Novel Function of Bluetongue Virus NS3 Protein in Regulation of the MAPK/ERK Signaling Pathway.

Kundlacz, Cindy; Pourcelot, Marie; Fablet, Aurore; Amaral Da Silva Moraes, Rayane; Léger, Thibaut; Morlet, Bastien; Viarouge, Cyril; Sailleau, Corinne; Turpaud, Mathilde; Gorlier, Axel; Breard, Emmanuel; Lecollinet, Sylvie; van Rijn, Piet A; Zientara, Stephan; Vitour, Damien; Caignard, Grégory

Bluetongue virus (BTV) is an arbovirus transmitted by blood-feeding midges to a wide range of wild and domestic ruminants. In this report, we showed that BTV, through its non-structural protein NS3 (BTV-NS3), is able to activate the MAPK/ERK pathway as assessed by phosphorylation levels of ERK1/2 and the translation initiation factor eIF4E. By combining immunoprecipitation of BTV-NS3 and mass spectrometry analysis from both BTV-infected and NS3-transfected cells, we identified the serine/threonine-protein kinase B-Raf (BRAF), a crucial player of the MAPK/ERK pathway, as a new cellular interactor of BTV-NS3. BRAF silencing led to a significant decrease of the MAPK/ERK activation by BTV supporting a model where BTV-NS3 interacts with BRAF to activate this signaling cascade. This positive regulation acts independently of the role of BTV-NS3 in counteracting the induction of the IFN-α/β response. Furthermore, the intrinsic ability of BTV-NS3 to bind BRAF and activate the MAPK/ERK pathway is conserved throughout multiple serotypes/strains but appears to be specific to BTV compared to other members of Orbivirus genus. Inhibition of MAPK/ERK pathway with U0126 reduced viral titers, suggesting that BTV manipulates this pathway for its own replication. Altogether, our data provide molecular mechanisms that unravel new essential functions of NS3 during BTV infection.IMPORTANCE Bluetongue Virus (BTV) is responsible of the arthropod-borne disease Bluetongue (BT) transmitted to ruminants by blood-feeding midges. In this report, we found that BTV, through its non-structural protein NS3 (BTV-NS3), interacts with BRAF, a key component of the MAPK/ERK pathway. In response to growth factors, this pathway promotes cell survival and increases protein translation. We showed that BTV-NS3 enhances the MAPK/ERK pathway and this activation is BRAF-dependent. Treatment of MAPK/ERK pathway with the pharmacologic inhibitor U0126 impairs viral replication, suggesting that BTV manipulates this pathway for its own benefit. Our results illustrate, at the molecular level, how a single virulence factor has evolved to target a cellular function to increase its viral replication.