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

Abstract

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.

Chicken eEF1α is a Critical Factor for the Polymerase Complex Activity of Very Virulent Infectious Bursal Disease Virus

“…Viral VP3 is a multifunctional "scaffold" protein and plays important roles in the virus life cycle [20,22,29,38]. Thus, it is identified as a potential target to disrupt virus-host interactions [30,31,39].…”

“…Interactions between pVP2 and VP3 drive capsid assembly [26][27][28]. Recently, it has been reported that both VP3 and VP1 are required to translation initiation of uncapped viral dsRNA [29]. VP3 also interacts with host factors to modulate viral replication.…”

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

“…IBDV dsRNA pulldown assay IBDV genomic dsRNA extraction was performed as previously described (12,40), and the purified IBDV genomic dsRNA was conjugated with biotin by UV irradiation (365 nm) as previously described (41). Biotin-labeled IBDV dsRNA was either added to whole cell lysates or incubated with E. coli-produced recombinant His-chSTAU1/(1-468).…”

“…VP3 interacts both with VP1 and viral dsRNA to assemble into RNP complexes (9,10) in which it significantly stimulates the VP1 RdRp activity (11). Furthermore, both VP1 and VP3 are required and sufficient for the translation initiation of IBDV uncapped genomic dsRNA (12).…”

Rapid Generation of Attenuated Infectious Bursal Disease Virus from Dual-Promoter Plasmids by Reduction of Viral Ribonucleoprotein Activity

“…The RNP complex is also involved in intracellular viral replication and translation (26), during which VP3 significantly stimulates the VP1 RdRp activity (27). Moreover, VP1 and VP3 have been identified to be the minimal set of trans-acting factors needed to render the infectivity to IBDV genomic dsRNA (28). In addition, both VP3 and VP4 are considered to be antagonists of interferon-␤ induction (28)(29)(30)(31), and VP3 has also been suggested to act as an antiapoptotic protein that prevents the activation of the cellular dsRNA-dependent protein kinase R (32,33).…”