The C-terminal 50 Amino Acid Residues of Dengue NS3 Protein Are Important for NS3-NS5 Interaction and Viral Replication

Tay, Moon Y. F.; Saw, Wuan Geok; Zhao, Yongxiang; Chan, Kitti Wing Ki; Singh, Daljit; Chong, Y. F.; Forwood, Jade K.; Ooi, Eng Eong; Grüber, Gerhard; Lescar, Julien; Luo, Dahai; Vasudevan, Subhash G.

doi:10.1074/jbc.m114.607341

Cited by 118 publications

(176 citation statements)

References 73 publications

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“…Alternatively, NS4B was reported to dissociate singlestranded RNA from the NS3 helicase in vitro, stimulating its RNAunwinding activity (16,38), which is critical for optimal RNA replication by the NS5 polymerase. Additionally, NS5 also associates with NS3 and modulates its NTPase/RNA helicase, as well as 5= RNA triphosphatase activities (39)(40)(41)(42). Therefore, it is tempting to speculate that NS4B coordinates the activity of the DENV replication complex.…”

Section: Figmentioning

confidence: 99%

A Combined Genetic-Proteomic Approach Identifies Residues within Dengue Virus NS4B Critical for Interaction with NS3 and Viral Replication

Chatel‐Chaix

Fischl

Scaturro

et al. 2015

J Virol

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Dengue virus (DENV) infection causes the most prevalent arthropod-borne viral disease worldwide. Approved vaccines are not available, and targets suitable for the development of antiviral drugs are lacking. One possible drug target is nonstructural protein 4B (NS4B), because it is absolutely required for virus replication; however, its exact role in the DENV replication cycle is largely unknown. With the aim of mapping NS4B determinants critical for DENV replication, we performed a reverse genetic screening of 33 NS4B mutants in the context of an infectious DENV genome. While the majority of these mutations were lethal, for several of them, we were able to select for second-site pseudoreversions, most often residing in NS4B and restoring replication competence. To identify all viral NS4B interaction partners, we engineered a fully viable DENV genome encoding an affinity-tagged NS4B. Mass spectrometry-based analysis of the NS4B complex isolated from infected cells identified the NS3 protease/helicase as a major interaction partner of NS4B. By combining the genetic complementation map of NS4B with a replication-independent expression system, we identified the NS4B cytosolic loopmore precisely, amino acid residue Q134 -as a critical determinant for NS4B-NS3 interaction. An alanine substitution at this site completely abrogated the interaction and DENV RNA replication, and both were restored by pseudoreversions A69S and A137V. This strict correlation between the degree of NS4B-NS3 interaction and DENV replication provides strong evidence that this viral protein complex plays a pivotal role during the DENV replication cycle, hence representing a promising target for novel antiviral strategies. IMPORTANCEWith no approved therapy or vaccine against dengue virus infection, the viral nonstructural protein 4B (NS4B) represents a possible drug target, because it is indispensable for virus replication. However, little is known about its precise structure and function. Here, we established the first comprehensive genetic interaction map of NS4B, identifying amino acid residues that are essential for virus replication, as well as second-site mutations compensating for their defects. Additionally, we determined the NS4B viral interactome in infected cells and identified the NS3 protease/helicase as a major interaction partner of NS4B. We mapped residues in the cytosolic loop of NS4B as critical determinants for interaction with NS3, as well as RNA replication. The strong correlation between NS3-NS4B interaction and RNA replication provides strong evidence that this complex plays a pivotal role in the viral replication cycle, hence representing a promising antiviral drug target. D engue virus (DENV) is an enveloped plus-strand RNA virus belonging to the genus Flavivirus of the family Flaviviridae. DENV infection causes the most prevalent arthropod-borne viral disease worldwide, with approximately 100 million symptomatic cases per year. In 0.5 to 1% of the cases, the infection manifests with severe symptoms that can deve...

