Biochemistry and Pharmacology - Theses
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ItemImpact of respiratory syncytial virus infection on host mitochondrial organisation and function( 2018)Respiratory syncytial virus (RSV), a leading cause of acute lower respiratory illness in infants, immunosuppressed adults and the elderly, is responsible for more deaths each year than influenza. Despite this, there are no freely available treatment options, making the development of safe and efficacious anti-RSV therapeutics a high priority. However, in order to achieve this, a deeper understanding of the RSV-host cell interaction is required. RSV infection has previously been found to induce global changes in the mitochondrial proteome and interfere with mitochondria-mediated antiviral signalling, but details of the RSV-host cell mitochondrial interaction are poorly understood. Therefore, the aim of this thesis is to explore the impact of RSV infection on host mitochondria and its role in viral pathogenesis in order to identify novel anti-RSV strategies. The results presented in this thesis reveal for the first time that RSV induces a staged, microtubule/dynein-dependent redistribution of mitochondria, concomitant with reduced mRNA levels of genes encoding mitochondrial proteins, compromised mitochondrial respiration, dissipated mitochondrial membrane potential (Δѱm), and increased generation of mitochondrial reactive oxygen species (ROS). It was also found that inhibiting mitochondrial redistribution or mitochondrial ROS production strongly suppressed RSV virus production, highlighting the RSV-mitochondrial interface as a potential antiviral target. Analysis of RSV proteins identified the matrix protein (M) is sufficient and necessary to induce mitochondrial perinuclear clustering, downregulation of mitochondrial genes, inhibition of mitochondrial respiration, loss of Δѱm, and accumulation of mitochondrial ROS in infection, while deletion and mutation studies identified its central nucleic acid-binding domain, and arginine/lysine residues 170/172 in particular, as essential for its remodelling ability in host cell mitochondria. Recombinant RSV carrying the arginine/lysine mutations in M was unable to elicit these effects on host mitochondria, and its replication in infected cells was severely impaired, underlining the importance of M-dependent effects on mitochondria to RSV infection. Importantly, clinically relevant human cell models of RSV infection were examined, highlighting the importance of RSV’s impact on host mitochondria to its infectious cycle, and its relevance to human disease. Further, inhibiting the accumulation of mitochondrial ROS in infected cells was confirmed as a viable anti-RSV approach in these systems, and work was extended to include a mouse model that showed significantly reduced RSV-related pathology as a result of treatment with a mitochondrial ROS scavenger. In summary, the studies presented in this thesis shed new light on the impact of RSV infection on host cell mitochondria by establishing the unique ability of RSV, facilitated by the M protein, to co-opt the host cell mitochondria to enhance virus production. In addition, the importance of RSV’s impact on host mitochondria for pathogenesis is explored in multiple disease models, highlighting it as a potential target for the development of anti-RSV treatments. Significantly, the studies reveal the inhibition of mitochondrial ROS levels for the first time as a viable approach to counteract RSV infection.
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ItemBiochemical mechanisms of biomineralization and elemental incorporation in otoliths: implications for fish and fisheries research( 2018)All vertebrates have small bioinorganic “earstones” in their inner ear labyrinth that are essential for hearing and balance. While otoliths play a vital anatomical role in fish, their true value to science is as biochronometers, largely due to their unique pattern of growth. Otoliths first form in embryo and continue to grow throughout the life of an individual, with a double-banded increment composed of a calcium carbonate-rich region and a protein-rich region being deposited daily. In addition to this, they are considered to be metabolically inert, and do not undergo remodelling or resorption. Consequently, otoliths are employed in a variety of ways in fish ecology. Firstly, an individual fish’s age and growth rate can be estimated through counting increments and measuring their widths. Secondly, analysis of increment trace element:calcium ratios, such as by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), can allow for the reconstruction of environmental histories, aiding in the determination of natal origin, movement, habitat use, diet and the impacts of climate change. The utility of specific trace elements as indicators of environmental change, however, is unclear as there is considerable uncertainty as to whether a given trace element is interacting with the mineral or protein components of an increment. This uncertainty is a consequence of otolith research having been largely focussed upon either microstructure or inorganic chemistry, with very few studies on the protein-rich regions of the otolith. As a result, very little is understood about the biochemical mechanisms of biomineralization or trace element incorporation. This is important, as the mechanisms that govern otolith formation and growth underpin the assumptions made in traditional increment analyses. In this thesis, I initially undertook a systematic review of all the literature pertaining to otolith biochemistry, revealing the significant gaps that exist in otolith biochemistry as a discipline. Importantly, I determined that fewer than a score of otolith proteins had been identified – a stark contrast to the hundreds or thousands of proteins that have been identified in comparable biomineral systems such as enamel or bone. Working on black bream (Acanthopagrus butcheri), an extensively studied species endemic to southern Australia, I used size exclusion chromatography coupled with ICP-MS to determine the trace element:protein interactions in endolymph, the inner ear fluid that otoliths are submerged in, and the source of all of its constituents. In this study, I assayed 22 elements, and determined that 12 were solely present in a protein-bound form, 6 were present as free ions, and 4 were present in both forms. This allowed me to make recommendations as to their utility in environmental reconstructions. In my next study, I created a unique, multi-disciplinary workflow that combined transcriptomics with proteomics. In this study, I sequenced the transcriptome of the black bream inner ear and used this to identify proteins from the separated organic phase of otoliths and endolymph from wild caught adult black bream. This resulted in the discovery of hundreds of previously unknown proteins, providing new insights into the likely biochemical mechanisms involved in otolith formation and growth. In my final study, I tested the utility of trace element ratios in environmental reconstructions. Specifically, I compared the ability of different cluster analysis approaches to resolve spatial and temporal differences in the likely spawning and larval nursery habitats of juvenile black bream in the Gippsland Lakes, Australia. The results from my thesis have allowed me to make recommendations as to the utility of trace elements in environmental reconstructions and have revealed exciting new avenues of research that fuse ecology and biochemistry.
