Analysis of protein-protein and protein-RNA interactions by severe acute respiratory syndrome nonstructural proteins and human karyopherin alpha 2
Miknis, Zachary Jason
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The severe acute respiratory syndrome (SARS) pandemic that began in late 2003 is a recent example of zoonotic transmission of a novel virus into human populations. The outbreak has highlighted the potential of coronaviruses (CoV) to produce more severe diseases of clinical significance and furthermore brought to attention the lack of information regarding coronavirus emergence from zoonotic sources, disease pathogenesis and replication. Replication of SARS-CoV requires the activity of 16 viral nonstructural proteins in order for the replication complex to function optimally, and several of these viral proteins have identified protein-protein and protein-RNA interactions. The work to be described in this dissertation involves several different investigations of protein-protein and protein-RNA interactions. The majority of work involves studies of nonstructural protein (nsp) 9, a RNA binding protein with non-specific binding capability and uncharacterized activity in the context of viral replication. In Chapters 2 and 3, work is described that investigates protein-protein and protein-RNA interactions by nsp9. Multiple experiments show that nsp9 forms a helix-helix homodimer in solution and that other crystallographically observed homodimers are not biologically relevant. The RNA binding surface of nsp9 was studied using mutagenesis of arginine and lysine residues exposed on the nsp9 surface. While no single residue was found that was wholly responsible for RNA binding, amino acid residues R10, R39, H48, R74 and R111 in combination were found to be important for RNA binding. The data suggest that the arginine and lysine residues act cooperatively in binding RNA through interactions with the phosphate groups of the RNA backbone. Nsp9 exhibits pH-dependent RNA binding, which is partially accounted for by the presence of a sole solvent-exposed histidine. In Chapter 4, small angle X-ray scattering (SAXS) was used to characterize the structure of two individual nonstructural proteins and two polyprotein forms of the nonstructural proteins. The SAXS model of nsp9 agrees well with the three homodimers that have been determined crystallographically and is likely the helix-helix homodimer which was shown in Chapter 2 to be biologically relevant. The SAXS model of nsp10 was determined and showed a globular structure that deviated from the crystal structure. Crystal structure analysis suggests rearrangements could be occurring in the N-terminus of nsp10. The SAXS data on polyprotein nsp7-8-9, a processing intermediate that may have biological function, showed that it is in an extended conformation that provides sufficient space to accommodate two copies each of nsp7, nsp8 and nsp9. Finally, the SAXS structure of polyprotein nsp9-10 was determined, revealing a globular structure for this processing intermediate. The SAXS model of nsp9-10 can accommodate an nsp9 dimer at its center, flanked on either end by a copy of nsp10. The electrostatic surface of the nsp9-10 model fit to the SAXS model shows large patches of exposed surface amenable for RNA binding and suggests that nsp9-10 could be functional in the context of viral replication. In Chapter 5, work is described that involves the structure determination of human karyopherin (importin) alpha 2. The hKPNα2 structure is highly homologous to previously determined structures of mouse and yeast KPNá2. hKPNá2 binds specifically to a SARS accessory protein, Orf6, and a model for that binding is proposed based on surface charges. The collective work presented in this dissertation shows that a number of protein-protein and protein-RNA interfaces are important for the SARS coronavirus to replicate and survive within the host.
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