Abstract
The endosymbiont Wolbachia is common among insects and known for the reproductive manipulations it exerts on hosts as well as inhibition of virus replication in their hosts. Recently, we showed that Wolbachia uses host microRNAs to manipulate host gene expression for its efficient maintenance in the dengue mosquito vector, Aedes aegypti. Cytosine methylation is mediated by a group of proteins called DNA (cytosine-5) methyltransferases, which are structurally and functionally conserved from prokaryotes to eukaryotes. The biological functions of cytosine methylation include host defense, genome stability, gene regulation, developmental promotion of organs, and lifespan regulation. Ae. aegypti has only one DNA methyltransferase gene (AaDnmt2) belonging to the cytosine methyltransferase family 2, which is the most deeply conserved and widely distributed gene among metazoans. Here, we show that in mosquitoes the introduced endosymbiont, Wolbachia, significantly suppresses expression of AaDnmt2, but dengue virus induces expression of AaDnmt2. Interestingly, we found that aae-miR-2940 microRNA, which is exclusively expressed in Wolbachia-infected mosquitoes, down-regulates the expression of AaDnmt2. Reversely, overexpression of AaDnmt2 in mosquito cells led to inhibition of Wolbachia replication, but significantly promoted replication of dengue virus, suggesting a causal link between this Wolbachia manipulation and the blocking of dengue replication in Wolbachia-infected mosquitoes. In addition, our findings provide an explanation for hypomethylation of the genome in Wolbachia-infected Ae. aegypti.
Keywords: endosymbiosis, Wolbachia pipientis
Dengue is endemic in more than 110 tropical countries, with 2.5–3.0 billion people living at risk for infection. Each year, it is reported to infect more than 50 million people worldwide (1). Dengue virus (DENV) from the genus Flavivirus is a positive-strand RNA virus, which is primarily transmitted to humans by female Aedes mosquitoes. Because there are currently neither specific antiviral drugs nor approved vaccines against DENV, insect vector control is still considered the best approach to prevent dengue transmission. In recent years, Wolbachia infections of mosquitoes have been proposed as a novel approach for area-wide dengue control that is inexpensive to implement and self-sustaining (2, 3).
Wolbachia, maternally inherited Gram-negative endosymbiotic bacteria, are estimated to occur naturally in more than 40% of all insect species and 28% of surveyed mosquito species (4, 5). However, with some exceptions, they have not been found in important species that are the vectors of human pathogens such as dengue and malaria. Recently, several strains of Wolbachia have been successfully introduced into Aedes aegypti (6, 7). Mosquitoes with wMelPop strain transinfection showed about a 50% reduction in adult female lifespan (8). Interestingly, Ae. aegypti infected with Wolbachia induces very strong resistance to several arboviruses including DENV, yellow fever, and Chikungunya viruses, Plasmodium, and filarial nematodes (9–12).
MicroRNAs (miRNAs) are an evolutionarily conserved class of small ∼22 nucleotides noncoding RNAs that regulate gene expression via partially or completely matching to their target genes (13). They play important roles in cellular processes including development, differentiation, apoptosis, and immunity. Until now, more than 25,000 miRNA sequences have been identified from different organisms including protozoans, plants, animals, and viruses (miRBase v19) (14). It has been reported that the expression levels of cellular miRNAs dramatically change in response to bacterial and viral infections in animals and plants (15–20). In human, a liver-specific miRNA, miR-122, can target the 5′ UTR of hepatitis C virus to enhance viral replication (21). Huang et al. (2007) (17) reported that HIV also uses host miRNAs to target its genes to its own advantage. Recently, we reported differential expression of several miRNAs in Wolbachia-infected Ae. aegypti mosquitoes with an miRNA, aae-miR-2940, which is exclusively expressed in Wolbachia-infected mosquitoes. By using the host aae-miR-2940, Wolbachia up-regulates the expression of a host metalloprotease gene, metalloprotease m41 ftsh, which is essential for the endosymbiont’s maintenance (18).
In this study, we identified another target gene of aae-miR-2940, AaDnmt2. AaDnmt2 belongs to cytosine methyltransferase family 2, which is the only DNA methyltransferase found in Diptera. In Drosophila, dDnmt2 is primarily localized in the cytoplasm and much less in the nucleus and could mediate both DNA and RNA methylations (22, 23). Here, we aimed to investigate the impact of Wolbachia and consequently up-regulation of aae-miR-2940 on AaDnmt2. In addition, we investigated the possible impact of regulation of the target gene on DENV replication. Our results suggest that AaDnmt2 plays an important role not only in the maintenance of Wolbachia infection in mosquito cells, but also in the resistance of mosquito cells to DENV infection.
