Examining the Role of Envelope Protein Mutations in Promoting the Dengue Viral Life Cycle

Abstract

Dengue viruses (DENVs), members of the Flaviviridae family, are a group of single stranded, positive sense RNA viruses transmitted to humans through mosquito vectors. DENVs present a significant global health burden, infecting an estimated 390 million people each year, yet vaccination and treatment efforts have been hindered by a lack of knowledge of the DENV life cycle. To gain insights into host-virus interactions governing this cycle, we adapted DENV strains by serial passaging to robustly infect lowly permissive human keratinocyte (HaCaT) cells. Through whole genome sequencing, common mutations were identified in the adapted strains that conferred a fitness advantage in cell culture, including two mutations in amino acid 327 (E327K, E327T) of the envelope (E) protein. To build on this discovery, we sought to investigate the impact of these mutations on the ability of the virus to carry out its infectious cycle. Firstly, the circular polymerase extension reaction was used to generate eight DENV viruses harboring the E327K or E327T mutations and in vitro growth curve analysis was conducted in both hepatic and keratinocyte cells to compare the mutant and wild-type phenotype. These mutations were found to increase viral fitness in keratinocytes in three closely related DENV4 strains, but not in the other DENV serotypes tested. To pinpoint which viral life cycle steps these mutations affect, binding and entry assays were performed to indicate that E327K mutants may demonstrate enhanced binding to HaCaT cells, while time point assays were conducted to suggest that the E327T mutation may be involved in promoting viral genome release. Alternatively, in vitro transcribed viral RNA was transfected to show that the genome replication rate remained constant. To investigate the impact of these mutations on viral assembly and egress, extracellular and intracellular virion quantities were compared by viral titration, suggesting an additional interaction between the E327 residue and host cell proteins in promoting this phase. Future research building on this project could potentially uncover host cell proteins involved in the genome release or viral egress stages of the DENV life cycle, a critical step towards therapeutic and vaccine development.