Characterization of Dengue Virus NS4A and NS4B Protein Interaction

Self Cite

The four serotypes of dengue virus (DENV-1 to -4) represent the most prevalent mosquito-borne viral pathogens in humans. No clinically approved vaccine or antiviral is currently available for DENV. Here we report a spiropyrazolopyridone compound that potently inhibits DENV both in vitro and in vivo. The inhibitor was identified through screening of a 1.8-million-compound library by using a DENV-2 replicon assay. The compound selectively inhibits DENV-2 and -3 (50% effective concentration [EC 50 ], 10 to 80 nM) but not DENV-1 and -4 (EC 50 , >20 M). Resistance analysis showed that a mutation at amino acid 63 of DENV-2 NS4B (a nonenzymatic transmembrane protein and a component of the viral replication complex) could confer resistance to compound inhibition. Genetic studies demonstrate that variations at amino acid 63 of viral NS4B are responsible for the selective inhibition of DENV-2 and -3. Medicinal chemistry improved the physicochemical properties of the initial "hit" (compound 1), leading to compound 14a, which has good in vivo pharmacokinetics. Treatment of DENV-2-infected AG129 mice with compound 14a suppressed viremia, even when the treatment started after viral infection. The results have proven the concept that inhibitors of NS4B could potentially be developed for clinical treatment of DENV infection. Compound 14a represents a potential preclinical candidate for treatment of DENV-2-and -3-infected patients. IMPORTANCE Dengue virus (DENV) threatens up to 2.5 billion people and is now spreading in many regions Emerging infectious pathogens represent a major threat to public health. Vaccines and therapeutics are two key countermeasures against these pathogens. Many viruses of the genus Flavivirus within the family Flaviviridae are arthropod-borne human pathogens, among which the four serotypes of dengue virus (DENV) alone cause 390 million human infections each year (1). Several promising DENV vaccines are currently in clinical development (2). The most advanced vaccine (CYD-TDV) exhibited good efficacy against DENV-1, -3, and -4 but weak protection against DENV-2 (3-5). For antiviral development, four compounds have been tested in dengue clinical trials, including balapiravir (a nucleoside inhibitor) (6), celgosivir (a cellular ␣-glucosidase inhibitor) (7), chloroquine (a malaria drug with antiviral and immunomodulatory activities) (8), and prednisolone (a corticosteroid drug) (9). None of them showed any antiviral activity or clinical benefits in dengue patients. Notably, all these compounds were repurposed from existing drugs or compounds previously developed for other viruses. Bona fide inhibitors specifically designed for DENV have never advanced to clinical trials (10).In this paper, we report the identification of a novel class of small-molecule anti-DENV agents, the spiropyrazolopyridones, using phenotypic screening. These inhibitors block DENV replication by targeting nonstructural protein 4B (NS4B), a nonenzymatic transmembrane protein functioning as an essential component of the viral r...

Section: Discussionmentioning

confidence: 67%

Discovery of Dengue Virus NS4B Inhibitors

Wang

Dong

Zou

et al. 2015

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“…Zou et al showed that DENV NS4B forms a dimer or multimer but is not N glycosylated in virusinfected cells (15). Moreover, the same report showed a single species (15) and a later report of the same research group showed two species from recombinant NS4B protein expression which were assumed to be 2K-4B unprocessed and processed forms (8).…”

Section: Discussionmentioning

confidence: 87%

“…Previous studies reported that mutations in DENV NS4B adversely affect viral genome replication, strongly supporting the fact that NS4B is a component of the replication complex (4)(5)(6). Additionally, NS4B was found to interact with other NS proteins, such as NS3 and NS4A in DENV (7)(8)(9). Re-cently, NS4B was shown to have genetic and physical interactions with NS4A and NS1 in the related flaviviruses Japanese encephalitis virus (JEV) and West Nile virus (WNV), and these interactions were essential for viral genome replication (10,11).…”

mentioning

confidence: 68%

Mutation of Putative N-Glycosylation Sites on Dengue Virus NS4B Decreases RNA Replication

