Birnaviridae Virus Factories Show Features of Liquid-Liquid Phase Separation and Are Distinct from Paracrystalline Arrays of Virions Observed by Electron Microscopy

Abstract

To gain more information about the nature of Birnaviridae virus factories (VFs), we used a recombinant infectious bursal disease virus (IBDV) expressing split-GFP11 tagged to the polymerase (VP1) that we have previously shown is a marker for VFs in infected cells expressing GFP1-10. We found that VFs co-localized with 5-ethynyl uridine in the presence of actinomycin, demonstrating they contained newly synthesized viral RNA, and VFs were visible in infected cells that were fixed and permeabilized with digitonin, demonstrating that they were not membrane bound. Fluorescence recovery after photobleaching (FRAP) a region of interest within the VFs occurred rapidly, recovering from approximately 25% to 87% the original intensity over 146 seconds, and VFs were dissolved by 1,6-hexanediol treatment, demonstrating they showed properties consistent with liquid-liquid phase separation. There was a lower co-localization of the VF GFP signal with the capsid protein VP2 (Manders’ coefficient (MC) 0.6), compared to VP3 (MC, 0.9), which prompted us to investigate the VF ultrastructure by transmission electron microscopy (TEM). In infected cells, paracrystalline arrays (PAs) of virions were observed in the cytoplasm, as well as discrete electron dense regions. Using correlative light and electron microscopy (CLEM), we observed that the electron dense regions correlated with the GFP signal of the VFs, which were distinct from the PAs. In summary, Birnaviridae VFs contain newly synthesized viral RNA, are not bound by a membrane, show properties consistent with liquid-liquid phase separation, and are distinct from the PAs observed by TEM. Importance Members of the Birnaviridae infect birds, fish and insects, and are responsible for diseases of significant economic importance to the poultry industry and aquaculture. Despite their importance, how they replicate in cells remains poorly understood. Here, we show that the Birnaviridae virus factories are not membrane bound, demonstrate properties consistent with liquid-liquid phase separation, and are distinct from the paracrystalline arrays of virions observed by transmission electron microscopy, enhancing our fundamental knowledge of virus replication that could be used to develop strategies to control disease, or optimize their therapeutic application.

On the Role of VP3-PI3P Interaction in Birnavirus Endosomal Membrane Targeting

“…In light of our results, it is conceivable to hypothesize that viral interaction with the EE membranes acts as a first step in the RCs biogenesis, leading to the growth of biomolecular condensates with LLPS characteristics around EE membranes, as recently proposed (Brodrick and Broadbent, 2023). In support of this model, both the RNA dependent RNA polymerase VP1 and the viral dsRNA associate to endosomes (6) and to the LLPS condensates (Delgui et al, 2013; Reddy et al, 2022). Further experiments are needed in order to link the association of VP3 to EE membranes with biomolecular condensate formation.…”

“…It has recently been reported that the RCs of IBDV exhibit liquid-liquid phase separation (LLPS) (Reddy et al, 2022). In LLPS, one or more biomolecules form a network of mild homo- and heterotypic interactions, resulting in the formation of a distinct liquid phase within a liquid medium (Banani et al, 2017).…”

Aurora-A condensation mediated by BuGZ aids its mitotic centrosome functions

“…Considering these methodology limits, we have been working on setting up the correlative light and electron microscopy (CLEM) platform, which has been widely performed recently in many LLPS investigations to confirm the phase separation property of candidate proteins in vivo . 76 , 77 , 78 It might be a powerful and effective tool to explore Aurora-A LLPS in depth.…”

Extending fluorescence anisotropy to large complexes using reversibly switchable proteins

“…In fact, STARSS measurements demonstrated the tendency of the Gag-homologous protein Arc to oligomerize even in crowded subcellular regions not measurable with conventional FCS due to the difficulties in detection of signal fluctuations. Considering the tendency of Arc to oligomerize and bind RNAs, it is tempting to speculate that these regions might represent liquid–liquid-phase-separated condensates resulting from the high density of proteins and RNAs, in analogy to viral factories 32 , 33 .…”