The Development and Application of Novel Reverse Genetics to Investigate the Life Cycle of Flavivirus

• Main Authors: Ren-Huang Wu, 吳仁煌
• Other Authors: Andrew Yueh
• Format: Others
• Language: en_US
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/29949950736504134047

博士 === 國防醫學院 === 生命科學研究所 === 103 === Reverse genetics is a powerful tool to study single-stranded RNA viruses and has led to important advances in our understanding of viral gene function. Previously, we had demonstrated that the cryptic ECP activity of viral genome in bacteria was significantly reduced by introducing silent mutations into the genome of DENV2 and JEV and we successfully obtained RNA-launched DENV2 and JEV infectious clone. However, the construction of DNA-launched flavivirus infectious clones in bacterial was unstable due to the toxicity of viral protein expression. Here we successfully propagated DNA-launched type 2 dengue virus (DENV2) and Japanese encephalitis virus (JEV) infectious cDNAs by introducing seven repeats of the tetracycline-response element (7XTRE) and a minimal cytomegalovirus (CMVmin) promoter upstream of the viral genome. Insertion of the 7XTRE-CMVmin sequence upstream of the DENV2 or JEV genome decreased the cryptic E. coli promoter (ECP) activity of the viral genome in bacteria, as measured using fusion constructs containing DENV2 or JEV segments and the reporter gene Renilla luciferase in an empty vector. The growth kinetics of recombinant viruses derived from DNA-launched DENV2 and JEV infectious cDNAs were similar to those of parental viruses. Similarly, RNA-launched DENV2 infectious cDNAs were generated by inserting 7XTRE-CMVmin, five repeats of the GAL4 upstream activating sequence (5XGAL4), or five repeats of BamHI linkers (5XBamHI) upstream of the DENV2 genome. All three tandem repeat sequences decreased the ECP activity of the DENV2 genome in bacteria. Notably 7XTRE-CMVmin stabilized RNA-launched JEV infectious cDNAs and reduced the ECP activity of the JEV genome in bacteria. The growth kinetics of recombinant viruses derived from RNA-launched DENV2 and JEV infectious cDNAs displayed patterns similar to those of the parental viruses. These results support a novel methodology for constructing flavivirus infectious cDNAs. To demonstrate the utility of our novel reverse genetics system, we used alanine scanning mutagenesis to study the functions of DENV2 NS2A protein in the context of DNA-launched infectious cDNAs. The NS2A protein of DENV has eight predicted transmembrane segments (pTMS1-8). NS2A has been shown to participate in RNA replication, virion assembly, and the host antiviral response. However, the role of the amino acid residues within the pTMS regions of NS2A during the virus life cycle is poorly understood. Here, we explore the function of DENV NS2A by introducing a series of double- or triple-alanine substitutions into the C-terminal half (pTMS4-8) of NS2A in the context of a DENV infectious clone or subgenomic replicon. Fourteen (eight within pTMS8) out of thirty-five NS2A mutants displayed a lethal phenotype due to impairment of RNA replication by replicon assay. Three NS2A mutants within pTMS7, CM20, 25, and 27, displayed similar phenotypes, low virus yields (>100-fold reduction), wild-type-like replicon activity, and low infectious virus-like particle yields by transient trans-packaging experiments, suggesting a defect in virus assembly/secretion. The sequencing of revertant viruses derived from CM20, 25, and 27 mutant viruses revealed a consensus reversion mutation, leucine (L)-to-phenylalanine (F), at codon 181 within pTMS7. The introduction of an L181F mutation into a full-length NS2A mutant, i.e., the CM20, 25, and 27 constructs, completely restored wild-type infectivity. Notably, L181F also substantially rescued the other severely RNA replication-defective mutants within pTMS4, 6, and 8, i.e., CM2, 3, 13, 31, and 32. In conclusion, the results revealed the essential roles of pTMS4-8 of NS2A in RNA replication and/or virus assembly/secretion. The intramolecular interaction between pTMS7 with pTMS4, 6, or 8 of the NS2A protein was also implicated.