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.

New Cell Lines Derived from European Tick Species

“…ticks in the Mediterranean and Balkan regions. Several studies on TBEV have used (directly or indirectly) the existing I. ricinus cell lines derived from UK and German ticks [7,[55][56][57][58][59][60]; the new I. ricinus lines derived from Spanish ticks will add a new dimension to research on TBEV-vector interactions. D. reticulatus is also suspected to be a natural vector of TBEV [57,61], but in vitro investigations have been hampered by the lack of any cell lines from this species.…”

Development and Validation of an ELISA for the Detection of Bluetongue Virus Serotype 4-Specific Antibodies

“…In short, 500 ng of pDONR207 vector was mixed with 2 µL of PCR product and 2 µL of BP Clonase™ in a final volume of 10 µL. The pDONR207 construct was amplified after transformation of E. coli DH5α (NEB) and extracted from bacteria using a NucleoSpin Plasmid kit (Macherey Nagel, Duren, Germany) following the manufacturer’s instructions [ 25 ].…”

Flavivirus–host interactions: an expanding network of proviral and antiviral factors

“…While progress has been made [ 23 , 24 ], genome annotations for tick species are still incomplete making MS-based methods of host factor discovery difficult. However, Lemasson et al [ 25 ] recently reported a yeast two-hybrid (Y2H)-based screen to identify protein–protein interactions of TBEV and louping ill virus (LIV) proteins in tick cells. Here, all TBEV and LIV proteins were screened against a cDNA library generated from Ixodes ricinus -derived cell lines.…”