doi: 10.1371/journal.ppat.1005202.
eCollection 2015 Oct.
Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein
Affiliations
- PMID: 26491875
- PMCID: PMC4619585
- DOI: 10.1371/journal.ppat.1005202
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Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein
Berlin Londono-Renteria et al.
PLoS Pathog.
.
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Abstract
Dengue 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.
Conflict of interest statement
DVe and ML are employed by L2 Diagnostics (CT). The other authors have declared that no competing interests exist. This does not alter our adherence to all PLOS Pathogens policies on sharing data and materials.
Figures
A. The mosquito genes listed in S1 Fig 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.
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.
A-E. Mosquitoes were intra-thoracically injected with either dsRNA against the coding region of CRVP379 or dsRNA against GFP 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. A. Levels of CRVP379 in select midguts where RNAi was successful, as compared to levels in control mosquitoes. P<0.01. B. Levels of DENV infection in select midguts where RNAi was successful, as compared to levels in control mosquitoes. P<0.01. Infection rates in midguts where CRVP379 RNAi was successful ranged from to .000000765–.0000315 ng DENV E/ng actin. C. Levels of CRVP379 in midguts where RNAi was both successful and unsuccessful, as compared to levels in control mosquitoes. D. Levels of DENV infection in midguts where RNAi was both successful and unsuccessful, as compared to levels in control mosquitoes. C-D. Squares represent midguts where RNAi did not knock down CRVP379 successfully, cirlces represent midguts where RNAi did knock down CRVP379 successfully. E. Levels of DENV infection correspond to levels of CRVP379 expression. Both midguts where RNAi did and did not knock down CRVP379 were analyzed for both CRVP379 expression and DENV infection by qRT-PCR. Data is plotted as ngs of DENV E versus levels of CRVP379, normalized to mosquito actin. Data correlated with Pearson, r = 0.6442, P<0.0001 F. Silencing CRVP379 reduces DENV infection in whole mosquitoes. Mosquitoes were intra-thoracically injected with either dsRNA against the coding region of CRVP379 or dsRNA against GFP as control. At 4 dpmi, mosquitoes were infected with DENV through blood feeding. At 7 dpi, homogenized whole mosquitoes were individually analyzed for gene expression with qRT-PCR analysis.
A. Prohibitin is required for DENV infection of Aedes aegypti. 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.
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. Ae. aegypti 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, Aedes aegypti salivary gland extract (SGE) and Aedes aegypti extracted saliva. O.D. values at 450nm are presented on the graph.
A/B. CRVP379 antisera inhibits DENV infection in mosquito cells. Aag2 (A) or Huh7 (B) cells were either incubated with antisera against CRVP379 or control preimmune sera for 2h at RT and then infected with DENV (pretreatment group) or antisera against CRVP379 or control preimmune sera was incubated with DENV for 1h at RT and then added to cells (simultaneous group). Infection was analyzed by qRT-PCR at 24 hpi. C/D. CRVP379 antisera inhibits DENV infection in mosquitoes. Ae. aegypti were fed a mixture of blood, DENV and either CRVP379 antisera or preimmune sera as indicated. Antisera were used at dilutions of 1/10 or 1/100 (C). Infection rates in midguts with CRVP379 antisera ranged from to .00008785–.07833 ng DENV E/ng actin. A separate group of mosquitoes was fed antisera against control mosquito proteins, MMP and PC (D). At 3 dpi, mosquito MG were dissected and qRT-PCR analysis done to quantify DENV infection. Results are shown as ng DENV E normalized to mosquito actin. Each data point represents one MG.
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