Determinants of the Nucleocytoplasmic Balance of the Cucumber Mosaic Virus and Peanut Stunt Virus 2b Proteins Regulate the Subcellular Localization and RNA Silencing Suppressor Activity

The 2b protein of cucumoviruses is an RNA silencing suppressor (RSS), and its activity is associated with its nucleocytoplasmic localization. However, the contribution of nuclear localization signals (NLSs), nuclear export signals (NESs), and their sequence context remains poorly understood. Here, we compare cucumber mosaic virus (CMV) and peanut stunt virus (PSV) 2b proteins and their NLS- and NES-containing regions using recombinant proteins. The subcellular localization of PSV 2b has not been characterized, and no protein structural model has been reported previously. CMV 2b displayed a balanced nucleocytoplasmic distribution and strong RSS activity, whereas PSV 2b showed predominantly nuclear localization and weaker RSS activity. Exchange of the N-terminal region and NLSs resulted in pronounced changes in localization and RSS activity. PPC 2b, containing the CMV C-terminal region preceded by PSV sequence, showed the highest RSS activity, while the corresponding recombinant virus caused the most severe symptoms. AlphaFold analysis demonstrated lower structural confidence and higher predicted flexibility in the NES-containing C-terminal regions of CMV 2b and PPC 2b, compared with PSV 2b and CCP 2b, containing the PSV C-terminal region preceded by CMV sequence. These differences may influence NES accessibility and nuclear export.