Coordination of Different Ligands to Copper(II) and Cobalt(III) Metal Centers Enhances Zika Virus and Dengue Virus Loads in Both Arthropod Cells and Human Keratinocytes

Trace elements such as copper and cobalt have been associated with virus-host interactions. However, studies to show the effect of conjugation of copper(II) or cobalt(III) metal centers to thiosemicarbazone ligand(s) derived from either food additives or mosquito repellent such as 2-acetylethiazole or citral, respectively, on Zika virus (ZIKV) or dengue virus (serotype 2; DENV2) infections have not been explored. In this study, we show that four compounds comprising of thiosemicarbazone ligand derived from 2-acetylethiazole viz., (E)-N-ethyl-2-[1-(thiazol-2-yl)ethylidene]hydrazinecarbothioamide (acetylethTSC) (compound 1), a copper(II) complex with acetylethTSC as a ligand (compound 2), a thiosemicarbazone ligand-derived from citral (compound 3) and a cobalt(III) complex with a citral-thiosemicarbazone ligand (compound 4) increased DENV2 and ZIKV replication in both mosquito C6/36 cells and human keratinocytes (HaCaT cells). Treatment of both cell lines with compounds 2 or 4 showed increased dengue viral titers at all three tested doses. Enhanced dengue viral plaque formation was also noted at the tested dose of 100 μM, suggesting higher production of infectious viral particles. Treatment with the compounds 2 or 4 enhanced ZIKV and DENV2 RNA levels in HeLa cell line and primary cultures of mouse bone marrow derived dendritic cells. Also, pre- or post treatments with conjugated compounds 2 or 4 showed higher loads of ZIKV or DENV2 envelope (E) protein in HaCaT cells. No changes in loads of E-protein were found in ZIKV-infected C6/36 cells, when compounds were treated after infection. In addition, we tested bis(1,10-phenanthroline)copper(II) chloride ([Cu(phen)2]Cl2, (compound 5) and tris(1,10-phenanthroline)cobalt(III) chloride ([Co(phen)3]Cl3, (compound 6) that also showed enhanced DENV2 loads. Also, we found that copper(II) chloride dehydrate (CuCl2·2H2O) or cobalt(II) chloride hexahydrate (CoCl2·6H2O) alone had no effects as “free” cations. Taken together, these findings suggest that use of Cu(II) or Co(III) conjugation to organic compounds, in insect repellents and/or food additives could enhance DENV2/ZIKV loads in human cells and perhaps induce pathogenesis in infected individuals or individuals pre-exposed to such conjugated complexes. Importance Mosquito-borne diseases are of great concern to the mankind. Use of chemicals/repellents against mosquito bites and transmission of microbes has been the topic of interest for many years. Here, we show that thiosemicarbazone ligand(s) derived from 2-acetylethiazole or citral or 1,10-phenanthroline upon conjugation with copper(II) or cobalt(III) metal centers enhances dengue virus (serotype 2; DENV2) and/or Zika virus (ZIKV) infections in mosquito, mouse and human cells. Enhanced ZIKV/DENV2 capsid mRNA or envelope protein loads were evident in mosquito cells and human keratinocytes, when treated with compounds before/after infections. Also, treatment with copper(II) or cobalt(III) conjugated compounds increased viral titers and number of plaque formations. These studies suggest that conjugation of compounds in repellents/essential oils/natural products/food additives with copper(II) or cobalt(III) metal centers may not be safe, especially in tropical and subtropical places, where several dengue infection cases and deaths are reported annually or in places with increased ZIKV caused microcephaly. © 2017 Elsevier B.V. All rights reserved

Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein

Citation: Londono-Renteria, B., Troupin, A., Conway, M. J., Vesely, D., Ledizet, M., Roundy, C. M., . . . Colpitts, T. M. (2015). Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein. Plos Pathogens, 11(10), 23. doi:10.1371/journal.ppat.1005202Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were >= 5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379), whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses

Inflammasome-activating nanoparticles as modular systems for optimizing vaccine efficacy,”

a b s t r a c t Innate immune system activation is a critical step in the initiation of an effective adaptive immune response; therefore, activation of a class of innate pathogen receptors called pattern recognition receptors (PRR) is a central feature of many adjuvant systems. It has recently been shown that one member of an intracellular PRR, the NLRP3 inflammasome, is activated by a number of classical adjuvants including alu- Inflammasome activation in vitro requires signaling of both the Toll-like receptor (TLR) and NLRP3 in antigen-presenting cells. Here we present a class of nanomaterials endowed with these two signals for rapid optimization of vaccine design. We constructed this system using a simple approach that incorporates lipopolysaccharides (LPS) onto the surface of nanoparticles constructed from a biocompatible polyester, poly(lactic-co-glycolic acid) (PLGA), loaded with antigen. We demonstrate that LPS-modified particles are preferentially internalized by dendritic cells compared to uncoated nanoparticles and the system, when administered to mice, elicits potent humoral and cellular immunity against a model antigen, ovalbumin. Wild-type macrophages pulsed with LPS-modified nanoparticles resulted in production of the proinflammatory cytokine IL-1␤ consistent with inflammasome activation. In comparison, NLRP3-deficient and caspase-1-deficient macrophages showed negligible production of IL-1␤. Furthermore, when endocytosis and lysosomal destabilization were inhibited, inflammasome activity was diminished, supporting the notion that nanoparticles rupture lysosomal compartments and behave as 'danger signals' [Hornung V, Bauernfeind F, Halle A, Samstad EO, Kono H, Rock KL, et al. Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nat Immunol 2008;9(August (8)):847-56]. The generality of this vaccination approach is tested by encapsulation of a recombinant West Nile envelope protein and demonstrated by protection against a murine model of West Nile encephalitis. The design of such an antigen delivery mechanism with the ability to stimulate two potent innate immune pathways represents a potent new approach to simultaneous antigen and adjuvant delivery

DENV infection optimally enhances CRVP379 expression.

