The Tudor protein Veneno assembles the ping-pong amplification complex that produces viral piRNAs in Aedes mosquitoes

Abstract PIWI-interacting RNAs (piRNAs) comprise a class of small RNAs best known for suppressing transposable elements in germline tissues. The vector mosquito Aedes aegypti encodes seven PIWI genes, four of which are somatically expressed. This somatic piRNA pathway generates piRNAs from viral RNA during infection with cytoplasmic RNA viruses through ping-pong amplification by the PIWI proteins Ago3 and Piwi5. Yet, additional insights into the molecular mechanisms mediating non-canonical piRNA production are lacking. TUDOR-domain containing (Tudor) proteins facilitate piRNA biogenesis in Drosophila melanogaster and other model organisms. We thus hypothesized that Tudor proteins are required for viral piRNA production and performed a knockdown screen targeting all A. aegypti Tudor genes. Knockdown of the Tudor genes AAEL012437, Vreteno, Yb, SMN and AAEL008101-RB resulted in significantly reduced viral piRNA levels, with AAEL012437-depletion having the strongest effect. This protein, which we named Veneno, associates directly with Ago3 in an sDMA-dependent manner and localizes in cytoplasmic foci reminiscent of piRNA processing granules of Drosophila. Veneno-interactome analyses reveal a network of co-factors including the orthologs of the Drosophila piRNA pathway components Vasa and Yb, which in turn interacts with Piwi5. We propose that Veneno assembles a multi-protein complex for ping-pong dependent piRNA production from viral RNA.

-  Figure  2 and S1B. (B) For genes with insufficient knockdown in the screen depicted in Figure 2, a second knockdown with different batches of dsRNA was performed. Numerical suffixes refer to dsRNA of different sequence (Yb 1-4 and Vasa 1-2), whereas AAEL008101-RB and -RC refer to the two splice variants of this gene. After knockdown, small RNA production of (+) strand Sindbis virus (SINV) and Histone H4 mRNA (H4)-derived piRNAs was assessed by northern blot. The heat map depicts relative changes in NSP4 and Capsid viral RNA levels and Tudor/Vasa/Piwi5 mRNA abundance as measured by RT-qPCR. (C) Relative abundance of targeted transcripts in the knockdown experiments shown in Figure 2 (experiment 1) and S1B (experiment 2). Expression levels were normalized to control samples treated with dsRNA targeting Firefly Luciferase (dsLuc). (D-E) Relative quantification of viral RNAs by RT-qPCR with primers located in the NSP4 (D) and Capsid (E) genes. Note that the Capsid sequence is present in both genomic and subgenomic RNA, whereas NSP4 is only present on genomic RNA. Bars in (C-E) are the mean +/-SD of three biological replicates. (F) No correlation was observed between the level of Capsid RNA expression as determined by RT-qPCR and the quantified vpiRNA signal on northern blots in Tudor knockdown experiments. Quantified vpiRNA signal was normalized to U6 snRNA signal, and expressed relative to dsLuc. Black and red circles correspond to the experiments shown in Figure 2 and S1B, respectively. The light blue circle indicates Veneno (Ven). The Pearson correlation coefficient (R2) was determined using GraphPad Prism 6. (G) Images of independent Ven-knockdown experiments used for signal quantification shown in (H); the vpiRNA signal is shown for each blot together with the signal used for normalization. Probing for miR2940-3p or U6 was used for normalization of two blots, whereas EtBr-stained rRNA served as loading control for the remainder. (H) Quantification of vpiRNAs produced upon Veneno knockdown (dsVen) and control knockdown (dsLuc). Bars are the mean +/-standard deviation of the quantified signals from the northern blots shown in (G), normalized to the dsLuc sample. Two-tailed student's T-test was used to determine statistical significance (****, P = 8.82 × 10 -6 ).      Figure S4. Revised gene annotation of AAEL012441/Veneno (Changed to AAEL012437 in the current genome annotation -AaegL5.1) (A) Schematic representation of the gene annotation for AAEL012441 as published by VectorBase (AaegL3.5, December 2017) and our revision based on sequencing of PCR fragments. In our revision, the sequences annotated as AAEL012437 and AAEL012441 in VectorBase are both part of the Veneno (Ven) transcript. Moreover, the sequence annotated in AaegL3.5 as the second intron of AAEL012441 is part of the coding sequence in our revised annotation. Finally, the carboxyl terminus of the coding sequence contains an additional guanosine that is not present in the AaegL3.5 annotation (indicated in red). Altogether, these revisions result in an increased protein size from 470 to 785 amino acids. Of note, the sequence that was annotated by VectorBase as AAEL012437 translates into the RNA recognition motif (RRM) that is now part of the Veneno gene.

