Infectious bursal disease virus (IBDV) is a bi-segmented double-strand RNA (dsRNA) virus of the family. While IBDV genomic dsRNA lacks a 5' cap, the means by which the uncapped IBDV genomic RNA is translated effectively is unknown. In this study, we describe a cap-independent pathway of translation initiation of IBDV uncapped RNA that relies on VP1 and VP3. We show that neither purified IBDV genomic dsRNA nor the uncapped viral plus-sense RNA transcripts was directly translated and rescued into infectious viruses in host cells. This defect in translation of the uncapped IBDV genomic dsRNA was rescued by-supplementation of the viral proteins VP1 and VP3, which was dependent on both the intact polymerase activity of VP1 and the dsRNA binding activity of VP3. Deletion analysis showed that both 5' - and 3' -UTRs of IBDV dsRNA were essential for the VP1/VP3-dependent translation initiation. Significantly, VP1 and VP3 could also mediate the recovery of infectious IBDV from the authentic minus-sense strand of IBDV dsRNA. Moreover, down-regulation or inhibition of the cap-binding protein eIF4E did not decrease, but rather enhanced the VP1/VP3-mediated translation of the uncapped IBDV RNA. Collectively, our findings for the first time reveal that VP1 and VP3 compensate for the deficiency of 5' cap and replace eIF4E to confer upon the uncapped IBDV RNA the ability to be translated and rescued into infectious viruses.A key point of control for virus replication is the viral translation initiation. The current study shows that the uncapped IBDV RNA cannot be translated into viral proteins directly by host translation machinery, and is thus noninfectious. Our results constitute the first direct experimental evidence that the VP1 and VP3 are required and sufficient to initiate translation of uncapped IBDV genomic RNA by acting as a substitute of cap and replacing the cap-binding protein eIF4E. Significantly, the VP1/VP3 mediate the recovery of infectious IBDV not only from the plus-sense but also from the minus-sense strand of the IBDV dsRNA. These findings provide not only new insights into the molecular mechanisms of the life cycle of IBDV, but also a new tool for an alternative strategy for the recovery of IBDV from both the plus- and the minus-sense strand of the viral genomic dsRNA.

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.

Summary. -As a paramyxovirus, Newcastle disease virus (NDV) has the ability to edit its P (phosphoprotein) gene to synthesize three kinds of viral protein (P, V and W). It is technically very diffi cult to diff erentiate P, V and W mRNAs, and little was known about NDV regulation of RNA-editing frequency. To investigate the rules of NDV RNA editing, the ratio of the P gene-derived transcripts (P, V and W) was determined by sequencing at diff erent time points post-infection. Th e results showed unstable ratio of V and W mRNA at diff erent time points, and the frequency of NDV editing was signifi cantly increased at the early period of infection (P <0.05). Along with the increase in editing frequency, the levels of V and W transcripts were obviously increased at 8 and 10 hours post infection (hpi). In addition, it was shown that the number of inserted G residues in P-derived transcripts was not limited to one or two: +3G, +4G, +5G and even +9G transcripts were identifi ed. Higher RNA editing frequency of NDV P gene occurred in the early period of infection, which may play a role in the process of viral infection.