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Section: Figmentioning

confidence: 99%

A Combined Genetic-Proteomic Approach Identifies Residues within Dengue Virus NS4B Critical for Interaction with NS3 and Viral Replication

Chatel‐Chaix

Fischl

Scaturro

et al. 2015

J Virol

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“…To examine the functional role of the conserved residue in the context of the virus life cycle, the same mutations as listed above were introduced into a DENV2 infectious cDNA clone (18). The viral RNA synthesis and infectious virus production capabilities of the sequence-confirmed clones and the WT were examined over the course of 5 days posttransfection by reverse transcription quantitative PCR (qPCR) and confirmed by immunofluorescence assay (IFA) staining of double-stranded RNA (dsRNA) and NS5 protein with specific antibodies (Fig.…”

Section: N Onstructural Protein 5 (Ns5) Is a Multifunctional Protein mentioning

confidence: 99%

“…3). Of the four analogous mutants in DENV2 (the I265G, I265P, IGG, and IPG mutants), only the I265P mutant (replacement of I265 with Pro) failed to show recovery of any viable viruses by plaque assay, similarly to the negative-control K330A mutant (18). Also, the I265P virus displayed significantly lower infectivity (Ͻ5%) than that of the WT (50 to 60%) on day 3, as indicated by the IFA staining of dsRNA and NS5.…”

Section: N Onstructural Protein 5 (Ns5) Is a Multifunctional Protein mentioning

confidence: 99%

Flexibility of NS5 Methyltransferase-Polymerase Linker Region Is Essential for Dengue Virus Replication

Zhao

Soh

Chan

et al. 2015

J Virol

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We examined the function of the conserved Val/Ile residue within the dengue virus NS5 interdomain linker (residues 263 to 272) by site-directed mutagenesis. Gly substitution or Gly/Pro insertion after the conserved residue increased the linker flexibility and created slightly attenuated viruses. In contrast, Pro substitution abolished virus replication by imposing rigidity in the linker and restricting NS5's conformational plasticity. Our biochemical and reverse genetics experiments demonstrate that NS5 utilizes conformational regulation to achieve optimum viral replication. N onstructural protein 5 (NS5) is a multifunctional protein essential for dengue virus (DENV) replication. NS5 consists of an N-terminal S-adenosyl-L-methionine (SAM)-dependent methyltransferase (MTase) domain (1) and a C-terminal RNAdependent RNA polymerase (RdRp) domain (2-5). The MTase domain catalyzes the formation of type I RNA cap, which is critical for efficient translation and subverting the host immune surveillance (1, 6-8). The MTase domain may also be responsible for guanylyltransferase activity (9, 10). The RdRp domain (NS5 RdRp) synthesizes both negative-strand RNA from the input genomic RNA (gRNA) and the progeny plus-strand gRNA (5, 11).Previous structural studies on DENV NS5 individual domains revealed electron density covering residues 6 to 262 (for the MTase domain) (PDB code 3P97) (12), and residues 273 to 882 (for RdRp) (PDB code 2J7U) (5). Limited proteolysis of full-length NS5 (NS5 FL) in combination with mass determination suggested that the residues 263 to 272 (5) form the interdomain linker, and sequence comparison across various flaviviruses showed that this region is poorly conserved (Fig. 1). Small-angle X-ray scattering (SAXS) studies suggested that NS5 could adopt different conformations permitted by a flexible linker (13). Interestingly, an Nterminally extended RdRp spanning residues from 265 to 900 improved thermostability and RdRp activity compared with a shorter RdRp spanning residues 273 to 900 (14). More recently, the structures of the full-length NS5 protein from DENV3 (15) and Japanese encephalitis virus (JEV) full-length NS5 (PDB code 4K6M) (16) have been resolved. Extensive comparisons between the two structures have been reported (15). Notably, unlike JEV NS5, the DENV NS5 structure revealed a hydrophilic interdomain interface and a linker region composed of a 3 10 -helix (amino acids [aa] 263 to 266) followed by a short loop (aa 267 to 272) ( Fig. 1A and B). In DENV3 full-length NS5, the 3 10 -helix linker adopts a compact conformation compared to JEV full-length NS5 or DENV2 MTase (1 to 296) (PDB code 1L9K) (Fig. 1C). Functionally, the MTase domain stimulates both de novo initiation and elongation activities of the RdRp domain, which implies a critical role for an interdomain linker in controlling NS5 RNA polymerization activities (17). Despite the various conformations adopted by NS5 in solution, position 264 (in DENV3 NS5) is found to be exclusively Val in flaviviruses, except for a homologous su...

