Both the genomic and antigenomic RNA strands of the arbovirus RVFV generate vsiRNAs.

<p>(A) RNA species produced during RVFV infection. (−) strand genomic segments and mRNAs are depicted in blue, (+) strand antigenomes and mRNAs in red. (B) RVFV vsiRNA size distribution (control library). (C) Distribution of 21 nt RVFV vsiRNAs across the three viral genomic segments. vsiRNAs mapping to genomic strand are depicted in blue, antigenomic strand in red. (D) RVFV vsiRNA size distribution between libraries depleted of RNase III enzymes. (E) Effect of RNase III enzyme depletion on 21 nt RVFV vsiRNAs. vsiRNAs from control (black), Dcr-1 (orange), Dcr-2 (green) and Drosha (blue) depleted cells are compared. (F) RVFV vsiRNA size distribution between libraries depleted of Argonaute proteins. (G) Effect of Argonaute depletion on 21 nt RVFV vsiRNAs. vsiRNAs from control (black), Ago1 (orange), and Ago2 (green) depleted cells are compared. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s001" target="_blank">Figures S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s002" target="_blank">S2</a>.</p

VACV terminal repeat-derived vsiRNAs are derived from long, repeat-containing precursors.

<p>(A) RNA secondary structure prediction of one of sixty 70-mer repeats located at the genomic termini. The abundant repeat-associated VACV vsiRNA is mapped in red. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s004" target="_blank">Figure S4</a>. (B) Expression analysis of VACV terminal repeat-associated transcripts in <i>Drosophila</i> DL1 cells and mouse embryonic fibroblasts (MEFs) by RT-PCR. The forward primer (red) lies within the 70-mer repeat sequence, while the reverse primer (green) binds a unique sequence outside of the repetitive region. The banding pattern of PCR products reflects the amplification of variable numbers of 70-mer repeats, as depicted in the diagram. M = DNA ladder.</p

DCV genomic strand RNA is preferentially targeted by antiviral RNAi.

<p>(A) RNA species produced during DCV infection. (+) strand genome is depicted in blue, (−) strand antigenome in red. (B) DCV vsiRNA size distribution (control library). (C) Distribution of 21 nt DCV-derived vsiRNAs across the viral genome. vsiRNAs mapping to genomic strand are depicted in blue, antigenomic strand in red. (D) DCV vsiRNA size distribution between libraries depleted of RNase III enzymes. (E) Effect of RNase III enzyme depletion on 21 nt DCV vsiRNAs. vsiRNAs from control (black), Dcr-1 (orange), Dcr-2 (green) and Drosha (blue) depleted cells are compared. (F) DCV vsiRNA size distribution between libraries depleted of Argonaute proteins. (G) Effect of Argonaute depletion on 21 nt DCV vsiRNAs. vsiRNAs from control (black), Ago1 (orange), and Ago2 (green) depleted cells are compared. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s001" target="_blank">Figures S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s002" target="_blank">S2</a>.</p

RNA transcripts produced by VACV are targeted by the <i>Drosophila</i> RNA silencing pathway.

<p>(A) The VACV genome is a dsDNA molecule with covalently closed identical termini. (B) VACV vsiRNA size distribution (control library). (C) Distribution of 21 nt VACV vsiRNAs across the viral genome. vsiRNAs mapping to the (+) strand are depicted in blue, (−) strand in red. Black arrows mark genomic termini. (D) VACV vsiRNA size distribution between libraries depleted of RNase III enzymes. (E) Effect of RNase III enzyme depletion on 21 nt VACV vsiRNAs. vsiRNAs from control (black), Dcr-1 (orange), Dcr-2 (green) and Drosha (blue) depleted cells are compared. (F) VACV vsiRNA size distribution between libraries depleted of Argonaute proteins. (G) Effect of Argonaute depletion on 21 nt VACV vsiRNAs. vsiRNAs from control (black), Ago1 (orange), and Ago2 (green) depleted cells are compared. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s001" target="_blank">Figures S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s002" target="_blank">S2</a>.</p

VSV vsiRNAs are concentrated at the 5′ genomic terminus.

<p>(A) RNA species produced during VSV infection. (−) strand genome is depicted in blue, (+) strand antigenome and mRNAs in red. (B) VSV vsiRNA size distribution (control library). (C) Distribution of 21 nt VSV vsiRNAs across the viral genome. vsiRNAs mapping to genomic strand are depicted in blue, antigenomic strand in red. (D) VSV vsiRNA size distribution between libraries depleted of RNase III enzymes. (E) Effect of RNase III enzyme depletion on 21 nt VSV vsiRNAs. vsiRNAs from control (black), Dcr-1 (orange), Dcr-2 (green) and Drosha (blue) depleted cells are compared. (F) VSV vsiRNA size distribution between libraries depleted of Argonaute proteins. (G) Effect of Argonaute depletion on 21 nt VSV vsiRNAs. vsiRNAs from control (black), Ago1 (orange), and Ago2 (green) depleted cells are compared. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s001" target="_blank">Figures S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s002" target="_blank">S2</a>.</p

A putative hairpin within the RVFV S segment generates abundant vsiRNAs in <i>Drosophila</i> and mosquito cells.

<p>(A) RNA secondary structure prediction of S segment IGR. The highly abundant vsiRNAs are mapped in red. (B) Northern blot analysis of RVFV-infected <i>Drosophila</i> DL1 cells, <i>Aedes aegypti</i> Aag2 cells, and <i>Aedes albopictus</i> C6/36 cells, probed for the S segment stem loop vsiRNAs and tRNA^val as a loading control.</p