Nuclear Inclusion Protein B-Driven In Vitro Replication System for Potato Virus Y

Potato virus Y (PVY) is a significant pathogen affecting potato and other crops globally. Current research on PVY primarily concentrates on strain analysis and protein interactions with the host. However, studies on PVY replication in vitro remain limited, primarily due to the absence of a dedicated in vitro replication system for PVY. This study aimed to develop an in vitro replication system mediated by the nuclear inclusion protein B (NIb) of PVY. Through IPTG gradient experiments, we determined that the optimal expression concentration of MBP-NIb is 0.2 mM. The results indicate that the optimal temperature for NIb expression in Escherichia coli is 37 °C, yielding approximately 10% solubility, which is sufficient for subsequent affinity chromatography using the MBP tag. The purified MBP-NIb specifically recognizes the 3′ terminal region of the positive-sense RNA and the 5′ terminal region of the negative-sense RNA of PVY in vitro, facilitating the synthesis of complementary strands of the viral RNAs. To our knowledge, this is the first successful establishment of an in vitro replication system for PVY. This system will advance the study of PVY replication mechanisms.