Results
Expression Profile of AaDnmt2 in Ae. Aegypti.
By using National Center for Biotechnology Information (NCBI) BLAST, RNAHybrid, and RNA22 software, a putative 5-cytosine DNA methyltransferase (AaDnmt2, GenBank ID: XM_001657505) from Ae. aegypti was identified as another target of a differentially expressed miRNA, aae-miR-2940, which was previously shown to up-regulate the transcript levels of a metalloprotease gene in Wolbachia-infected mosquitoes (18). Further analysis of the sequence showed that AaDnmt2 is a homolog of pmt1 from fission yeast (24) and Dnmt2 from human, Drosophila, and other organisms (25–27). AaDnmt2 is a 344-aa protein with 47% identity to the Drosophila Dnmt2 protein. Like other Dnmt2 proteins, AaDnmt2 contains all 10 diagnostic and catalytic motifs of active DNA methyltransferases. In addition, similar to Drosophila, there is only one Dnmt gene present in Ae. aegypti genome.
In Drosophila, human, and zebrafish, the expression of Dnmt2 is developmentally and tissue-specifically regulated (22, 23, 25, 28). In this study, we investigated the expression pattern of AaDnmt2 in Ae. aegypti by RT-PCR using AaDnmt2 gene–specific primers. We found that the transcript levels of AaDnmt2 gene were not detectable in any of the larval stages, but could be detected in adults (Fig. 1A). The transcript levels of AaDnmt2 were lower in 1-d-old adults compared with 4-d-old adults (Fig. 1A). We also found that AaDnmt2 is mainly expressed in the abdomen of both male and female mosquitoes, which suggests that AaDnmt2 could be specifically expressed in some organs in the abdomen (Fig. 1B). Tissue-specific RT-quantitative PCR (qPCR) analysis of four tissues (ovary, salivary glands, midgut, and thoracic muscles) from Ae. aegypti females at 4 and 12 d after emergence confirmed that the highest transcript levels of AeDnmt2 were found in the ovaries (see below).
Fig. 1.
AaDnmt2 expression profile. (A) RT-PCR analysis was performed using the total RNA samples from Ae. aegypti mosquito larvae and adults. (B) RT-PCR analysis was performed with head + thorax (HT) and abdomen (Ab) of mosquito females and males. Rps17 gene was used as control to show the integrity of RNA. +C, positive control using Aag2 genomic DNA (gDNA); RT-C, RT-PCR control reactions in which reverse transcriptase was not added.
Ae. aegypti is the host and vector of an important human pathogen, DENV. Recently, the insect endosymbiont Wolbachia was successfully introduced into Ae. aegypti and found to inhibit the ability of pathogens such as DENV to infect this mosquito species (10, 11). Based on this, we investigated the expression profile of AaDnmt2 in mosquitoes infected with Wolbachia or DENV. In Wolbachia-infected mosquitoes at 4 d after emergence, AaDnmt2 was not detected by RT-PCR, but it could be detected in its tetracycline-cured (tet-cured) counterpart mosquitoes (Fig. 2A). AaDnmt2 was mainly expressed in the abdomen of tet-cured mosquitoes (Fig. 2B). Interestingly, in 3-d-old females infected with Wolbachia, AaDnmt2 could not be detected in either the head and thorax or the abdomen (Fig. 2B). Consistently, when specific tissues from 4- and 12-d-old female mosquitoes were analyzed using RT-qPCR, in general, about twofold less AeDnmt2 transcripts were found in Wolbachia-infected mosquito tissues (Fig. 3). In tet-cured mosquitoes infected with DENV, RT-PCR results showed that AaDnmt2 was expressed at much higher levels compared with the mosquitoes without DENV (Fig. 2C).
Fig. 2.
