| Abstract: | The NS2A protein of Dengue virus (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. IMPORTANCE: The reported characterization of the C-terminal half of dengue virus NS2A is the first comprehensive mutagenesis study to investigate the function of flavivirus NS2A involved in the steps of the virus life cycle. In particular, detailed mapping of the amino acid residues within the predicted transmembrane segments (pTMSs) of NS2A involved in RNA replication and/or virus assembly/secretion was performed. A revertant genetics study also revealed that L181F within pTMS7 is a consensus reversion mutation that rescues both RNA replication- and virus assembly/secretion-defective mutants within the other three pTMSs of NS2A. Collectively, these findings elucidate the role played by NS2A during the virus life cycle, possibly through the intricate intramolecular interaction between pTMS7 and other pTMSs within the NS2A protein. |
