Interactions of Vank proteins from Microplitis bicoloratus bracovirus with host Dip3 suppress eIF4E expression

Highlights

• Vank-Dip3 complexes inhibited eIF4E during Bracovirus-mediated immunosuppression.

• Vank proteins bound to full-length Dip3, including the MADF, BESS, and non-motif.

• Dip3 rescued eIF4E transcription from inhibition by MbBV infection.

• RNAi-mediated dip3 silencing inhibited eIF4E, leading to immunosuppression.

Abstract

Microplitis bicoloratus bracovirus (MbBV) inhibits the immune response of the host Spodoptera litura by disrupting nuclear factor (NF)-κB signaling and downstream gene expression. However, the underlying molecular mechanisms are not well understood. Herein, we report that viral ankyrin (Vank) proteins interacted with host dorsal-interacting protein 3 (Dip3) to selectively inhibit the transcription of eukaryotic translation initiation factor 4 E (eIF4E). Dip3 and Vank proteins were co-expressed and colocalized in the nucleus. Furthermore, ectopic expression of Dip3 rescued the transcription of some NF-κB-dependent genes suppressed by Vank proteins, including eIF4E. Co-immunoprecipitation and pull-down assays confirmed that Vank proteins interacted with and bound to full-length Dip3, which including MADF, DNA-binding protein, BESS, and protein-protein interaction motifs as well as non-motif sequences. In vivo, RNAi-mediated dip3 silencing decreased eIF4E levels and was accompanied by an immunosuppressive phenotype in S. litura. Our results provided novel insights into the regulation of host transcription during immune suppression by viral proteins that modulate nuclear NF-κB signaling.

Introduction

Disruption of the nuclear factor (NF)-κB signaling pathway and downstream gene expression or inhibition of eukaryotic initiation factors (e.g., eukaryotic translation initiation factor 4 E [eIF4E]) promotes immunosuppression (Dong et al., 2017a; Fiore et al., 2018). Recent data have shown that these key regulatory proteins are expressed at various levels; however, the mechanism through which an abundance of eIF4E promotes translation cannot be fully explained by the disruption of NF-κB inhibitory activity alone (Yu et al., 2016). Interactions between host and viral proteins may play important roles in strongly inhibiting these functions. In both vertebrates and invertebrates, NF-κB family members contain a conserved N-terminal Rel homology domain (RHD) responsible for DNA binding, homo- and heterodimerization, nuclear localization, and IκB binding (Hayden and Ghosh, 2008; Hetru and Hoffmann, 2009).

The transcription factor dorsal-interacting protein 3 (Dip3) is required for optimal resistance to immune challenges. Dip3 contains two domains; the MADF domain binds to DNA in a sequence-specific manner, and the BESS domain interacts with the RHD domain of NF-κB proteins. Therefore, Dip3 can heterodimerize with a TATA-binding protein-associated factor or interact with dorsal protein in the nucleus to bind to specific elements in target promoters and synergistically activate gene transcription (Bhaskar and Courey, 2002; Rao et al., 2016; Ratnaparkhi et al., 2008; Shukla et al., 2014). Moreover, cactus functions as an inhibitor that binds to dorsal and prevents it from entering the nucleus (Kidd, 1992), and its co-localization with dorsal in the nucleus represses dorsal-dependent transcriptional activity (Harari-Steinberg et al., 2007) and extends the dynamic range of the dorsal gradient (O'Connell and Reeves, 2015).

The wasp Microplitis bicoloratus, which is associated with the polydnavirus Microplitis bicoloratus bracovirus (MbBV), is an endoparasitoid of the tobacco cutworm Spodoptera litura. M. bicoloratus induces immunosuppression via MbBV-induced hemocyte apoptosis and a reduced head capsule width, a hallmark of inhibition of host development (Kou et al., 2017; Luo et al., 2007b). Previous studies have shown that viral ankyrin (Vank) proteins, which contain ankyrin repeat domains and are homologous to the NF-κB inhibitor IκB, are thought to negatively impact immune NF-κB signaling in insect hosts (Bitra et al., 2012; Gueguen et al., 2013). In particular, MbBV downregulates the NF-κB signaling pathway and decreases NF-κB-mediated expression of the antimicrobial defense gene attacin in M. bicoloratus (Li et al., 2014; Xu et al., 2017). Another study has shown that Vank proteins form a stable complex with the RHD domains of dorsal, Dif, and relish and sequester these proteins in the cytoplasm (Thoetkiattikul et al., 2005). However, whether Vank proteins compete for the RHD domain post translocation to the nucleus (Han and Ip, 1999; Hedengren-Olcott et al., 2004) remains to be determined. We have previously reported increased mRNA levels for several Vank genes, including vank86, vank92, and vank101, in hemocytes from S. litura hosts parasitized by M. bicoloratus (Li et al., 2014). Moreover, previous studies have shown that MbBV inhibits translation by decreasing the activity of the eIF4E/eIF4A axis (Cui et al., 2019; Dong et al., 2017a), thus raising questions regarding the mechanisms underlying viral protein translation in cells infected with MbBV. We speculated that viral IκB-like proteins may directly influence the transcriptional function of Dip3, thereby impairing the expression of genes downstream of NF-κB.

To test this hypothesis, we examined the roles of Dip3 in immunosuppression using RNAi and characterized its role in activating other gene expression. Our results demonstrated distinct roles for MbBV IκB-like proteins in modulating host transcription and provide new insights into the mechanisms through which viral targeting of coactivators directly triggers immune suppression.