title: 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.
authors.affiliation: From the Program in Emerging Infectious Diseases, Duke-National University of Singapore Graduate Medical School, 8 College Road, Singapore 169857, Singapore.
authors.forename: Moon Y F
authors.initials: MY
authors.lastname: Tay
authors.affiliation: the School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.
authors.forename: Wuan Geok
authors.initials: WG
authors.lastname: Saw
authors.affiliation: From the Program in Emerging Infectious Diseases, Duke-National University of Singapore Graduate Medical School, 8 College Road, Singapore 169857, Singapore, the NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, 28 Medical Drive, Singapore 117456, Singapore.
authors.forename: Yongqian
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: From the Program in Emerging Infectious Diseases, Duke-National University of Singapore Graduate Medical School, 8 College Road, Singapore 169857, Singapore.
authors.forename: Kitti W K
authors.initials: KW
authors.lastname: Chan
authors.affiliation: From the Program in Emerging Infectious Diseases, Duke-National University of Singapore Graduate Medical School, 8 College Road, Singapore 169857, Singapore.
authors.forename: Daljit
authors.initials: D
authors.lastname: Singh
authors.affiliation: From the Program in Emerging Infectious Diseases, Duke-National University of Singapore Graduate Medical School, 8 College Road, Singapore 169857, Singapore.
authors.forename: Yuwen
authors.initials: Y
authors.lastname: Chong
authors.affiliation: the School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, New South Wales 2650, Australia.
authors.forename: Jade K
authors.initials: JK
authors.lastname: Forwood
authors.affiliation: From the Program in Emerging Infectious Diseases, Duke-National University of Singapore Graduate Medical School, 8 College Road, Singapore 169857, Singapore.
authors.forename: Eng Eong
authors.initials: EE
authors.lastname: Ooi
authors.affiliation: the School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Grüber
authors.affiliation: the Division of Structural Biology and Biochemistry, School of Biological Sciences, Nanyang Technological University, Singapore 138673, Singapore, and.
authors.forename: Julien
authors.initials: J
authors.lastname: Lescar
authors.affiliation: the Lee Kong Chian School of Medicine, Nanyang Technological University, 61 Biopolis Drive, Proteos Building, 07-03, Singapore 138673, Singapore.
authors.forename: Dahai
authors.initials: D
authors.lastname: Luo
authors.affiliation: From the Program in Emerging Infectious Diseases, Duke-National University of Singapore Graduate Medical School, 8 College Road, Singapore 169857, Singapore, the NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, 28 Medical Drive, Singapore 117456, Singapore, subhash.vasudevan@duke-nus.edu.sg.
authors.forename: Subhash G
authors.initials: SG
authors.lastname: Vasudevan
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry; D001665:Binding Sites; D002460:Cell Line; D045744:Cell Line, Tumor; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D003716:Dengue Virus / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008239:Lysine / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000737:chemistry*; D012324:RNA-Dependent RNA Polymerase; D012542:Scattering, Radiation; D012697:Serine Endopeptidases / Q000737:chemistry*; D017361:Viral Nonstructural Proteins / Q000737:chemistry*; D014779:Virus Replication; D000085242:Viral Proteases
chemical_list: D000998:Antiviral Agents; D018076:DNA, Complementary; D008239:Lysine; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C582223:NS5 protein, dengue virus; C504449:NS3 protease, dengue virus; D000085242:Viral Proteases
keywords: Dengue Virus; Flavivirus; Nonstructural Protein Interaction; Plus-stranded RNA Virus; Protein-Protein Interaction; Replication Complex; Viral Helicase; Viral Polymerase; Viral Protein; Viral Replication