GBNV encoded movement protein (NSm) remodels ER network via C-terminal coiled coil domain

Abstract

Plant viruses exploit the host machinery for targeting the viral genome-movement protein complex to plasmodesmata (PD). The mechanism by which the non-structural protein m (NSm) of Groundnut bud necrosis virus (GBNV) is targeted to PD was investigated using Agrobacterium mediated transient expression of NSm and its fusion proteins in Nicotiana benthamiana. GFP:NSm formed punctuate structures that colocalized with mCherry:plasmodesmata localized protein 1a (PDLP 1a) confirming that GBNV NSm localizes to PD. Unlike in other movement proteins, the C-terminal coiled coil domain of GBNV NSm was shown to be involved in the localization of NSm to PD, as deletion of this domain resulted in the cytoplasmic localization of NSm. Treatment with Brefeldin A demonstrated the role of ER in targeting GFP NSm to PD. Furthermore, mCherry:NSm co-localized with ER-GFP (endoplasmic reticulum targeting peptide (HDEL peptide fused with GFP). Co-expression of NSm with ER-GFP showed that the ER-network was transformed into vesicles indicating that NSm interacts with ER and remodels it. Mutations in the conserved hydrophobic region of NSm (residues 130-138) did not abolish the formation of vesicles. Additionally, the conserved prolines at positions 140 and 142 were found to be essential for targeting the vesicles to the cell membrane. Further, systematic deletion of amino acid residues from N- and C-terminus demonstrated that N-terminal 203 amino acids are dispensable for the vesicle formation. On the other hand, the C-terminal coiled coil domain when expressed alone could also form vesicles. These results suggest that GBNV NSm remodels the ER network by forming vesicles via its interaction through the C-terminal coiled coil domain. Interestingly, NSm interacts with NP in vitro and coexpression of these two proteins in planta resulted in the relocalization of NP to PD and this relocalization was abolished when the N-terminal unfolded region of NSm was deleted. Thus, the NSm interacts with NP via its N-terminal unfolded region and the NSm-NP complex could in turn interact with the ER membrane via the C-terminal coiled coil domain of NSm to form vesicles that are targeted to PD and there by assist the cell to cell movement of the viral genome complex.

Intracellular Localization, Interactions and Functions of Capsicum Chlorosis Virus Proteins

“…Interactions of NSm and N proteins have also been reported for several tospoviruses other than TSWV (Dietzgen et al, 2012; Leastro et al, 2015; Singh and Savithri, 2015; Tripathi et al, 2015a). Therefore, we expected that CaCV NSm might interact with cognate N protein.…”

“…However, BiFC assays did not show such an interaction in either orientation in repeated experiments, despite NSm being expressed and showing self-interaction. This may be due to steric hindrance or limited access to NSm N-terminus in the fusion protein as observed for some other tospoviruses previously studied (Leastro et al, 2015; Singh and Savithri, 2015). These studies showed NSm-N dimer formation of BeNMV, GBNV, and TSWV was restricted to certain combinations of fluorescent protein pairs tested.…”

“…For instance, TSWV N protein has been shown to traffic on the actin/ER network, a novel property of a plant virus capsid protein (Feng et al, 2013). Studies on groundnut bud necrosis virus (GBNV) unraveled a novel feature of tospovirus NSm, which remodels ER networks to form vesicles to facilitate viral movement (Singh and Savithri, 2015). CaCV is an emerging tospovirus which is serologically and phylogenetically distinct (belongs to a different clade) from TSWV (McMichael et al, 2002; Oliver and Whitfield, 2016).…”

“…Feng et al (2013) showed cellular actin/ER membrane transport networks are involved in intracellular movement of TSWV N inclusions and ER network is involved in intercellular translocation of NSm and virus replication complexes where host factors are likely to play an important role (Feng et al, 2016). On the other hand, Singh and Savithri (2015) described vesicle-mediated intracellular transport of GBNV NSm. GBNV NSm directly interacts with ER membranes via a coiled-coil domain at the C-terminus, which remodels ER network to form vesicles that translocate NSm to PD.…”

“…In host plants, viral RNPs in association with NSm are transported from initially infected cells to neighboring cells through plasmodesmata (PD), assisting progression of the infection (Storms et al, 1995; Soellick et al, 2000). Recently, molecular interactions of viral and host proteins have been reported for TSWV and several other tospoviruses (Paape et al, 2006; Feng et al, 2013, 2016; Singh and Savithri, 2015). These findings point to significant complexity in the tospovirus infection process and unexplored unique characteristics for some tospovirus proteins beyond TSWV.…”