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

Abstract Previously we reported that the multifunctional cylindrical inclusion (CI) protein of turnip mosaic virus (TuMV) is targeted to endosomes through the interaction with the medium subunit of adaptor protein complex 2 (AP2β), which is essential for viral infection. Although several functionally important regions in the CI have been identified, little is known about the determinant(s) for endosomal trafficking. The CI protein contains seven conserved acidic dileucine motifs [(D/E)XXXL(L/I)] typical of endocytic sorting signals recognized by AP2β. Here, we selected five motifs for further study and identified that they all were located in the regions of CI interacting with AP2β. Coimmunoprecipitation assays revealed that alanine substitutions in the each of these acidic dileucine motifs decreased binding with AP2β. Moreover, these CI mutants also showed decreased accumulation of punctate bodies, which enter endocytic‐tracking styryl‐stained endosomes. The mutations were then introduced into a full‐length infectious clone of TuMV, and each mutant had reduced viral replication and systemic infection. The data suggest that the acidic dileucine motifs in CI are indispensable for interacting with AP2β for efficient viral replication. This study provides new insights into the role of endocytic sorting motifs in the intracellular movement of viral proteins for replication.

Intracellular membrane traffic relies, to a large extent, on the interactions between adaptor protein (AP) complexes (AP1 to AP5) and the transmembrane cargoes (Owen et al., 2004). AP complexes orchestrate the formation of vesicles destined for transport by distinct intracellular pathways (Park & Guo, 2014). AP complex 2 (AP2) sorts in the endocytic pathway, while AP1 and AP4 facilitate sorting in post-Golgi compartments. Recognition of either tyrosine-based (YXXΦ) or dileucine-based [(D/E)XXXL(L/I)] motifs within the cargo protein by subunits of the AP complex mediates these interactions (X is any amino acid and Φ is a bulky hydrophobic amino acid) (Owen et al., 2004). The AP2 complex is a heterotetramer involved in clathrin-mediated endocytosis of cargo proteins from the plasma membrane in animal cells. In plants, the AP2 complex is a pentamer consisting of two large (α), one medium (β), and two small (σ and μ) subunits (Fan et al., 2015). The AP2 complex recognizes both the YXXΦ and (D/E)XXXL(L/I) motifs for endocytosis (Bonifacino & Traub, 2003). Dileucine-based sorting motifs often harbour an acidic residue D/E at position −9, −5, −4, −3 or −2 from the first leucine pair (Lebrun et al., 2018;Park & Guo, 2014;Xiao et al., 2018). The acidic residue D/E is not as crucial as the LL residues for binding to AP2 (Kelly et al., 2008). AP2 complexes play important roles in floral organ development and plant reproduction (Yamaoka et al., 2013).
The YXXΦ motif present in the cargo proteins is involved in effector-triggered immunity (Geldner & Robatzek, 2008;Hatsugai et al., 2016), and little is known about the biological relevance of the interaction of AP2 and the (D/E)XXXL(L/I) motif in cargo proteins.
In addition, the AP complexes have also been implicated in viral infections, particularly human and animal viral infections (Strazic Geljic et al., 2021). Very few studies have been devoted to unravel the involvement of AP2 in plant viral infections. In the case of the plant DNA virus cauliflower mosaic virus (CaMV), it has been reported that its movement protein (MP) contains three YXXΦ motifs that interact with AP2μ, and at least one of these motifs is essential for the localization of MP to endosomes, for tubule assembly, and for viral infection (Carluccio et al., 2014). More recently, we have shown that the medium unit of AP2 (AP2β) from Arabidopsis recognizes the replication proteins of turnip mosaic virus (TuMV), a plant RNA virus, as cargoes for endocytosis, endosomal trafficking, and viral infection (Wu et al., 2020).
TuMV is one of the most prevalent pathogens worldwide, causing major losses in economically important vegetable, oilseed, biofuel, forage, and ornamental crops, particularly in the family Brassicaceae (Yang et al., 2021). Turnip mosaic virus belongs to the genus Potyvirus, and has a single-stranded, positive-sense, (ss [+]) RNA genome of c.10 kb that contains a long open reading frame (ORF) and a short ORF resulting from RNA polymerase slippage in the P3 coding sequence (Olspert et al., 2015;Wylie et al., 2017). On translation, the two polyproteins are processed by three viral proteases into 11 mature proteins. Among these, the cylindrical inclusion (CI) protein is a multipartner and multifunctional protein, which has ATPase and RNA helicase activities, and participates in viral genome replication and viral intercellular movement (Revers & Garcia, 2015;Sorel et al., 2014). In a recent study, we reported that AP2β is essential for TuMV replication and mediates the trafficking of TuMV CI in endosomes (Wu et al., 2020). This study was directed to further understand the mechanism underlying the TuMV CI and AP2β interaction for viral endosome trafficking and replication.
We hypothesized that TuMV proteins might bind to AP2 to mediate intracellular traffic for viral replication and therefore we first aimed to identify the region(s) in CI involved in AP2β binding.
