Juvenile Hormone-Sensitive Ribosomal Activity Enhances Viral Replication in Aedes aegypti

Abstract

Zika virus (ZIKV; Flaviviridae) is a devastating virus transmitted to humans by the mosquito Aedes aegypti. The interaction of the virus with the mosquito vector is poorly known. The double-stranded RNA (dsRNA)-mediated interruption or activation of immunity-related genes in the Toll, IMD, JAK-STAT, and short interfering RNA (siRNA) pathways did not affect ZIKV infection in A. aegypti. Transcriptome-based analysis indicated that most immunity-related genes were upregulated in response to ZIKV infection, including leucine-rich immune protein (LRIM) genes. Further, there was a significant increment in the ZIKV load in LRIM9-, LRIM10A-, and LIRM10B-silenced A. aegypti, suggesting their function in modulating viral infection. Further, gene function enrichment analysis revealed that viral infection increased global ribosomal activity. Silencing of RpL23 and RpL27, two ribosomal large subunit genes, increased mosquito resistance to ZIKV infection. In vitro fat body culture assay revealed that the expression of RpL23 and RpL27 was responsive to the Juvenile hormone (JH) signaling pathway. These two genes were transcriptionally regulated by JH and its receptor methoprene-tolerant (Met) complex. Silencing of Met also inhibited ZIKV infection in A. aegypti. This suggests that ZIKV enhances ribosomal activity through JH regulation to promote infection in mosquitoes. Together, these data reveal A. aegypti immune responses to ZIKV and suggest a control strategy that reduces ZIKV transmission by modulating host factors. IMPORTANCE Most flaviviruses are transmitted between hosts by arthropod vectors such as mosquitoes. Since therapeutics or vaccines are lacking for most mosquito-borne diseases, reducing the mosquito vector competence is an effective way to decrease disease burden. We used high-throughput sequencing technology to study the interaction between mosquito Aedes aegypti and ZIKV. Leucine-rich immune protein (LRIM) genes were involved in the defense in response to viral infection. In addition, RNA interference (RNAi) silencing of RpL23 and RpL27, two JH-regulated ribosomal large subunit genes, suppressed ZIKV infection in A. aegypti. These results suggest a novel control strategy that could block the transmission of ZIKV.

Keywords: LRIM; antiviral; juvenile hormone; mosquito; ribosome.

FIG 1

Toll, JAK-STAT, IMD, and RNAi pathways do not affect ZIKV infectivity in A. aegypti. (A) Merged list of selected genes for RNAi screening research. The RNAi efficiency of 5 to 10 mosquitoes was measured at 7 dpi, shown as means from 2 to 5 replicates. (B to E) dsRNA was microinjected into the thorax of 1-day-old female mosquitoes. After a 3-day recovery period, these females were fed blood meal containing 1.0 × 10⁶ PFU/ml ZIKV. At 7 dpi, total RNA of a single mosquito was isolated, and the viral load was determined by qPCR. The viral load was normalized against the A. aegypti actin gene. EGFP dsRNA-treated mosquitoes were the control group. The top of each column shows the ratio of the number of infected mosquitoes to the total number of mosquitoes. Data are means ± SEM. The result shown is pooled from two independent experiments. A Mann-Whitney test was used for the statistical analysis. ns, not significant.

FIG 2

Immunity-related gene expression in the midgut and fat body are distinct. (A) Heatmap analysis of immunity-related DEGs following viral infection. According to their functions, immune genes were divided into three categories: recognition molecules, signaling regulators, and immune effectors. The FPKM values from three replicates were used to plot the heatmap. (B) Venn diagram analysis of immunity-related DEGs that are regulated by ZIKV infection in two tissues. The direction of gene changes is indicated by up and down arrows. Mg, midgut; Fb, fat body. (C) LRIMs are involved in the anti-ZIKV defense. After gene silencing, female A. aegypti organisms were fed blood meal containing 1.0 × 10⁶ PFU/ml ZIKV. At 7 dpi, the ZIKV viral load was tested by qPCR and normalized against the A. aegypti actin gene. EGFP dsRNA-treated females were the control group. One dot represents 1 mosquito, and the number of mosquitoes used for testing is shown in brackets. The experiment was repeated three times with similar results. Data are means ± SEM, and the P value was determined by a Mann-Whitney test. *, P < 0.05; **, P < 0.01.

FIG 3

Functional classification of the different expression genes. (A) GO enrichment analysis of midgut transcripts significantly regulated by ZIKV infection. The GO terms corresponding to biological process were analyzed. The GO terms most associated with the upregulated and downregulated enriched genes are shown. Statistical significance was determined at the false discovery rate. *, adjusted P value (P_adj), <0.05; **, P_adj < 0.01; ns, not significant. (B) KEGG functional classification of midgut and fat body transcripts significantly regulated by ZIKV infection. The KEGG pathways most associated with the upregulated and downregulated enriched genes are shown in the red box (P_adj < 0.05) or in the red box with an asterisk (P_adj < 0.01). Statistical significance was determined at the false discovery rate. Up, upregulated genes; dn, downregulated genes.

