Cell Biology of Reovirus Infection

University Of Pittsburgh At Pittsburgh, Pittsburgh PA

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Abstract

Most viruses that replicate in the cytoplasm form neoorganelles that serve as sites of viral genome replication and particle assembly. These highly specialized viral factory (VF) structures concentrate viral replication proteins and nucleic acids, limit activation of cell-intrinsic defenses, and coordinate release of progeny particles. Despite the importance of VFs in viral replication, there are key gaps in knowledge about how these organelles form and function. Reovirus is a genetically tractable double-stranded RNA (dsRNA) virus that has been linked to celiac disease and shows promise for oncolytic applications. The proposed research will define mechanisms of reovirus factory formation, genome replication and translation, and progeny particle release. In common with other dsRNA viruses, including important pathogens of animals (orbiviruses) and humans (rotaviruses), reovirus factories are nucleated by viral nonstructural proteins that recruit viral structural proteins for genome replication and capsid assembly. We have discovered that (i) nonstructural protein µNS forms VF-like structures that resemble biomolecular condensates formed by liquid-liquid phase-separation (LLPS), (ii) nonstructural protein σNS interacts with RNA polymerase λ3 to promote RNA synthesis, and µNS interacts with host protein ATXN2L to enhance viral translation, and (iii) progeny particles are transported from factories and released using a vesicular sorting mechanism dependent on modified lysosomes and actin-like filaments. Three integrated specific aims are proposed to fill knowledge gaps about reovirus factory biogenesis, replication and translation, and progeny particle egress. In Specific Aim 1, VF properties of µNS will be elucidated using biochemical and cell-imaging assays established to define LLPS condensates. The structure of µNS will be determined using X- ray crystallography and cryo-electron microscopy (cryo-EM). Host proteins that contribute to factory maturation will be examined using rapid proximity-dependent biotin identification (TurboID) of µNS-interacting partners. In Specific Aim 2, mechanisms by which reovirus factories facilitate viral replication and translation will be evaluated. The function of σNS in viral genome replication will be defined by determining the structure of σNS in complex with λ3 and RNA along with assays of RNA polymerase activity. The function of ATXN2L in synthesis of viral proteins will be determined using ribosomal sequencing and cellular and in vitro translation assays. In Specific Aim 3, the process by which mature virions are transported to the cell surface and released will be visualized using single-particle cryo-EM and cryo-electron tomography. Host proteins required for nonlytic reovirus egress will be identified using candidate and unbiased screening approaches and validated using replication and egress assays. These studies will enhance an understanding of mechanisms by which pathogenic viruses alter cellular architecture to engineer replication organelles, coordinate key replication functions, and exit infected cells. This information may foster development of antiviral drugs that impede these essential viral replication steps and improve the use of reovirus as an oncolytic therapeutic.