Abstract
Four serotypes of mosquito-borne dengue virus (DENV), evolved from a common ancestor, are human pathogens of global significance for which there is no vaccine or antiviral drug available. The N-terminal domain of DENV NS5 has guanylyltransferase and methyltransferase (MTase), and the C-terminal region has the polymerase (POL), all of which are important for 5'-capping and RNA replication. The crystal structure of NS5 shows it as a dimer, but the functional evidence for NS5 dimer is lacking. Our studies showed that the substitution of DENV2 NS5 MTase or POL for DENV4 NS5 within DENV2 RNA resulted in a severe attenuation of replication in the transfected BHK-21 cells. A replication-competent species was evolved with the acquired mutations in the DENV2 and DENV4 NS5 MTase or POL domain or in the DENV2 NS3 helicase domain in the DENV2 chimera RNAs by repeated passaging of infected BHK-21 or mosquito cells. The linker region of seven residues in NS5, rich in serotype-specific residues, is important for the recovery of replication fitness in the chimera RNA. Our results, taken together, provide genetic evidence for a serotype-specific interaction between NS3 and NS5 as well as specific interdomain interaction within NS5 required for RNA replication. Genome-wide RNAseq analysis revealed the distribution of adaptive mutations in RNA quasispecies. Those within NS3 and NS5 are located at the surface and/or within the NS5 dimer interface, providing a functional significance to the crystal structure NS5 dimer.
Highlights
Four serotypes of mosquito-borne dengue virus (DENV), evolved from a common ancestor, are human pathogens of global significance for which there is no vaccine or antiviral drug available
Dengue virus (DENV)2 is a member of the Flavivirus genus that includes a large number of human pathogens transmitted by mosquitoes, such as yellow fever virus (YFV), Japanese encephalitis virus (JEV), West Nile virus (WNV), the recently emerging and rapidly spreading Zika virus, or by ticks, such as tick-borne encephalitis virus (TBEV)
We showed that the DENV2 chimera virus containing DENV4 MTase-K74I and DENV2 NS3 helicase (NS3hel)-V226I mutations gained the ability to replicate (Fig. 6A, row 3), caused cytopathic effect (CPE), and formed plaques (Fig. 6, B and D)
Summary
We sought to analyze the two substitutions of the DENV2 POL domain with the corresponding region of DENV4 POL. The supernatants of DENV2 chimera RNA containing DENV4 NS5(264 –270aa) linker-transfected cells at 14 days post-transfection, and sequence analysis showed no acquired mutation (Table 2). The culture supernatants from the two DENV2 chimera RNAs containing the DENV4 POL(264- 900aa) and POL(271–900aa) domains were used to infect C6/36 cells, and their replication fitness was analyzed by qRT-PCR (Fig. 4A). The recovered DENV4 MTase(1–270aa) chimera virus containing either the K74I adaptive mutation in the DENV4 MTase domain or the D290N mutation in the DENV2 NS3hel domain (ϳ30-day supernatants of BHK-21 cells [33]) was used to infect C6/36 cells Both the DENV2 POL and the MTase chimera viruses replicated in a similar manner, both were still slower than the WT (Fig. 4A). Amino acid alterations gained within NS5 and NS3 genes of chimera viruses in BHK-21 cells
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