The antiviral effect of <i>Hig</i> in JEV infection of <i>C</i>. <i>pipiens pallens</i>.

<p>(A-C) Inoculation of <i>Culex pipiens pallens Hig</i> (<i>CpHig</i>) dsRNA significantly decreased the <i>CpHig</i> expression in the whole mosquitoes and heads of <i>C</i>. <i>pipiens pallens</i> at both the mRNA (A and B) and protein (C) levels. The <i>CpHig</i> abundance was assessed by SYBR Green qPCR (A and B) and immuno-blotting with an AaHig antibody (C) at 6 days post dsRNA microinjection. (D-G) Silencing <i>CpHig</i> enhanced JEV infection in <i>C</i>. <i>pipiens pallens</i>. 10 M.I.D.₅₀ JEV were inoculated at 3 days post <i>CpHig</i> dsRNA inoculation. The viral load of whole bodies (D and E) and heads (F and G) was assessed at 3 days and 6 days post-infection via Taqman qPCR and normalized with <i>Culex actin</i>. (H-K) Immuno-blockade of CpHig enhanced the JEV replication in <i>C</i>. <i>pipiens pallens</i>. The murine AaHig antibody, crossreacting with CpHig, was premixed with 10 M.I.D.₅₀ JEV to co-inoculate into the <i>Culex</i> mosquitoes thorax. The treated mosquitoes were sacrificed to examine the viral load in the mosquito bodies (H and J) and heads (I and K) at 3 and 6 days post-infection by TaqMan qPCR and normalized against <i>Culex actin</i>. (D-K) The primers and probes of Taqman qPCR were described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004848#ppat.1004848.s022" target="_blank">S1 Table</a>. The experiments were repeated 3 times with similar results. One dot represents 1 mosquito and the horizontal line represents the median of the results. The data were statistically analyzed by the non-parametric <i>Mann-Whitney</i> test.</p

A Neuron-Specific Antiviral Mechanism Prevents Lethal Flaviviral Infection of Mosquitoes

<div><p>Mosquitoes are natural vectors for many etiologic agents of human viral diseases. Mosquito-borne flaviviruses can persistently infect the mosquito central nervous system without causing dramatic pathology or influencing the mosquito behavior and lifespan. The mechanism by which the mosquito nervous system resists flaviviral infection is still largely unknown. Here we report that an <i>Aedes aegypti</i> homologue of the neural factor <i>Hikaru genki</i> (<i>AaHig</i>) efficiently restricts flavivirus infection of the central nervous system. <i>AaHig</i> was predominantly expressed in the mosquito nervous system and localized to the plasma membrane of neural cells. Functional blockade of AaHig enhanced Dengue virus (DENV) and Japanese encephalitis virus (JEV), but not Sindbis virus (SINV), replication in mosquito heads and consequently caused neural apoptosis and a dramatic reduction in the mosquito lifespan. Consistently, delivery of recombinant AaHig to mosquitoes reduced viral infection. Furthermore, the membrane-localized AaHig directly interfaced with a highly conserved motif in the surface envelope proteins of DENV and JEV, and consequently interrupted endocytic viral entry into mosquito cells. Loss of either plasma membrane targeting or virion-binding ability rendered AaHig nonfunctional. Interestingly, <i>Culex pipien pallens</i> Hig also demonstrated a prominent anti-flavivirus activity, suggesting a functionally conserved function for Hig. Our results demonstrate that an evolutionarily conserved antiviral mechanism prevents lethal flaviviral infection of the central nervous system in mosquitoes, and thus may facilitate flaviviral transmission in nature.</p></div

AaHig directly recognizes dengue virus.

