Exploration of binary protein-protein interactions between tick-borne flaviviruses and Ixodes ricinus

Abstract

Background: Louping ill virus (LIV) and tick-borne encephalitis virus (TBEV) are tick-borne flaviviruses that are both transmitted by the major European tick, Ixodes ricinus. Despite the importance of I. ricinus as an arthropod vector, its capacity to acquire and subsequently transmit viruses, known as vector competence, is poorly understood. At the molecular scale, vector competence is governed in part by binary interactions established between viral and cellular proteins within infected tick cells.

Methods: To investigate virus-vector protein-protein interactions (PPIs), the entire set of open reading frames for LIV and TBEV was screened against an I. ricinus cDNA library established from three embryonic tick cell lines using yeast two-hybrid methodology (Y2H). PPIs revealed for each viral bait were retested in yeast by applying a gap repair (GR) strategy, and notably against the cognate protein of both viruses, to determine whether the PPIs were specific for a single virus or common to both. The interacting tick proteins were identified by automatic BLASTX, and in silico analyses were performed to expose the biological processes targeted by LIV and TBEV.

Results: For each virus, we identified 24 different PPIs involving six viral proteins and 22 unique tick proteins, with all PPIs being common to both viruses. According to our data, several viral proteins (pM, M, NS2A, NS4A, 2K and NS5) target multiple tick protein modules implicated in critical biological pathways. Of note, the NS5 and pM viral proteins establish PPI with several tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins, which are essential adaptor proteins at the nexus of multiple signal transduction pathways.

Conclusion: We provide the first description of the TBEV/LIV-I. ricinus PPI network, and indeed of any PPI network involving a tick-borne virus and its tick vector. While further investigation will be needed to elucidate the role of each tick protein in the replication cycle of tick-borne flaviviruses, our study provides a foundation for understanding the vector competence of I. ricinus at the molecular level. Indeed, certain PPIs may represent molecular determinants of vector competence of I. ricinus for TBEV and LIV, and potentially for other tick-borne flaviviruses.

Fig. 1

Strategy of TBEV and LIV protein screening. a Schematic representation of TBEV and LIV genome; pink and orange boxes represent structural and non-structural constructs, respectively, used for protein–protein interaction (PPI) screening. Grey circles represent the GAL4 DNA-binding domain used in the yeast two-hybrid assay. b Pipeline describing yeast two-hybrid screening and gap repair validation to define virus–tick PPIs

Fig. 2

Summary of data from Y2H and GR screens. a, c, e Venn diagrams showing the number of protein–protein interactions (PPIs) and tick proteins obtained by Y2H screens for louping ill virus (LIV) and tick-borne encephalitis virus (TBEV); the number indicated in the intersection represents the PPIs or the tick proteins common to LIV and TBEV and identified using Y2H (a) and the PPIs or tick proteins confirmed by GR for LIV (c) and TBEV (e). b, d, f Tables indicating the PPIs identified for each viral protein identified in Y2H (b), for LIV (d) and TBEV (f). g Histogram indicating the number of PPIs identified for each viral bait for LIV and TBEV

Fig. 3

The TBEV/LIV-tick PPI network. Network of PPIs identified by yeast two-hybrid (Y2H) screens and gap repair (GR). LIV and TBEV proteins are represented in orange and red hexagons, respectively. Green nodes indicate tick (Ixodes ricinus) proteins identified in our screens. Edges represent PPIs. Solid lines denote interactions identified by both Y2H and GR. Dashed lines denote interactions only identified by GR. The line width represents the concentration of 3-amino-1,2,4-triazole used for the screen

Fig. 4

Functional enrichment analysis of tick cellular functions targeted by LIV and TBEV. a Histogram indicating statistical enrichment for specific biological processes (BP) determined by Gene Ontology analysis with PANTHER and REVIGO webserver. *Represents the 7 most specific biological processes according to the REVIGO (b) PPI network of Ixodes ricinus proteins identified by our screen and clustered into the 7 functional modules according to enriched GO terms of REVIGO. Yellow hexagons represent viral proteins, circle nodes represent I. ricinus proteins and rectangles indicate the enriched biological processes

Fig. 5

Clustering of viral bait according to GO enrichment ratio for biological processes. Clustering and visualization were performed using clusterMaker2 and JTree TreeView, respectively. 2K: 2K peptide, M: membrane protein, NS4a: Nonstructural protein 4a, NS5: Nonstructural protein 5, pM: pre-membrane protein