Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 May 3;2(3):e00144-17.
doi: 10.1128/mSphere.00144-17. eCollection 2017 May-Jun.

Aedes aegypti Piwi4 Is a Noncanonical PIWI Protein Involved in Antiviral Responses

Margus Varjak  1 , Kevin Maringer  2 , Mick Watson  3 , Vattipally B Sreenu  1 , Anthony C Fredericks  2 , Emilie Pondeville  1 , Claire L Donald  1 , Jelle Sterk  1 , Joy Kean  1 , Marie Vazeille  4 , Anna-Bella Failloux  4 , Alain Kohl  1 , Esther Schnettler  1
Affiliations

Aedes aegypti Piwi4 Is a Noncanonical PIWI Protein Involved in Antiviral Responses

Margus Varjak et al. mSphere. .

Abstract

The small interfering RNA (siRNA) pathway is a major antiviral response in mosquitoes; however, another RNA interference pathway, the PIWI-interacting RNA (piRNA) pathway, has been suggested to be antiviral in mosquitoes. Piwi4 has been reported to be a key mediator of this response in mosquitoes, but it is not involved in the production of virus-specific piRNAs. Here, we show that Piwi4 associates with members of the antiviral exogenous siRNA pathway (Ago2 and Dcr2), as well as with proteins of the piRNA pathway (Ago3, Piwi5, and Piwi6) in an Aedes aegypti-derived cell line, Aag2. Analysis of small RNAs captured by Piwi4 revealed that it is predominantly associated with virus-specific siRNAs in Semliki Forest virus-infected cells and, to a lesser extent, with viral piRNAs. By using a Dcr2 knockout cell line, we showed directly that Ago2 lost its antiviral activity, as it was no longer bound to siRNAs, but Piwi4 retained its antiviral activity in the absence of the siRNA pathway. These results demonstrate a complex interaction between the siRNA and piRNA pathways in A. aegypti and identify Piwi4 as a noncanonical PIWI protein that interacts with members of the siRNA and piRNA pathways, and its antiviral activities may be independent of either pathway. IMPORTANCE Mosquitoes transmit several pathogenic viruses, for example, the chikungunya and Zika viruses. In mosquito cells, virus replication intermediates in the form of double-stranded RNA are cleaved by Dcr2 into 21-nucleotide-long siRNAs, which in turn are used by Ago2 to target the virus genome. A different class of virus-derived small RNAs, PIWI-interacting RNAs (piRNAs), have also been found in infected insect cells. These piRNAs are longer and are produced in a Dcr2-independent manner. The only known antiviral protein in the PIWI family is Piwi4, which is not involved in piRNA production. It is associated with key proteins of the siRNA and piRNA pathways, although its antiviral function is independent of their actions.

Keywords: Aedes aegypti; PIWI; RNA interference; antiviral response; arbovirus; innate immunity.