<p>(A) Expression and purification of AaHig from <i>Drosophila</i> S2 cells. The full length <i>AaHig</i> was cloned into the pMT/BiP/V5-His A expression vector. The recombinant plasmid was transfected into <i>Drosophila</i> S2 cells, and the expression was probed using an anti-V5 mAb. The supernatant from mock-transfected S2 cells was used as the mock control (Right Panel). Recombinant AaHig protein, produced in <i>Drosophila</i> cells, was purified using a Ni-His column (Left Panel). (B) AaHig interacted with DENV-2 E protein in a co-immunoprecipitation (co-IP) assay. The purified AaHig (V5) and DENV-2 E (FLAG) proteins were used to investigate the interaction of the proteins. We reproduced the experiment 3 times. (C) AaHig captured DENV-2 virions in an ELISA assay. The binding was probed using the flavivirus E mAb 4G2. The data were presented as the mean ± standard error. The experiment was reproduced 3 times. (D) AaHig interfaced with DENV virions in the infected mosquito cells. pAc-AaHig was transfected in Aag2 cells, and subsequently the cells were infected by 5 M.O.I. DENV-2 at 12 hrs post transfection. The uninfected cells transfected by pAc-AaHig were used as a mock control. After 48 hrs infection, the cells were lysated and an anti-flaviviral E 4G2 mAb was added into the lysate for the pull-down assay. We reproduced the experiment 3 times. (E) The co-staining between AaHig and DENV-2 in the <i>A</i>. <i>aegypti</i> brain. The mosquito brains were dissected from uninfected mocks and infected mosquitoes at 6 days post infection to undergo immunofluorescence staining. AaHig was stained with anti-mouse IgG Alexa-546 (Red), and the DENV-2 E protein was stained using anti-human IgG Alexa-488 (Green). Nuclei were stained with To-Pro-3 iodide (Blue). Images were examined using the 63×objective lens of a Zeiss LSM 780 meta confocal. (F) The expression of ectodomains in the DENV-2 E protein. The genes of ED1+ED2 (1-296AA) and ED3 (297-400AA) of DENV-2 E protein were cloned into pMT/BiP/V5-His A vector and the encoded peptides were expressed in <i>Drosophila</i> S2 cells. The supernatant from empty vector-transfected S2 cells was used as a mock. The recombinant peptides were detected with an anti-FLAG antibody via western blotting. (G) The ectodomains of DENV-2 E protein interacted with AaHig in a co-IP assay. The protein complex was pulled down with an anti-FLAG antibody and detected using an anti-V5-HRP antibody. We reproduced the experiment 3 times. (H) The ectodomains of DENV-2 E protein interfaced with AaHig by an ELISA assay. The binding was probed using an anti-V5-HRP antibody. The data were presented as the mean ± standard error. The experiment was reproduced 3 times. (I) The interaction between AaHig and the linear motifs of ED1/ED2. The linear motifs of ED1 and ED2 were sequentially deleted from the ectodomains. The five truncations were cloned into the pMT/BiP/V5-His A vector, and subsequently expressed in the S2 cell supernatant. The protein complex was pulled down with an anti-FLAG antibody and detected using an anti-V5-HRP antibody. We reproduced the experiment 3 times. (J) Inoculation of AaHig impaired DENV-2 infectivity in <i>A</i>. <i>aegypti</i>. Two laddered concentrations of purified AaHig protein were premixed with 10 M.I.D.₅₀ of DENV-2 for mosquito thoracic microinjection. The infected mosquitoes were sacrificed at 3 (i) and 6 (ii) days post infection. The viral load was determined by Taqman qPCR and normalized by <i>A</i>. <i>aegypti actin</i>. The results were combined from 3 independent experiments. One dot represents 1 mosquito and the horizontal line represents the median value in the figures. The statistical analysis was done with the <i>Mann-Whitney</i> test.</p

The high expression of AaHig in the brain of <i>A</i>. <i>aegypti</i>.