Detection of TUDOR orthologs from Drosophila and Ae. aegypti
The Drosophila proteome was scanned using the conserved TUDOR multi-domain sequence (pfam00567: LPEGSTIDVVVSHIESPSHFYIQPVSDSKKLEKLTEELQEYYASKPPESLPPAVGDGCVAAFSE DGKWYRARVTESLDDGLVEVLFIDYGNTETVPLSDLRPLPPEFESLPPQAIKCSLAG) in HHpred 2.18 (cutoff E ≤ 0.01) (4). Homologous sequences were subsequently used as input for iterative searches using Jackhmmer 2.7 to predict all D. melanogaster and Ae. Aegypti TUDOR domains (5). We aligned identified TUDOR-domain sequences using T-Coffee to generate a neighbor joining tree based on sequence identity without correcting for multiple substitutions (6). For Tudor genes that contain multiple TUDOR domains (e.g. AAEL007841), the phylogenetic tree was used to guide identification of the domains that had the highest degree of similarity with a D. melanogaster TUDOR domain. Using only these TUDOR domain sequences, a new neighbor joining tree was generated with T-coffee, which is shown in Figure 1. A combination of SMART-, HHPred-, Pfam-and Hmmscan-mediated domain prediction as well as the available literature was used to determine protein domain composition (5,7,8). The prion-like amino acid composition (PLAAC) web application (http://plaac.wi.mit.edu/) was used to scan the Ven protein sequence for sequences with low amino acid complexity (9).

Cells and viruses
Aag2 cells were cultured at 25°C in Leibovitz's L-15 medium (Invitrogen) supplemented with 10% fetal bovine serum (Gibco), 50 U/ml Penicillin, 50 μg/mL Streptomycin (Invitrogen), 1x Non-essential Amino Acids (Invitrogen) and 2% Tryptose phosphate broth solution (Sigma). The viruses used in this study are recombinant Sindbis viruses that contain a second subgenomic promoter located downstream of the structural genes from which GFP (SINV-GFP) is expressed when indicated. Viruses were produced in BHK-21 cells as previously described (10).

Stable cell lines
We generated plasmids containing a polyubiquitin promoter driving the expressing of GFP-tagged proteins linked to a puromycin resistance gene via a T2A polyprotein self-cleavage site. Approximately 3 × 10 6 Aag2 cells were transfected with 5μg of plasmid. Three hours post-transfection, medium was refreshed and 48 hours later it was replaced with medium containing puromycin (2 μg/mL) as a selection marker. The medium was replaced every 3-4 days, and cells were kept under puromycin pressure throughout. All cell lines were polyclonal.

Generation of plasmids
Wildtype and mutant Veneno as well as Vasa (AAEL004978) sequences were sub-cloned into the pAGW, pARW (Carnegie Gateway vector collection) or pUGW expression vectors, using Gateway cloning (Invitrogen). The pUGW expression vector was derived from the pUbB-GW vector (kindly provided by Gorben Pijlman, University of Wageningen), which was generated by exchanging the OpIE2 promoter from pIB-GW (Thermo Scientific) by the poly-ubiquitin promoter from pGL3-Pub (11). A PCR product of pPUb was created with BspHI and SacI sites, which was then ligated into the BspHI and SacI-digested pIB-GW vector. To generate the pUGW vector, the GFP sequence for Nterminal tagging of proteins was ligated into the pPUbB-GW vector using the SacI restriction sites. AttB1 and AttB2 recombination sites flanking indicated Ven-and Vasa-sequences were added during PCR amplification from Aag2 complementary DNA (cDNA). Donor vectors were generated through BP-recombination of the produced PCR products with pDONR/Zeo (Invitrogen). Subsequently, gene fragments were cloned into the expression vectors by LR-recombination (Invitrogen). Point mutations in Ven-expression plasmids were introduced by site-directed mutagenesis using In-fusion HD Cloning (Takara). V5-3xflag tagged Yb (AAEL001939) was cloned into an expression vector based on the pAc5.1 (Invitrogen) backbone using In-fusion. The following primers were used for cloning (boldcase refers to restriction sites, lowercase to AttB1/AttB2 recombination sites, and additional stopcodons are shown in italic):