Naik

2015

Dengue virus (DENV) nonstructural protein 4B (NS4B) is an endoplasmic reticulum (ER) membrane-associated protein, and mutagenesis studies have revealed its significance in viral genome replication. In this work, we demonstrated that NS4B is an N-glycosylated protein in virus-infected cells as well as in recombinant protein expression. NS4B is N glycosylated at residues 58 and 62 and exists in two forms, glycosylated and unglycosylated. We manipulated full-length infectious RNA clones and subgenomic replicons to generate N58Q, N62Q, and N58QN62Q mutants. Each of the single mutants had distinct effects, but the N58QN62Q mutation resulted in dramatic reduction of viral production efficiency without affecting secretion or infectivity of the virion in mammalian and mosquito C6/36 hosts. Real-time quantitative PCR (qPCR), subgenomic replicon, and trans-complementation assays indicated that the N58QN62Q mutation affected RNA replication possibly by the loss of glycans. In addition, four intragenic mutations (S59Y, S59F, T66A, and A137T) were obtained from mammalian and/or mosquito C6/36 cell culture systems. All of these second-site mutations compensated for the replication defect of the N58QN62Q mutant without creating novel glycosylation sites. In vivo protein stability analyses revealed that the N58QN62Q mutation alone or plus a compensatory mutation did not affect the stability of NS4B. Overall, our findings indicated that mutation of putative N-glycosylation sites affected the biological function of NS4B in the viral replication complex. IMPORTANCE This is the first report to identify and reveal the biological significance of dengue virus (DENV) nonstructural protein 4B (NS4B) posttranslation N-glycosylation to the virus life cycle. The study demonstrated that NS4B is N glycosylated in virus-infected cells and in recombinant protein expression. NS4B is modified by glycans atAsn-58 and Asn-62. Functional characterization implied that DENV NS4B utilizes the glycosylation machinery in both mammalian and mosquito hosts. Four intragenic mutations were found to compensate for replication and subsequent viral production deficiencies without creating novel N-glycosylation sites or modulating the stabilities of the protein, suggesting that glycans may be involved in maintaining the NS4B protein conformation. NS4B glycans may be necessary elements of the viral life cycle, but compensatory mutations can circumvent their requirement. This novel finding may have broader implications in flaviviral biology as the most likely glycan at Asn-62 of NS4B is conserved in DENV serotypes and in some related flaviviruses. D engue virus (DENV) belongs to the Flaviviridae family of theFlavivirus genus, exists in four serotypes (DENV1 to -4), and is transmitted to humans by Aedes mosquitoes. Incidences of dengue have grown dramatically around the globe in recent decades and are endemic to tropical and subtropical countries. According to the latest report of the World Health Organization (WHO) (February 2015), approximately 50% of th...

“…Residue K330 from the thumb region of the NS5 polymerase was shown to interact with the NS3 helicase (35). The accompanying paper by Zou et al (37) characterizes the NS4A and NS4B protein interaction, and further studies in the presence of RNA are now needed to clarify the detailed network of intermolecular contacts established between NS2B, NS4A, NS4B, and NS5 at various stages of RNA replication.…”

Section: Discussionmentioning

confidence: 99%

Mapping the Interactions between the NS4B and NS3 Proteins of Dengue Virus

Zou

Lee

Wang

et al. 2015

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Flavivirus RNA synthesis is mediated by a multiprotein complex associated with the endoplasmic reticulum membrane, named the replication complex (RC). Within the flavivirus RC, NS4B, an integral membrane protein with a role in virulence and regulation of the innate immune response, binds to the NS3 protease-helicase. NS4B modulates the RNA helicase activity of NS3, but the molecular details of their interaction remain elusive. Here, we used dengue virus (DENV) to map the determinants for the NS3-NS4B interaction. Coimmunoprecipitation and an in situ proximity ligation assay confirmed that NS3 colocalizes with NS4B in both DENV-infected cells and cells coexpressing both proteins. Surface plasmon resonance demonstrated that subdomains 2 and 3 of the NS3 helicase region and the cytoplasmic loop of NS4B are required for binding. Using nuclear magnetic resonance (NMR), we found that the isolated cytoplasmic loop of NS4B is flexible, with a tendency to form a three-turn ␣-helix and two short ␤-strands. Upon binding to the NS3 helicase, 12 amino acids within the cytoplasmic loop of NS4B exhibited line broadening, suggesting a participation in the interaction. Sequence alignment showed that 4 of these 12 residues are strictly conserved across different flaviviruses. Mutagenesis analysis showed that three (Q134, G140, and N144) of the four evolutionarily conserved NS4B residues are essential for DENV replication. The mapping of the NS3/NS4B-interacting regions described here can assist the design of inhibitors that disrupt their interface for antiviral therapy. IMPORTANCENS3 and NS4B are essential components of the flavivirus RC. Using DENV as a model, we mapped the interaction between the viral NS3 and NS4B proteins. The subdomains 2 and 3 of NS3 helicase as well as the cytoplasmic loop of NS4B are critical for the interaction. Functional analysis delineated residues within the NS4B cytoplasmic loop that are crucial for DENV replication. Our findings reveal molecular details of how flavivirus NS3 protein cooperates with NS4B within the RC. In addition, this study has established the rationale and assays to search for inhibitors disrupting the NS3-NS4B interaction for antiviral drug discovery. Dengue is a major and emerging public health problem: 50 to 100 million dengue cases occur every year, and over 40% of the world population is at risk (1). According to a 2014 update by WHO, "an estimated 500,000 people with severe dengue are hospitalized each year including a large proportion of children," and of those, about 2.5% die (2). The disease is caused by infection with one of the four serotypes of dengue virus (DENV), transmitted by Aedes mosquitoes. There is currently no clinically approved antiviral therapeutics. The recent phase IIb clinical trials in Thailand revealed that the CYD tetravalent vaccine failed to protect against DENV serotype 2 (DENV2) although the vaccine showed efficacy against the other three serotypes of DENV (3). Before effective vaccine and antiviral treatments become available, dengue pat...