Replication and trafficking of a plant virus are coupled at the entrances of plasmodesmata

lant viruses use movement proteins (MPs)to modify intercellular pores called plasmodesmata (PD)to cross the plant cell wall. Many viruses encode a conserved set of three MPs, known as the triple gene block (TGB), typified by Potato virus X (PVX). In this paper, using live-cell imaging of viral RNA (vRNA)and virus-encoded proteins, we show that the TGB proteins have distinct functions during movement. TGB2 and TGB3 established endoplasmic reticulum-derived mem-branous caps at PD orifices. These caps harbored the PVX replicase and nonencapsidated vRNA and repre-sented PD-anchored viral replication sites. TGB1 medi-ated insertion of the viral coat protein into PD, probably by its interaction with the 5?end of nascent virions, and was recruited to PD by the TGB2/3 complex. We pro-pose a new model of plant virus movement, which we term coreplicational insertion, in which MPs function to compartmentalize replication complexes at PD for local-ized RNA synthesis and directional trafficking of the virus between cells.