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

Abstract

26Bluetongue virus (BTV) is an arbovirus transmitted by blood-feeding midges to a wide 27 range of wild and domestic ruminants. In this report, we showed that BTV, through its virulence 28 non-structural protein NS3 (BTV-NS3), is able to activate the MAPK/ERK pathway. In 29 response to growth factors, the MAPK/ERK pathway activates cell survival, differentiation, 30 proliferation and protein translation but can also lead to the production of several inflammatory 31 cytokines. By combining immunoprecipitation of BTV-NS3 and mass spectrometry analysis 32 from both BTV-infected and NS3-transfected cells, we identified the serine/threonine-protein 33 kinase B-Raf (BRAF), a crucial player of the MAPK/ERK pathway, as a new cellular interactor 34 of BTV-NS3. BRAF silencing led to a significant decrease of the MAPK/ERK activation by 35 BTV supporting a model where BTV-NS3 interacts with BRAF to activate this signaling 36 cascade. Furthermore, the intrinsic ability of BTV-NS3 to bind BRAF and activate the 37 MAPK/ERK pathway is conserved throughout multiple serotypes/strains but appears to be 38 specific to BTV compared to other members of Orbivirus genus. Inhibition of MAPK/ERK 39 pathway with U0126 reduced viral titers, suggesting that BTV manipulates this pathway for its 40 own replication. Therefore, the activation of the MAPK/ERK pathway by BTV-NS3 could 41 benefit to BTV replication by promoting its own viral protein synthesis but could also explain 42 the deleterious inflammation associated with tissue damages as already observed in severe cases 43 of BT disease. Altogether, our data provide molecular mechanisms to explain the role of BTV-44 NS3 as a virulence factor and determinant of pathogenesis. 45 author/funder. All rights reserved. No reuse allowed without permission. Importance 46 47Bluetongue Virus (BTV) is responsible of the non-contagious arthropod-borne disease 48 Bluetongue (BT) transmitted to ruminants by blood-feeding midges. Despite the fact that BTV 49 has been extensively studied, we still have little understanding of the molecular determinants 50 of BTV virulence. In this report, we found that the virulence protein NS3 interacts with BRAF, 51a key component of the MAPK/ERK pathway. In response to growth factors, this pathway 52 promotes cell survival, increases protein translation but also contributes to the production of 53 inflammatory cytokines. We showed that BTV-NS3 enhances the MAPK/ERK pathway and 54 this activation is BRAF-dependent. Our results demonstrate, at the molecular level, how a 55 single virulence factor has evolved to target a cellular function to ensure its viral replication. 56On the other hand, our findings could also explain the deleterious inflammation associated with 57 tissue damages as already observed in severe cases of BT disease. 58 author/funder. All rights reserved. No reuse allowed without permission.

Evaluation of Bluetongue Virus (BTV) Antibodies for the Immunohistochemical Detection of BTV and Other Orbiviruses

“…Bluetongue virus NS3/3a proteins are also highly conserved across BTV serotypes, with significant residue conservation with other orbiviruses at both the N- and C-termini [ 20 ]. Despite the conservation of NS3 peptide among orbiviruses, BTV and EHDV share higher levels of amino acid similarity than that between BTV and AHSV [ 48 ]. In one study, a NS3/3a mAbs that recognized the region between amino acid positions 82 to 95 of BTV NS3/3a cross-reacted with 24 serotypes of BTV as well as Ibaraki virus (a strain of EHDV) [ 20 ].…”

The VP3 Protein of Bluetongue Virus Associates with the MAVS Complex and Interferes with the RIG-I-Signaling Pathway

“…HEK293T and HeLa cells were transfected using Polyplus transfection (JetPRIME), according to the manufacturer’s instructions. ORFs-encoding sequences from BTV8 WT strain were cloned into dedicated destination vectors as described in [ 24 ]. GST-tag and 3xFlag-tag fusions were achieved by Gateway cloning using pDEST27 (Thermo Fisher Scientific) and pCI-neo-3xFlag vector, respectively.…”

Multiple Routes of Bluetongue Virus Egress

“…In mammalian cells, the expression of outer capsid proteins alone is sufficient to trigger apoptosis [ 25 ]. The mitogen activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, a key mediator of apoptosis, can be modulated by BTV in a strain-dependent manner [ 26 , 27 ]. Along with VP2, NS3 is a major determinant of BTV virulence [ 28 ].…”

“…Recently, it has been shown that NS3 is responsible for the activation of the MAPK/ERK pathway by interacting with the serine/threonine-protein kinase B-Raf. Although beneficial for the virus replication, this phenomenon is likely to contribute to increased expression of pro-inflammatory factors that would cause tissue damage [ 26 ].…”