The C terminus of infectious bursal disease virus (IBDV) VP3 encodes a predicted intrinsically disordered region (IDR), which promotes the formation of cytoplasmic puncta and modulates their physical properties.

Abstract

The virus factories (VFs) of infectious bursal disease virus (IBDV) form through liquid-liquid phase separation (LLPS). A major component of the IBDV VF is the nonstructural protein VP3. Here, we predicted the full-length structure of the VP3 monomer and homodimer and employed molecular dynamics simulations to characterize their behavior. We identified the 36 amino acid carboxy(C)-terminus as a highly dynamic intrinsically disordered region (IDR). We then compared the cytoplasmic puncta that were made in the presence of the wild type (wt) VP3 with those made with a VP3 that lacked the C-terminus (VP3{Delta}C). Using live-cell imaging with fluorescent reporter tagged proteins, we found that VP3{Delta}C puncta were significantly less numerous (p<0.0001), smaller (p<0.0001), and more irregular in shape than puncta formed in the presence of wt VP3, demonstrating that the VP3 C terminal IDR promoted their formation. Moreover, by fluorescence recovery after photobleaching (FRAP), the VP3{Delta}C puncta had a significantly reduced mobile fraction (0.29) as compared to full-length VP3 puncta (0.70) (p<0.001), demonstrating that the VP3 C terminal IDR modulated their physical properties. However, the VP3{Delta}C puncta still exhibited liquid-like fusion events in the cytoplasm and were sensitive to treatment with aliphatic diols. Moreover, VP3 did not form puncta when expressed alone, and the removal of the C terminus did not abolish puncta formation completely. We propose that VP3 forms part of a higher order complex with other biomolecules to drive LLPS, and that the VP3 C terminal IDR modulates the physical properties of the resultant LLPS structures. ImportanceLLPS is a phenomenon of growing interest in cell biology. It is a part of the replication cycles of diverse viruses, but our understanding of the molecular basis that underpins the mechanism of phase separation is incomplete. We previously demonstrated that the birnavirus IBDV, a major agricultural pathogen, exploits LLPS in the formation of its VFs. Here, we have characterized the C-terminal 36 amino acid region of IBDV VP3 bioinformatically and by molecular dynamics simulations and found that it encodes a highly dynamic intrinsically disordered region (IDR). Furthermore, we found this region to promote the formation of cytoplasmic puncta and modulate their physical properties. This work contributes to a more detailed understanding of birnavirus replication at the molecular level, and to the study of LLPS as a phenomenon.