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ItemNew pathogenic mechanisms in SCA1 neurodegenerative disease revealed by the ataxin-1 interactome( 2018)Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disease in which a marked atrophy of neurons in the cerebellum and brainstem causes coordination and movement disabilities and ultimately death within 10-20 years of symptom onset. The molecular cause is an expanded polyglutamine (polyQ) sequence in the ataxin-1 protein. The resulting accumulation of the mutant ataxin-1 (polyQ-ataxin-1) protein as distinctive nuclear bodies (NBs) has been proposed as contributing to neuronal toxicity and/or dysfunction, but little is known about the biophysical properties of these NBs and their ultimate impact on neuronal cells. The studies presented in this thesis address these issues initially with proteomics approaches in Neuro-2a neuronal cells to identify the interacting protein partners of polyQ-ataxin-1, i.e. the polyQ-ataxin-1 interactome. The results from proximity labelling and affinity purification approaches were combined to improve confidence in the resulting lists of partner proteins. Further bioinformatics analysis identified enrichment of several protein functional groups; nuclear transport proteins and RNA helicases were prioritised for further study by biochemical and advanced imaging techniques. The expression of polyQ-ataxin-1 in Neuro-2a cells was shown to disrupt the localisation of multiple nuclear transport proteins. Nuclear transporters importin-α2, importin-β1, importin- 13, Hikeshi, exportin-1, and nucleoporin NUP98 have been mislocalized and partially co- localized with ataxin-1 NBs. The observations of altered nuclear/cytoplasmic distributions of model cargo proteins were also consistent with the disruption of the processes of nuclear transport in the presence of polyQ-ataxin-1. The physical properties of the polyQ-ataxin-1 NBs were also assessed, with specific consideration of the contributions by RNA/RNA helicases. Under standard conditions these NBs showed rapid exchange of the ataxin-1 protein consistent with dynamic liquid droplets; the down-regulation of RNA helicases DDX42, DDX46, and DHX15, the decreased ATP level, or altered environmental conditions were shown to slow exchange. These results thus reveal the phase transition to a less dynamic hydrogel or fibrillar phase and emphasize the tunable dynamics of these polyQ-ataxin-1 NBs as RNA/protein droplets. Taken together, these studies have revealed new insights into the impact and regulation of polyQ-ataxin-1 made possible by the identification of new proximal or interacting partners for polyQ-ataxin-1, and thus suggest new strategies for future interventions in the treatment of SCA1 and other neurodegenerative diseases.
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ItemUnderstanding biological signalling in the βc cytokine receptor family( 2018)The beta-common ( βc) family of cytokines, namely granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin (IL)-3 and IL-5, are modulators of cell survival, differentiation and proliferation during normal and malignant haematopoiesis. They have recently been shown to act outside the haematopoietic system, playing roles in the nervous system. Aberrant signalling from the bc cytokines has been linked to many diseases, such as leukaemia and asthma. These cytokines signal by binding to heteroreceptors made of a cytokine specific a-subunit and the b-subunit, which interacts with all three cytokines (bc subunit). As such, the shared use of the bc subunit by GM- CSF, IL-3 and IL-5 makes it an attractive target of investigation for novel therapeutics that would disrupt the interaction of all three cytokines with their receptors. The studies undertaken have focussed on investigating the assembly mechanism of the bc receptor complexes, as well as identifying small molecule inhibitors and monoclonal antibodies targeting the interaction interfaces formed within the signalling complex. These inhibitors will aid in understanding the downstream signalling pathways activated by the βc family of cytokines and may elucidate new targets for drug discovery. Investigating the interaction between the βc subunit and putative binding partners such as the erythropoietin receptor (EPOR), janus kinase 2 (JAK2) and the 14-3-3 family has also been part of these studies. The βc subunit and EPOR have been reported to form an innate repair receptor complex under stress and I have demonstrated that the extracellular regions of the two receptors are not sufficient to mediate a direct interaction. The first steps towards understanding how two main intracellular pathways activated by the βc cytokines, the JAK-STAT and the PI3K pathways, are triggered 2 through the interaction of the βc subunit with JAK2 and the 14-3-3 family have also been undertaken. Throughout my PhD studies, I have utilised a wide array of biophysical techniques and crystallisation methods. The outcomes of this research will expand the understanding of the signalling of the family of βc cytokines, opening new avenues for the development of therapeutics.
