Diverging Affinity of Tospovirus RNA Silencing Suppressor Proteins, NSs, for Various RNA Duplex Molecules

Abstract

The tospovirus NSs protein was previously shown to suppress the antiviral RNA silencing mechanism in plants. Here the biochemical analysis of NSs proteins from different tospoviruses, using purified NSs or NSs containing cell extracts, is described. The results showed that all tospoviral NSs proteins analyzed exhibited affinity to small double-stranded RNA molecules, i.e., small interfering RNAs (siRNAs) and micro-RNA (miRNA)/miRNA* duplexes. Interestingly, the NSs proteins from tomato spotted wilt virus (TSWV), impatiens necrotic spot virus (INSV), and groundnut ringspot virus (GRSV) also showed affinity to long double-stranded RNA (dsRNA), whereas tomato yellow ring virus (TYRV) NSs did not. The TSWV NSs protein was shown to be capable of inhibiting Dicer-mediated cleavage of long dsRNA in vitro. In addition, it suppressed the accumulation of green fluorescent protein (GFP)-specific siRNAs during coinfiltration with an inverted-repeat-GFP RNA construct in Nicotiana benthamiana. In vivo interference of TSWV NSs in the miRNA pathway was shown by suppression of an enhanced GFP (eGFP) miRNA sensor construct. The ability to stabilize miRNA/miRNA* by different tospovirus NSs proteins in vivo was demonstrated by increased accumulation and detection of both miRNA171c and miRNA171c* in tospovirus-infected N. benthamiana. All together, these data suggest that tospoviruses interfere in the RNA silencing pathway by sequestering siRNA and miRNA/miRNA* molecules before they are uploaded into their respective RNA-induced silencing complexes. The observed affinity to long dsRNA for only a subset of the tospoviruses studied is discussed in light of evolutional divergence and their ancestral relation to the animal-infecting members of the Bunyaviridae.

The specific binding to 21-nt double-stranded RNAs is crucial for the anti-silencing activity of Cucumber vein yellowing virus P1b and perturbs endogenous small RNA populations

“…Other RSSs, as potyviral HCPro and tombusviral P19, show strong sizepreference for small RNAs of 21 nt, and act by preventing small RNA loading in the RNA-induced silencing complex (RISC) (Silhavy et al 2002;Lakatos et al 2004;Hemmes et al 2007). Moreover, it has been suggested that some RSSs, as the protein B2 of the nodavirus Flock house virus and the NSs proteins of some tospoviruses could use both strategies to achieve a more efficient suppression (Chao et al 2005;Schnettler et al 2010). By in vitro EMSA analyses we showed that CVYV P1b interacts specifically with dsRNA molecules (Fig.…”

Noncoding Flavivirus RNA Displays RNA Interference Suppressor Activity in Insect and Mammalian Cells

“…2C) or mammalian cells (Fig. 2D), in contrast to the positive controls, TSWV-NSs and RHBV-NS3, which are potent RSSs used in plant and mammalian cells, respectively (57,58). In both assays, the expression of WNV NS1, NS3, and NS5 was confirmed by Western blot analysis (data not shown).…”

“…The binding reaction was performed as previously described (24,58). A total of 0.5 nM radiolabeled RNA was incubated for 20 min at room temperature with ϳ2 g of total protein from the cell extract.…”

“…PCR products were gel purified and used for dsRNA production with the RNAi MEGAscript kit (Ambion). Experiments investigating the effect of Ago1 or Ago2 knockdown on the miR1 sensor constructs in S2 cells were basically performed as previously described (58). S2 cells were mixed with 200 ng of dsRNA of either Ago1, Ago2, or eGFP (unspecific) during seeding.…”

“…dsRNA production and electromobility shift assays (EMSAs). Radioactively labeled 114-nt dsRNA, siRNA, and miRNA molecules were produced as previously described (58). Briefly, the 114-nt dsRNA was generated by T7 RNA polymerase (Promega) transcription from an eGFP PCR product in the presence of [␣-…”