AaDnmt2 expression profile in Wolbachia or DENV-infected mosquitoes. (A) RT-PCR analysis was performed using total RNA samples from 4-d-old Wolbachia-infected (Wol+) and uninfected (Wol−) female mosquitoes. (B) RT-PCR analysis was performed using total RNA samples from Wol+ and Wol− head + thorax (HT) and abdomen (Ab) of female mosquitoes at 2 and 3 d after emergence. (C) RT-PCR analysis was performed using total RNA samples from 3-d DENV-infected and uninfected mosquitoes (without Wolbachia). Rps17 gene was used as control to show the integrity of RNA. +C in A serves as positive control for C because the reactions were done at the same time.
Fig. 3.
Tissue-specific expression of AeDnmt2 in female Ae. aegypti mosquitoes. RT-qPCR analysis of AeDnmt2 transcript levels in the ovary, midgut, salivary glands, and thoracic muscle tissues (muscle) of 4- and 12-d-old Wol+ and Wol− female mosquitoes. Transcript levels of AeDnmt2 were also compared in the ovaries and salivary glands in the samples (bottom two graphs). There are statistically significant differences between groups with different letters at P < 0.0001 (one-way ANOVA with Tukey’s post-hoc test).
aae-miR-2940 Down-regulates the Expression of AaDnmt2.
The target sequences of aae-miR-2940 were predicted in the coding region of AaDnmt2 (nucleotides 1011–1035) with significant complementarity to aae-miR-2940 (Fig. 4A). In Ae. aegypti Aag2 cells as well as wMelPop-CLA–infected Aag2 cells (aag2.wMelPop-CLA), AaDnmt2 transcripts could not be detected (Fig. 2B). The reason for lack of detection of AaDnmt2 in Aag2 cells, compared with Wolbachia-free mosquitoes (Fig. 2), is the expression of aae-miR-2940 in noninfected mosquito cell lines compared with whole mosquitoes, although at a lower level compared with Wolbachia-infected cells (18). To confirm this, the Dicer-1 gene was silenced in Aag2 cells using RNAi. After confirming gene silencing, RT-PCR results showed that the transcript levels of AaDnmt2 were considerably higher compared with mock-transfected Aag2 cells (Fig. 4B), which confirmed that inhibition of AaDnmt2 expression in Aag2 cells is mediated by miRNAs. To further confirm that aae-miR-2940 contributes in this inhibition, we transfected Ae. aegypti Aag2 cells with synthetic aae-miR-2940–specific and aae-miR-2940*–specific inhibitors, respectively. RT-PCR results showed increased transcript levels of AaDnmt2 in cells transfected with aae-miR-2940–specific inhibitor compared with cells transfected with control aae-miR-2940*–specific inhibitor and mock-transfected cells (Fig. 4B). These results suggest that aae-miR-2940 down-regulates the transcript levels of AaDnmt2, which is consistent with the expression pattern of AaDnmt2 gene in mosquitoes with or without Wolbachia (Figs. 2 A and B and 3).
Fig. 4.
AaDnmt2 transcription is down-regulated by aae-miR-2940. (A) Schematic diagram showing AaDnmt2 mRNA and its target sequences with complete complementarity of aae-miR-2940 seed region (bold and underlined) with the sequences. (B) RT-PCR analysis of RNA extracted from mock-transfected Aag2 and aag2.wMelPop-CLA (Pop) cells, and Aag2 cells transfected with Dicer-1 dsRNA, aae-miR-2940, and aae-miR-2940* (control) synthetic inhibitors. Rps17 gene was used as control to show the integrity of RNA.
AaDnmt2 Suppresses Wolbachia Replication.
In a previous study, we reported that aae-miR-2940 up-regulates the transcript levels of the host metalloprotease, which is required for Wolbachia replication and maintenance in mosquito cells (18). Considering that aae-miR-2940 down-regulates the transcript levels of AaDnmt2, we investigated whether AaDnmt2 has any effect on Wolbachia replication. For this, AaDnmt2 was overexpressed in aag2.wMelPop-CLA cells and the density of Wolbachia was analyzed by PCR. Two different amounts of pIZ/AaDnmt2 were used to transfect aag2.wMelPop-CLA cells resulting in one line expressing the gene at a higher level (L1) compared with the other (L2) (Fig. 5A). PCR results with wsp gene–specific primers revealed that Wolbachia density was considerably lower in cells transfected with the pIZ/AaDnmt2 vector compared with cells transfected with the empty vector (pIZ) (Fig. 5B). This effect was found to be dose-dependent because PCR results showed that Wolbachia density was much lower in L1, where AaDnmt2 is expressed at a higher level compared with L2, the lower overexpressing AaDnmt2 line (Fig. 5B). However, Wolbachia density was lower in L2 compared with pIZ empty vector transfected cells (Fig. 5B). A qPCR analysis further confirmed that overexpression of AaDnmt2 leads to lower Wolbachia density (Fig. 5C). These results suggest that AaDnmt2 suppresses Wolbachia replication in the cell line and the suppression is concentration-dependent, which is consistent with the expression profile that AaDnmt2 is detected at considerably lower levels in Wolbachia-infected mosquitoes compared with tetracycline-cured counterpart mosquitoes (Figs. 2A and 3).
