Molecular Recognition Mechanisms Between Nonstructural Protein 1 of the 1918 Influenza A Virus and Host Proteins
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Influenza A virus (IAV) is the leading cause of seasonal flu epidemics. The Centers for Disease Control and Prevention (CDC) estimates that influenza viral infections cause 12,000 to 79,000 annual deaths in the United States. IAV also causes global flu pandemics, such as the 1918 flu pandemic (a. k. a. Spanish flu), which was responsible for an estimated 50 million deaths. Despite its importance, molecular determinants of the high virulence of 1918 IAV remain elusive. The virulence of influenza virus is a multigenic trait, which is dependent on the interactions between several viral and host factors. Of the viral factors, nonstructural protein 1 (NS1) is considered particularly important because it counteracts the host antiviral immune system. NS1 comprises an RNA binding domain (RBD), an effector domain (ED), and a long-disordered C-terminal tail (CTT). RBD binds viral double-stranded RNA (dsRNA), sequestering the viral RNA from the host innate immune response. The ED and CTT bind a wide range of host proteins, including 30 kDa subunit of cleavage and polyadenylation specificity factor (CPSF30), tripartite motif-containing proteins (TRIM), and polyadenylate-binding protein II (PABII), suppressing type-I interferon (IFN) expression. Recently, NS1 was also found to activate phosphatidylinositol 3-kinase (PI3K) and subsequently suppresses cellular apoptosis, resulting in enhanced viral replication. The intrinsically disordered CTT in NS1 contains several short linear motifs that interact with diverse host proteins. One of the most notable functional differences between the 1918 NS1 and those of other human IAVs is that the 1918 NS1 CTT contains a Thr to Pro mutation at residue 215 (T215P). This mutation enables the interaction between 1918 NS1 and members of the host CT10 regulator of kinase (CRK) protein family, which is not observed in other NS1 proteins. CRKs are signaling adaptor proteins, which are involved in a variety of cellular signaling pathways involved in cell migration, immune response, cancer metastasis and invasion. Therefore, 1918 NS1 interferes with the interactions between CRK and its cellular binding partners such as c-Jun N-terminal kinase (JNK) and Abelson (ABL) tyrosine kinase, resulting in an enhanced cytopathogenic effect. Moreover, recent studies have indicated that hijacked CRK plays an important role in hyperactivation of PI3K by 1918 NS1. However, the mechanism of how 1918 NS1 exploits hijacked CRK to hyperactivate PI3K remains to be determined. To reveal the molecular mechanism by which 1918 NS1 hyperactivates PI3K using CRK, this dissertation aims to (1) characterize the structures of 1918 NS1 and its complex with PI3K and CRK using nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography, (2) elucidate the thermodynamic and kinetic mechanisms of its binary (1918 NS1-CRK and 1918 NS1-PI3K) and ternary interactions (CRK-1918 NS1-PI3K), using fluorescence, isothermal titration calorimetry (ITC), and bio-layer interferometry (BLI). Moreover, CRK proteins are involved in human cancer metastasis and invasion. The interaction between CRK and BCRABL, an oncogenic mutant of ABL kinase, plays important roles in chronic myeloid leukemia, suggesting that inhibiting the interaction of CRK with BCR-ABL might attenuate cancer metastasis. This dissertation demonstrates a rational strategy for the development of potent CRK inhibitors using a CRK-binding motif contained in 1918 NS1. The designed peptide binds to the N-terminal Src-homology-3 (nSH3) domain of CRK with a 10 nM affinity. An in vitro biochemical assay demonstrated that the peptide effectively inhibits the phosphorylation of CRK by ABL kinase.
Shen, Qingliang (2019). Molecular Recognition Mechanisms Between Nonstructural Protein 1 of the 1918 Influenza A Virus and Host Proteins. Doctoral dissertation, Texas A&M University. Available electronically from