<p>Aag2 cells were transfected with an insect expression vector encoding CRVP379 (AcCRVP379) or GFP (AcGFP) and A. CRVP379 expression was measured by qRT-PCR at 48 h post-transfection. B. Cells were infected with DENV (MOI of 1.0) at 48 h post-transfection and infection levels were measure by qRT-PCR at 24 hpi.</p

CRVP379 tandem affinity purification assay results.

<p>CRVP379 tandem affinity purification assay results.</p

Silencing select virally-up-regulated genes reduces DENV infection in mosquito cells.

<p>A. The mosquito genes listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005202#ppat.1005202.s001" target="_blank">S1 Fig</a> were knocked down in Aag2 cells using RNAi and the effects on DENV infection were analyzed. The genes that reduced infection below 60% of control are shown. Aag2 cells were infected with DENV (MOI of 1.0) 72 h post-knockdown and analyzed for infection by qRT-PCR 24h post-infection. Data is displayed as percent control infection (using scrambled siRNA). Both DENV infection and qRT-PCR analysis were done in triplicate, data is pooled and error bars indicate standard deviation. B. DENV infection increases CRVP379 in Aag2 cells over time. Aag2 cells were infected with DENV (MOI of 1.0) and infection was measured using qRT-PCR analysis at the timepoints indicated. P<0.01. C. Expression of CRVP379 during RNAi knockdown. CRVP379 siRNA was transfected into Aag2 cells and gene expression was analyzed by qRT-PCR. Samples were taken at 24, 48 and 72h post-knockdown. Expression after transfection of GFP control siRNA is also indicated. D. Reduction of CRVP379 reduces DENV infection over time. Either siRNA against CRVP379 or GFP was transfected into Aag2 cells and cells were infected with DENV (MOI of 1.0) at 72 h post-knockdown. Cells were analyzed for infection by qRT-PCR at the timepoints indicated. E. DENV infection increases CRVP379 expression during siRNA knockdown. Either siRNA against CRVP379 or GFP was transfected into Aag2 cells and cells were infected with DENV at 72 h post-knockdown. Gene expression was analyzed by qRT-PCR at the timepoints indicated. Data is expressed as the fold change in CRVP379 expression in cells with GFP siRNA versus cells with CRVP379 siRNA during DENV infection. B-E. Data is pooled from 6 separate experiments, error bars indicate standard deviation.</p

CRVP379 interacts with mosquito prohibitin during DENV infection.

<p>A. Prohibitin is required for DENV infection of <i>Aedes aegypti</i>. Mosquitoes were intra-thoracically injected with either dsRNA against the coding region of prohibitin (Prohdsrna) or dsRNA against GFP (gfpdsrna) as control. At 4 dpmi, mosquitoes were infected with DENV through blood feeding. At 4 dpi, midgut tissues were dissected and individually analyzed for gene expression with qRT-PCR analysis. Each data point represents one mosquito midgut. P<0.01. B. Aag2 cells were transfected with His-tagged CRVP379 and/or infected with DENV. At 48h post-infection, antibodies against the His tag were used to pull the His-tagged CRVP379 protein out of cell lysates. The precipitated solution was run on SDS-PAGE gel and Western blot analysis was performed using antibody against prohibitin. C. CRVP379 and prohibitin co-localize in mosquito cells during DENV infection. Aag2 cells were transfected with His-tagged CRVP379 and infected with DENV 48h post-transfection. Cells were fixed in 4% paraformaldehyde 24h post-infection and stained with antibodies against the His-tag (green) and against prohibitin (red). DAPI was used to localize the nucleus. Representative images are shown.</p

Antibodies against CRVP379 recognize native protein in <i>Ae</i>. <i>aegypti</i>.

<p>A. CRVP379 antisera binds recombinant protein. An SDS-PAGE gel was run using rCRVP379 and Western blot analysis was done using the CRVP379 antisera. B. Antibodies were made against mosquito CRVP379 and used to bind endogenous protein in the Aag2 cell line. Aag2 cells were infected with DENV and cell fixed 24 hpi for staining. A representative image is shown at 20X. C. CRVP379 antibodies bind endogenous CRVP379 in mosquito midguts. <i>Ae</i>. <i>aegypti</i> MG were dissected and fixed in 4% paraformaldehyde before staining. Both preimmune sera and sera from CRVP-379 injected mice were used for staining (green). Phalloidin594 was used to highlight midgut structure (red). D. Antisera against CRVP379 recognizes the CRVP379 protein in mosquito cells. Aag2 cells were transfected with an expression construct encoding His-tagged CRVP379 protein. Cells were fixed 48h post-transfection and stained with both CRVP379 antisera and antibody against the His tag. Secondary antibodies were used as indicated. E. CRVP379 is increased in both MG and SG of DENV-infected mosquitoes. Mosquitoes were either infected with DENV or mock solution and organs dissected at 1, 2 and 7 dpi. CRVP379 gene expression was analyzed by qRT-PCR analysis and is shown as ng CRVP379 normalized to actin. DENV was used at 10⁵ PFU/mL for infection in mosquitoes. F. CRVP379 antisera binds mosquito cells and tissues. ELISA analysis was done with Aag2 cell lysate, <i>Aedes aegypti</i> salivary gland extract (SGE) and <i>Aedes aegypti</i> extracted saliva. O.D. values at 450nm are presented on the graph.</p