Serotype-specific interactions among functional domains of dengue virus 2 nonstructural proteins (NS) 5 and NS3 are crucial for viral RNA replication

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.

Positive epistasis between viral polymerase and the 3′ untranslated region of its genome reveals the epidemiologic fitness of dengue virus

“…Thus, we focused our attention on the significance of these NS5 mutations in altering the epidemiologic fitness of DENV2. Indeed, previous studies have shown that mutations in NS5 compromise virus replication (35,36).…”

Infection of Aedes albopictus Mosquito C6/36 Cells with the w Melpop Strain of Wolbachia Modulates Dengue Virus-Induced Host Cellular Transcripts and Induces Critical Sequence Alterations in the Dengue Viral Genome

“…RNAseq alignment analysis of DENV2 sequences. Raw Fastq files described above were used for analysis of frequencies and locations of nucleotide changes in DENV2 RNA using Geneious (version 9.1) software (Biomatters) as previously described (91).…”

“…Extracted viral RNAs from supernatants of infected cells were used for viral cDNA synthesis and PCR amplification with DENV2 sequence-specific primers (91). PCR products were purified with an agarose gel (Zymo Research), and DNA sequencing was performed by GENEWIZ Inc. (South Plainfield, NJ).…”

Cross-serotypically conserved epitope recommendations for a universal T cell-based dengue vaccine

“…Most of the identified conserved epitopes (45 out of 55) were derived from NS5 and NS3 (25 and 20, respectively) (Fig 3), highlighting the potential biological importance of targeting these non-structural proteins. Both NS5 and NS3 are large DENV proteins, comprising about 900 and 618 residues respectively (lengths slightly vary across serotypes), which are multifunctional enzymes playing a critical role in viral replication [50][51][52]. The high conservation of the identified NS5 and NS3 epitopes across serotypes (Fig 3) indicates that the protein regions associated with them may be structurally/functionally important for DENV.…”