FIG 4

Global ribosome genes are induced after early infection. (A and B) The upregulated ribosomal protein transcripts from 1-dpi midgut and 7-dpi fat body were collected for analysis. (A) Heatmap analysis revealed the induction of ribosomal protein transcripts after viral infection. The corresponding 7-dpi midgut transcripts were the control. The log₂ ratio (read number in the virus-infected group/read number in the mock group) was used to plot the heatmap. Mg, midgut; Fb, fat body. (B) Venn diagram analysis of ribosomal protein genes significantly induced by ZIKV infection. Mg_1 d_up, upregulated genes from 1-dpi midgut; Fb_7 d_up, upregulated genes from 7-dpi fat body. (C) qPCR analysis of selected ribosome-related genes. Unpaired Student’s t tests were used for statistical analysis. The result shown is the mean ± SEM from three independent experiments. *, P < 0.05; **, P < 0.01.

FIG 5

Ribosomal component proteins RpL23 and RpL27 are critical for ZIKV infection. (A to E) After gene silencing, female A. aegypti was fed blood meal containing 1.0 × 10⁶ PFU/ml ZIKV. EGFP dsRNA-treated mosquitoes were the control group. (A) The role of translation-related genes in ZIKV infection. At 7 dpi, the viral load of a single mosquito was tested by qPCR and normalized against the A. aegypti actin gene. The top of each column shows the ratio of the number of infected mosquitoes to the total number of mosquitoes. Data are means ± SEM. The result shown is representative of three independent experiments. P value was determined by a Mann-Whitney test. ***, P < 0.001; ns, not significant. (B to E) Silencing A. aegypti RpL23 or RpL27 impairs ZIKV infection. (B and C) Thirty midguts (B) and 60 salivary glands (C) were pooled for viral load detection by qPCR, as mentioned above. Data are mean ± SEM from three independent experiments. Unpaired Student’s t tests were used for statistical analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D and E) At 7 dpi, midguts (D) and salivary glands (E) of mosquitoes were dissected, and the viral infection was detected by immunofluorescence assay using anti-E 4G2 antibody (red). Nuclei were stained by Hoechst 33258 (blue). The bar shows 200 μm. The result shown is representative of two independent experiments.

FIG 6

Ribosomal protein genes are potentially responsive to juvenile hormones. (A) Venn diagram comparing the upregulated genes in 7-dpi fat body transcriptome with the LPE gene cluster. LPE, late posteclosion. (B) Functional classification of 196 shared genes from panel A using the inNOG database.

FIG 7

JH-receptor complex regulates the transcription of RpL23 and RpL27. (A to D) Effects of JH and Met RNAi knockdown (iMet) on the expression of RpL23 and RpL27 in cultured fat bodies in vitro. The fat body isolated from female A. aegypti was cultured for 8 h under different treatment conditions. Kr-h1 (AAEL002390) was the positive control. JH, medium containing 10 μM JH; acetone, medium containing acetone. The expression level was normalized against the A. aegypti actin gene. The result shown is the mean ± SEM from three biological replicates. Unpaired Student’s t tests were used for statistical analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (E) Dual luciferase reporter assay. Aag2 cells were cotransfected with pGL4.10/RpL23 −2287 to −721, together with either a pAc5.1b empty vector or an expression vector for pAc5.1/Met-V5 or pAc5.1/Tai-V5 as indicated. At 42 h posttransfection, 20 μM JH was added to the wells as indicated. The expression of Met-V5 and/or Tai-V5 in Aag2 cells was confirmed by Western blots. GAPDH was the loading control. The columns with the letters a, b, and c show significantly different groups (P < 0.05, one-way ANOVA). (F) EMSA confirmed the binding of JH-Met-Tai complex to the RpL23 probe. The DNA probe (5′-CTCAAAGGAACACGCGATTGGAGGCT-3′) contains the Met putative binding motif from the RpL23 promoter region. Unlabeled probe (50× cold probe) identified the binding specificity, and anti-Met antibody confirmed the presence of Met protein. The DNA-protein complex disappeared after the E-box-like motif (CACGCG) was mutated to TCAATA. Assays were tested with fat body nuclear extract of female A. aegypti at 72 h posteclosion. The arrow indicates the specific DNA-protein complex. (G) Depletion of Met impaired ZIKV infection. Female A. aegypti was fed blood meal containing 1.0 × 10⁶ PFU/ml ZIKV. At 7 dpi, the viral load of a single mosquito was tested by qPCR and normalized with the A. aegypti actin. iEGFP, EGFP RNAi knockdown; iMet, Met RNAi knockdown. Data are mean ± SEM. The result shown is representative of three independent experiments, and P value was determined by a Mann-Whitney test. **, P < 0.01.

FIG 8

Model for the function of ribosomal protein and LRIMs during ZIKV infection. At early stages of infection, ZIKV regulates many ribosomal protein genes (including RpL23 and RpL27) through JH signal. JH-Met-Tai acts on the promoters of RpL23 and RpL27 to activate their transcription, which facilitates the translation of viral proteins and promotes viral infection. Moreover, ZIKV induces fat body to secrete LRIMs, including LRIM9, LRIM10A, and LRIM10B, which act as antagonists to inhibit infection.