Exploration of Binary Protein-Protein Interactions Between Tick-Borne Flaviviruses and Ixodes Ricinus

Abstract

BackgroundLouping 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.MethodsTo 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.ResultsFor 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.ConclusionWe 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.

Integrated protein-protein interaction and RNA interference screens reveal novel restriction and dependency factors for a tick-borne flavivirus in its human host

“…Various proteins have been shown to physically interact with dynein light chains (for review see 19 ). Our previous study disclosed a physical interaction between the prM’ moiety of TBEV and the DYNLL1 ortholog in the tick Ixodes ricinus 20 , suggesting that the interaction, and possibly functional role, is conserved across in the arboviral life cycle between mammalian host and arthropod vector.…”

“…Of note, proteins of several viruses have been found to interact with ubiquilin family members 27,28,29 , in some cases providing benefit for viral replication 29 . In our previous study, the tick I. ricinus ortholog of ubiquilin-1 (UBQLN1 gene), was found by Y2H to be an interactor of TBEV NS4A 20 , suggesting that co-opting of ubiquilin by TBEV may be conserved in its mammalian host and arthropod vector.…”

“…The Hypr strain of TBEV (GenBank accession number U39292.1), isolated in the Czech Republic from a child with TBE 53 , was propagated and titrated in Vero cells grown in DMEM supplemented with antibiotics as described above and 2% FCS. Viral stocks were prepared as described in Lemasson et al 20 and quantified by endpoint dilution assay on Vero cells, as visualized by the virus-induced cytopathic effect (CPE), as previously described 54 . Briefly, 1.5 × 10 4 Vero cells were seeded in 100 μL of DMEM supplemented with antibiotics and 2% FCS in wells of a 96-well plate the day prior to infection with 25 μL of serial dilutions of virus in medium as above (6 wells per dilution).…”

“…To this end, the coding sequences of all open reading frames of TBEV Hypr were cloned and recombined into the pPC97 plasmid (Invitrogen) downstream of the DNA-binding domain of the Gal4 transcription factor (Gal4-DB; Fig. 1), as described in Lemasson et al 20 . Of note, ORFs encoding the cleavage products of full-length prM, namely, the mature M protein and the cleaved Nterminal portion (prM') of prM, rather than the entire prM protein, were used separately for screening.…”

“…The TBEV-H. sapiens screen was part of a broader screen in which viral bait proteins from not only TBEV but also from the highly related Louping Ill virus (LIV) were used to screen cDNA libraries from not only H. sapiens, but also Bos taurus species. The cloning of LIV baits and the derivation of the B. taurus cDNA library have been described in Lemasson et al 20 and Fablet et al 57 , respectively.…”