title: Serotype-specific interactions among functional domains of dengue virus 2 nonstructural proteins (NS) 5 and NS3 are crucial for viral RNA replication.
abstract: Four serotypes of mosquito-borne dengue virus (DENV), evolved from a common ancestor, are human pathogens of global significance for which there is no vaccine or antiviral drug available. The N-terminal domain of DENV NS5 has guanylyltransferase and methyltransferase (MTase), and the C-terminal region has the polymerase (POL), all of which are important for 5'-capping and RNA replication. The crystal structure of NS5 shows it as a dimer, but the functional evidence for NS5 dimer is lacking. Our studies showed that the substitution of DENV2 NS5 MTase or POL for DENV4 NS5 within DENV2 RNA resulted in a severe attenuation of replication in the transfected BHK-21 cells. A replication-competent species was evolved with the acquired mutations in the DENV2 and DENV4 NS5 MTase or POL domain or in the DENV2 NS3 helicase domain in the DENV2 chimera RNAs by repeated passaging of infected BHK-21 or mosquito cells. The linker region of seven residues in NS5, rich in serotype-specific residues, is important for the recovery of replication fitness in the chimera RNA. Our results, taken together, provide genetic evidence for a serotype-specific interaction between NS3 and NS5 as well as specific interdomain interaction within NS5 required for RNA replication. Genome-wide RNAseq analysis revealed the distribution of adaptive mutations in RNA quasispecies. Those within NS3 and NS5 are located at the surface and/or within the NS5 dimer interface, providing a functional significance to the crystal structure NS5 dimer.
authors.affiliation: From the Department of Microbiology and Immunology, School of Medicine, Georgetown University, Washington, D. C. 20057 and.
authors.forename: Tadahisa
authors.initials: T
authors.lastname: Teramoto
authors.affiliation: From the Department of Microbiology and Immunology, School of Medicine, Georgetown University, Washington, D. C. 20057 and.
authors.forename: Anuradha
authors.initials: A
authors.lastname: Balasubramanian
authors.affiliation: the Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555-0156.
authors.forename: Kyung H
authors.initials: KH
authors.lastname: Choi
authors.affiliation: From the Department of Microbiology and Immunology, School of Medicine, Georgetown University, Washington, D. C. 20057 and rp55@georgetown.edu.
authors.forename: Radhakrishnan
authors.initials: R
authors.lastname: Padmanabhan
mesh_terms: D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D009033:Culicidae; D003716:Dengue Virus / Q000502:physiology*; D006801:Humans; D000072417:Protein Domains; D020365:RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D065288:Serogroup; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014779:Virus Replication / Q000502:physiology*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D020365:RNA Helicases; D012367:RNA, Viral; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C582223:NS5 protein, dengue virus
keywords: RNA helicase; RNA methyltransferase; RNA polymerase; RNA virus; RNA-protein interaction; dengue virus (DENV); protein-protein interaction; viral polymerase; viral replication