P3N-PIPO Interacts with P3 via the Shared N-Terminal Domain To Recruit Viral Replication Vesicles for Cell-to-Cell Movement

Abstract

P3N-PIPO, the only dedicated movement protein (MP) of potyviruses, directs cylindrical inclusion (CI) protein from the cytoplasm to the plasmodesma (PD), where CI forms conical structures for intercellular movement. To better understand potyviral cell-to-cell movement, we further characterized P3N-PIPO using Turnip mosaic virus (TuMV) as a model virus. We found that P3N-PIPO interacts with P3 via the shared P3N domain and that TuMV mutants lacking the P3N domain of either P3N-PIPO or P3 are defective in cell-to-cell movement. Moreover, we found that the PIPO domain of P3N-PIPO is sufficient to direct CI to the PD, whereas the P3N domain is necessary for localization of P3N-PIPO to 6K2-labeled vesicles or aggregates. Finally, we discovered that the interaction between P3 and P3N-PIPO is essential for the recruitment of CI to cytoplasmic 6K2-containing structures and the association of 6K2-containing structures with PD-located CI inclusions. These data suggest that both P3N and PIPO domains are indispensable for potyviral cell-to-cell movement and that the 6K2 vesicles in proximity to PDs resulting from multipartite interactions among 6K2, P3, P3N-PIPO, and CI may also play an essential role in this process. IMPORTANCE Potyviruses include numerous economically important viruses that represent approximately 30% of known plant viruses. However, there is still limited information about the mechanism of potyviral cell-to-cell movement. Here, we show that P3N-PIPO interacts with and recruits CI to the PD via the PIPO domain and interacts with P3 via the shared P3N domain. We further report that the interaction of P3N-PIPO and P3 is associated with 6K2 vesicles and brings the 6K2 vesicles into proximity with PD-located CI structures. These results support the notion that the replication and cell-to-cell movement of potyviruses are processes coupled by anchoring viral replication complexes at the entrance of PDs, which greatly increase our knowledge of the intercellular movement of potyviruses.

Association between flower stalk elongation, anArabidopsisdevelopmental trait, and the subcellular location and movement dynamics of the nonstructural protein P3 ofTurnip mosaic virus

“…At 1 dpi, co-expression of P3[UK1]-GFP together with 6K2[UK1]-ChFP revealed the joint perinuclear presence of both proteins and chloroplasts (Figure 8a-d (Figure 8i-p, Videos S15 and S16). 6K2 forms chloroplast-interacting ER-derived vesicles (Wei et al, 2010(Wei et al, , 2013Grangeon et al, 2012;Cui et al, 2017), and UK 1 P3 is present in these membranous structures, as described for TEV and TuMV by other authors (Cui et al, 2010(Cui et al, , 2017Chai et al, 2020), but the behaviour of JPN 1 P3 in relation to its 6K2 partner has not been previously described.…”

“…P3 may or may not play a role in the formation of the VRC, but it would be its carrier, although at different rates depending on viral strains. Very recently a model for the role of TuMV P3 protein in the viral cell-to-cell movement has been proposed (Chai et al, 2020).…”

“…It was also proposed to interact with itself (Choi et al, 2000) and with no other viral protein (Kang et al, 2004). Very recently, the interaction of P3 with the fusion protein P3N-PIPO has been shown for TuMV (Chai et al, 2020).…”

Small hydrophobic viral proteins involved in intercellular movement of diverse plant virus genomes

“…During infection, RNA viruses re-organize cell membranes into ‘viral factories’ that are intracellular virus-specific compartments serving as sites of virus replication. These virus replication compartments (VRCs) contain viral RNA as well as virus and host proteins involved in genomic RNA replication [66] – [72] . In potyviruses, almost all virus polypeptides forming VRCs are involved in intercellular RNA genome movement as well.…”

“…In potyviruses, a small viral membrane protein 6K2 is involved in the local spatial re-arrangement of the ER membrane and the formation of vesicular VRC at the ER exit sites [59] , [65] , [73] – [76] . Several other viral proteins including CI (replicative SF2 RNA helicase), 6K1, 6K2, NIa, HC-Pro, P3, and P3N-PIPO have been shown to be functional protein elements of VRC in addition to NIb [65] , [72] , [77] , [78] . The VRC with incorporated viral CP may work as the motile vesicles moving along actin filaments and involved in virus replication, the genome intercellular movement and localization on chloroplasts [42] , [65] , [76] , [79] , [80] .…”