Fig. 5.
AaDnmt2 reduces Wolbachia replication. (A) RT-PCR analysis was performed to confirm that AaDnmt2 was overexpressed in pIZ/AaDnmt2 plasmid-transfected aag2.wMelPop-CLA cells. L1 and L2 refer to two transfected lines established using two different concentrations of pIZ/AeDnmt2; L1 reflects a concentration of 2 µg; L2 a concentration of 1 µg. Rps17 primers were used as control to show the integrity of RNA. (B) PCR analysis of Wolbachia density 72 h after transfection with pIZ or pIZ/AaDnmt2 plasmid in L1 and L2 (as in A) in aag2.wMelPop-CLA cells using primers specific to the Wolbachia wsp gene. Rps17 primers were used as control to show the integrity of DNA. (C) qPCR analysis of DNA extracted from aag2.wMelPop-CLA cells 72 h after transfection with pIZ or pIZ/AaDnmt2. Three biological with three technical replicates were carried out for each transfection. Asterisks indicate a significant difference between mock and AaDnmt2 transfections (P < 0.001; t test).
AaDnmt2 Enhances DENV Replication.
In both Ae. aegypti and mosquito cell lines, Wolbachia was found to limit replication of DENV (10, 11), which could be due to manipulation of the host genes expression via miRNAs by Wolbachia. To explore the effect of AaDnmt2 on DENV replication, we overexpressed AaDnmt2 in Aag2 cells that were subsequently infected with DENV. Total RNA at 72 h after viral infection was isolated and analyzed by RT-PCR with DENV-specific primers. The results showed that the relative abundance of DENV was significantly higher in the cells overexpressing AaDnmt2 compared with cells transfected with the pIZ empty vector (Fig. 6A). This result was also confirmed by RT-qPCR (Fig. 6B). These results suggest that AaDnmt2 enhances replication of DENV in cells, which is consistent with the expression profile that AaDnmt2 transcripts levels were induced in mosquitoes infected with DENV (Fig. 2C). However, when aag2.wMelPop-CLA cells were transfected with pIZ/AaDnmt2 and then infected with DENV, virus replication was not enhanced by AaDnmt2 compared with cells transfected with the pIZ empty vector (Fig. 6C). Consistently, when wMelPop-CLA cells were transfected with aae-miR-2940–specific inhibitor and subsequently infected with DENV, virus replication increased over time compared with wMelPop-CLA cells transfected with the control inhibitor; however, the effect was moderate (1.5 times at 72 h) and not statistically significant (P > 0.05; Fig. 7). The moderate effect could be due to regulation of AeDnmt2 transcripts by more than one miRNA; therefore, when aae-miR-2940 is specifically inhibited, another miRNA may still down-regulate AeDnmt2 transcript levels leading to only a partial up-regulation of AeDnmt2 transcript levels resulting from aae-miR-2940 inhibition. In fact, when Dicer-1 was silenced in Aag2 cells, which affects miRNA biogenesis in general, considerably more AeDnmt2 transcripts were amplified compared with when aae-miR-2940 was specifically inhibited using the miRNA-specific inhibitor (Fig. 4B; compare Dicer 1 RNAi with 2940 inhibitor), which further supports the assumption. It has already been shown that one mRNA can be a target of more than one miRNA (29–31). Our preliminary bioinformatics search showed that a number of other Ae. aegypti miRNAs (Fig. S1), which are differentially expressed in Wolbachia-infected mosquitoes (18), could potentially target AeDnmt2; however, their interactions with AeDnmt2 need to be experimentally validated.
Fig. 6.