<p>(A-B) The expression of <i>AaHig</i> in various tissues of <i>A</i>. <i>aegypti</i>. The abundance of AaHig was assessed via SYBR Green qPCR (A) and immuno-blotting with an AaHig antibody (B). (A) The total RNA was isolated from mosquito tissues to determine <i>AaHig</i> expression by SYBR Green qPCR and normalized by <i>A</i>. <i>aegypti actin</i> (<i>AAEL011197</i>). The qPCR primers were described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004848#ppat.1004848.s022" target="_blank">S1 Table</a>. Data were represented as the mean ± standard error. (B) A variety of tissues were dissected from female <i>A</i>. <i>aegypti</i>. 50 μg of total protein from tissue lysates was loaded into each lane. The detection of <i>A</i>. <i>aegypti</i> actin was used as the internal control. (C) Secretory property of AaHig. The full length of AaHig gene (1bp-2436bp) was inserted into the expression vector pAc5.1/V5-His A with a V5 tag. The recombinant DNA plasmid (pAc-AaHig) was transfected into S2 cells. AaHig expression was detected with anti-V5 antibody in the cell lysate and supernatant. (D) AaHig staining in <i>A</i>. <i>aegypti</i> Aag2 cells. Aag2 is an <i>A</i>. <i>aegypti</i> cell lineage of embryonic origin. The pAc-AaHig recombinant plasmid was transfected into Aag2 cells. AaHig was stained by anti-V5 antibody and anti-mouse IgG Alexa-546 (Red). The plasma membrane was stained by the Wheat Germ Agglutinin (WGA) conjugated with Alexa-488 (Green). Nuclei were stained with To-Pro-3 iodide (Blue). Images were examined using the 63×objective lens of a Zeiss LSM 780 meta confocal. (E) AaHig is localized on the cell surface of neural cells of the <i>A</i>. <i>aegypti</i> brain. The brains were dissected from female <i>A</i>. <i>aegypti</i> for the <i>in situ</i> staining. An anti-HRP rabbit polyclonal antibody with anti-rabbit IgG Alexa-488 was used for surface staining of mosquito neural cells (Green). The AaHig protein was detected by an anti-AaHig mouse polyclonal antibody and anti-mouse IgG Alexa-546 (Red). Nuclei were stained blue with To-Pro-3 iodide. Images were examined using the 10× and 63×objective lens of a Zeiss LSM 780 meta confocal. AL, antennal lobes; OL, optic lobes.</p

AaHig interrupts flaviviral endocytosis into mosquito cells.

<p>(A-B) Viral attachment assay. The serial concentration of purified AaHig protein was premixed with 5 M.O.I. DENV-2 on ice, and then the Aag2 (A) and C6/36 (B) cells were consequently preadsorbed with the mixture for a time course at 4oC. The cells were washed 5 times by cold PBS and collected at certain time points for total RNA isolation. (C-D) Viral entry assay. The serial concentration of purified AaHig protein was premixed with 5 M.O.I. DENV-2 on ice, and then the mixture was transferred into the cultured Aag2 cells (C) at 28°C and C6/36 (D) cells at 30°C respectively. The cells were stringently washed 5 times by PBS at room temperature, and then collected at serial time points for detection. (A-D) For the assay at 48 hours, the cells were washed 5 times after 1 hr incubation at 4°C (A and B) or 28°C /30°C (C and D), and consequently cultured at 28°C (Aag2) or 30°C (C6/36) for an additional 48 hrs. The viral genome was determined by Taqman qPCR and normalized by <i>A</i>. <i>aegypti actin</i>. The data were presented as the mean ± standard error. The result was combined from 3 independent experiments. *, <i>p<0</i>.<i>05</i>; **, <i>p<0</i>.<i>01</i>; ***, <i>p<0</i>.<i>001</i>. (E) Detection of AaHig-mediated entry interruption by structured illumination microscopy imaging. The Aag2 cells were seeded in a class bottle cell culture dish. The purified AaHig protein premixed with 10 M.O.I. DENV-2 was added into the cells. The equal amount of BSA was used as a negative control. The treated cells were collected and analyzed at a time course. DENV-2 was stained by the 4G2 mouse mAb and anti-mouse IgG Alexa Fluor-488 (Green). The early endosome marker, Rab5, was stained by a rabbit polyclonal antibody and anti-rabbit IgG Alexa Fluor-546 (Red). Images were examined using the 100×objective lens of a Zeiss ELYRA PS.1 structured illumination microscopy.</p

Bioinformatic comparison and phylogenetic analysis of <i>Hikaru genki</i> (<i>Hig</i>) in insects.

