Browsing by Author "Zhang, Jim Lu"

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    Structure-function studies of the nematode-infecting Orsay virus
    (2022-07-01) Zhang, Jim Lu; Tao, Yizhi J
    Viruses are ubiquitously distributed about the biosphere, serving as potent agents of disease, moderators of ecosystems, and drivers of evolution. While understandings of virus structure and infection have greatly improved, there remains an unfulfilled niche for an accessible, eukaryotic host-virus model system compatible with high-throughput experimentation. The non-enveloped, RNA-based Orsay virus, which naturally infects Caenorhabditis elegans, provides a promising opportunity to develop a platform for studying viral pathogenesis in an accessible, well-characterized model organism. Previous studies surrounding Orsay have successfully resulted in structures describing the recombinant icosahedral capsid and a truncated form of the δ fiber protein. The δ fiber, which can be expressed alone or as a CP-δ fusion protein, has since been implicated as a required component for viral infection and exit. However, how CP-δ is ultimately incorporated into the native, infectious particle remained unknown. Here, a method supporting the amplification and purification of Orsay virus from C. elegans is presented, providing a valuable means for native-condition studies of the intact, infectious virion. Biochemical and structural analyses via immunoblotting and electron microscopy demonstrate that CP-δ is indeed incorporated within the mature virus as a pentamer about the icosahedral five-fold axis of symmetry. Subsequent antibody-based neutralization assays confirm incorporated CP-δ is ultimately essential for viral entry and infection, while purified virion fractions were additionally used to examine and possibly simulate viral uncoating in vitro. Finally, the expression and purification of a putative host cell receptor for viral infection is discussed, with preliminary assays indicating possible, but inconclusive δ-binding activity. Broadly, the methods and work presented here marks the first reported study of the infectious Orsay virion and supports future characterizations of its viral replication cycle. Doing so will greatly augment the continued development of this promising model virus and its application with C. elegans as a platform for studying viral pathogenesis.