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“…Reproducing the inherent flexibility of biomolecules is a challenge and recently flexibility analysis in solution by small-angle X-ray scattering (SAXS) has become a central tool in characterizing multi-state systems [11,42] where crystallography, NMR and electron microscopy methods have limited scope [50]. Flexibility analysis using SAXS ensemble methods has been successfully applied to several biological systems such as, polynucleotide kinase [51], Lys-63 linked tetraubiquitin [52], Hck tyrosine kinase [53], ubiquitin-PCNA complex [54], endosome-associated ESCRT-III CHMP3 domain [55], non-structured proteins of Dengue [56], and the vacuolar ATPase [31].…”

Section: Intrinsic Dynamics Of Ecahpf and The Substrate Induced Compamentioning

confidence: 99%

Crystallographic and solution studies of NAD+- and NADH-bound alkylhydroperoxide reductase subunit F (AhpF) from Escherichia coli provide insight into sequential enzymatic steps

Kamariah

Manimekalai

Nartey

et al. 2015

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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Redox homeostasis is significant for the survival of pro- and eukaryotic cells and is crucial for defense against reactive oxygen species like superoxide and hydrogen peroxide. In Escherichia coli, the reduction of peroxides occurs via the redox active disulfide center of the alkyl hydroperoxide reductase C subunit (AhpC), whose reduced state becomes restored by AhpF. The 57kDa EcAhpF contains an N-terminal domain (NTD), which catalyzes the electron transfer from NADH via an FAD of the C-terminal domain into EcAhpC. The NTD is connected to the C-terminal domain via a linker. Here, the first crystal structure of E. coli AhpF bound with NADH and NAD(+) has been determined at 2.5Å and 2.4Å resolution, respectively. The NADH-bound form of EcAhpF reveals that the NADH-binding domain is required to alter its conformation to bring a bound NADH to the re-face of the isoalloxazine ring of the flavin, and thereby render the NADH-domain dithiol center accessible to the NTD disulfide center for electron transfer. The NAD(+)-bound form of EcAhpF shows conformational differences for the nicotinamide end moieties and its interacting residue M467, which is proposed to represent an intermediate product-release conformation. In addition, the structural alterations in EcAhpF due to NADH- and NAD(+)-binding in solution are shown by small angle X-ray scattering studies. The EcAhpF is revealed to adopt many intermediate conformations in solution to facilitate the electron transfer from the substrate NADH to the C-terminal domain, and subsequently to the NTD of EcAhpF for the final step of AhpC reduction.

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The C-terminal 50 Amino Acid Residues of Dengue NS3 Protein Are Important for NS3-NS5 Interaction and Viral Replication

Cited by 118 publications

References 73 publications

A Combined Genetic-Proteomic Approach Identifies Residues within Dengue Virus NS4B Critical for Interaction with NS3 and Viral Replication

A Combined Genetic-Proteomic Approach Identifies Residues within Dengue Virus NS4B Critical for Interaction with NS3 and Viral Replication

Flexibility of NS5 Methyltransferase-Polymerase Linker Region Is Essential for Dengue Virus Replication

Crystallographic and solution studies of NAD+- and NADH-bound alkylhydroperoxide reductase subunit F (AhpF) from Escherichia coli provide insight into sequential enzymatic steps

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