Browsing by Author "Tao, Yizhi J"

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    Discovery and Characterization of Lipid Disruption Activity in the Human Astrovirus Capsid
    (2022-12-02) Ykema, Matthew Ryan; McNew, James; Tao, Yizhi J
    Astroviruses (AstVs) are non-enveloped, positive single stranded RNA viruses that cause a wide range of inflammatory diseases in mammalian and avian hosts. The T=3 viral capsid is unique in its ability to infect host cells is a process driven by host proteases. Intercellular protease cleavages allow for viral egress from a cell, while extracellular cleavages allow for the virus to enter a new host cell to initiate infection. High resolution models of the capsid core indicate a large, exposed region enriched with protease cleavage sites. During maturation, the capsid goes through significant structural changes including the loss of many surface spikes. However, there is still a lack of structural information surrounding the immature VP90 capsid assembly, along with the matured VP70 and VP34/27/25 assemblies. I have expressed the VP90 protein and correlated the maturation of the viral protein to a previously uncharacterized function, disruption of lipid membranes in vitro. The original goal of this project was to model the stages of the capsid maturation cycle at high resolution. I was able to purify the VP90 state of the virus through expression and purification in a bacterial system, however full capsid assemblies were unable to be obtained. These purified VP90 proteins were used as a template for functional assays. It has been previously proposed that the AstV capsid can function as a toxic protein that causes an increased cellular membrane flux and disruption, but this function has not been observed in other conditions. I developed an assay to use artificially generated liposomes to test the lipid-disruption activity of AstV protein in vitro. I was the first to observe that the AstV VP90 protein has high levels of lipid-disruption activity only after maturation by trypsin treatment. This liposome assay was used to test many AstV proteins and lipid conditions. I have concluded that the lipid-disruption activity originates from the cleavage of trypsin sites from amino acids 299 to 313 in the AstV Outer Core domain, and the activity is dependent on the types of lipids present in the liposomes. Further understanding of the viral capsid structure and maturation process can contribute to characterizing the toxic properties of the AstV capsid, gastric therapeutics, and viral engineering applications.
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    Regulation of the neurovascular patterning by growth factors and cytokines during anterior ocular development
    (2015-12-16) Ojeda Cardenas, Ana; Bennett, George N; Jacot, Jeffrey G; McNew, James A; Tao, Yizhi J
    The cornea is a transparent, avascular, and one of the most innervated tissue of the body. Corneal diseases including injuries, neovascularization, congenital eye defects and degenerations, represent a major public health burden. Although, studies have been focused on understanding the basis of transparency, innervation, and neovascularization of the adult cornea, little is known about the molecular mechanisms that lead to this specialized structure results in a highly innervated but avascular tissue during embryogenesis. The purpose of this work was to identify molecular regulators of the neurovascular patterning during cornea development. First, Sema3A, a well-known chemorepulser of axons, was identified as a key modulator in the establishment of cornea avascularity in both, avian and murine models. Moreover, I demonstrated that chemokines, initially described for their function in controlling immune cell migration, also play an important role in axon guidance and vasculogenesis during ocular development. Examination of the expression of the chemokine CXCL14 by in situ hybridization and immunohistochemistry revealed novel patterns of localization in the corneal stroma, iris, lens epithelium, retina and trigeminal ganglion. Comparison in the expression of CXCL14 and CXCL12 shows that they are expressed in complementary patterns in most tissues during ocular development, suggesting an interactive regulation of these chemokines. Visual examination of Retrovirus-mediated Knockdown of CXCL14 embryos revealed relatively smaller eyes compared to controls, and immunohistochemical analysis of ocular nerves indicated exacerbated projection of sensory nerves into the corneal stroma, corneal epithelium and iris, which subsequently elevated nerve density in these tissues. In vitro analyses revealed that CXCL14 has an inhibitory effect on CXCL12-induced axon growth of trigeminal ganglion sensory neurons. Furthermore, Knockdown of CXCL14 in Tg(tie1: H2B:eYFP) transgenic Japanese quail embryos resulted in ectopic migration of YFP fluorescently labeled angioblasts into the cornea and exogenous CXCL14 inhibits VEGF- and CXCL12-induced angioblast migration into the cornea. This is the first time that CXCL14 has been shown to have a critical function during embryogenesis that may be mediated through inhibition of CXCL12 signaling. Collectively, these results demonstrate that neurovascular patterning of the anterior eye during development depends on an intricate process and fine balance of growth factors and cytokines. These findings will contribute to a better understanding of the molecular mechanisms involved in pathological conditions such as cornea neovascularization, anterior segment ocular dysgeneses and wound healing, where angiogenesis and nerve regeneration are critically compromised.
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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.