antiviral therapy is urgently needed for flaviviruses.The flavivirus genome is a single strand, plus-sense RNA of about 11 kb in length. The genomic RNA contains a 5Ј-untranslated region (UTR), a single open reading frame, and a 3Ј-UTR. The single open reading frame encodes a long polyprotein that is processed by viral and host proteases into 10 mature viral proteins (2). Three structural proteins (capsid, pre-membrane, and envelope) are primarily involved in virus particle formation, and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) are mainly responsible for viral replication (2). Among these, NS3 and NS5 possess enzymatic activities and have been targeted for antiviral development. NS3 functions as a protease (with NS2B as a cofactor), helicase, 5Ј-RNA triphosphatase, and nucleoside triphosphatase (3-5). NS5 is the largest and the most conserved viral protein. The N-terminal part of NS5 is a methyltransferase that methylates the N-7 and 2Ј-O positions of the viral RNA cap structure (6 -9);...

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“…Current literature highlights the development of effective nucleoside analogues tested in vitro that target RNA replication by inhibiting incorporation of nucleotides into the newly synthesized RNA strand (Cameron & Castro, 2001;Yin et al, 2009) or targeting the active site of the enzyme such as the S-adenosyl methionine (SAM) pocket of the MTase which is essential for methylation (Selisko et al, 2010). It has also been suggested that targeting interactions of nonstructural proteins in the replication complex is another approach to designing specific antivirals (Loregian et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

An interaction between the methyltransferase and RNA dependent RNA polymerase domains of the West Nile virus NS5 protein

Tan

Hobson‐Peters

Stoermer

et al. 2013

Journal of General Virology

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The flavivirus nonstructural protein 5 (NS5) is a large protein that is structurally conserved among members of the genus, making it an attractive target for antiviral drug development. The protein contains a methyltransferase (MTase) domain and an RNA dependent RNA polymerase (POL) domain. Previous studies with dengue viruses have identified a genetic interaction between residues 46-49 in the aA3-motif in the MTase and residue 512 in POL. These genetic interactions are consistent with structural modelling of these domains in West Nile virus (WNV) NS5 that predict close proximity of these regions of the two domains, and potentially a functional interaction mediated via the aA3-motif. To demonstrate an interaction between the MTase and POL domains of the WNV NS5 protein, we co-expressed affinity-tagged recombinant MTase and POL proteins in human embryonic kidney cells with simian virus 40 large T antigen (HEK293T cells) and performed pulldown assays using an antibody to the flag tag on POL. Western blot analysis with an anti-MTase mAb revealed that the MTase protein was specifically coimmunoprecipitated with POL, providing the first evidence of a specific interaction between these domains. To further assess the role of the aA3 helix in this interaction, selected residues in this motif were mutated in the recombinant MTase and the effect on POL interaction determined by the pulldown assay. These mutations were also introduced into a WNV infectious clone (FLSDX) and the replication properties of these mutant viruses assessed. While none of the aA3 mutations had a significant effect on the MTase-POL association in pulldown assays, suggesting that these residues were not specific to the interaction, an E46L mutation completely abolished virus viability indicating a critical requirement of this residue in replication. Failure to generate compensatory mutations in POL to rescue replication, even after several passages of the transfection supernatant in Vero cells, precluded further conclusion of the role of this residue in the context of MTase-POL interactions.

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An adenosine nucleoside inhibitor of dengue virus

Cited by 324 publications

References 18 publications

Dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro

Dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro

Functional Analysis of Two Cavities in Flavivirus NS5 Polymerase

An interaction between the methyltransferase and RNA dependent RNA polymerase domains of the West Nile virus NS5 protein

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