The stress granule component G3BP is a novel interaction partner for the nuclear shuttle proteins of the nanovirus pea necrotic yellow dwarf virus and geminivirus abutilon mosaic virus
Graphical abstract
Introduction
Stress granules (SGs) are cytoplasmic-localized dynamic structures that quickly form when external stresses are applied, which leads to a general decline in translation. On the other hand, SGs disperse and gene expression resumes, when cellular stress conditions abate (reviewed in Buchan and Parker, 2009). The scenario most often described for SG formation follows when oxidative, nutrient or heat stress activates one of the eIF2α kinases, which phosphorylate the alpha subunit of eIF2 and abortive translation initiation complexes form around mRNA molecules (Kedersha et al., 1999). Assembly of the granules is dependent on the RNA-binding ability of the Ras-GAP SH3 domain–binding protein (G3BP; Tourrière et al., 2003). G3BP is a multifunctional RNA-binding protein that is present in three forms in humans, G3BP-1, -2a and -2b. SGs have been shown to be induced by a number of viral infections and have been implicated in cellular defense against infection. In many cases, viral gene products were shown to inhibit SG assembly such that their formation is transient or undetectable in wild-type virus infections (Beckham and Parker, 2008, Lloyd, 2012, White et al., 2007). McInerney et al. (2005) reported that early events in Semliki Forest virus (SFV) infection induce the formation of SGs in the cytoplasm of infected cells, but that the SGs are rapidly disassembled in the vicinity of newly formed viral RNA replication complexes as the infection progresses. The C-terminal domain of the viral nonstructural protein 3 (nsP3) of SFV forms a complex with G3BP and sequesters it into viral RNA replication complexes in a manner that inhibits the formation of SG on viral mRNAs (Panas et al., 2014, Panas et al., 2012). Emerging evidence indicates that plant cells utilize SGs for posttranscriptional gene control similar to mammalian cells. SG-like structures were identified by cellular localization studies of eukaryotic initiation factor 4E (eIF4E), oligouridylate-binding protein 1 (UBP1), poly-A binding protein (PABP) and small ribosome subunit proteins (Pomeranz et al., 2010, Weber et al., 2008). In Arabidopsis, a tandem zinc finger protein, AtTZF1, shuttles into SG-like structures. TZF proteins recruit and activate the mRNA decay machinery in mammalian cells (Lykke-andersen and Wagner, 2005) and may nucleate mRNA processing bodies (PBs) in conjunction with silencing of mRNAs containing AU-rich elements. Pomeranz et al. (2010) found that AtTZF1 co-localizes with plant SG components and also binds both DNA and RNA. Reverse genetic analyses indicated that AtTZF1 acts as a positive regulator of sugar and abscisic acid (ABA) −mediated stress response, and as a negative regulator of giberellic acid (GA) −dependent growth response. Plants over-expressing AtTZF1 are more compact than wild type, flower later, and exhibit superior cold- and drought-tolerance (Pomeranz et al., 2010).
There are only a few studies, which link plant virus to RNA granules (PBs and SGs). For instance, the replication cycle of brome mosaic virus (BMV), which belongs to the alphavirus-like superfamily, has been studied in yeast and PB constituent proteins were shown to affect its replication. PBs are cytoplasmic protein complexes that are also involved in degradation and translational arrest of mRNA and are functionally linked to SGs (Beckham et al., 2007). Recently, it was demonstrated that the helper component-proteinase (HCpro) of potato virus A (PVA), the potyviral suppressor of RNA silencing, induces the formation of RNA granules (Hafrén et al., 2015). Presence of argonaute 1 (AGO1), UBP1, varicose (VCS) and eIF4E in these potyvirus-induced RNA granules was shown. All these proteins are also components of SGs and PBs. To date, this is the only study that describes the co-localization of a plant SG component (UBP1) and a plant virus protein (HC-Pro) (Hafrén et al., 2015).