PubMed Disclaimer

Figures

FIG 1
FIG 1
Stable expression of tagged RNAi proteins in A. aegypti-derived Aag2 cells. (A) Schematic representation of constructs used for production of stable cell lines. Polycistronic mRNA was expressed from the polyubiquitin promoter (PUb), and zeocin (Zeo) was cleaved from the gene of interest via 2A autoprotease activity of Thosea asigna virus. Linearized plasmid was transfected into Aag2 cells, followed by zeocin selection. (B) Immunoblot analysis of cell line extracts for the expression of V5-tagged siRNA and piRNA pathway proteins and eGFP (control). Tubulin was used as a loading control.
FIG 2
FIG 2
Analysis of RNAi protein associations in Aag2 cells. V5-tagged protein-expressing Aag2 cell lines were mock transfected (pPUb) or transfected with a myc-tagged protein expression construct; cell lysates were prepared 48 h p.t. and subjected to immunoprecipitation (IP) with anti-myc antibodies. Samples were analyzed by Western blotting. Immunoprecipitated samples were probed for the presence of V5 and myc tags, and cell lysates were analyzed for V5 tag. Associations between Piwi4 and Piwi5 (A, G, and M), Piwi6 (B, H, and N), Ago3 (C, I, and O), Ago2 (D, J, and P), or Dcr2 (E, K, and Q) and between Ago2 and Dcr2 (F, L, and R) are shown.
FIG 3
FIG 3
Characterization of SFV-specific small RNAs bound by Piwi4 and Ago2 in Aag2 cells. V5-eGFP-, V5-Piwi4-, or V5-Ago2-expressing cells were infected with SFV at an MOI of 10. At 24 h p.i., V5-tagged proteins were pulled down to further isolate and sequence the protein-bound small RNAs. Properties of Piwi4-bound (left) and Ago2-bound (right) small RNAs are shown. (A) Length distributions (in nucleotides) are shown for small RNAs mapping to the SFV genome (red) or antigenome (green) bound to either Piwi4 or Ago2. (B) Distribution of Ago2- and Piwi4-captured small RNAs of 21 nt in length along the SFV genome (red; positive numbers on the y axis) or antigenome (green; negative numbers on the y axis). (C) Relative nucleotide frequency and conservation for the position of the 24- to 29-nt SFV-specific small RNAs mapping either to the genome or antigenome. Because a DNA template was used for sequencing, U is represented by T in the sequences. The experiments shown here were repeated independently three times; results of the third experiment are shown.
FIG 4
FIG 4
Characterization of a Dcr2 knockout Aag2 cell line. (A) Validation of Dcr2 knockout by cotransfection of FFLuc and Rluc expression constructs together with dsRNA against Rluc (dsRluc) into parental AF5 clone and the derived Dcr2 knockout line, AF319. dsRNA against eGFP (dseGFP) was used as a control. At 24 h p.t., cells were lysed to determine the luciferase levels; relative luciferase levels (Rluc/FFluc; with that with cells transfected with dseGFP set to 1) are shown on the y axis. (B) As an alternative silencing inducer, siRNAs against FFLuc (siFFLuc) or hygromycin B resistance gene (siHyg control) were transfected; relative luciferase levels (FFluc/Rluc; with cells transfected with siHyg set to 1) are given on the y axis. (C) Repeat of experiments described above, but in AF319 cells, which included in addition to FFLuc and RLuc expression plasmids and dsRNAs (dsRluc or dseGFP) a V5-tagged Dcr2-expressing plasmid (V5-Dcr2) or pPUb (control). (D) Similarly, the effect of expression of V5-Dcr2 in AF319 cells on FFLuc silencing with siRNAs (siFFLuc, siHyg) is shown. (E) Detection of V5-tagged Dcr2 following cotransfection of FFLuc, Rluc, and pPUb or V5-Dcr2 expression plasmids with dsRluc or siFFLuc into AF319 Dcr2 KO cells using anti-V5 antibody. The bottom panel shows Western blot detection of tubulin as the loading control. (F) AF319 Dcr2 KO and parental AF5 cells were infected with SFV(3H)-FFLuc at an MOI of 0.01 and lysed at 48 h p.i., and luciferase activity was measured. (G) Effect of expression of Dcr2 in AF319 Dcr2 KO cells on SFV(3H)-FFLuc (MOI of 0.01) replication by transfection of pPUb-V5-Dcr2 or pPUb 24 h prior to viral infection; cells were lysed at 48 h p.i. Mean values of three (panels A to E) or four (panels F and G) independent experiments performed in triplicate are presented, with standard errors. *, significant (P < 0.05) by Student’s t test. (H) Northern blot analysis of small RNA fractions isolated from SFV-infected (MOI of 10, 24 h p.i.) AF5 and AF319 cells. Following size separation, RNAs were probed with a combination of radioactively labeled oligonucleotides to detect siRNAs and piRNAs.
FIG 5
FIG 5
