Structural basis of the interaction between the C-terminal domain of rabies virus phosphoprotein and human STAT1
AuthorHossain, Md Alamgir
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2020-08-27.
© 2018 Dr Md Alamgir Hossain
The archetype rabies virus belongs to the genus lyssavirus which collectively are the causative agents of rabies disease. The deadly disease has a 100% case-fatality rate in humans, killing over 61,000 people a year and also resulting in a financial burden worldwide of approximately US$6 billion. Rabies virus possesses a unique mechanism to evade the interferon (IFN) mediated host immunity during infection. Specifically, the virus genome encodes a multifunctional phosphoprotein (P) which has the binding site for the IFN-activated downstream signaling molecules, the signal transducer and activator of transcription (STAT) family. Through direct binding of P protein to both STAT1 and STAT2, it prevents the expression of IFN-stimulated genes (ISGs) necessary to establish an anti-viral state. The globular C-terminal domain (CTD) of P protein comprises the binding site for human STAT1. Recent research poses that the residues Trp265 and Met287, which form the W-hole on the hydrophobic patch of the P protein CTD, constitute the site of interaction. Furthermore, mutating these two residues within the viral genome resulted in non-pathogenic viruses that could still replicate. In a yeast-2-hybrid study the binding site for P protein was identified within the coiled-coil domain (CCD) and DNA-binding domain (DBD) of STAT1. However, no biophysical studies have been conducted to prove the interaction in vitro. Therefore we aimed to validate the composition of the binding interface using biophysical and structural approaches. To study the molecular details of the interaction we have optimized the expression and purification of the P protein CTD and STAT1. Although purification of CTD was quite straightforward, purification of soluble STAT1 was more difficult. Using the GB1 fusion tag as a solubilisation enhancement tag we have been able to markedly improve the yield of STAT1 from 5 mg to 30 mg from each liter of bacterial culture. We have successfully purified four versions of STAT1: STAT1-full-length, and truncates of the N-terminal domain STAT1-ND, the coiled-coil domain (STAT1-CCD) and both the coiled-coil and DNA binding domain (STAT1-CCD-DBD) with improved yield and solubility and shown they are structurally intact by Circular Dichroism spectroscopy. Analytical Ultracentrifugation sedimentation velocity (AUC SV) analysis indicated that the purified GB1-STAT1-full-length is predominantly dimeric while GB1-STAT1-CCD-DBD is a monomer confirming no effect of GB1 to STAT1 oligomeric state. Using AUC SV analysis and fluorescence detection, GFP-fused NiP-CTD were titrated against GB1-STAT1-full-length and GB1-STAT1-CCD-DBD, to show a clear complex formed with an estimated Kd of ~10 µM. In an attempt to define the binding site an NMR titration monitored by 15N Heteronuclear Single Quantum Coherence (HSQC) was performed using GB1-STAT1 constructs and 15N-labelled P protein CTD. Upon titration global broadening was observed for GB1-STAT1-full-length and GB1-STAT1-CCD-DBD, but not GB1-STAT1-ND or GB1-STAT1-CCD. While these experiments indicate the DNA-binding domain (DBD) of STAT1 is critical for the interaction with P protein CTD, the site of interaction could not be defined. To validate and define the STAT1 binding site on the P protein CTD transferred cross-saturation (TCS) measurement were performed on GB1-STAT1-CCD-DBD and GB1-STAT1-full-length. These experiments showed two regions, spanning Ile200 to Phe210 and Asn233 to Lys239, were attenuated by both STAT1 constructs, while a third region Leu276 to Val278 was attenuated with GB1-STAT1-full-length. Importantly, these experiments proposed Trp265 and Met287 are not a part of the STAT1 binding site. To validate the newly proposed binding site, site-directed mutagenesis coupled with cell based luciferase reporter assays were conducted. Mutation of two residues (Phe209 and Asp235) within this new binding site resulted in loss of STAT1 binding both in NMR experiments as well as cell-based co-immunoprecipitation assays. Further biophysical and structural characterization of P protein CTD double mutants (W265G/M287V and F209A/D235A) revealed that, a change in the structure of NiP-CTD caused by W265G/M287V mutation alters its ability to interact with STAT1 while mutation on F209A/D235A resulted P protein CTD has minimal structural perturbation. These data support that mutation of Trp265 and Met287 indirectly perturbs STAT1 binding. Furthermore, as the STAT1 binding regions are conserved amongst the lyssaviruses, mutations such as F209A/D235A mutant, may show promise as novel targets to design live attenuated vaccines of the lyssavirus for the control of rabies.
KeywordsSTAT1; protein expression; P protein; viral protein interactions; NMR; cross-saturation measurement
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