title: Construction of an infectious cDNA clone for a Brazilian prototype strain of dengue virus type 1: characterization of a temperature-sensitive mutation in NS1.
abstract: To help understand the mechanism of pathogenesis of dengue virus (DV), we set out to create an infectious cDNA of the Brazilian prototype strain of DV serotype 1 (DV1-BR/90). PCR-amplified fragments of DV1-BR/90 cDNA were readily assembled into a subgenomic cDNA that could be used to produce replicating RNAs (replicons), lacking the structural protein-encoding regions of the genome. However, assembly of a cDNA capable of producing infectious virus was only possible using a bacterial artificial chromosome plasmid, indicating that DV1 sequences were especially difficult to propagate in E. coli. While characterizing our cDNA we discovered a fortuitous temperature-sensitive mutation in the NS1 encoding region. Using our infectious cDNA and a renilla luciferase-expressing replicon we were able to demonstrate that this mutation produced a defect in RNA replication at 37 degrees C, demonstrating that the DV1 NS1 protein plays an essential role in RNA replication.
authors.affiliation: Department of Pathology, University of Texas Medical Branch, 3.206B Mary Moody Northen Pavilion, 301 University Boulevard, Galveston, TX 77555-0436, USA.
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Suzuki
authors.forename: Luana
authors.initials: L
authors.lastname: de Borba
authors.forename: Claudia N
authors.initials: CN
authors.lastname: Duarte dos Santos
authors.forename: Peter W
authors.initials: PW
authors.lastname: Mason
mesh_terms: D000328:Adult; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D022202:Chromosomes, Artificial, Bacterial; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D003715:Dengue / Q000821:virology; D003716:Dengue Virus / Q000235:genetics* / Q000502:physiology; D020043:Genes, Essential / Q000235:genetics; D016679:Genome, Viral / Q000235:genetics*; D006801:Humans; D008297:Male; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D020815:Polyproteins / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013696:Temperature; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D014779:Virus Replication
chemical_list: D018076:DNA, Complementary; D004279:DNA, Viral; C082515:NS1 protein, dengue-1 virus; D020815:Polyproteins; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins