The Potyvirus NIb Multifunctional Protein Suppresses Antiviral Defense by Interacting with Several Protein Components of the RNA Silencing Pathway

Potyviruses express their genome as a single large open reading frame, translated into a polyprotein that is post-translationally cleaved by three virus-encoded proteases into 10 functional proteins. Several of these potyviral proteins, including nuclear inclusion protein b (NIb), are multifunctional. Here, using the classic GFP silencing in Nicotiana benthamiana gfp-transgenic plants model, we show that NIb, in addition to its canonical role as the viral RNA-dependent RNA polymerase (RdRP), functions as a suppressor of RNA silencing. Mutational analyses revealed a previously unreported NIb nuclear localization signal (NLS) consisting of a triple-lysine motif, and NIb suppression of RNA silencing activity was lost when this novel NLSs were mutated, suggesting that nuclear localization is required for NIb suppression of RNA silencing activity. Analysis of sequenced GFP siRNAs revealed three prominent siRNA hotspots at ≈nt 175, ≈320–330, and 560–700 nt. These data showed that there were differences in the positional distribution of the siRNAs between samples expressing NIb and those expressing NIbDel3x2, the NIb null mutant that does not suppress RNA silencing. However, there was no increase in the transcript‑wide siRNA burden between the two treatments. Furthermore, NIb was found to interact with four key RNA silencing pathway proteins—AGO4, HSP70, HSP90, and SGS3. Except for HSP90, each of these proteins showed degradation products that were absent in NIb mutants that did not suppress RNA silence. These findings support a role for NIb in countering host defense during virus infection.