Research ArticleDENGUE VIRUS

A T164S mutation in the dengue virus NS1 protein is associated with greater disease severity in mice

See allHide authors and affiliations

Science Translational Medicine  26 Jun 2019:
Vol. 11, Issue 498, eaat7726
DOI: 10.1126/scitranslmed.aat7726
  • Fig. 1 Conservation, spatial location, and phylogenetic distribution of dengue virus NS1 residue 164.

    (A) Organization of the dengue virus genome indicating structural (C, capsid; prM, premembrane; E, envelope) and nonstructural (NS1 to NS5) genes flanked by 5′ and 3′UTRs (untranslated regions). Sequence alignment of the connector (Co) subdomain (residues 150 to 180) is shown for DENV1 to DENV4 (represented by selected strains with the following GenBank accession numbers: EU081230.1, EU081177.1, EU081190.1, and GQ398256.1, respectively). The percent (%) conservation of residues T164 and S164 across dengue virus serotypes was estimated using the total number (n) of publicly available NS1 sequences for DENV1 (n = 1868), DENV2 (n = 1590), DENV3 (n = 1020), and DENV4 (n = 243) in the Virus Pathogen Database and Analysis Resource [ViPR database (10) as of 28 March 2017]. The 3D structure of the dengue virus NS1 dimer [Protein Data Bank (PDB) 4O6B] (11) showing one monomer in color (β-roll, yellow; β-ladder, purple; wing domain, blue) and the other monomer in gray is depicted. The greasy finger loop (residues 159 to 162, shaded in pink for both monomers) is based on the West Nile virus NS1 dimer (PDB 4O6D), with residue T164 indicated (red stick) for both monomers. ER, endoplasmic reticulum. (B) Evolutionary relationship of DENV2 strains showing that the T164S mutation is distributed exclusively within the American clade of DENV2 genotype III. (C) Bayesian molecular clock showing the spatiotemporal distribution of the T164S mutation within the Southeast (SE) Asian/American genotype III clade.

  • Fig. 2 Characterization of the T164S mutation in NS1 of dengue virus.

    (A) Dengue virus serotype 2 3295 strain infectious clone (GenBank accession number EU081177.1) indicating standard genetic manipulations for introduction of the T164S mutation into NS1 (asterisks). (B) Real-time PCR quantification of intracellular viral RNA at the indicated time points after electroporation of BHK-21 cells with wild-type or T164S mutant virus. The dashed line represents the detection level for the mock-transfected control BHK-21 cells. WT, wild type. (C) Virus titer in infected BHK-21 cell supernatants at 24 hours after transfection with either wild-type or T164S mutant virus, which was determined using a plaque assay (left axis). The GE shown on the right axis is the ratio of extracellular RNA GCs measured by RT-PCR to infectious virus measured by plaque assay (pfu). (D) Real-time PCR quantification of wild-type or T164S mutant virus genome replication at an MOI of 1 or 10 in infected HuH-7 liver cells. (E) Supernatants from infected HuH-7 cells 24 hours after infection were subjected to plaque quantification (pfu/ml). (F) Nonreducing SDS–polyacrylamide gel electrophoresis (SDS-PAGE) of the cell lysates from (D). (G) Intracellular NS1 (iNS1) quantification in cell lysates from (D) measured by ELISA. (H) Quantification of sNS1 in the supernatants from cells in (D) measured by ELISA. Data are presented as means ± SD from duplicate of two independent experiments, and differences between wild-type and T164S mutant virus groups were compared by Mann-Whitney test (*P < 0.05).

  • Fig. 3 Replication kinetics of wild-type or T164S mutant dengue virus in the A. aegypti mosquito vector.

