Discrete Virus Factories Form in the Cytoplasm of Cells Coinfected with Two Replication-Competent Tagged Reporter Birnaviruses That Subsequently Coalesce over Time

Abstract

The Birnaviridae family, responsible for major economic losses to poultry and aquaculture, is composed of nonenveloped viruses with a segmented double-stranded RNA (dsRNA) genome that replicate in discrete cytoplasmic virus factories (VFs). Reassortment is common; however, the underlying mechanism remains unknown given that VFs may act as a barrier to genome mixing. In order to provide new information on VF trafficking during dsRNA virus coinfection, we rescued two recombinant infectious bursal disease viruses (IBDVs) of strain PBG98 containing either a split GFP11 or a tetracysteine (TC) tag fused to the VP1 polymerase (PBG98-VP1-GFP11 and PBG98-VP1-TC). DF-1 cells transfected with GFP1-10 prior to PBG98-VP1-GFP11 infection or stained with a biarsenical derivative of the red fluorophore resorufin (ReAsH) following PBG98-VP1-TC infection, had green or red foci in the cytoplasm, respectively, that colocalized with VP3 and dsRNA, consistent with VFs. The average number of VFs decreased from a mean of 60 to 5 per cell between 10 and 24 h postinfection (hpi) (P < 0.0001), while the average area increased from 1.24 to 45.01 μm2 (P < 0.0001), and live cell imaging revealed that the VFs were highly dynamic structures that coalesced in the cytoplasm. Small VFs moved faster than large (average 0.57 μm/s at 16 hpi compared to 0.22 μm/s at 22 hpi), and VF coalescence was dependent on an intact microtubule network and actin cytoskeleton. During coinfection with PBG98-VP1-GFP11 and PBG98-VP1-TC viruses, discrete VFs initially formed from each input virus that subsequently coalesced 10 to 16 hpi, and we speculate that Birnaviridae reassortment requires VF coalescence. IMPORTANCE Reassortment is common in viruses with segmented double-stranded RNA (dsRNA) genomes. However, these viruses typically replicate within discrete cytoplasmic virus factories (VFs) that may represent a barrier to genome mixing. We generated the first replication competent tagged reporter birnaviruses, infectious bursal disease viruses (IBDVs) containing a split GFP11 or tetracysteine (TC) tag and used the viruses to track the location and movement of IBDV VFs, in order to better understand the intracellular dynamics of VFs during a coinfection. Discrete VFs initially formed from each virus that subsequently coalesced from 10 h postinfection. We hypothesize that VF coalescence is required for the reassortment of the Birnaviridae. This study provides new information that adds to our understanding of dsRNA virus VF trafficking.

Rotavirus Replication Factories Are Complex Ribonucleoprotein Condensates

“…We propose that RV replication factories represent a unique case of specialized RNP granules that promote accumulation of viral transcripts to minimise spurious promiscuous RNA-RNA interactions with noncognate host cell RNAs 44 . Our findings have several important ramifications for future studies of rotavirus replication mechanisms, posing many outstanding questions that arise from recognition of the LLPS-driven assembly of viroplasms, and potentially of other viral replication factories in segmented dsRNA viruses that exhibit similar liquidlike behavior in cells 75,76 . The proposed LLPS-driven mechanism of viroplasm formation offers a unified model that explains multiple results from previous efforts to explain their assembly, and establishes LLPS as an attractive target for antiviral intervention 77 .…”

The Novel Genetic Background of Infectious Bursal Disease Virus Strains Emerging from the Action of Positive Selection

“…This lower fidelity could give an adaptive advantage to these types of strains. It has been recently uncovered that IBDV’s replication takes place thorough a mechanism of viral factories [ 60 ], which favors the stamping machine replication mode that generates a lower level of heterogeneity compared to the geometric replication mechanism [ 61 ]. Hence, the IBDV quasispecies cloud composition relies more on the capacity of the polymerase to generate the mutant spectra than the mechanism of replication per se.…”

Transcriptomic analysis reveals that severity of infectious bursal disease in White Leghorn inbred chicken lines is associated with greater bursal inflammationin vivoand more rapid induction of pro-inflammatory responses in primary bursal cells stimulatedex vivo

“…The virus was also titrated in the immortalised B cell-line DT40 (14). At 3 dpi, wells were fixed in 4% paraformaldehyde and stained with a mouse monoclonal antibody against the IBDV VP3 protein (15) that co-localises with IBDV virus factories (16) and a goat anti-mouse secondary antibody conjugated to Alexa-Fluor 488 (Invitrogen). Wells were scored as positive or negative based on the presence of infected cells, and the titre was expressed as tissue-culture infectious dose-50 (TCID 50 ) (17).…”