AaDnmt2 enhances DENV replication in cells without Wolbachia. (A) RT-PCR analysis of RNA using DENV-specific primers 72 h after transfection with pIZ or pIZ/AaDnmt2 in Aag2 cells. Rps17, primers to the Rps17 gene showing the integrity of RNA. (B) RT-qPCR analysis of DENV abundance 72 h after transfection with pIZ or pIZ/AaDnmt2 in Aag2 cells. (C) RT-qPCR analysis of RNA using DENV-specific primers in aag2.wMelPop-CLA cells 72 h after transfection with pIZ or pIZ/AaDnmt2. For B and C, three biological replicates with three technical replicates were carried out for each transfection. Asterisks indicate a significant difference between mock and AaDnmt2 transfections (P < 0.001; t test); NS indicates no significant differences between mock and pIZ/AaDnmt2 transfections (P > 0.05; t test).
Fig. 7.
Effect of inhibition of aae-miR-2940 on DENV replication. aag2.wMelPop-CLA cells were transfected with synthetic aae-miR-2940 inhibitor (2940In) and control inhibitor (Inhibitor-C) 48 h before infection with DENV. RNA samples were extracted from cells at (A) 24 h and (B) 72 h after infection and analyzed by RT-qPCR using specific primers to DENV. Three biological with three technical replicates were carried out for each transfection. NS, no significant differences between Inhibitor-C and 2940In (P > 0.05; t test).
Discussion
In a recent study, we reported that wMelPop strain of Wolbachia altered the expression of a number of host miRNAs, including aae-miR-2940 in the host mosquito Ae. aegypti (18). In turn, aae-miR-2940 up-regulated the transcript levels of a target gene, metalloprotease ftsh. Metalloprotease was found to be crucial for efficient replication and maintenance of the endosymbiont in Ae. aegypti (18). However, the role of induced miRNAs in the immune response to DENV and other microorganisms has not been explored. The present study identified AaDnmt2, a methyltransferase, as another target gene of aae-miR-2940 in Ae. aegypti, which is highly expressed in the ovaries. We found that in the absence of the miRNA, either by Dicer-1 silencing or specifically using a synthetic inhibitor of aae-miR-2940, AeDnmt2 was up-regulated, suggesting that aae-miR-2940 negatively regulates the transcripts of AeDnmt2. In addition, by investigating the expression patterns, we found that the transcription levels of AaDnmt2 were significantly suppressed by Wolbachia in mosquitoes and by aae-miR-2940 in the mosquito cell line. This trend was also observed in specific tissues of the mosquitoes tested. The transcription levels of AaDnmt2 were increased by DENV infection in mosquitoes. Interestingly, overexpression of AaDnmt2 in aag2.wMelPop-CLA cells resulted in a significant decline in Wolbachia density. Overexpression and coinfection with DENV in Aag2 cells showed a significant increase in DENV replication, but not in aag2.wMelPop-CLA cells. These results suggest that by inducing the expression of aae-miR-2940, Wolbachia could down-regulate AaDnmt2, which in turn inhibits replication of DENV. This describes a potential mechanism of suppression of DENV replication by Wolbachia.
Insects use a variety of immune responses such as RNA interference, the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) (JAK/STAT), Toll, and Immune Deficiency (IMD) pathways and autophagy to contain viral infections (32–36). In their original host Drosophila, some strains of Wolbachia could induce resistance to various viruses such as Drosophila C virus, cricket paralysis virus, Nora virus, flock house virus, and West Nile virus (37–39). Drosophila synergistically uses several pathways to control viral infections (40–42). Recently, some strains of Wolbachia were successfully introduced into various mosquito species such as Ae. aegypti, Aedes albopictus, and Anopheles gambiae (somatic infection) in an effort to limit replication and spread of various vector-borne pathogens such as DENV and malaria (6, 7, 43). Wolbachia effectively suppress DENV, Chikungunya virus, yellow fever virus, and Plasmodium in their new hosts (9–12, 43, 44). Studies have shown that in Ae. aegypti, the symbionts up-regulate the expression of a large number of host immune genes such as defensins, cecropins, JAK-STAT, and Toll pathway genes (10, 33, 35, 43, 44). These suggest that similar to Drosophila, Wolbachia-infected Ae. aegypti may also use multiple pathways activated by Wolbachia to control pathogen infections. AaDnmt2 down-regulation might be used synergistically to suppress DENV by Ae. aegypti.