Recently, (Panas et al., 2015) proposed further candidates of virus proteins with the ability to bind to G3BP and thereby potentially inhibiting SG function. This assumption is based on the similarity to the G3BP-binding motif ‘FGDF’ in the nsP3 of SFV, and the authors suggested that several plant viruses may have the potential to bind to G3BP, e. g. the M-Rep of subterranean clover stunt virus (SCSV) that contains an ‘FGEF’-motif. SCSV is a member of the Nanoviridae family, plant virus pathogens with a multipartite genome of circular single-stranded (ss) DNAs (Vetten et al., 2012). The observation by Panas et al. (2015) that the M-Rep of subterranean clover stunt virus contained an ‘FGEF’-motif cannot be extended to the M-Rep proteins of other nanoviruses as their sequences vary at this position. Available data on the three-dimensional structure of the faba bean necrotic yellow virus M-Rep DNA binding domain show that amino acids at this position are part of a conserved beta-sheet in the replication initiator proteins of ssDNA viruses (Vega-Rocha et al., 2007), which would make their implication in G3BP-binding difficult to reconcile. Therefore, we looked for the presence of other potential G3BP-binding motifs in plant virus proteins and found that the NSPnanovirus harbor a well conserved ‘F(N/T)GSF’-motif in the central part of the protein and NSPbegomovirus a ‘(F/Y)VS(F/Y)’-motif at the C-terminal end. Hence the question arose whether the NSPs have the ability to bind to the plant homologues of G3BP.
First we wanted to see if plant SGs are functionally similar to their mammalian counterparts and then test if the NSP of pea necrotic yellow dwarf virus (PNYDV), a nanovirus identified in commercially grown pea (Pisum sativum L.) in Germany (Grigoras et al., 2010) and the NSP of abutilon mosaic virus (AbMV) have the ability to bind to potential G3BP homologues from plants.
Section snippets
Microorganisms, plants, and general methods
Material for the construction of PYNDV NSP and AbMV NSP expression vectors were kindly provided by Bruno Gronenborn, Tatjana Kleinow and Holger Jeske, respectively. Material for the construction of GFP_nsP3_31 and GFP:HsG3BP expression vectors were kindly provided by Gerald M. McInerney. N. benthamiana plants were grown in an insect-free S1 greenhouse with 16 h supplementary illumination. Recombinant DNA techniques were performed according to (Sambrook and Russell, 2001). Restriction
Cellular localization studies of C-terminal domain of SFV nsP3 fused to GFP (GFP:nsP3_31) in Nicotiana benthamiana epidermal cells
It was shown that HsG3BP binds to an FGDF followed by a 2 charged aa-motif, a motif within the C-terminal domain of SFV nsP3. The last 31aa of nsP3 were sufficient to bind to HsG3BP (Panas et al., 2014). Several G3BP-like proteins have been annotated for plants, but none has been identified as a plant SGs component. We hypothesized that the SFV nsP3 C-terminal domain fused to GFP (GFP:nsP3_31; pEGFP-nsP3-31 in Panas et al., 2014) would also bind to a G3BP plant homologue. GFP and GFP:nsP3_31
Discussion
SGs are protein-mRNA aggregates that are formed in response to biotic and abiotic stress, resulting in stalled translation (Buchan and Parker, 2009). Essential for SG formation is the cellular protein G3BP (Kedersha et al., 2016), which has not yet been characterized in plants. The observation that human G3BP expressed in plant tissue co-localized with a plant SG marker protein TZF1 (Suppl. Fig. 6) indicated that the molecular mechanisms of SG formation seems to be conserved among eukaryotes.
Conclusion
In summary, we propose a new plant interaction partner for the nanovirus and begomovirus nuclear shuttle proteins, the G3BP-like protein. We also speculate that the interplay of G3BP/USP10/Caprin1 in mammalian cells and the interference with this mechanism by viruses is conserved in plants. G3BP-mediated SG assembly is regulated by mutually exclusive binding of Caprin1 and USP10. Caprin1 binding to G3BP promotes SG formation, whereas USP10 binding to G3BP inhibits SG assembly (Kedersha et al.,
Acknowledgement
This work was funded by grants of the Deutsche Forschungsgemeinschaft (SFB796 subproject AP2). The authors would like to thank the gardener Christine Hösl for taking care of experimental plants. We are very grateful to Bruno Gronenborn (Institute for Integrative Biology of the Cell [CNRS, CEA, Université Paris Sud], 91198 Gif sur Yvette, France) and Frederik Börnke (Leibniz Institute of Vegetable and Ornamental Crops, Großbeeren) for providing plasmids and support on the BiFC analysis. We would
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2021, Current Opinion in VirologyCitation Excerpt :G3BP1 and Upf1 interact via base-paired RNA sequences that do not overlap with Stau1 binding sites as determined by CLIP-seq [95,96••]. G3BP1 forms stress granule cores during stress leading to sequestration and translational repression of cellular transcripts [97–100]. G3BP1 has several pro-viral (Table 1) and anti-viral roles in virus lifecycles.