Mimicking immune signatures of flavivirus infection with targeted adjuvants improves dengue subunit vaccine immunogenicity.

Neutralizing antibodies (nAbs) are a critical component for protection against dengue virus (DENV) infection, but little is known about the immune mechanisms governing their induction and whether such mechanisms can be harnessed for vaccine development. In this study, we profiled the early immune responses to flaviviruses in human peripheral blood mononuclear cells and screened a panel of toll-like receptor (TLR) agonists that stimulate the same immune signatures. Monocyte/macrophage-driven inflammatory responses and interferon responses were characteristics of flavivirus infection and associated with induction of nAbs in humans immunized with the yellow fever vaccine YF-17D. The signatures were best reproduced by the combination of TLR agonists Pam3CSK4 and PolyI:C (PP). Immunization of both mice and macaques with a poorly immunogenic recombinant DENV-2 envelope domain III (EDIII) induced more consistent nAb and CD4+ T-cell responses with PP compared to alum plus monophosphoryl lipid A. Induction of nAbs by PP required interferon-mediated signals in macrophages in mice. However, EDIII + PP vaccination only provided partial protection against viral challenge. These results provide insights into mechanisms underlying nAb induction and a basis for further improving antigen/adjuvant combinations for dengue vaccine development

Discovery of Dengue Virus Host Factors

Ph.DDOCTOR OF PHILOSOPH

Correction: G3BP1, G3BP2 and CAPRIN1 Are Required for Translation of Interferon Stimulated mRNAs and Are Targeted by a Dengue Virus Non-coding RNA

<p>Correction: G3BP1, G3BP2 and CAPRIN1 Are Required for Translation of Interferon Stimulated mRNAs and Are Targeted by a Dengue Virus Non-coding RNA</p

G3BP1, G3BP2 and CAPRIN1 Are Required for Translation of Interferon Stimulated mRNAs and Are Targeted by a Dengue Virus Non-coding RNA

<div><p>Viral RNA-host protein interactions are critical for replication of flaviviruses, a genus of positive-strand RNA viruses comprising major vector-borne human pathogens including dengue viruses (DENV). We examined three conserved host RNA-binding proteins (RBPs) G3BP1, G3BP2 and CAPRIN1 in dengue virus (DENV-2) infection and found them to be novel regulators of the interferon (IFN) response against DENV-2. The three RBPs were required for the accumulation of the protein products of several interferon stimulated genes (ISGs), and for efficient translation of PKR and IFITM2 mRNAs. This identifies G3BP1, G3BP2 and CAPRIN1 as novel regulators of the antiviral state. Their antiviral activity was antagonized by the abundant DENV-2 non-coding subgenomic flaviviral RNA (sfRNA), which bound to G3BP1, G3BP2 and CAPRIN1, inhibited their activity and lead to profound inhibition of ISG mRNA translation. This work describes a new and unexpected level of regulation for interferon stimulated gene expression and presents the first mechanism of action for an sfRNA as a molecular sponge of anti-viral effectors in human cells.</p></div

DENV-2 3′UTR downregulates ISG protein expression through G3BP1, G3BP2 and CAPRIN1 binding.

<p>(A) Schematics of DENV-2 3′UTR RNAs synthesized to mimic sfRNAs: DENV-2 3′UTR WT, DENV-2 3′UTR YFSLE replaced SL-II/PKSLII with the equivalent structure in YFV-17D (YFV-17D SLE, see panel B) in the DENV-2 3′UTR background, and DENV-2 3′UTR YFSLE-ST4 which contains an additional four point mutations in the middle stem of SL-IV. The corresponding structures are shown in (B). Nucleotides implicated in pseudoknot formation are highlighted in red and substitutions in DENV-2 SL-IV in blue. (C) <i>In vitro</i> transcribed DENV-2 3′UTR RNAs were transfected into HuH-7 cells and their binding to G3BP1 was interrogated by G3BP1 IP. Results are presented as mean ± SEM of three independent experiments, normalized to control IgG IP. (D–G) HuH-7 cells were transfected with 5 ng, 50 ng or 500 ng of <i>in vitro</i> transcribed DENV-2 3′UTR or DENV-2 3′UTR YFSLE, and treated with 100 UI/ml IFN-β for 4 h. (D–E) Levels of viral 3′UTR and IFITM2 RNAs were measured by quantitative real-time PCR and normalized to intracellular GAPDH mRNA levels. (F–G) Levels of IFITM2 protein were determined by western blots and quantified by analysis of fluorescence intensity relative to GAPDH. All results are presented as mean ± SEM of three independent experiments in triplicate. One representative western blot used for IFITM2 protein quantification is shown.</p

G3BP1, G3BP2 and CAPRIN1 interact with DENV-2 gRNA and sfRNA in infected cells.

