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The Induction of Infectivity in Human Astrovirus in Response to Capsid

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ABSTRACT The Induction of Infectivity in Human Astrovirus in Response to Capsid Proteolysis by Justin Harper Astrovirus is a non-enveloped, T=3, positive-sense RNA virus that presents with self-limiting gastroenteritis; however, it has been additionally associated with serious presentations such as nephritis, hepatitis, and encephalitis, which is compounded by its propensity to engage in cross-species penetrations. Astrovirus undergoes a complex capsid maturation process mediated by host proteases in which an inert, immature capsid composed of VP90 is sequentially cleaved to yield a highly infectious particle composed of VP34 and VP27/VP25, which form the capsid shell and spikes, respectively. By overexpressing a VP9070-418 truncate in insect cells, we have demonstrated that the shell domain alone cannot support particle assembly, implying a crucial role for the dimeric contacts within the spike. Various monomeric, shell domain truncates (i.e. VP9071-252, VP9071-283, VP9071-313, and VP9071-415) have been successfully expressed and purified, but none yielded useful crystals, suggesting the structural context of the capsid lattice may be needed to stabilize their conformational flexibility. Acknowledgments There are many people deserving of my gratitude, without which this work would have been nigh impossible. Foremost, I need to acknowledge my thesis adviser Jane Tao whose guidance and knowledge of structural virology has been critical in providing direction and spurring ideas regarding novel ways to surmount my encountered difficulties. There are also the members of the Tao and Shamoo lab who have provided an array of technical expertise, troubleshooting, and conversations that have guided this research: Haijiang Chen, Aaron Collier, Milya Davlieva, Yangyang Dong, Yusong Guo, Tom Guu, Yukimatsu Toh, and Wenjie Zheng. Many obstacles were also surmounted via conversations with the members of my thesis committee: James McNew, Charles Stewart, and Timothy Palzkill. Much of this work would have been severely impeded without external support. I would like to thank Carlos Arias at UNAM for providing HAstV-8 VP90 sequences, viral samples, and polyclonal antibodies; Mark Yeager at UVa for sharing his Yuc8 cryo-EM reconstructions; and Vikram Vakharia at UMBC for providing TAstV genomic samples. Additionally, I need to acknowledge Wenhua Guo at the Rice SEA for providing assistance with all things pertinent to TEM. This work was made possible through funding from the Robert A. Welch Foundation (C-1565), the Nanobiology Interdisciplinary Graduate Training Program (NIGTP) offered through the Gulf Coast Consortia (T32EB009379), and the NIH Ruth L. Kirchstein National Research Service Predoctoral Fellowship (NRSA) given through NIAID (F31AI104300). Contents Acknowledgments ..................................................................................................... iii Contents ................................................................................................................... iv List of Figures ........................................................................................................... viii List of Tables ............................................................................................................. xi Nomenclature ............................................................................................................ 1 Background and Significance ...................................................................................... 6 1.1. Clinical Significance .................................................................................................. 7 1.1.1. Host Range ......................................................................................................... 7 1.1.2. Canonical Pathology .......................................................................................... 8 1.1.3. Novel Neurological Manifestations ................................................................... 9 1.1.4. Evidence for Recombination and Tropism Expansion ..................................... 11 1.1.5. Viral-host Codon Matching .............................................................................. 12 1.2. Astrovirus Genomic Architecture ........................................................................... 13 1.2.1. Sub-genomic Fragment Expression of ORF2 ................................................... 14 1.2.2. VP90 Structural Domains ................................................................................. 15 1.2.3. Capsid Assembly and Maturation .................................................................... 18 1.2.4. The Crystal Structure of VP25 and HEV Homology Modeling ......................... 20 1.3. Goals and Impact .................................................................................................... 23 Experimental Methodology ...................................................................................... 25 2.1. Molecular Cloning .................................................................................................. 25 2.1.1. Templates ........................................................................................................ 27 2.1.2. Cloning Primers ................................................................................................ 28 2.1.3. PCR Amplification ............................................................................................ 29 2.1.4. Endonuclease Digestion ................................................................................... 31 2.1.5. Ligation Reactions ............................................................................................ 31 2.1.6. Chemical Transformation ................................................................................ 32 2.1.7. Insert Screening ............................................................................................... 33 2.2. Baculovirus Vector Generation .............................................................................. 34 v 2.2.1. Transposition and Blue-white Screening ......................................................... 34 2.2.2. Bacmid Isolation .............................................................................................. 35 2.2.3. Insect Cell Transfection .................................................................................... 36 2.2.4. Viral Stock Amplification .................................................................................. 37 2.3. Protein Expression .................................................................................................. 38 2.3.1. Bacterial-based Expression .............................................................................. 38 2.3.2. Insect Cell-based Expression............................................................................ 39 2.3.2.1. γMH68 ORF75C ......................................................................................... 40 2.4. Protein Purification ................................................................................................ 40 2.4.1. Lysis .................................................................................................................. 40 2.4.2. Affinity Chromatography ................................................................................. 42 2.4.2.1. Gravity Flow Ni-NTA .................................................................................. 42 2.4.2.2. Talon Metal Affinity .................................................................................. 43 2.4.2.3. HisTrap FF crude ........................................................................................ 43 2.4.2.4. HiTrap Benzamidine FF ............................................................................. 44 2.4.3. Ion Exchange Chromatography ....................................................................... 44 2.4.4. Gel Filtration Chromatography ........................................................................ 45 2.4.5. Ultracentrifugation .......................................................................................... 45 2.5. Crystallization and X-ray Crystallography .............................................................. 46 2.5.1. Protein Crystallization ...................................................................................... 46 2.5.2. X-ray Crystallography ....................................................................................... 47 2.5.3. Lysine Methylation .......................................................................................... 47 2.6. Assorted Biochemical and Biophysical Techniques ............................................... 48 2.6.1. in vitro Proteolysis ........................................................................................... 48 2.6.1.1. Trypsin Digestion of C-6xHis VP9071-415..................................................... 49 2.6.2. Western Blot .................................................................................................... 49 2.6.2.1. VP90 Polyclonal Antibodies ...................................................................... 50 2.6.3. N-terminal Sequencing .................................................................................... 51 2.6.4. Negative-staining Electron Microscopy ........................................................... 52 2.6.5. Circular Dichroism ...........................................................................................
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