Zika Virus Antagonizes Type I Interferon Responses during Infection of Human Dendritic Cells

<div><p>Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that is causally linked to severe neonatal birth defects, including microcephaly, and is associated with Guillain-Barre syndrome in adults. Dendritic cells (DCs) are an important cell type during infection by multiple mosquito-borne flaviviruses, including dengue virus, West Nile virus, Japanese encephalitis virus, and yellow fever virus. Despite this, the interplay between ZIKV and DCs remains poorly defined. Here, we found human DCs supported productive infection by a contemporary Puerto Rican isolate with considerable variability in viral replication, but not viral binding, between DCs from different donors. Historic isolates from Africa and Asia also infected DCs with distinct viral replication kinetics between strains. African lineage viruses displayed more rapid replication kinetics and infection magnitude as compared to Asian lineage viruses, and uniquely induced cell death. Infection of DCs with both contemporary and historic ZIKV isolates led to minimal up-regulation of T cell co-stimulatory and MHC molecules, along with limited secretion of inflammatory cytokines. Inhibition of type I interferon (IFN) protein translation was observed during ZIKV infection, despite strong induction at the RNA transcript level and up-regulation of other host antiviral proteins. Treatment of human DCs with RIG-I agonist potently restricted ZIKV replication, while type I IFN had only modest effects. Mechanistically, we found all strains of ZIKV antagonized type I IFN-mediated phosphorylation of STAT1 and STAT2. Combined, our findings show that ZIKV subverts DC immunogenicity during infection, in part through evasion of type I IFN responses, but that the RLR signaling pathway is still capable of inducing an antiviral state, and therefore may serve as an antiviral therapeutic target.</p></div

ZIKV infection minimally activates human DCs.

<p><b>(A)</b> moDCs were left uninfected (“Mock”) or infected with PR-2015, P6-1966, MR-1947, or Dak-1984 at MOI of 1 (n = 6–8 donors). Cells were collected at 48hpi and labeled for ZIKV E protein and indicated DC activation markers. Cells were categorized as being viral E protein- or viral E protein+ and activation marker surface expression quantitated by flow cytometry. Values are represented as median fluorescence intensity (MFI) for each individual donor with uninfected and ZIKV infected samples from the same donor connected with a line. Statistical significance (p< 0.05) was determined using a Friedman test with comparisons made to donor-paired, uninfected cells. <b>(B)</b> moDCs infected with PR-2015 at MOI of 1 were stratified into “low” (n = 3 donors) and “high” (n = 5 donors) infection on the basis of viral E protein staining. MFIs are shown as the mean +/- SD. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006164#ppat.1006164.s003" target="_blank">S3 Fig</a>.</p

ZIKV infection induces an antiviral state within human DCs.

<p>moDCs were infected with ZIKV PR-2015, P6-1966, MR-1947, or Dak-1984 at MOI of 1 (n = 6–8 donors). Cells were collected at indicated hours post-infection and antiviral gene expression was determined by qRT-PCR. Gene expression was normalized to <i>GAPDH</i> transcript levels in each respective sample and represented as the averaged log₂ normalized fold increase above donor and time-point matched uninfected cells. The averaged log₁₀ normalized levels of infectious virus (FFU/mL) at each time point is depicted beneath the gene expression heat map. <b>(A)</b> RLR gene expression. <b>(B)</b> Antiviral effector gene expression. <b>(C)</b> moDCs were left untreated (“Mock”), treated with RIG-I agonist (10ng/1e5 cells), or infected with ZIKV PR-2015 (MOIs of 1 and 10) or MR-1947 (MOI 1). After 18hrs of agonist treatment or at 48hpi with ZIKV, whole-cell lysates were collected for western blot analysis of host antiviral effector protein expression. Western blots are shown for a single donor and are representative of data obtained from two donors. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006164#ppat.1006164.s005" target="_blank">S5 Fig</a>.</p

Differential infection of human DCs by evolutionarily distinct ZIKV strains.

