The Barley stripe mosaic virus γb protein promotes viral cell-to-cell movement by enhancing ATPase-mediated assembly of ribonucleoprotein movement complexes.

Nine genera of viruses in five different families use triple gene block (TGB) proteins for virus movement. The TGB modules fall into two classes: hordei-like and potex-like. Although TGB-mediated viral movement has been extensively studied, determination of the constituents of the viral ribonucleoprotein (vRNP) movement complexes and the mechanisms underlying their involvement in vRNP-mediated movement are far from complete. In the current study, immunoprecipitation of TGB1 protein complexes formed duringBarley stripe mosaic virus(BSMV) infection revealed the presence of the gamma b protein in the products. Further experiments demonstrated that TGB1 interacts with gamma bin vitroandin vivo, and that gamma b-TGB1 localizes at the periphery of chloroplasts and plasmodesmata (PD). Subcellular localization analyses of the gamma b protein inNicotiana benthamianaepidermal cells indicated that in addition to chloroplast localization, gamma b also targets the ER, actin filaments and PD at different stages of viral infection. By tracking gamma b localization during BSMV infection, we demonstrated that gamma b is required for efficient cell-to-cell movement. The N-terminus of gamma b interacts with the TGB1 ATPase/helicase domain and enhances ATPase activity of the domain. Inactivation of the TGB1 ATPase activity also significantly impaired PD targeting.In vitrotranslation together with co-immunoprecipitation (co-IP) analyses revealed that TGB1-TGB3-TGB2 complex formation is enhanced by ATP hydrolysis. The gamma b protein positively regulates complex formation in the presence of ATP, suggesting that gamma b has a novel role in BSMV cell-to-cell movement by directly promoting TGB1 ATPase-mediated vRNP movement complex assembly. We further demonstrated that elimination of ATPase activity abrogates PD and actin targeting ofPotato virus X(PVX) andBeet necrotic yellow vein virus(BNYVV) TGB1 proteins. These results expand our understanding of the multifunctional roles of gamma b and provide new insight into the functions of TGB1 ATPase domains in the movement of TGB-encoding viruses. Author summary Plant viruses employ varied movement strategies to mediate local and systemic infections. Viral ribonucleoprotein (vRNP) movement complexes comprising either the hordei- or potex-like triple gene block (TGB) and viral RNAs represent important models for plant virus movement. However, the constituents of viral ribonucleoprotein (vRNP) movement complexes as well as their biological significance in vRNP assembly and subsequent interactions are far from complete. Additionally, the mechanistic roles of the highly conserved TGB1 ATPase domain in vRNP-mediated movement remain an enigma. Here, we demonstrate that the gamma b protein acts as a novel positive regulator of BSMV cell-to-cell movement by directly interacting with the TGB1 protein.In vitrobiochemical assays verified an essential role of TGB1 ATPase-mediated ATP hydrolysis in assembly of vRNP movement complexes, a process that can be further enhanced by the gamma b protein. We also extend our studies of BSMV TGB1 ATPase to those of PVX and BNYVV, and suggest a model for an evolutionally conserved mode of energy-coupled vRNP movement complex assembly among different TGB-encoding viruses. Our results address the knowledge gap between TGB1 ATPase activity and vRNP movement complex assembly and expand our understanding of the multifaceted roles of gamma b in BSMV infection.