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ItemCharacterisation of the human TIM22 mitochondrial import translocase( 2018)Mitochondria are essential cellular organelles for cell viability due to their fundamental role in ATP production, programmed cell death and biosynthetic pathways. Dysfunctional mitochondria are implicated in various pathologies including cancers, cardiovascular diseases and neurodegenerative diseases. Mitochondrial function relies on ~1500 mitochondrial proteins, which are predominately nuclear-encoded and must be imported into mitochondria. Protein trafficking to mitochondria is executed by sophisticated multimeric protein import machines, termed, Translocases. One such machine, the Translocase of the Inner Membrane 22 (TIM22) mediates the import of an important class of hydrophobic proteins, the carrier proteins, which facilitate chemical exchange across the inner membrane, contributing to cellular metabolism and bioenergetics. While TIM22 has been well-characterised using Baker’s yeast, very little is known about the human complex. This study has focussed on disentangling the molecular composition of the human TIM22 complex, and mechanisms underpinning the carrier import pathway. Using immunoprecipitation/mass-spectrometric analysis, we uncovered two novel, metazoan-specific subunits of the human TIM22 complex, termed Tim29 and Acylglycerol kinase (AGK). This is the first report of additional subunits of the human TIM22 complex since the initial reports of the human complex in 1999 (Bauer et al., 1999b). We showed that Tim29 is involved in the assembly of the TIM22 complex and creates contacts with the general entry gate of the outer membrane, the TOM complex, providing a novel mechanism for the translocation and import of hydrophobic proteins. Dissecting the molecular function of AGK revealed a lipid kinase-independent role of this previously described mitochondrial lipid kinase in mediating carrier protein import via the TIM22 complex. Identification of AGK at TIM22 also shed additional insight into the pathomechanisms underpinning Sengers syndrome, a mitochondrial disorder uniquely associated with AGK mutations, providing an unexpected link between mitochondrial protein import and Sengers syndrome. The TIM22 complex is also linked to a neurodegenerative disease, Mohr-Tranebjaerg syndrome (MTS), which is caused by mutations in the TIMM8A gene. To date, the pathomechanism underlying this disease remain unknown. Using CRISPR/Cas9-genome editing and label-free quantitative proteomics, we analysed two distinct cell lines (HEK293 and SH-SY5Y) and revealed a cell-specific function of hTim8a in Complex IV biogenesis in neuronal mitochondria. Our findings indicate that loss of hTim8a leads to mitochondrial dysfunctions which amplifies cytochrome c levels in mitochondria and sensitises cells to death. This research has contributed new insights into understanding of the human TIM22 translocase and carrier protein biogenesis. It has revealed novel features and knowledge on the pathomechanism of two TIM22-associated mitochondrial diseases – Sengers syndrome and Mohr-Tranebjaerg syndrome.
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ItemTranscriptional regulation and functional differences driven by STAT3 isoforms, STAT3α and STAT3β( 2018)The Signal transducer and activator of transcription 3 (STAT3) protein, a member of the STAT family of transcription factors, plays important roles in the regulation of critical biological processes. STAT3 is best known for its role in the JAK-STAT pathway, mediating the transcriptional regulation of target gene expression following its activation by extracellular signal, and continues to be the subject of intense research efforts due to its essential roles in crucial physiological processes, such as embryonic development and inflammation, as well as its involvement in numerous types of cancer. While the genome-wide DNA-binding and transcriptional regulation activities of the full-length STAT3α have been widely studied, those of a naturally occurring shorter STAT3β spliceform are less understood. Despite having identical DNA-binding domains, the STAT3 spliceforms display significant differences in their transcriptional regulation activities. Furthermore, STAT3α and STAT3β can drive opposing oncogenic and tumour suppression outcomes, respectively. Thus, the major focus of this study is to explore STAT3α- and STAT3β-specific genome-wide DNA-binding, transcriptional regulation of non-coding micro-RNAs (miRNAs), and functional outcomes in cancer cell biology. By employing ChIP-seq and miRNA expression profiling assays, this study presents results revealing that STAT3α and STAT3β display clear differences in genome-wide DNA-binding and regulation of miRNA expression. Bioinformatic analyses of transcription factor binding sites also uncovered the possible roles of co-transcription factors in the distinct STAT3 spliceform-specific transcriptional regulation activities. In addition, a Morpholino-directed splicing modulation approach to drive STAT3 knockdown and STAT3α-to-β splicing switch in cancer cells showed that the STAT3 spliceforms can differentially alter the tumourigenic properties of cancer cells, with STAT3β expression being associated with tumour suppression outcomes by driving the suppression of cell proliferation, survival and migration. Taken together, this study highlights the distinct properties of the STAT3 spliceforms in genome-wide DNA-binding which possibly underlie their different gene transcriptional regulation activities in both protein-coding and non-coding genes, and further provides evidence that STAT3β can drive distinct tumour suppression outcomes.
