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.

Identification of Mutated Cyclization Sequences That Permit Efficient Replication of West Nile Virus Genomes: Use in Safer Propagation of a Novel Vaccine Candidate

“…For studies requiring a full-length WNV cDNA, a previously described WNV cDNA derived from a human WNV isolate obtained in Texas in 2002 (36) was introduced into a bacterial artificial chromosome (BAC) plasmid to enhance its stability (41). A BAC-propagated cDNA derivative of our previously described single-cycle WNV lacking a functional C gene (28), designated RepliVAX WN.2, has been described (44).…”

Development of a Novel Dengue-1 Virus Replicon System Expressing Secretory <i>Gaussia</i> luciferase for Analysis of Viral Replication and Discovery of Antiviral Drugs

“…The DNA-based replicons are more stable than RNA-based constructs and the replicons can be directly transfected (thus without in vitro transcription) (5,6). Reporter replicon systems have been used for the analysis of flavivirus replication mechanisms and for conducting high-throughput screening for antiviral compounds (8)(9)(10)(11)(12)(13)(14)(15). The incorporation of firefly and Renilla luciferase reporter genes into DENV replicon constructs has facilitated the study of this virus.…”

A two-plasmid strategy for engineering a dengue virus type 3 infectious clone from primary Brazilian isolate

“…Several infectious cDNA clones have been described for flaviviruses: YFV (Bredenbeek et al 2003, Rice et al 1989, WNV (Yamshchikov et al 2001), JEV (Pu et al 2011, Sumiyoshi et al 1992, Yun et al 2003, DENV1 (Puri et al 2000, Suzuki et al 2007), DENV2 (Kapoor et al 1995, Polo et al 1997, Pu et al 2011), DENV3 (Blaney et al 2004, Messer et al 2012) and DENV4 (Lai et al 1991). Subsequently, self-replicating RNAs termed replicons were also developed (Jones et al 2005, Khromykh andWestaway 1997).…”

“…The reasons of this toxicity are not completely understood, but some reports have suggested that the genome sequence at the E/NS1 region or the expression of a deleterious product from it causes instability in E. coli (Blaney et al 2004, Polo et al 1997. Distinct approaches have been assessed to overcome genome instability, including (i) the cloning of the genome in separate fragments and recovering a full-length infectious clone by in vitro ligation (Kapoor et al 1995, Messer et al 2012, Sumiyoshi et al 1992; (ii) the use of low-copy number vectors (Bredenbeek et al 2003) or (iii) Bacterial Artificial Chromosomes (BACs) (Suzuki et al 2007); (iv) the use of different E. coli strains (Bredenbeek et al 2003, Sriburi et al 2001 or (v) the cloning in yeast cells (Polo et al 1997, Puri et al 2000.…”