Comparison of SFV-derived small RNAs in Aag2 cells. RNA from SFV4-infected (MOI, 10) parental AF5 (A), Dcr2 KO line AF319 (B), or AF319 cells transfected with the V5-Dcr2 expression construct (C) was isolated at 24 h p.i., and small RNAs were sequenced and characterized. (Left panels) Size distribution of small RNA sequences mapping to the SFV genome (red) or antigenome (green) (as percentages of the total reads). (Right) Distribution of 21-nt small RNAs along the SFV genome (red; positive numbers on the y axis) or antigenome (green; negative numbers on the y axis).
FIG 6
FIG 6
Effects of Piwi4 and Ago2 knockdown on SFV replication in Dcr2 knockout cells. (A) AF319 (Dcr2 KO) cells transfected with gene-specific siRNA (Ago2, Piwi4, or eGFP) were infected with wild-type SFV4 (MOI, 0.01) to measure relative amounts of viral genomic RNA (at 24 h p.i. and 48 h p.i.) by using qRT-PCR. Mean relative RNA levels are shown from four independent experiments in which ribosomal S7 was used as a housekeeping gene; error bars show errors of means. (B) Relative FFluc levels in siRNA-transfected (Ago2, Piwi4, or eGFP as control) cells (Dcr2 KO AF319 cells or parental cell line AF5) at 48 h p.i. with SFV(3H)-FFLuc (MOI, 0.01). The mean of three independent experiments performed in triplicate are shown, with standard errors. (C) AF5 or AF319 cells transfected with siRNAs against eGFP, Ago2, or Piwi4 were infected with wild-type SFV (MOI, 0.01), and medium was harvested at 48 h p.i. to measure PFU. Means of 5 independent experiments are shown, and error bars indicate standard deviations. (D) Detection of relative Ago2 and Piwi4 transcript levels in AF5 and AF319 cells by qRT-PCR at 48 h p.t. for siRNAs targeting Ago2, Piwi4, or eGFP (siAgo2, siPiwi4, or sieGFP). *, significant difference (P < 0.05) by Student’s t test.
FIG 7
FIG 7
SFV-specific small RNAs bound by Piwi4 or Ago2 in SFV-infected AF319 cells. V5-eGFP-, V5-Piwi4-, or V5-Ago2-expressing AF319 cells were infected with SFV at an MOI of 10. At 24 h p.i., V5-tagged protein was pulled down and RNA was isolated in order to characterize protein-associated small RNAs. Characteristics of V5-Piwi4-captured (left) and V5-Ago2-captured (right) small RNAs are shown. (A) Length distributions of small RNAs that align to the SFV genome (red) or antigenome (green). (B) Relative nucleotide frequencies and conservation for the positions of the 24- to 29-nt-long SFV-specific small RNAs that align to the genome or antigenome. As DNA was used as the template for sequencing, T represents U. Experiments shown were repeated twice independently. Results of the second series of experiments are shown.
FIG 8
FIG 8
Effects of silencing Ago3 and Piwi5 on SFV replication. (A) Relative Ago3 and Piwi5 transcript levels in AF5 and AF319 cells, as determined by qRT-PCR 24 h p.t., of siRNAs targeting Ago3, Piwi5, or eGFP (siAgo3, siPiwi5, or sieGFP). (B) Transfected cells were infected with SFV(3H)-FFLuc (MOI, 0.01) to determine luciferase expression levels at 24 h and 48 h p.i. Means (on a log scale) are from four independent experiments conducted in triplicate, and standard errors are shown. *, significant difference (P < 0.05) by Student’s t test.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Blair CD, Olson KE. 2015. The role of RNA interference (RNAi) in arbovirus-vector interactions. Viruses 7:820–843. doi:10.3390/v7020820. - DOI - PMC - PubMed
    1. Campbell CL, Keene KM, Brackney DE, Olson KE, Blair CD, Wilusz J, Foy BD. 2008. Aedes aegypti uses RNA interference in defense against Sindbis virus infection. BMC Microbiol 8:47. doi:10.1186/1471-2180-8-47. - DOI - PMC - PubMed
    1. Keene KM, Foy BD, Sanchez-Vargas I, Beaty BJ, Blair CD, Olson KE. 2004. RNA interference acts as a natural antiviral response to O’nyong-nyong virus (alphavirus; Togaviridae) infection of Anopheles gambiae. Proc Natl Acad Sci U S A 101:17240–17245. doi:10.1073/pnas.0406983101. - DOI - PMC - PubMed
    1. McFarlane M, Arias-Goeta C, Martin E, O’Hara Z, Lulla A, Mousson L, Rainey SM, Misbah S, Schnettler E, Donald CL, Merits A, Kohl A, Failloux AB. 2014. Characterization of Aedes aegypti innate-immune pathways that limit Chikungunya virus replication. PLoS Negl Trop Dis 8:e2994. doi:10.1371/journal.pntd.0002994. - DOI - PMC - PubMed
    1. Sánchez-Vargas I, Scott JC, Poole-Smith BK, Franz AWE, Barbosa-Solomieu V, Wilusz J, Olson KE, Blair CD. 2009. Dengue virus type 2 infections of Aedes aegypti are modulated by the mosquito’s RNA interference pathway. PLoS Pathog 5:e1000299. doi:10.1371/journal.ppat.1000299. - DOI - PMC - PubMed

LinkOut - more resources