It is well known that proteins that are intrinsically disordered can obtain the required structure when they bind to their interactors (Charon et al., 2016;Dyson & Wright, 2005). Therefore, the disordered region(s) of the CI were predicted using the PONDR (Predictor of Natural Disordered Regions) website ( Figure 1a). The output of three PONDR algorithms predicted four disordered regions: amino acid residues 1-100, residues 101-300, residues 301-500, and residues 501-644. CI was then split accordingly into these four regions ( Figure 1b) using the primers listed in Table S1. A bimolecular fluorescence complementation (BiFC) assay was performed in Nicotiana benthamiana leaves by agroinfiltration to confirm the interaction of each of these four regions of CI with AtAP2β (see File S1 for experimental procedures). AtAP2β and CI fragments were fused to the Cterminus of either the N-proximal or C-proximal region of the yellow fluorescent protein (YFP) (Tian et al., 2011), respectively. Consistent with our recent report (Wu et al., 2020), there was a positive interaction between full-length CI and AtAP2β, which occurred in the cytoplasm, showing as punctate bodies around the plasma membrane (PM) (Figure 1c). All four CI fragments also interacted with AtAP2β.
CI 101-300 and CI 501-644 bound with AtAP2β mainly at the PM, while CI 1-100 interacted at several punctate bodies in the cytoplasm and the CI 301-500 -AtAP2β complex formed several ring-like structures in the cytoplasm (Figure 1c; inset). No interaction signals were observed in the negative controls ( Figure S1).
We therefore focused on another endocytic motif (D/E)XXXL(L/I).
Inspection of the primary amino acid sequence of TuMV CI revealed seven classical (D/E)XXXL(L/I) sorting motifs in which an acidic residue D or E is located at the −2 to −10 position relative to the first leucine pair or LL residues: 79D-83LL84, 81D-83LL84, 181D-190LL191, 263D-268LL269, 435D-445LL446, 550D-552LI553, and 602E-608LL609 ( Figure S2). Here, we mainly focused on the acidic D/E and LL residues of the CI protein in TuMV infection. The D81 residue in the CI protein has been identified to be important for interaction with the coat protein (CP) and TuMV intercellular movement previously (Deng et al., 2015), thus the 81D-83LL84 motif was not included here. Five (D/E)XXXLL sorting motifs (79D-83LL84, 181D-190LL191, 263D-268LL269, 435D-445LL446, and 602E-608LL609, which we named i to v) were selected for further study.
Notably, these five motifs are located in the four candidate binding regions (Figures 1c and S2). Multiple alignments of the CI amino acid sequences among potyviruses indicated that these motifs are conserved among multiple potyviruses (Figure 1d), which could indicate that they play a relevant and conserved role, potentially involved in the interaction with AtAP2β. These results are therefore consistent with the hypothesis that acidic dileucine motifs in the four disordered regions of the CI protein are involved in binding with AtAP2β.
To further dissect the role of these acidic dileucine motifs in binding with AtAP2β, we generated a double mutant (DM) with D/E-LL to D/E-AA substitution and a triple mutant (TM) with D/E-LL to A-AA substitution for each of the motifs, as shown in Table 1 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 × Myctagged construct, and wild-type CI and its mutants were tagged by an N-terminal YFP tag. These fusions were transiently coexpressed in N. benthamiana leaves, followed by co-IP. As shown in Figure 2b, and consistent with our previous report, the CI protein could be immunoprecipitated with AtAP2β. All 10 CI mutants were able to interact with AtAP2β, but the interaction was weaker than with wildtype CI, especially for the five triple mutants. Taken together, these data suggest that the absence of just one of the motifs significantly weakens the protein-protein interaction between CI and AtAP2β.
The protein mapping and mutagenesis analysis revealed that alanine substitutions in the dileucine motifs altered the intracellular distribution of the CI protein when interacting with AtAP2β (Figures 1   and 2). A key question emerging from this observation is whether the acidic dileucine motifs in TuMV CI are functional internalization motifs. To this end, we investigated the subcellular localization of these CI mutants. Wild-type CI protein and its mutants fused to an N-terminal YFP tag were transiently expressed in N. benthamiana In mammals and yeast, LL residues are recognized by APs 1-3 with a combination of two subunits: AP1 γ-σ1, AP2 α-σ2, and AP3 δ-σ3 hemicomplexes (Doray et al., 2007;Janvier et al., 2003). The binding specificity of plant AP complexes has yet to be elucidated. The acidic dileucine motif of Arabidopsis tonoplast-localized ion transporter VTI1, EKQTLL, interacts with AP1γ1/2 and σ1/2 subunits, but not with AP3 δ and σ (Wang et al., 2014), raising the possibility that these subunits could also be interacting partners of TuMV CI besides AP2β.
Although we cannot exclude the possibility that the acidic dileucine motif in CI may be important for binding of other host proteins, our study addresses the function of these motifs in the intracellular targeting of TuMV CI, which is dependent on its interaction with AP2β.
Finally, the role of these acidic dileucine motifs of CI in TuMV infection was investigated by alanine mutagenesis of a pCambia-TuMV::GFP infectious clone of TuMV (Cotton et al., 2009) 79  83  84  181  190  191  263  268  269  435  445  446  602  608 609 residue plays a predominant role in infection compared with the LL in this motif. Motif iii is also located in the RNA helicase domain of CI (Sorel et al., 2014). Previously, alanine-scanning mutagenesis of conserved amino acids of TuMV CI revealed that residues at positions 261 and 263, which partially overlap with motif iii in this study, are required for TuMV replication (Deng et al., 2015), suggesting that motif iii might also have an RNA helicase role.
In summary, this work uncovers novel CI functional motifs, as well as molecular mechanisms, underlying the role of the acidic di-

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that supports the findings of this study are available in the supplementary material of this article.