The Culicoides sonorensis inhibitor of apoptosis 1 protein protects mammalian cells from apoptosis induced by infection with African horse sickness virus and bluetongue virus

Abstract

African horse sickness virus (AHSV) and bluetongue virus (BTV) are arboviruses of the genus Orbivirus that are transmitted to their vertebrate hosts by Culicoides biting midges. These orbiviruses exhibit lytic infection (apoptosis) in mammalian cells, but cause persistent infection with no cytopathic effects in Culicoides sonorensis cells. Although regulation of apoptosis could thus be integral for establishing persistent virus infection in midge cells, nothing is known about the presence and function of apoptosis pathways in Culicoides midges and their derived cell lines. Here, we report the cloning and functional characterization of an inhibitor of apoptosis protein (IAP), designated CsIAP1, from C. sonorensis cells. The CsIAP1 protein contains two baculoviral IAP repeat (BIR) domains and a RING domain. Silencing of the Cs iap1 gene in C. sonorensis cells caused apoptosis, indicating that CsIAP1 plays a role in cell survival. Stable expression of the CsIAP1 protein in BSR mammalian cells suppressed apoptosis induced by AHSV-4 and BTV-10 infection, and biochemical data indicated that CsIAP1 is an inhibitor of mammalian caspase-9, an initiator caspase in the intrinsic apoptotic pathway. Mutagenesis studies indicated that the BIR2 and RING domains are required for the anti-apoptotic activity of CsIAP1. The results suggest that the mechanism by which CsIAP1 suppresses apoptosis in insect cells may involve inhibition of a Culicoides caspase-9 homologue through a mechanism that requires both the BIR2 and RING domains. This study provides the first evidence that the CsIAP1 protein is a key negative regulator of apoptosis in C. sonorensis cells.

Multiple Routes of Bluetongue Virus Egress

“…This suggests virion particles are released from insect cells by a non-lytic mechanism [ 62 ]. In contrast to mammalian cells, Culicoides vector cells respond to BTV infection using different mechanisms involving apoptotic pathway inhibition and RNA interference [ 63 , 64 ]. Here, virus budding is the norm for egress, a situation which is mirrored by the requirement of NS3 for non-lytic release of recombinant VLPs produced in Spodoptera frugiperda (caterpillar) insect cells using recombinant baculoviruses.…”

Infection of Mammals and Mosquitoes by Alphaviruses: Involvement of Cell Death

“…The balance between a rapid apoptosis and a delayed, secondary necrosis may explain in part the differences between tolerant and resistant mosquitoes' strains. For more details see [177,[189][190][191][192][193][194][195][196] and Figure 3 for suggested mechanisms which may be involved in alphavirus infection in sensitive versus resistant mosquitoes, extrapolated from other arboviruses. In addition to the virus' ability to penetrate the cells of a particular mosquito (specific receptor, lipid membrane composition), the difference between resistant and tolerant strains may also lie in the rapidity of cell response to viral infection in the first targeted tissue, i.e., the mosquito gut.…”

“…Sensitive mosquitoes may tolerate viral infection through IAP-dependent apoptosis inhibition, as shown for the arbovirus bluetongue virus (BTV) [191,192]. Another tolerance mechanism to DENV seems to rely on resistance to oxidative stress through CAT protection in mosquito gut [193] or p53 isoforms regulation leading to cell survival in drosophila [194] and mosquito [195,196].…”

Evaluation of potential reference genes in the biting midge Culicoides sonorensis for real-time quantitative PCR analyses

“…The EF1b gene was subsequently used as a reference gene in studies of humoral immune responses 14 , EHDV infection 11 , 15 , and RNA interference (RNAi) studies 16 , 17 . Other reference genes used in C. sonorensis studies include heat shock protein 60 ( HSP60 ), cytochrome ( CytB5 ), RpL13 , RpL21 , RpS8 , and vacuolar type ATPase ( V-ATPase ) subunits 18 , 19 . Only one study appears to have used the widely accepted analysis tools NormFinder, geNorm, and BestKeeper to determine suitability of reference genes for normalization of RT-qPCR data in C. sonorensis , and this study focused on the vector competence of susceptible and refractory individuals 19 .…”