<p>(A) Percentage of amino acid identity between AaHig and <i>Drosophila melanogaster</i> Hig (DmHig). (B) Schematic representation of AaHig and DmHig. The functional modules were predicted using the SMART (<a href="http://smart.embl-heidelberg.de/smart/set_mode.cgi?GENOMIC=1" target="_blank">http://smart.embl-heidelberg.de/smart/set_mode.cgi?GENOMIC=1</a>) and Pfam (<a href="http://pfam.sanger.ac.uk/" target="_blank">http://pfam.sanger.ac.uk/</a>) websites. (C) Unrooted phylogenetic tree of insect Higs. The tree was constructed using the neighbour-joining (NJ) method based on the alignment of insect Hig protein sequences. The bootstrap values of 5000 replicates are indicated on the branch nodes. <i>Drosophila</i> spp. (<i>Dr</i>.); <i>Ceratitis</i> spp. (<i>Ct</i>.); <i>Musca</i> spp. (<i>Mu</i>); <i>Anopheles</i> spp. (<i>An</i>.); <i>Aedes</i> spp. (<i>Ae</i>.); <i>Culex</i> spp. (<i>Cx</i>); <i>Tribolium</i> spp. (<i>Tr</i>.); <i>Dendroctonus</i> spp. (<i>De</i>.); <i>Nasonia</i> spp. (<i>Na</i>.); <i>Cerapachys</i> spp. (<i>Cp</i>.); <i>Camponotus</i> spp. (<i>Ca</i>.); <i>Acromyrmex</i> spp. (<i>Ac</i>.); <i>Solenopsis</i> spp. (<i>So</i>.); <i>Megachile</i> spp. (<i>Me</i>.); <i>Bombus spp</i>. (<i>Bu</i>.); <i>Apis spp</i>. (<i>Ap</i>.); <i>Danaus spp</i>. (<i>Da</i>.); <i>Bombyx spp</i>. (<i>By</i>.).</p

The role of <i>AaHig</i> in flaviviruses infection of <i>A</i>. <i>aegypti</i>.

<p>Immuno-blockade of AaHig enhanced the DENV-2 (A-B) and JEV (C-D) infections in the whole bodies (i) and heads (ii) of mosquitoes. The murine AaHig antibody, in the 10-fold serial dilutions, was premixed with 10 M.I.D.₅₀ viruses to co-microinject into the thorax of mosquitoes. The treated mosquitoes were sacrificed to examine the viral load in the whole mosquito bodies (i) and heads (ii) at 3 (A, C) and 6 (B, D) days post-infection by TaqMan qPCR and normalized against <i>A</i>. <i>aegypti actin</i>. The results were reproduced by 3 times. One dot represents 1 mosquito/head and the horizontal line represents the median of the results. The data were analyzed statistically using the non-parametric <i>Mann-Whitney</i> test.</p

Both the viral interaction and membrane bound of AaHig are essential for the AaHig antiviral activity.