<p>moDCs were infected with PR-2015, P6-1966, MR-1947, or Dak-1984 at MOI of 1 and assessed for viral replication at the indicated hours post-infection. <b>(A)</b> Infectious virus release into the supernatant was determined by FFA. Shown as the mean +/- SEM from 6–9 donors. <b>(B)</b> Infectious virus release for 6 of the individual donors summarized in panel A. <b>(C)</b> Percent infected cells assessed by ZIKV E protein staining and flow cytometry. Shown as the mean +/- SEM from 6–9 donors. <b>(D)</b> Percent infected cells in 6 of the individual donors summarized in panel C. <b>(E)</b> Cell viability of infected moDCs assessed by Ghost Red 780 (Tonbo) viability staining and flow cytometry. Shown as the mean +/- SEM from 6–9 donors. Statistical significance (p< 0.05) was determined using a two-way ANOVA with comparisons made to mock-infected cells. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006164#ppat.1006164.s007" target="_blank">S1 Table</a>.</p

Innate immune signaling restricts ZIKV viral replication within human DCs.

<p><b>(A)</b> moDCs were infected with PR-2015, P6-1966, MR-1947, or Dak-1984 at MOI of 1 (n = 4 donors). After viral attachment and entry at 1hpi, cells were treated with RIG-I agonist (10ng/1e5 cells), human IFNβ (100 IU/mL), or left untreated. <b>(B)</b> Supernatants were collected at 48hpi and assessed for infectious virus release by FFA. Values for each individual donor are shown with the mean +/- SD. Statistical significance (p< 0.05) was determined using a Friedman test with comparisons made to donor-paired, untreated, ZIKV-infected cells. The assay limit of detection is indicated with a dashed line.</p

Significant residue diversity between CO clades.

<p>Significant residue diversity between CO clades.</p

Maximum likelihood tree of CHIKV Asian genotype.

<p>The major nodes with bootstrap values above 70% are indicated. The CO clade, supported by bootstrap value 96.6%, is colored in red. Amino acid substitutions that contribute to clade diversity are mapped to the branches of the tree. CO-clade-specific amino acid substitutions are marked in red. Monophyletic groups within CO clade and associated amino acid changes are marked in blue. The scale bar represents genetic distance.</p

Phylogeographic clustering of Caribbean/Americas epidemic CHIKV strains.

<p>The Bayesian maximum clade credibility (MCC) tree of CHIKV Asian genotype was annotated to reflect the geographic origin of samples collected in the Americas. The CO clade was split into three parts, magnified, and indicated by gray shading: subclade CO1 (right side of the tree), subclade CO2 (left middle panel), and non-CO1/CO2 strains (left lower panel). Sequences from different regions are colored in branches as described in the key. The original countries of infections (either indigenous or travel-related) are depicted with colored ovals next to the complete strain names and described in the key as well.</p

Phylodynamics of CHIKV Asian genotype epidemics.

<p>Time-scaled maximum clade credibility (MCC) tree of the CHIKV Asian genotype was inferred by Bayesian analysis. Sequences from different regions are colored as described in the key. The Caribbean outbreak (CO) clade is supported by Bayesian posterior probabilities (BPP) as 1 in BMCMC analysis and bootstrap values 96% in the maximum likelihood phylogeny. Subclades CO1 and CO2, supported by BPP over 0.98, are also marked in the tree. A small group of Nicaraguan sequences that are relatively distinct from other Nicaraguan sequences are highlighted by a red hash symbol (#). The major ancestral geographic states (state probabilities over 0.5) at the backbone of phylogenetic tree are colored by region and labeled in the tree. The nodes with state probabilities greater than 0.9 are marked by asterisks. The mean and 95% highest posterior density (HPD) of the most recent common ancestor (tMRCA) of the entire Asian genotype and tMRCAs of virus movement between regions are also indicated. The scale bar represents chronological time (in years).</p

Maximum likelihood phylogenetic tree of complete coding nucleotide sequences of CHIKV.

<p>Well-supported nodes by bootstrap values over 70% are marked in the tree. Three genotypes (West African, ECSA, and Asian) and clades (Indian Ocean and Caribbean outbreaks) are described in the trees. New sequences sampled from the Americas are colored in red. The phylogenetic tree is midpoint-rooted, and the scale bar represents the number of nucleotide substitutions per site. Arrows indicate known recent travel of the human hosts to Africa or Asia in the ECSA genotype.</p