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The C-terminal 50 amino acid residues of dengue NS3 protein are important for NS3-NS5 interaction and viral replication.

ABSTRACT: Dengue virus multifunctional proteins NS3 protease/helicase and NS5 methyltransferase/RNA-dependent RNA polymerase form part of the viral replication complex and are involved in viral RNA genome synthesis, methylation of the 5'-cap of viral genome, and polyprotein processing among other activities. Previous studies have shown that NS5 residue Lys-330 is required for interaction between NS3 and NS5. Here, we show by competitive NS3-NS5 interaction ELISA that the NS3 peptide spanning residues 566-585 disrupts NS3-NS5 interaction but not the null-peptide bearing the N570A mutation. Small angle x-ray scattering study on NS3(172-618) helicase and covalently linked NS3(172-618)-NS5(320-341) reveals a rigid and compact formation of the latter, indicating that peptide NS5(320-341) engages in specific and discrete interaction with NS3. Significantly, NS3:Asn-570 to alanine mutation introduced into an infectious DENV2 cDNA clone did not yield detectable virus by plaque assay even though intracellular double-stranded RNA was detected by immunofluorescence. Detection of increased negative-strand RNA synthesis by real time RT-PCR for the NS3:N570A mutant suggests that NS3-NS5 interaction plays an important role in the balanced synthesis of positive- and negative-strand RNA for robust viral replication. Dengue virus infection has become a global concern, and the lack of safe vaccines or antiviral treatments urgently needs to be addressed. NS3 and NS5 are highly conserved among the four serotypes, and the protein sequence around the pinpointed amino acids from the NS3 and NS5 regions are also conserved. The identification of the functionally essential interaction between the two proteins by biochemical and reverse genetics methods paves the way for rational drug design efforts to inhibit viral RNA synthesis.

SUBMITTER: Tay MY

PROVIDER: S-EPMC4303688 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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The C-terminal 50 amino acid residues of dengue NS3 protein are important for NS3-NS5 interaction and viral replication.

Tay Moon Y F MY Saw Wuan Geok WG Zhao Yongqian Y Chan Kitti W K KW Singh Daljit D Chong Yuwen Y Forwood Jade K JK Ooi Eng Eong EE Grüber Gerhard G Lescar Julien J Luo Dahai D Vasudevan Subhash G SG

The Journal of biological chemistry 20141208 4

Dengue virus multifunctional proteins NS3 protease/helicase and NS5 methyltransferase/RNA-dependent RNA polymerase form part of the viral replication complex and are involved in viral RNA genome synthesis, methylation of the 5'-cap of viral genome, and polyprotein processing among other activities. Previous studies have shown that NS5 residue Lys-330 is required for interaction between NS3 and NS5. Here, we show by competitive NS3-NS5 interaction ELISA that the NS3 peptide spanning residues 566- ...[more]

PMID: 25488659

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Project description:Dengue fever is a virus spread by mosquitoes that has no effective treatment or vaccination. Several dengue cases combined with the current COVID-19 pandemic, exacerbates this problem. Two proteins, NS5 methyltransferase and NS2B/NS3 primary protease complexes, are crucial for dengue viral replication and are the target sites for antiviral development. Thus, this study screened published literature and identified 162 marine fungus-derived compounds with active bioavailability. Following Lipinski's rules and antiviral property prediction, 41 compounds were selected for docking with NS5 methyltransferase and NS2B/NS3 protease (PDB ID: 6IZZ and 2FOM) to evaluate compounds that could stop the action of dengue viral protein complexes. To find the best candidates, computational ADME, toxicity, and drug target prediction were performed to estimate the potential of the multi-targeting fungal-derived natural compounds. Analyzing the result from 41 compounds, Chevalone E (-13.5 kcal/mol), Sterolic acid (-10.3 kcal/mol) showed higher binding energy against dengue NS2B/NS3 protease; meanwhile, Chevalone E (-12.0 kcal/mol), Brevione K (-7.4 kcal/mol), had greater binding affinity against NS5 methyltransferase. Consequently, this study suggests that Chevalone E is an effective inhibitor of NS5 methyltransferase and NS2B/NS3 protease. Ligand-based virtual screening from DrugBank was utilized to predict biologically active small compounds against dengue virus NS2B/NS3 major protease and NS5 methyltransferase. Both licensed medications, estramustine (DB01196) and quinestrol (DB04575), were found to be similar to Chevalone E, with prediction scores of 0.818 and 0.856, respectively. In addition, cholic acid (DB02659), acitretin (DB00459), and mupirocin (DB00410) are similar to Sterolic acid, zidovudine (DB00495), imipenem (DB01598), and nadolol (DB01203) are similar to Brocazine A, and budesonide (DB01222) and colchicine (DB01394) are related to Brevione K. These findings suggest that these could be feasible dengue virus treatment options, meaning that more research is needed.

Project description:UNLABELLED:Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. Due to the limited coding capacity of the viral genome and the complexity of the viral life cycle, host cell proteins play essential roles throughout the course of viral infection. Host RNA-binding proteins mediate various aspects of virus replication through their physical interactions with viral RNA. Here we describe a technique designed to identify such interactions in the context of infected cells using UV cross-linking followed by antisense-mediated affinity purification and mass spectrometry. Using this approach, we identified interactions, several of them novel, between host proteins and dengue viral RNA in infected Huh7 cells. Most of these interactions were subsequently validated using RNA immunoprecipitation. Using small interfering RNA (siRNA)-mediated gene silencing, we showed that more than half of these host proteins are likely involved in regulating virus replication, demonstrating the utility of this method in identifying biologically relevant interactions that may not be identified using traditional in vitro approaches. IMPORTANCE:Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. Viral RNA molecules physically interact with cellular RNA-binding proteins (RBPs) throughout the course of infection; the identification of such interactions will lead to the elucidation of the molecular mechanisms of virus replication. Until now, the identification of host proteins bound to dengue viral RNA has been accomplished using in vitro strategies. Here, we used a method for the specific purification of dengue viral ribonucleoprotein (RNP) complexes from infected cells and subsequently identified the associated proteins by mass spectrometry. We then validated a functional role for the majority of these proteins in mediating efficient virus replication. This approach has broad relevance to virology and RNA biology, as it could theoretically be used to purify any viral RNP complex of interest.

Dataset Information

The C-terminal 50 amino acid residues of dengue NS3 protein are important for NS3-NS5 interaction and viral replication.

Publications

The C-terminal 50 amino acid residues of dengue NS3 protein are important for NS3-NS5 interaction and viral replication.

Similar Datasets

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