Reductions in AaDnmt2, the only methyltransferase in Ae. aegypti, by Wolbachia via induction of aae-miR-2940, would imply hypomethylation of the genome in Wolbachia-infected mosquitoes, which has significant implications in epigenetic regulation of gene expression. Role of miRNAs in regulation of methylation via controlling DNA methyltransferases has been documented. For example, miR-148 down-regulates Dnmt3B in human HeLa cells by targeting its transcripts in the coding region (45) and Dnmt1 in both human cholangiocarcinoma and systemic lupus erythematosus cell lines by targeting either 3′UTR or the coding region (46, 47). Overexpression of miR-148 decreased Dnmt1, which in turn promoted DNA hypomethylation and enhanced the expression of various methylation-sensitive genes. Considering substantial changes in the transcriptome profile of Wolbachia-infected mosquitoes relative to noninfected ones (12), it is conceivable that some of these changes are due to differential/hypo methylation of the mosquito genome. Indeed, a recent report demonstrated that in wMelPop-infected Ae. aegypti mosquitoes the pattern of cytosine methylation of the genome is widely disrupted leading to an overall hypomethylation of the genome in Wolbachia-infected mosquitoes compared with noninfected mosquitoes (48).
Taken together, our studies suggest that aae-miR-2940 plays an important role not only in the maintenance of Wolbachia infection in mosquito cells, but also contributes to the resistance to DENV infection in mosquito cells. Our findings describe a mechanism for Wolbachia to regulate host gene expression by affecting methylation and limit DENV infection by using host miRNAs in Ae. aegypti. Our results demonstrated a critical functional link between Wolbachia, miRNAs, and DENV, which furthers our understanding of Wolbachia–virus–mosquito interactions.
Materials and Methods
Mosquitoes, Insect Cell Lines, and DENV Infection.
Ae. aegypti infected with wMelPop-CLA strain of Wolbachia (Wol+) and a Wolbachia-free strain, tet-cured line (Wol−), were the stocks as previously described (7). Adults and larvae were reared at 25 °C with 80% relative humidity and a 12-h light regimen. Larvae were maintained with fish food pellets (Tetramin, Tetra) at a density of 50 larvae per liter of water in flat trays; adults were offered 10% (wt/vol) sucrose solution, ad libitum. Ae. aegypti Aag2 cells and those infected with Wolbachia (denoted as aag2.wMelPop-CLA) (49) were maintained in a media with a 1:1 mixture of Mitsuhasgi-Maramorosch and Schneider’s insect media (Invitrogen) supplemented with 10% FBS.
RNA Extraction, cDNA Synthesis, and PCR.
Total RNA from mosquitoes and cell lines was isolated using Tri-Reagent (Ambion Inc.), and subsequently treated with DNase I before being used for reverse transcription. The first-strand cDNA for AaDnmt2 was synthesized by RT with AaDnmt2 gene-specific primers (forward: 5′-ATGAGTGCTACCGACGTA-3′; reverse: 5′-TCACCAGTCCATCTCATC-3′). In each RT reaction, ∼2 µg of total RNA was used as template in a total volume of 20 μL. Amplification following cDNA synthesis was carried out in a total reaction volume of 25 μL with 2 μL of RT products. The amplification was performed at 94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, 56 °C for 30 s, 68 °C for 1 min, and a final extension at 68 °C for 10 min. The ribosomal protein S17 (RPS17) gene was used for normalizing data as described previously (18).
Quantitative PCR of Wolbachia Density.
Total genomic DNA was extracted from cells as described previously (50). Wolbachia density in cells was determined by qPCR using the wsp gene-specific primers as described previously (18). qPCR was performed using Platinum SYBR Green Mix (Invitrogen) with 20 ng of total genomic DNA in a Rotor-Gene thermal cycler (Qiagen) under the following conditions: 95 °C hold for 30 s, then 40 cycles of 95 °C for 15 s, 50 °C for 15 s, and 72 °C for 20 s, following by the melting curve (68–95 °C). For this experiment, three biological replicates with three technical replicates were analyzed. The RPS17 gene was used for normalization of DNA templates. The Student t test was used to compare the differences in means between different treatments.
qRT-PCR.
Total RNA was extracted and reverse transcribed as described previously using a specific reverse primer to DENV (reverse, CCATCCCGTACCAGCATCCG). Following RT, qPCR with DENV gene-specific primers (forward, GTGGTGGTGACTGAGGACTG) was performed to determine DENV density in cells. This amplified a 138-nt fragment from nucleotides 3273–3409 of the DENV genome. Platinum SYBR Green Mix (Invitrogen) was used for qPCR with 1 μL of RT products as described previously. For this experiment, three biological replicates with three technical replicates were analyzed. The RPS17 gene was used for normalization of RNA templates. The Student t test was used to compare the differences in means.
