THE COMPOSITION, PROPERTIES, AND PHASE SEPARATING BEHAVIOR OF INFECTIOUS BURSAL DISEASE VIRUS (IBDV) VIRUS FACTORIES

Abstract

Infectious bursal disease virus (IBDV), a member of the family Birnaviridae, is a significant agricultural pathogen of chickens and turkeys, and is the causative agent of infectious bursal disease (Gumboro disease) in chickens. As a double-stranded RNA (dsRNA) virus, IBDV exhibits molecular biology similar to other dsRNA viruses, such as reovirus and rotavirus, including the formation of cytoplasmic inclusions during infection. For all these viruses, the cytoplasmic inclusions are sites of certain aspects of viral replication; in the case of IBDV, they are termed virus factories (VFs). Previous work has shown that VFs form via liquid-liquid phase separation (LLPS), a biophysical phenomenon in which biomolecules segregate into distinct phases without a membranous barrier. The LLPS is also the mechanism by which well-studied cellular structures such as P-bodies, stress granules, and the nucleolus are formed, and is exploited by viruses from multiple families as part of their replicative strategies. IBDV, and birnaviruses more broadly, are understudied pathogens. As such, the molecular biology of IBDV infection, including the formation, properties, and behavior of VFs, is not fully understood. The objective of this work was to resolve several unanswered questions regarding the composition and properties of IBDV VFs – namely, I aimed to develop a suite of biochemical and analytical tools for the quantitative measurement of VFs in infected cells, to identify the minimal set of biomolecules required to induce VF formation, and to identify how the physical properties of the structures are modulated. I successfully developed a biological and computational toolkit for the quantitative analysis of VFs and discovered that IBDV VFs exhibit increased liquidity at late time points, a behavior markedly different from that of analogous inclusions formed by rotavirus. Furthermore, I have identified VP3, VP1, and viral RNA as the minimal components for IBDV VF phase separation, which will be useful for VF phase separation research. Finally, our study of VP3 revealed an intrinsically disordered region (IDR) at the carboxy terminus of the protein, which is frequently identified as essential for phase separation. Interestingly, the VP3 IDR is both an important driver of VF phase separation and contributes to the liquidity of VFs, although it is not essential to their formation. These findings address several gaps in the understanding of IBDV molecular virology and IBDV VFs, thereby contributing to research on viral replicative structures and LLPS biology.