Turnip mosaic virus co-opts the vacuolar sorting receptor VSR4 to promote viral genome replication in plants by targeting viral replication vesicles to the endosome

Abstract

Accumulated experimental evidence has shown that viruses recruit the host intracellular machinery to establish infection. It has recently been shown that the potyvirus Turnip mosaic virus (TuMV) transits through the late endosome (LE) for viral genome replication, but it is still largely unknown how the viral replication vesicles labelled by the TuMV membrane protein 6K2 target LE. To further understand the underlying mechanism, we studied the involvement of the vacuolar sorting receptor (VSR) family proteins from Arabidopsis in this process. We now report the identification of VSR4 as a new host factor required for TuMV infection. VSR4 interacted specifically with TuMV 6K2 and was required for targeting of 6K2 to enlarged LE. Following overexpression of VSR4 or its recycling-defective mutant that accumulates in the early endosome (EE), 6K2 did not employ the conventional VSR-mediated EE to LE pathway, but targeted enlarged LE directly from cis-Golgi and viral replication was enhanced. In addition, VSR4 can be N-glycosylated and this is required for its stability and for monitoring 6K2 trafficking to enlarged LE. A non-glycosylated VSR4 mutant enhanced the dissociation of 6K2 from cis-Golgi, leading to the formation of punctate bodies that targeted enlarged LE and to more robust viral replication than with glycosylated VSR4. Finally, TuMV hijacks N-glycosylated VSR4 and protects VSR4 from degradation via the autophagy pathway to assist infection. Taken together, our results have identified a host factor VSR4 required for viral replication vesicles to target endosomes for optimal viral infection and shed new light on the role of N-glycosylation of a host factor in regulating viral infection.

StREM1.3 REMORIN Protein Plays an Agonistic Role in Potyvirus Cell-to-Cell Movement in N. benthamiana

“…The cylindrical inclusion (CI) of TuMV is a multifunctional scaffolding protein crucial for potyvirus movement [ 5 ] and is the component of the characteristic pinwheel-shaped inclusion bodies, a unique feature shared by all Potyviridae [ 8 ]. Here, we showed that TuMV CI interacts with StREM1.3 in SuY2H, an interaction that was not observed with AtREM1.2 by [ 29 ] using the same SuY2H assays ([ 28 ] Figure S11), highlighting another discrepancy between AtREM1.2 and StREM1.3 properties. In our study, the interaction between TuMV CI and StREM1.3 occurs at the level of PM but is not predominantly associated with PD.…”

“…By contrast with the results of [ 42 ], which showed that NbREM1 is downregulated during RSV infection in N. benthamiana , we showed here that the level of endogenous NbREMS was not modified by TuMV infection, as described during PVX infection [ 36 ]. Although in one study [ 29 ], the authors showed that in N. benthamiana , transiently expressed HA-AtREM1.2 is degraded during TuMV infection ([ 28 ] Figure S10), there is no indication concerning the effect of TuMV infection on the endogenous NbREMs protein levels.…”

“…A few plant factors involved in potyvirus cell-to-cell movement were identified using virus-target-based approaches (such as yeast two-hybrid cDNA library screening and in planta pull-down assays using viral baits), forward-genetics, and bottom-up proteomic approaches. Those factors are diverse and correspond to PM-associated proteins targeted to PD [ 19 ], cell-wall-loosening protein [ 20 ], HSP70-related chaperones [ 21 ], proteins essential for endomembrane remodelling, endoplasmic reticulum bending, inter-organelle vesicular exchanges [ 13 , 17 , 22 , 23 , 24 , 25 ], and proteins involved in endocytic processes [ 26 , 27 , 28 ]. Those factors identified in diverse plant–potyvirus pathosystems were shown to be proviral movement factors.…”

Autophagy in plant viral infection

“…It has been reported that late endosome (LE) serves as a replication site for TuMV, during TuMV infection, viral replication vesicles target to LE [77]. Very recently, Wu et al [78] found that TuMV replication depends on functional vesicle transport from cis-Golgi to the enlarged LE pathway, which is mediated by a specific vacuolar sorting receptor protein VSR4. Nglycosylation of VSR4 is required to modulate the targeting of TuMV replication vesicles to enlarged LE and enhance viral replication.…”

Acidic dileucine motifs in the cylindrical inclusion protein of turnip mosaic virus are crucial for endosomal targeting and viral replication

“…To confirm these protein–protein interactions, we conducted a coimmunoprecipitation (co‐IP) assay as previously described (Win et al, 2011 ; Wu et al, 2022 ). AtAP2β was cloned into pBA‐FLAG‐4 × Myc‐DC vector (Zhu et al, 2011 ) to yield the FLAG‐4 × Myc‐tagged construct, and wild‐type CI and its mutants were tagged by an N‐terminal YFP tag.…”