    A. aegypti mosquitoes were infected with wild-type or T164S mutant virus (5 × 105 pfu/ml) through a blood meal, and midgut viral loads were compared to those of whole carcasses. (A) Average viral load in whole mosquito carcasses obtained by summing the midgut and carcass viral loads (n = 30). (B) Scatter plot showing the viral load of individual mosquito midguts from (A) quantified by real-time PCR. (C) Average infectious viral titer of mosquito midgut homogenates at day 3 after infection based on the number of midgut homogenates with a virus titer >10 pfu. (D) Scatter plot of the viral loads in individual mosquito salivary glands and carcasses [from (A)] on day 7 after infection measured by real-time PCR. (E) Western blot of NS1 and NS3 proteins in homogenates from 50 mosquito midguts. (F) The band intensity of NS1 from (E) was normalized to that of β-actin using ImageJ. (G) The total NS1 in the mosquito gut homogenates was quantitated by ELISA. Mean values for wild-type and mutant NS1 were compared by Mann-Whitney test. Mean values of mosquito gut viral loads between the wild-type and T164S mutant virus groups were compared by the Mann-Whitney test. Data are presented as means ± SD from three independent experiments, and significance is indicated as *P < 0.05.

  • Fig. 4 Infection of human PBMCs ex vivo with wild-type or T164S mutant dengue virus in the absence or presence of humanized 4G2 antibody.

    Human PBMCs (n = 3 donors) were infected with 107 pfu (MOI = 10) of wild-type or T164S mutant virus either alone or in an immune complex with 0.05 μg of the humanized 4G2 antibody (Ab) (17). Supernatants were collected at 24 hours after infection and were analyzed as follows: (A) infectious virus titer quantification by plaque assay, (B) sNS1 measurement by ELISA, and (C) IL-6 and (D) TNFα concentrations measured by ELISA. (E) Native SDS-PAGE Western blot of wild-type and T164S mutant virus sNS1 purified from BHK-21 cell supernatants. (F) IL-6 and (G) TNFα concentrations were measured in the supernatants of human PBMCs incubated with purified wild-type or T164S mutant sNS1 (either 1 or 10 μg/ml) for 24 hours. Data are presented as scatter plots or bar graphs and show means ± SD from three independent experiments. Differences between the wild-type virus and T164S mutant virus were compared by Mann-Whitney test (*P < 0.05).

  • Fig. 5 Expression of inflammation-associated genes after infection of human PBMCs with wild-type or T164S mutant dengue virus.

    Human PBMCs (n = 2 donors) were infected with 107 pfu (MOI = 10) of wild-type or T164S mutant virus either alone or in an immune complex with 0.05 μg of the humanized 4G2 antibody (17). Total RNA was extracted from human PBMCs at 6 and 24 hours after infection, and gene expression analysis was performed using custom probes (table S1). Gene expression was normalized to that of the panel’s internal housekeeping genes. Normalized gene expression of the T164S mutant virus was then expressed as log2 fold change with respect to wild-type virus. Clusters of gene expression in the heat maps are based on KEGG (Kyoto Encyclopedia of Genes and Genomes)/Gene Ontology analysis. (A) Heat map of expression of MyD88-dependent/TLR signaling pathway genes. (B) Heat map of expression of cytokine/IFN-mediated signaling pathway genes. (C) Heat map of expression of complement cascade genes. (D) STRING analysis showing interaction network of the complement cascade genes in the gene expression panel generated by Cytoscape (43). Genes in red are those that are highly up-regulated (log2 fold change ≥ 1.5) in human PBMCs after infection by the T164S mutant virus. Data presented are from two independent experiments.

  • Fig. 6 Nonlethal infection of AG129 mice with wild-type or T164S mutant virus.

    (A) Schematic showing the timeline of nonlethal infection of AG129 mice with wild-type or T164S mutant virus and daily sampling for analysis of virus replication and proinflammatory markers. AG129 mice (n = 6 per group) were given 50 μg of mouse 4G2 antibody intraperitoneally 1 day before intravenous injection of 107 pfu (~5 × 1011 GE) of wild-type or T164S mutant virus. (B) Viremia kinetics were measured by PCR in pooled serum collected from mice infected with wild-type virus or T164S mutant virus. (C) Average viremia on day 1 after infection of mice infected with wild-type or T164S mutant virus. (D) The average infectious titer on day 1 after infection was measured in mouse serum by a plaque assay. (E) The amount of sNS1 in mouse serum was measured by ELISA. (F) Average amount of sNS1 produced on day 1 after infection. (G to K) Mouse serum concentrations of proinflammatory cytokines measured by ELISA for (G and H) IL-6, (I and J) TNFα, and (K) complement C3. Mouse serum concentrations for day 1 after infection for (H) IL-6 and (J) TNFα. Differences in viremia kinetics and serum complement C3 concentrations between wild-type virus– and T164S mutant virus–infected mouse groups were compared by two-way analysis of variance (ANOVA) with Bonferroni correction. Data are presented as means ± SD from two independent experiments, and differences between wild-type virus– and T164S mutant virus–infected mouse groups were compared by Mann-Whitney test (*P < 0.05 and **P < 0.01). i.p., intraperitoneally; i.v., intravenously.

