The Formation and Function of Birnaviridae Virus Factories

Abstract

The use of infectious bursal disease virus (IBDV) reverse genetics to engineer tagged reporter viruses has revealed that the virus factories (VFs) of the Birnaviridae family are biomolecular condensates that show properties consistent with liquid-liquid phase separation (LLPS). Although the VFs are not bound by membranes, it is currently thought that viral protein 3 (VP3) initially nucleates the formation of the VF on the cytoplasmic leaflet of early endosomal membranes, and likely drives LLPS. In addition to VP3, IBDV VFs contain VP1 (the viral polymerase) and the dsRNA genome, and they are the sites of de novo viral RNA synthesis. Cellular proteins are also recruited to the VFs, which are likely to provide an optimal environment for viral replication; the VFs grow due to the synthesis of the viral components, the recruitment of other proteins, and the coalescence of multiple VFs in the cytoplasm. Here, we review what is currently known about the formation, properties, composition, and processes of these structures. Many open questions remain regarding the biophysical nature of the VFs, as well as the roles they play in replication, translation, virion assembly, viral genome partitioning, and in modulating cellular processes.

Keywords: Birnaviridae; biomolecular condensate; birnavirus; infectious bursal disease virus (IBDV); liquid–liquid phase separation (LLPS); virus factory (VF).

Figure 1

Simplified model of the IBDV virion and vRNP. (A) The IBDV virion is comprised of an icosahedral T = 13 capsid of VP2, containing genomic vRNPs and free VP1. (B) The structure of an IBDV genomic vRNP, comprising dsRNA associated with VP3, and VP1 linked to the 5′ end of each genomic strand (VPg). Created with BioRender (www.Biorender.com).

Figure 2

The IBDV reproductive cycle. (A) Viral attachment and entry, followed by uptake into an endosome. (B) Decreasing pH and calcium ion concentration within the endosome promotes capsid disassembly and release of pore-forming viral peptide pep46. (C) vRNPs exit the endosome and seed the formation of a virus factory (VF) (green), which are initially associated with the cytoplasmic leaflet of endosomal membranes. (D) VFs may detach from endosomal membranes and move through the cytoplasm, gradually coalescing. (E) Virions are assembled, and form PVAs. The mechanism of PVA formation remains unknown. Created with BioRender (www.Biorender.com).

Figure 3

Model of IBDV VF formation in association with PI3P-enriched EEs. Following viral capsid breakdown and pep46 induced pore formation, vRNPs exit the endosome and VP3 binds PI3P molecules on the cytoplasmic leaflet of EE membranes. VP3 recruits the vRNA and other viral proteins. Further protein and RNA synthesis contribute to the growth of these EE-associated complexes. Created with BioRender (www.Biorender.com).

Figure 4

Model of IBDV VF growth. (A) The EE-associated VF grows due to continued viral protein and RNA synthesis. The growing VF recruits host client proteins (purple and orange). (B) The VF (green) may detach from the EE and move through the cytoplasm, coalescing with other VFs. Created with BioRender (www.Biorender.com).

Figure 5

Possible mechanisms of IBDV assembly. (A) Simultaneous assembly–VFs (green) may reach a critical concentration of viral components, and spontaneously form virions (blue hexagons). (B) Assembly and ejection—virions may be assembled within the VF, and ejected due to an incompatibility with the VF liquid phase. (C) Off-site assembly—viral components (vRNPs and proteins) may be exported to the cytoplasm or another cellular compartment, where virion assembly occurs. Created with BioRender (www.Biorender.com).

Figure 6

Model for nucleated IBDV VF assembly. (A) Following attachment and entry, intact IBDV resides within an early endosome (EE), which contains PI3P exposed to the cytoplasm. (B) The reduction of calcium ion concentration in the endosome induces disassembly of the IBDV capsid, exposing vRNPs comprised of VP1, VP3, and dsRNA, and freeing a viral peptide that ruptures the endosome. (C) IBDV VP3 molecules accumulate on the cytoplasmic leaflet of the EE by binding to PI3P. VP3 binds the VP1 polymerase and viral genome replication begins. (D) Continued viral genome replication, protein synthesis, and recruitment of host client proteins (purple and orange) causes the VFs to grow, and when their critical concentration is reached, they separate into a distinct liquid phase. (E) After nucleation, VFs might detach from EEs, and move through the cytosol. (F) Fluorescence micrograph of a DF-1 cell infected with IBDV strain PBG98, with cytoplasmic inclusions enriched in VP3 (VP3 green; nucleus blue). Created with BioRender (www.Biorender.com).