Abstract
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 during Barley stripe mosaic virus (BSMV) infection revealed the presence of the γb protein in the products. Further experiments demonstrated that TGB1 interacts with γb in vitro and in vivo, and that γb-TGB1 localizes at the periphery of chloroplasts and plasmodesmata (PD). Subcellular localization analyses of the γb protein in Nicotiana benthamiana epidermal cells indicated that in addition to chloroplast localization, γb also targets the ER, actin filaments and PD at different stages of viral infection. By tracking γb localization during BSMV infection, we demonstrated that γb is required for efficient cell-to-cell movement. The N-terminus of γb 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 vitro translation together with co-immunoprecipitation (co-IP) analyses revealed that TGB1-TGB3-TGB2 complex formation is enhanced by ATP hydrolysis. The γb protein positively regulates complex formation in the presence of ATP, suggesting that γb 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 of Potato virus X (PVX) and Beet necrotic yellow vein virus (BNYVV) TGB1 proteins. These results expand our understanding of the multifunctional roles of γb and provide new insight into the functions of TGB1 ATPase domains in the movement of TGB-encoding viruses.
Highlights
Plant RNA viruses typically replicate on host endomembranes and require one or several movement proteins (MPs) for assembly and transport of progeny viral (v) RNAs to adjacent cells through plasmodesmata (PD) [1, 2]
Because the coat protein (CP) is dispensable for cell-to-cell movement mediated by Barley stripe mosaic virus (BSMV) triple gene block (TGB) [37], RNAβ CP expression was eliminated by mutating the start codon of the CP ORF from AUG to UUG to produce RNAβmCP)
A small proportion of the IP products were separated by SDS-PAGE followed by silver staining, which indicated that substantial proteins were co-precipitated with the 3xFlag-TGB1 proteins compared with untagged TGB1 (S2 Fig)
Summary
Plant RNA viruses typically replicate on host endomembranes and require one or several movement proteins (MPs) for assembly and transport of progeny viral (v) RNAs to adjacent cells through plasmodesmata (PD) [1, 2]. Based on the presence or absence of long N-terminal domains in TGB1 proteins and variable requirements of the coat protein (CP) for viral cell-to-cell movement, the TGB modules fall into two groups: hordei-like (class I) and potex-like (class II) [9,10,11]. Coordinated actions of the three TGB proteins are essential for viral ribonucleoprotein (vRNP) movement complex assembly and subsequent viral cell-to-cell movement [12,13,14]. The assembly and regulations of vRNP movement complexes, and recycling of plant-viral movement proteins by the endocytic pathway remain unclear at the molecular level
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