Bamboo mosaic virus (BaMV) is a positive-sense RNA virus belonging to the genus Potexvirus. Open reading frame 1 (ORF1) encodes the viral replication protein that consists of a capping enzyme domain, a helicase-like domain (HLD), and an RNA-dependent RNA polymerase domain from the N to C terminus. ORF5 encodes the viral coat protein (CP) required for genome encapsidation and the virus movement in plants. In this study, application of a yeast-two hybrid assay detected an interaction between the viral HLD and CP. However, the interaction did not affect the NTPase activity of the HLD. To identify the critical amino acids of CP interacting with the HLD, a random mutational library of CP was created using error-prone PCR, and the mutations adversely affecting the interaction were screened by a bacterial two-hybrid system. As a result, the mutations A209G and N210S in CP were found to weaken the interaction. To determine the significance of the interaction, the mutations were introduced into a BaMV infectious clone, and the mutational effects on viral replication, movement, and genome encapsidation were investigated. There was no effect on accumulations of BaMV CP and genomic RNAs within protoplasts; however, the virus cell-to-cell movement in plants was restricted. Sequence alignment revealed that A209 of BaMV CP is conserved in many potexviruses. Mutation of the corresponding residue in Foxtail mosaic virus CP also reduced the viral HLD-CP interaction and restricted the virus movement, suggesting that interaction between CP and a widely conserved HLD in the potexviral replication protein is crucial for viral trafficking through plasmodesmata.To spread throughout hosts, plant viruses have evolved a number of pathways to allow their progeny to pass across plasmodesmata into neighboring cells and travel along the vascular system (8, 26). The virus-encoded movement proteins play a pivotal role through diverse mechanisms in these cellto-cell and vascular transports. Ancillary proteins, for example, the viral coat proteins (CPs) in some cases, and host factors may also participate in these processes. Numerous studies have been conducted to elucidate the movement mechanisms. Many of the results have been summarized in a number of recent reviews (23,28,30). They provided in-depth discussions on issues such as the identification and characterization of the involved viral and host proteins and the transport models for some exemplified viruses, such as Tobacco mosaic virus (TMV) and Potato virus X (PVX). Despite these efforts, many details of the processes remain elusive.Members of the genus Potexvirus have a positive-strand RNA genome that contains five open reading frames (ORFs), a 5Ј methyl cap, and a 3Ј poly(A) tail. ORF1 encodes the viral replication protein, consisting of a capping enzyme domain, a helicase-like domain (HLD), and an RNA-dependent RNA polymerase domain (RdRp) from the N terminus to the C terminus (16,17). The HLD has RNA 5Ј-triphosphatase and nucleoside triphosphatase (NTPase) activities (18). With the co...