  • Fig. 7 Lethal infection of AG129 mice with wild-type or T164S mutant dengue virus.

    (A) The schematic shows the timeline of the experiment. AG129 mice were given 50 μg of 4G2 mouse antibody intraperitoneally and were injected intravenously 1 day later with 108 pfu (~ 5 × 1012 GE) of wild-type or T164S mutant virus (n = 5 or 6 per group). Mouse survival was monitored for 10 days after infection (27). In addition, two to three mice were independently infected and sacrificed at ~85 hours after infection, and their tissue viral load and pathological markers were measured. (B) Survival plots (log-rank Mantel-Cox test) for AG129 mice infected with wild-type or T164S mutant virus. (C) Mouse serum sNS1 was measured at 85 hours after infection by ELISA. (D) Viral load in mouse tissues [small intestine (S. int), liver (Liv), and spleen (Spl)] was measured by PCR, and the viral RNA expression was normalized to actin expression. (E to G) Concentrations of (E) IL-6, (F) TNFα, and (G) complement C3 were measured in mouse tissue homogenates by ELISA. Data points shown as scatter plots (D to G) are means ± SD from two independent experiments. Differences between wild-type virus and T164S mutant virus were compared by Mann-Whitney test (*P < 0.05).

  • Fig. 8 Molecular simulations and lipid analysis of wild-type and mutant hexameric NS1.

    (A) Snapshot simulations of wild-type NS1 (left) and T164S mutant NS1 (right) in a lipid-free system with amino acid residue T164 labeled. (B) Zoomed-in initial (0 ns, left) and final (200 ns, right) snapshots of the wild-type (top) and T164S mutant (bottom) NS1 hexamer highlighting the hydrophobic interface between adjacent NS1 dimers (shown in wireframe format with labeling of the amino acids at the dimer interface involved in hydrophobic interactions). (C) Distribution of protein backbone root mean square deviations (RMSD) with respect to the x-ray structure as well as the minimum distance between interdimer hydrophobic clusters for wild-type (black) and T164S mutant (red) NS1. The NS1 monomer (top left), NS1 dimer (top right), and NS1 hexamer (bottom left) are shown. The bottom right panel shows the interdimer distance of wild-type (black) and T164S mutant (red) NS1. (D) Relative abundance of major lipid classes extracted from immunoaffinity-purified wild-type or T164S mutant sNS1 determined by LC-MS/MS. DG, diacylglycerols; PI, phosphatidylinositols; PE, phosphatidylethanolamines; PC, phosphatidylcholines; CE, cholesterol esters; LPC-O, ether lysophosphatidylcholines; PC-O, ether phosphatidylcholines; SM, sphingomyelins. (E) Relative abundance of short-chain phosphatidylethanolamine (PE) subclasses (carbon length of 32 to 34 atoms) for immunoaffinity-purified wild-type and T164S mutant sNS1 [blue box in (D)]. Data are presented as means ± SD from three independent experiments and were compared by unpaired two-tailed Student t test (*P < 0.05 and ***P < 0.001).

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/498/eaat7726/DC1

    Materials and Methods

    Fig. S1. Genome equivalent (GE), infectivity and plaque morphology of T164S mutant virus.

    Fig. S2. Infectivity of wild-type and T164S mutant virus in the mosquito vector A. aegypti.