<p>(A-B) Construction and expression of recombinant AaHig truncations. The schematic representation of AaHig truncations with sequential depletion of modules is shown in the panel A. The fragments of <i>AaHig</i> truncated genes were constructed into pAc5.1/V5-His A vector to express in <i>Drosophila</i> S2 cells (pAc-AaHig-A ~ pAc-AaHig-F). The S2 cells transfected by the empty vector were used as a mock control. The expression in the supernatant was detected by western blotting with anti-V5 mAb (B). (C) The interaction between AaHig truncated peptides and the DENV-2 E protein in a co-IP assay. The cell supernatant was individually incubated with purified DENV-2 E protein. The supernatant from the pAc-GFP transfected cells was used as a mock control. The protein complex was pulled down with an anti-V5 antibody and probed using an anti-FLAG-HRP antibody. The experiment was reproduced 3 times. (D) The detection of binding capacity between AaHig fragments and DENV-2 E protein by ELISA. The supernatant of pAc-AaHigs and pAc-GFP transfected cells was used to assess the binding activity with DENV-2 E proteins. The binding was probed using the anti-V5 mAb. The data were presented as the mean ± standard error. The experiment was repeated 3 times with the similar result. (E) AaHig resists DENV-2 infection in <i>A</i>. <i>aegypti</i> Aag2 cells. The pAc-AaHig-Full recombinant plasmid was transfected into Aag2 cells. The pAc-GFP was used as negative control. After 48 hrs, the 0.01 M.O.I. DENV-2 was added into the cells and the viral load was determined by Taqman qPCR and normalized by <i>A</i>. <i>aegypti actin</i>. The data were presented as the mean ± standard error. The result was combined from 3 independent experiments. (F) The role of AaHig truncated peptides in DENV-2 infection of Aag2 cells. A variety of AaHig truncations were expressed in Aag2 cells. After 0.01 M.O.I. DENV-2 infection, the viruses were determined by Taqman qPCR and normalized against <i>A</i>. <i>aegypti actin</i>. The data were presented as the mean ± standard error. The result was combined from 2 independent experiments. (G) The schematic representation of AaHig-G truncation. (H) The interaction between AaHig-G peptide and the DENV-2 E protein in a co-IP assay. The gene of <i>AaHig-G</i> was cloned into pMT/BiP/V5-His A and expressed in S2 cells. The cell supernatant with AaHig-G was individually incubated with purified DENV-2 E protein. The supernatant from the pMT/BiP/V5-His A vector transfected cells was used as a mock control. The transfection of pMT-AaHig-Full served as a positive control. The protein complex was pulled down with an anti-V5 antibody and probed using an anti-FLAG-HRP antibody. The experiment was reproduced 3 times. (I) The role of AaHig-G in DENV-2 infection of Aag2 cells. Both <i>AaHig-G</i> and <i>AaHig-Full</i> cloned in pMT/BiP/V5-His A vector were ectopically expressed in S2 cells. The conditional medium premixed with 0.01 M.O.I. DENV-2 was added into mosquito Aag2 cells for infection. The infectivity were determined by Taqman qPCR and normalized against <i>A</i>. <i>aegypti actin</i>. The data were presented as the mean ± standard error. The result was combined from 3 independent experiments.</p

Complement-Related Proteins Control the Flavivirus Infection of <i>Aedes aegypti</i> by Inducing Antimicrobial Peptides

<div><p>The complement system functions during the early phase of infection and directly mediates pathogen elimination. The recent identification of complement-like factors in arthropods indicates that this system shares common ancestry in vertebrates and invertebrates as an immune defense mechanism. Thioester (TE)-containing proteins (TEPs), which show high similarity to mammalian complement C3, are thought to play a key role in innate immunity in arthropods. Herein, we report that a viral recognition cascade composed of two complement-related proteins limits the flaviviral infection of <i>Aedes aegypti</i>. An <i>A. aegypti</i> macroglobulin complement-related factor (AaMCR), belonging to the insect TEP family, is a crucial effector in opposing the flaviviral infection of <i>A. aegypti</i>. However, AaMCR does not directly interact with DENV, and its antiviral effect requires an <i>A. aegypti</i> homologue of scavenger receptor-C (AaSR-C), which interacts with DENV and AaMCR simultaneously <i>in vitro</i> and <i>in vivo</i>. Furthermore, recognition of DENV by the AaSR-C/AaMCR axis regulates the expression of antimicrobial peptides (AMPs), which exerts potent anti-DENV activity. Our results both demonstrate the existence of a viral recognition pathway that controls the flaviviral infection by inducing AMPs and offer insights into a previously unappreciated antiviral function of the complement-like system in arthropods.</p></div

Regulation of antimicrobial peptides abundance in DENV-2 infection of <i>A. aegypti</i>.

<p>Regulation of antimicrobial peptides abundance in DENV-2 infection of <i>A. aegypti</i>.</p