Abstract
Infectious bursal disease virus (IBDV) is an avian pathogen responsible for an acute immunosuppressive disease that causes major losses to the poultry industry. Despite having a bipartite dsRNA genome, IBDV, as well as other members of the Birnaviridae family, possesses a single capsid layer formed by trimers of the VP2 capsid protein. The capsid encloses a ribonucleoprotein complex formed by the genome associated to the RNA-dependent RNA polymerase and the RNA-binding polypeptide VP3. A previous report evidenced that expression of the mature VP2 IBDV capsid polypeptide triggers a swift programmed cell death response in a wide variety of cell lines. The mechanism(s) underlying this effect remained unknown. Here, we show that VP2 expression in HeLa cells activates the double-stranded RNA (dsRNA)-dependent protein kinase (PKR), which in turn triggers the phosphorylation of the eukaryotic initiation factor 2α (eIF2α). This results in a strong blockade of protein synthesis and the activation of an apoptotic response which is efficiently blocked by coexpression of a dominant negative PKR polypeptide. Our results demonstrate that coexpression of the VP3 polypeptide precludes phosphorylation of both PKR and eIF2α and the onset of programmed cell death induced by VP2 expression. A mutation blocking the capacity of VP3 to bind dsRNA also abolishes its capacity to prevent PKR activation and apoptosis. Further experiments showed that VP3 functionally replaces the host-range vaccinia virus (VACV) E3 protein, thus allowing the E3 deficient VACV deletion mutant WRΔE3L to grow in non-permissive cell lines. According to results presented here, VP3 can be categorized along with other well characterized proteins such us VACV E3, avian reovirus sigmaA, and influenza virus NS1 as a virus-encoded dsRNA-binding polypeptide with antiapoptotic properties. Our results suggest that VP3 plays a central role in ensuring the viability of the IBDV replication cycle by preventing programmed cell death.
Highlights
Virus replication entails a complex set of interactions between the host cell machinery and viral products that has the potential to alter cell homeostasis
VP2 expression leads to PKR and eukaryotic initiation factor 2a (eIF2a) phosphorylation We have previously shown that VP2 expression induces a potent shut off of protein synthesis that is followed by a Programmed cell death (PCD) response [26]
To gain further insight into the molecular events leading to the PCD response induced by the expression of the Infectious bursal disease virus (IBDV) VP2 gene we have taken advantage of the vaccinia virus (VACV) expression system
Summary
Virus replication entails a complex set of interactions between the host cell machinery and viral products that has the potential to alter cell homeostasis. The intrinsic pathway, known as mitochondrial pathway, is set off following the detection of different types of cellular stress by specific sensor proteins belonging to the BH3-only member of the Bcl-2 family. This results in the formation of the apoptosome that prompts the activation of effector proteases, namely caspase-3 and -7 [3]. The extrinsic pathway is activated by ligation of receptors containing death domains located at the cell membrane This leads to the formation of the death-induced signaling complex (DISC) that triggers the activation of the initiator caspase-8 [4]. Caspase-8 activation triggers a direct activation of the caspase-3 and -7 effectors, whilst in others it requires the amplification of death signals through stimulation of the mitochondrial pathway via the Bid sensor protein, a member of the Bcl-2 family [5]
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