For tissue-specific analysis of AaDnmt2 transcript levels, total RNA was extracted from ovaries, thoracic muscle tissue, salivary glands, and midgut dissected from 4- and 12-d-old Wol+ and Wol− female mosquitoes as described previously. RT-qPCR reactions were carried out as described previously using primers specific to AaDnmt2 gene (forward, GTAGTATCAGCAATTGACATCA; reverse, TGGGGGTGACATGAGAATAAG). Similarly, three biological replicates with three technical replicates were analyzed for each tissue and mosquito type. Each biological replicate consisted of a pull of total RNA extracted from specific tissues of five mosquitoes. The RPS17 gene was also used for normalization of RNA templates.
Overexpression of AaDnmt2 in Insect Cell Lines.
The cDNA of AaDnmt2 gene (GenBank accession no. XM_001657505) was synthesized by RT-PCR using gene-specific primers with KpnI and SacII restriction sites (forward: 5′-GGTACCATGGGGAGTGCTACCGACGTA-3′; reverse: 5′-CCGCGGCCAGTCCATCTCATC-3′; restriction sites are underlined). AaDnmt2 was cloned into pIZ/V5-His vector containing an insect-specific promoter (Life Technologies). Two micrograms of the produced plasmid with AaDnmt2 gene was transfected into Aag2 or aag2.wMelPop-CLA cell lines using Cellfectin (Life Technologies) as a transfection reagent. Cells transfected with the empty pIZ/V5-His vector were used as controls. Cells were collected at 72 h after transfection, and total RNA was isolated and analyzed by RT-PCR and/or RT-qPCR with gene-specific primers as required.
Inhibition of aae-miR-2940 in Wolbachia-Infected Cells.
aae-miR-2940 (5′-GCCUCGACAGAUAAGAUAAACCA-3′) and aae-miR-2940* (5′-AGUGAUUUAUCUCCCUGUCGAC-3′, as a control) inhibitors were synthesized by Genepharma. In this experiment, 100 ng of the aae-miR-2940 and aae-miR-2940* inhibitors were transfected into aag2.wMelPop-CLA cells using the Cellfectin transfection reagent. Cells were collected at 72 h after transfections, total RNA was extracted, and RT-PCR analyses were performed with AaDnmt2 gene–specific primers as previously.
RNA-Mediated Gene Silencing.
For RNAi-based experiments, dsRNA was synthesized in vitro by using the T7 Megascript transcription kit according to the manufacturer’s instructions (Ambion Inc.). T7 promoter sequences (TAATACGACTCACTATAGGG) were incorporated in both forward and reverse primers designed to amplify a 450 bp of the Ae. aegypti Dicer-1 gene (forward: 5′-CCCGGACCAAGTCCTAGTA-3′; reverse: 5′-CAACTCTTTCGGCACGTAA-3′). About 1 μg of the PCR product was used in a 4-h incubation at 37 °C for dsRNA synthesis. After DNase treatment, RNA was precipitated by lithium chloride. About 500 ng of dsRNA was used for transfection of Aag2 cells, which were transfected again at 48 h after the first transfection. Cells were collected for RNA isolation for analysis 48 h after the second transfection. Gene silencing was confirmed by RT-qPCR with Dicer-1–specific primers.
Prediction of Cellular Targets of aae-miR-2940.
NCBI BLAST, RNAHybrid, and RNA22 software were used to identify the potential targets of aae-miR-2940. tBLASTN was performed to search for homologous sequences. The BioEdit program was used to align sequences and to calculate the identities and similarities of sequences.
Supplementary Material
Acknowledgments
We thank Guangjin Lu from Brian Kay’s laboratory (Queensland Institute of Medical Research) for providing mosquito larval samples. This work was supported by Australian Research Council Discovery Grant DP110102112 (to S.A.), Australian Research Council Discovery Early Career Researcher Award fellowship DE120101512 (to M.H.), and a grant from the Foundation of the National Institutes of Health through the Grand Challenges in Global Health initiative of the Bill and Melinda Gates Foundation (to S.L.O.).
Footnotes
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1303603110/-/DCSupplemental.
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