Abstract
Birnaviruses are unconventional members of the icosahedral double-stranded (dsRNA) RNA virus group. The main differential birnavirus trait is the lack of the inner icosahedral transcriptional core, a ubiquitous structure conserved in all other icosahedral dsRNA viruses, that shelters the genome from cellular dsRNA sensors and provide the enzymatic machinery to produce and extrude mature messenger RNAs. In contrast, birnaviral particles enclose ribonucleoprotein (RNP) complexes formed by the genome segments, the dsRNA-binding VP3 polypeptide and the virus-encoded RNA polymerase (RdRp). The presence of RNPs suggests that the birnavirus replication program might exhibit significant differences with respect to those of prototypal dsRNA viruses. However, experimental evidences supporting this hypothesis are as yet scarce. Of particular relevance for the understanding of birnavirus replication is to determine whether RNPs act as intracellular capsid-independent transcriptional units. Our study was focused to answer this question using the infectious bursal disease virus (IBDV), the best characterized birnavirus, as model virus. Here, we describe the intracellular assembly of functional IBDV RNPs in the absence of the virus-encoded VP2 capsid polypeptide. Recombinant RNPs are generated upon coexpression of the IBDV VP1 and RdRp polypeptides and transfection of purified virus dsRNA. Presented data show that recombinant RNPs direct the expression of the IBDV polypeptide repertoire and the production of infectious virus in culture cells. Results described in this report constitute the first direct experimental evidence showing that birnaviral RNPs are intracellularly active in the absence of the virus capsid. This finding is consistent with presented data indicating that RNP formation precedes virus assembly in IBDV-infected cells, and supports the recently proposed IBDV replication model entailing the release of RNPs during the initial stages of the infection. Indeed, results presented here also support the previously proposed evolutionary connection between birnaviruses and positive-strand single-stranded RNA viruses.
Highlights
Virus capsids are designed to accurately deliver the viral genome to a correct destination within the host cell
We describe the development of a cell-based infectious bursal disease virus (IBDV) RNP assembly system devoid of the capsid protein that efficiently triggers the expression of IBDV polypeptides and the production of infectious IBDV
These results demonstrate that RNPs assembled in the absence of the capsid polypeptide are fully capable of initiating a productive IBDV replication process
Summary
Virus capsids are designed to accurately deliver the viral genome to a correct destination within the host cell. +ssRNA virions lack virus-encoded RNAdependent RNA polymerases (RdRp), and their gene expression is initiated by the release of functional virus-encoded mRNAs upon virus entry These viruses assemble their RNA replication/ transcription complexes in close association with membranous cell compartments that provide an effective shield against dsRNA sensors [2]. T = 2 capsids from dsRNA viruses with life cycles including extracellular phases, usually enclosed by one or two additional capsid shells, are released into the cell endosome upon virus entry, and remain intact throughout the entire replication cycle In these viruses, RNA replication is associated to virus morphogenesis and takes place inside assembling previrion particles, precluding the detection of the newly synthesized dsRNA and the subsequent activation of innate antiviral host-cell responses [4,5]
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