<p>(A) The DENV-2 NGC 3′UTR variable region (VR) is predicted to contain five stem-loops (SL-I to SL-V) and two highly conserved pseudoknots PKSL-II and PKSL-IV. Sequences shared by DENV-2 gRNA and sfRNA are highlighted in grey. To relatively quantify these two RNAs species, a differential real-time RT-PCR strategy was designed in which one primer pair, QG, detects DENV-2 gRNA only, while the other primer pair, QGSF, amplifies sequences shared by the gRNA and sfRNA. sfRNA levels are obtained by subtraction of absolute levels of amplicons obtained from both primer pairs calculated against a standard curve (for more details refer to <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004242#ppat.1004242.s007" target="_blank">Figure S7</a></b>). (B–C) HuH-7 cells were infected with DENV-2 at MOI = 1 for 24 h and binding of host RBPs to viral and cellular RNAs lysates was analyzed by RNA immunoprecipitation (IP). (B) A representative western blot shows robust enrichment of G3BP1 and KSRP in the specific IP. (C) Pellet fractions from IP with anti-G3BP1 or anti-KSRP antibodies were analyzed for DENV-2 gRNA and sfRNA as described above. Results are presented as mean ± SEM of the ratio of aforementioned RNAs over GAPDH mRNA in the pellet fraction, normalized to same value for control α-IgG IP. (D–E) RNAs containing a 5′ terminal binding aptamer were used to identify sequences required for G3BP1, G3BP2 and CAPRIN1 binding. RNA matrices DENV-2 3′UTR, the same deleted of SL-II (dSLII), or containing two point mutations in the terminal loop of SLII, which are predicted to disrupt PKSLII (D) were incubated with uninfected cell lysates and bound host RBPs eluted as previously described. The DENV-2 NS2A ORF was used as a negative control <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004242#ppat.1004242-Ward1" target="_blank">[17]</a>. The binding of TIAR (TIAL1), DDX6, G3BP1, G3BP2, or CAPRIN1 was interrogated using specific antibodies. DDX6, which was shown to bind DENV-2 DB region, was used as a positive control <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004242#ppat.1004242-Ward1" target="_blank">[17]</a>. TIAR, which was shown not to interact with DENV-2 positive strand RNA, was used as a negative control <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004242#ppat.1004242-Emara1" target="_blank">[64]</a>. One representative western blot (E) of three done is shown.</p

G3BP1, G3BP2 and CAPRIN1 depletion specifically inhibits translation of reporters under the control of ISG UTRs.

<p>(A) Schematic representation of IFN-stimulated response element (ISRE)-driven firefly luciferase reporters under the control of ELF2, GAPDH, IFITM2 or PKR UTRs. (B to I) HuH-7 cells stably transfected with the above constructs were treated with control siGFP or siG12C#1 siRNAs, induced with 1000 UI/ml of IFN-β for 10 h and firefly luciferase mRNA determined by quantitative real-time RT-PCR and normalized to GAPDH mRNA levels (B–E). Firefly luciferase protein levels were determined by measuring luciferase activity and normalized to total protein concentration (F–I). Both mRNA and protein activity are expressed as fold induction from control, untreated cells (siGFP, IFN-). Fluc measurements for the GAPDH-Fluc, IFITM2-Fluc and PKR-Fluc constructs were derived from 5 independent experiments in triplicate (n = 15). Fluc measurements for the ELF2-Fluc were derived from 3 independent experiments in triplicate (n = 9). All Fluc mRNA levels were measured in two of these independent experiments (n = 6).</p

G3BP1, G3BP2 and CAPRIN1 are required for IFN-β mediated antiviral activity against DENV-2.

<p>(A) HuH-7 cells were treated with siGFP or one of two independent sets of siRNAs (siG12C#1 and siG12C#2) targeting G3BP1, G3BP2 and CAPRIN1, and knockdown was confirmed by western blot analysis. (B) Indirect immunofluorescence was used to detect dsRNA-containing replication complexes (red) and CAPRIN1 (green) in HuH-7 cells treated with control siGFP or siRNA targeting G3BP1, G3BP2 and CAPRIN1 (siG12C#1) and pretreated or not with 100 UI/ml IFN-β before infection with DENV-2 at MOI = 1 for 24 h. The percentage of cells with a replication complex (i.e., infected cells) for each condition is indicated in the lower left corner of each image. (C and D) Cells treated with siGFP, siG12C#1 or siG12C#2 were incubated with increasing concentrations of IFN-β for 16 h before DENV-2 infection at MOI = 1. DENV-2 infectivity was determined at 24 h post-infection by indirect immunofluorescence (C) and DENV-2 infectious particles production (D). Asterisks indicate values below detection levels. (E and F) HuH-7 cells treated with control or siG12C#1 siRNAs, pretreated with IFN-β as above and infected with DENV-2 (E) or YFV-17D (F) at MOI = 1. Viral RNAs levels were determined at 24 h post-infection by quantitative real-time RT-PCR and normalized to intracellular GAPDH mRNA levels.</p

Model of the DENV-2 sfRNA antagonizing IFN action.

<p>IFNs are produced by infected cells and released in the extracellular space. Binding of IFN to IFNAR on neighboring naïve cells activates a cascade of signaling events leading to selective transcriptional activation of ISGs, which contain an interferon-sensitive response element (ISRE). Host RBPs G3BP1, G3BP2 and CAPRIN1 are required for translation of antiviral ISGs proteins and as a consequence for establishment of the antiviral state. In infected cells, high levels of non-coding sfRNA are produced and act as a RNA sponge, binding to host RNA-binding proteins. G3BP1, G3BP2 and CAPRIN1 bound to DENV-2 sfRNA are prevented to exert their activity in post-transcriptional regulation, leading to downregulation of ISGs, thus protecting DENV-2 replication against IFN antiviral effects.</p

Summary of mRNA induction, protein induction and translation efficiency for each Fluc reporter construct.

<p>Results presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004242#ppat-1004242-g004" target="_blank">Figure 4</a> were compiled and for each construct, the relative translation efficiency was calculated as the ratio of the average firefly luciferase activity induction (normalized to siGFP, untreated cells set as 1) over to the average firefly luciferase mRNA induction (normalized to siGFP, untreated cells set as 1). The fold difference between siGFP + IFN and siG12C +IFN is shown in bold in the fourth column. The p-values for the differences in mRNA and Fluc induction between siGFP + IFN-β and siG12C + IFN-β conditions are indicated: ns, non significant;</p><p>* p<0.05;</p><p>** p<0.01;</p><p>***p<0.005.</p