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

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

Self Cite

“…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%

“…We speculate that loss of glycans may affect the structure of NS4B, most probably in the N-terminal luminal domains, and the compensatory abilities of the aromatic amino acid (tyrosine or phenylalanine) within the Asn-58 glycosylation acceptor motif, the alanine at the amino acid (aa) 66 position, or the threonine at the aa 137 position play a significant role in correcting the structural defect of NS4B N58QN62Q. A recent study showed the presence of an interaction between the cytosolic loop of NS4B (aa 129 to 165) and NS3 protein (9). It is possible that NS4B N58QN62Q mutation alters the conformation of the cytosolic loop and disrupts NS4B-NS3 interaction, and different compensatory mutations might correct the conformation of the cytosolic loop, rebuilding the interaction with NS3.…”

Section: Discussionmentioning

confidence: 99%

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...

“…(iv) WNV NS4A regulates the ATPase activity of NS3 helicase (25), while DENV NS4B interacts with the helicase domain of NS3 and dissociates it from singlestrand RNA (26). As noted in the accompanying article by Zou et al (27), the DENV NS3-NS4B interaction was mapped to the NS3 helicase (subdomains 2 and 3) and the NS4B cytoplasmic region (amino acids 129 to 165). (v) NS4A and NS4B genetically interact with NS1 to modulate viral replication.…”

mentioning

confidence: 96%

Characterization of Dengue Virus NS4A and NS4B Protein Interaction

Zou

Xie

Wang

et al. 2015

Self Cite

127

107

Flavivirus replication is mediated by a membrane-associated replication complex where viral membrane proteins NS2A, NS2B, NS4A, and NS4B serve as the scaffold for the replication complex formation. Here, we used dengue virus serotype 2 (DENV-2) as a model to characterize viral NS4A-NS4B interaction. NS4A interacts with NS4B in virus-infected cells and in cells transiently expressing NS4A and NS4B in the absence of other viral proteins. Recombinant NS4A and NS4B proteins directly bind to each other with an estimated K d (dissociation constant) of 50 nM. Amino acids 40 to 76 (spanning the first transmembrane domain, consisting of amino acids 50 to 73) of NS4A and amino acids 84 to 146 (also spanning the first transmembrane domain, consisting of amino acids 101 to 129) of NS4B are the determinants for NS4A-NS4B interaction. Nuclear magnetic resonance (NMR) analysis suggests that NS4A residues 17 to 80 form two amphipathic helices (helix ␣1, comprised of residues 17 to 32, and helix ␣2, comprised of residues 40 to 47) that associate with the cytosolic side of endoplasmic reticulum (ER) membrane and helix ␣3 (residues 52 to 75) that transverses the ER membrane. In addition, NMR analysis identified NS4A residues that may participate in the NS4A-NS4B interaction. Amino acid substitution of these NS4A residues exhibited distinct effects on viral replication. Three of the four NS4A mutations (L48A, T54A, and L60A) that affected the NS4A-NS4B interaction abolished or severely reduced viral replication; in contrast, two NS4A mutations (F71A and G75A) that did not affect NS4A-NS4B interaction had marginal effects on viral replication, demonstrating the biological relevance of the NS4A-NS4B interaction to DENV-2 replication. Taken together, the study has provided experimental evidence to argue that blocking the NS4A-NS4B interaction could be a potential antiviral approach. IMPORTANCEFlavivirus NS4A and NS4B proteins are essential components of the ER membrane-associated replication complex. The current study systematically characterizes the interaction between flavivirus NS4A and NS4B. Using DENV-2 as a model, we show that NS4A interacts with NS4B in virus-infected cells, in cells transiently expressing NS4A and NS4B proteins, or in vitro with recombinant NS4A and NS4B proteins. We mapped the minimal regions required for the NS4A-NS4B interaction to be amino acids 40 to 76 of NS4A and amino acids 84 to 146 of NS4B. NMR analysis revealed the secondary structure of amino acids 17 to 80 of NS4A and the NS4A amino acids that may participate in the NS4A-NS4B interaction. Functional analysis showed a correlation between viral replication and NS4A-NS4B interaction, demonstrating the biological importance of the NS4A-NS4B interaction. The study has advanced our knowledge of the molecular function of flavivirus NS4A and NS4B proteins. The results also suggest that inhibitors of the NS4A-NS4B interaction could be pursued for flavivirus antiviral development.T he four serotypes of dengue virus (DENV-1 to DENV-4) are the causati...