    Fig. S3. Ex vivo human PBMC infection with wild-type or T164S mutant virus in the absence or presence of humanized 4G2 antibody.

    Fig. S4. Heat map expression profile of inflammation-associated genes after infection of human PBMCs with wild-type or T164S mutant virus.

    Fig. S5. Heat map expression profile of inflammation-associated genes after infection of human PBMCs with wild-type or T164S mutant virus in the presence of humanized 4G2 antibody.

    Fig. S6. Heat map expression profile of clustered inflammation-associated genes after infection of human PBMCs with wild-type or T164S mutant virus.

    Fig. S7. Heat map expression profile of clustered inflammation-associated genes after infection of human PBMCs with wild-type or T164S mutant virus in the presence of humanized 4G2 antibody.

    Fig. S8. Viremia and proinflammatory cytokine production in AG129 mice infected with DENV2 wild-type or T164S mutant virus.

    Fig. S9. Pairwise growth competition assay between wild-type and T164S mutant virus.

    Fig. S10. Proposed mechanism of the increased secretion of the T164S NS1 hexamer.

    Table S1. List of add-on genes in the NanoString Human Inflammation v2 codeset.

    Table S2. Mass spectrometry parameters used for determining the lipid composition of the NS1.

    Table S3. Summary of lipid species and their relative abundance extracted from immunoaffinity-purified wild-type or T164S mutant NS1 proteins.

    Data file S1. Individual-level data for characterization of the T164S mutation in NS1 of dengue virus.

    Data file S2. Individual-level data for Infection of human PBMCs ex vivo with wild-type or T164S mutant dengue virus in the absence or presence of humanized 4G2.

    Data file S3. Individual-level data for nonlethal infection of AG129 mice with wild-type or T164S mutant dengue virus.

    Data file S4. Individual-level data for lethal infection of AG129 mice with wild-type or T164S mutant dengue virus.

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Genome equivalent (GE), infectivity and plaque morphology of T164S mutant virus.
    • Fig. S2. Infectivity of wild-type and T164S mutant virus in the mosquito vector A. aegypti.
    • Fig. S3. Ex vivo human PBMC infection with wild-type or T164S mutant virus in the absence or presence of humanized 4G2 antibody.
    • Fig. S4. Heat map expression profile of inflammation-associated genes after infection of human PBMCs with wild-type or T164S mutant virus.
    • Fig. S5. Heat map expression profile of inflammation-associated genes after infection of human PBMCs with wild-type or T164S mutant virus in the presence of humanized 4G2 antibody.
    • Fig. S6. Heat map expression profile of clustered inflammation-associated genes after infection of human PBMCs with wild-type or T164S mutant virus.
    • Fig. S7. Heat map expression profile of clustered inflammation-associated genes after infection of human PBMCs with wild-type or T164S mutant virus in the presence of humanized 4G2 antibody.
    • Fig. S8. Viremia and proinflammatory cytokine production in AG129 mice infected with DENV2 wild-type or T164S mutant virus.
    • Fig. S9. Pairwise growth competition assay between wild-type and T164S mutant virus.
    • Fig. S10. Proposed mechanism of the increased secretion of the T164S NS1 hexamer.
    • Table S1. List of add-on genes in the NanoString Human Inflammation v2 codeset.
    • Table S2. Mass spectrometry parameters used for determining the lipid composition of the NS1.
    • Table S3. Summary of lipid species and their relative abundance extracted from immunoaffinity-purified wild-type or T164S mutant NS1 proteins.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Individual-level data for characterization of the T164S mutation in NS1 of dengue virus.
    • Data file S2 (Microsoft Excel format). Individual-level data for Infection of human PBMCs ex vivo with wild-type or T164S mutant dengue virus in the absence or presence of humanized 4G2.
    • Data file S3 (Microsoft Excel format). Individual-level data for nonlethal infection of AG129 mice with wild-type or T164S mutant dengue virus.
    • Data file S4 (Microsoft Excel format). Individual-level data for lethal infection of AG129 mice with wild-type or T164S mutant dengue virus.

Stay Connected to Science Translational Medicine

Navigate This Article