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Comparative Study
. 2012;7(1):e30861.
doi: 10.1371/journal.pone.0030861. Epub 2012 Jan 24.

Arbovirus-derived piRNAs Exhibit a Ping-Pong Signature in Mosquito Cells

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Free PMC article
Comparative Study

Arbovirus-derived piRNAs Exhibit a Ping-Pong Signature in Mosquito Cells

Nicolas Vodovar et al. PLoS One. .
Free PMC article

Abstract

The siRNA pathway is an essential antiviral mechanism in insects. Whether other RNA interference pathways are involved in antiviral defense remains unclear. Here, we report in cells derived from the two main vectors for arboviruses, Aedes albopictus and Aedes aegypti, the production of viral small RNAs that exhibit the hallmarks of ping-pong derived piwi-associated RNAs (piRNAs) after infection with positive or negative sense RNA viruses. Furthermore, these cells produce endogenous piRNAs that mapped to transposable elements. Our results show that these mosquito cells can initiate de novo piRNA production and recapitulate the ping-pong dependent piRNA pathway upon viral infection. The mechanism of viral-piRNA production is discussed.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Aedes albopictus U4.4 cells are Dcr-2 competent and produce two populations of viral small RNAs.
A. Dicer assay in uninfected U4.4 cells. Lane 3 shows processing of a 113-bp dsRNA substrate into 21-nt siRNAs after incubation in a U4.4 cell extract. Synthetic siRNA (21-nt) and input dsRNA (113-nt) are used as size markers in lanes 1 and 2, respectively. B. RNAi reporter assay. Co-transfection of firefly luciferase specific dsRNA with reporter plasmids encoding firefly and Renilla luciferase into U4.4 cells results in silencing of the firefly luciferase reporter. GFP dsRNA was used as non-specific dsRNA control. Renilla luciferase activity was used as internal control to normalize the firefly luciferase activity. Error bars represent the standard deviations of three individual samples. C. Size distribution of the small RNA reads that match the genome of SINV-GFP with 0 mismatches.
Figure 2
Figure 2. U4.4 cells produce vsiRNAs and vpiRNAs through a ping-pong mechanism upon (+) ssRNA arbovirus infection.
Profile of 21 nt vsiRNAs (A) and 25–29 nt (B) SINV-GFP-derived small RNAs allowing 0 mismatch during alignment. Viral small RNAs that mapped to the sense and antisense strand of the SINV-GFP genome are shown in red and blue, respectively. C. Conservation and relative nucleotide frequency per position of 25–29 nt SINV-GFP-derived reads that mapped to the sense (top) and the antisense (bottom) strands of the SINV-GFP genome. The overall height of the nucleotide stack indicates the sequence conservation; the height of the nucleotides within each stack represents their relative frequency at that position. n indicates the number of reads used to generate each logo. D. Frequency map of the distance between 25–29 nt small RNAs that mapped to opposite strands of the SINV-GFP genome. The peak at position 9 on the sequence (the first nucleotide being position 0) indicates the position of maximal probability of finding the 5′ end of a complementary small RNA.
Figure 3
Figure 3. Aedes albopictus C6/36 cells produce ping-pong dependent vpiRNA upon (-) RNA virus infection.
A, B, and C. Conservation and relative nucleotide frequency per position of the 25–29 nt LACV-derived reads that mapped to the antigenomic sense (top) and genomic antisense (bottom) strands of the LACV genome segments L, M and S, respectively. n indicates the number of reads used to generate each logo. D, E, and F. Frequency map of the distance between 25–29 nt reads that mapped to opposite strands of the LACV genome segments L, M and S, respectively. The peak at position 9 on the sequence (the first nucleotide being position 0) indicates the position of maximal probability of finding the 5′ end of a complementary small RNA.
Figure 4
Figure 4. Aedes aegypti Aag2 cells produce vsiRNA and vpiRNA with a ping-pong signature upon arbovirus infection.
A. Size distribution of the small RNA reads that match the genome of SINV-GFP with 0 mismatches. Profile of 21 nt vsiRNAs (B) and 25–29 nt (C) SINV-GFP-derived small RNAs allowing 0 mismatch during alignment. Viral small RNA that mapped to the sense and antisense strand of the SINV-GFP genome are shown in red and blue, respectively. D. Conservation and relative nucleotide frequency per position of 25–29 nt SINV-GFP-derived reads that mapped to the sense (top) and antisense (bottom) strands of the SINV-GFP genome. n indicates the number of reads used to generate each logo. E. Frequency map of the distance between 25–29 nt small RNAs that mapped to opposite strands of the SINV-GFP genome. The peak at position 9 on the sequence (the first nucleotide being position 0) indicates the position of maximal probability of finding the 5′ end of a complementary small RNA. F. Expression of PIWI family members in Aag2 cells analyzed by RT-PCR. cDNA synthesis was performed in the presence (+) or absence (−) of reverse transcriptase (RT). The -RT samples are included as controls for contamination of RNA preparations with chromosomal DNA. The coding sequences of Piwi1 and Piwi3 are 95% identical at the nucleotide level. Two different primer sets that amplify both Piwi1 and Piwi3 were used (a and b). A higher exposure was used for the gel image with Piwi1to Piwi3. A 100 bp ladder was used as a size marker (M). The asterisk indicates a non-specific PCR amplification product.
Figure 5
Figure 5. Aedes aegypti Aag2 cells produce transposon-derived piRNAs with a ping-pong signature.
A. Size distribution of the small RNA reads that match with 0 mismatches against an Aedes aegypti transposon dataset that contain full-length non-composite transposons sequences (TEfam: http://tefam.biochem.vt.edu/tefam/index.php). B. Heat map for 25–29 nt small RNAs that mapped to individual retrotransposons with more than 1000 reads. Read count and log-transformed ratios of antisense/sense small RNAs are presented. C. Profile of 25–29 nt reads that mapped to the transposon Copia Ele56 (TF000691) allowing 0 mismatch during alignment. Transposon-derived piRNAs that mapped to the sense and antisense strand of the transposon sequence are shown in red and blue, respectively. D. Conservation and relative nucleotide frequency per position of 25–29 nt reads that mapped to the sense (top) and the antisense (bottom) strands of the entire transposon dataset. n indicates the number of reads used to generate each logo.

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