Infectious Bursal Disease Virus Activates c-Src To Promote α4β1 Integrin-Dependent Viral Entry by Modulating the Downstream Akt-RhoA GTPase-Actin Rearrangement Cascade

Abstract

While the entry of infectious bursal disease virus (IBDV) is initiated by the binding of the virus to the two major receptors integrin and HSP90, the signaling events after receptor binding and how they contribute to virus entry remain elusive. We show here that IBDV activates c-Src by inducing the phosphorylation of the Y416 residue in c-Src both in DF-1 chicken fibroblasts and in vivo in the bursa of Fabricius from specific-pathogen-free (SPF) chickens. Importantly, inactivated IBDV fails to stimulate c-Src Y416 phosphorylation, and a very virulent IBDV strain induces a much higher level of c-Src Y416 phosphorylation than does an attenuated strain. Inhibition of c-Src activation by an Src kinase inhibitor or expression of a c-Src dominant negative mutant results in a significant decrease in the internalization of IBDV but has little effect on virus adhesion. Furthermore, short hairpin RNA (shRNA) downregulation of integrin, either the ␣4 or ␤1 subunit, but not HSP90 remarkably attenuates IBDV-induced c-Src Y416 phosphorylation, resulting in a decrease in IBDV internalization but not virus adhesion. Moreover, interestingly, inhibition of either c-Src downstream of the phosphatidylinositol 3-kinase (PI3K)/Akt-RhoA signaling cascade or actin rearrangement leads to a significant decrease in IBDV internalization irrespective of the IBDV-induced high levels of c-Src phosphorylation. Cumulatively, our results suggest a novel feed-forward model whereby IBDV activates c-Src for benefiting its cell entry via an integrin-mediated pathway by the activation of downstream PI3K/Akt-RhoA signaling and cytoskeleton actin rearrangement.IMPORTANCE While IBDV-caused immunosuppression is highly related to viral invasion, the molecular basis of the cellular entry of IBDV remains elusive. In this study, we demonstrate that IBDV activates c-Src by inducing the phosphorylation of the Y416 residue in c-Src to promote virus internalization but not virus adhesion. The ability to induce the level of c-Src Y416 phosphorylation correlates with the pathogenicity of an IBDV strain. IBDV-induced c-Src Y416 activation is ␣4␤1 integrin but not HSP90 dependent and involves the activation of the downstream PI3K/Akt-RhoA GTPase-actin rearrangement cascade. Thus, our findings provide new insights into the IBDV infection process and the potential for c-Src as a candidate target for the development of IBDV therapeutic drugs.

VP1 and VP3 Are Required and Sufficient for Translation Initiation of Uncapped Infectious Bursal Disease Virus Genomic Double-Stranded RNA

“…RNA interference. Two tested target of eIF4E (GGATGGTATTGAGCCTATG and AAGCAAACCTGCGG CTGATCT) (49) were selected to generate the shRNA plasmids (sheIF4E-1 and sheIF4E-2) as described previously (50). The knockdown of expression of eIF4E in HEK 293T cells was performed by transient transfection of shRNA plasmids, and the shRNA targeting EGFP (shEGFP) was used as a negative control.…”

“…Western blotting. Western blot analysis was performed as described previously (50). Briefly, wholecell lysate was extracted by using lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, and 1% sodium deoxycholate) at 4°C for 30 min, followed by centrifugation at 12,000 ϫ g at 4°C for 30 min.…”

“…Plaque assay. A plaque assay for IBDV on DF-1 cells was performed as described previously (50). Briefly, a 10-fold dilution series of a virus-containing sample (0.1-ml aliquots of each dilution) was inoculated onto the monolayer of DF-1 cells in six-well plates.…”

Discrete virus factories form in the cytoplasm of cells co-infected with two strains of the segmented dsRNA virus, infectious bursal disease virus (IBDV), that subsequently coalesce

“…Interestingly, VFs have also been observed co-localising with actin in cells infected with Fowlpox [34] and Bunyamwera virus [35], and actin is involved in the internalisation, replication, and non-lytic egress of rotavirus [36, 37]. IBDV internalisation into the cell is also dependent on an intact actin network [32], [38]. In order to distinguish between the involvement of actin in IBDV entry and VF movement, cultures were treated with cytochalasin D from 2 hours pi, to allow IBDV time to enter the cells prior to treatment.…”

STAU1 binds to IBDV genomic double‐stranded RNA and promotes viral replication via attenuation of MDA5‐dependent β interferon induction

“…Rabbit anti-STAU1 and anti-G3BP1 antibodies were supplied by Proteintech (Wuhan, China). Mouse anti-VP1 and anti-VP3 polyclonal antibodies were generated as previously described (37). Alexa Fluor-568-labeled goat anti-mouse IgG was purchased from Thermo Fisher Scientific, and DAPI was purchased from Merck Millipore (Billerica, MA, USA).…”