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ItemAnterograde sorting and trafficking of the β-amyloid precursor protein and β-Secretase in Alzheimer’s disease( 2018)Alzheimer’s disease (AD) is characterized by the extracellular deposition of amyloid plaques in the brain. Amyloid plaques are derived from the aggregation of pathogenic β-amyloid (Aβ) peptide, which is generated from the sequential amyloidogenic processing of the β-amyloid precursor protein (APP) by membrane-bound proteases, β-secretase (BACE1) and γ-secretase. Several AD susceptibility alleles have been associated with membrane trafficking of APP and BACE1, and the regulation of Aβ production. Although the endocytic pathway has received considerable attention, early studies indicated that the secretory/anterograde pathway can also generate Aβ. However, the anterograde transport of APP and BACE1, and particularly the mechanism of sorting of these proteins in the Golgi, has not been well-defined yet is highly relevant for understanding the pathogenesis of AD. My thesis aimed to define (1) the trafficking machineries that are required for regulating the Golgi export of APP and BACE1; (2) the post-Golgi trafficking itineraries of newly synthesized APP and BACE1; (3) the relevance of my findings for the trafficking of APP and BACE1 in primary neurons; and (4) the importance of trafficking machineries in regulating the intracellular processing of APP in the Golgi. As newly synthesized APP and BACE1 are sorted at the trans-Golgi network (TGN), dysregulation in the sorting of APP and mature BACE1 is likely to enhance convergence of the two proteins to promote amyloidogenic processing in this compartment. I investigated the sorting and trafficking of newly synthesized APP and BACE1 from the Golgi using a range of approaches including RNA interference, immunofluorescence microscopy, flow cytometry, and the retention using selective hooks (RUSH) system. In Chapter 3, I have shown that Arl5b is required for the efficient recruitment of AP4 to the TGN which regulates the export of APP, but not BACE1, in HeLa cells. From the Golgi, newly synthesized APP traffics directly to the early endosomes then the late endosomes/lysosomes. In contrast, I have demonstrated in Chapter 4 that newly synthesized BACE1 is transported directly to the PM from the TGN in HeLa cells. Moreover, the TGN export of BACE1 is regulated by AP1/Arf1/Arf4 transport machineries, which are distinct from the transport machinery required for the post-Golgi transport of APP. Depletion of either AP4/Arl5b or AP1/Arf1/Arf4 export machineries in the presence of a γ-secretase inhibitor (DAPT) increased the level of β- CTF/C99, the product of APP cleavage by BACE1, confirming that the direct cleavage of APP by endogenous BACE1 was enhanced when the Golgi export of either APP or BACE1 was impaired. In the absence of DAPT, only low levels of β-CTF/C99 were detected following depletion of the Golgi export machineries for either APP or BACE1. These findings indicate that β-CTF/C99 is rapidly processed by γ-secretase to liberate Aβ and that the biogenesis of Aβ probably occurs in the TGN. Therefore, the accumulation of APP or BACE1 in the TGN after the depletion of AP4/Arl5b or AP1/Arf1/Arf4 post- Golgi export machineries, respectively, resulted in enhanced amyloidogenic processing of APP. To determine the significance of the post-Golgi trafficking studies of APP and BACE1 conducted in HeLa cells, I generated shRNA lentivirus and successfully depleted AP4 and AP1 in primary neurons and analyzed the impact on endogenous APP trafficking. I have shown that AP4, but not AP1, is required for the trafficking of endogenous APP from the TGN in neurons. Moreover, the depletion of AP4 in primary neurons resulted in enhanced processing of APP by endogenous BACE1 in the TGN. The intracellular sites of APP cleavage by 𝛼-secretase along the protective nonamyloidogenic pathway have not been well-defined. In Chapter 5 and 6, I have demonstrated that APP can be processed by endogenous 𝛼-secretase in the TGN of both HeLa and neuronal cells. These findings are significant as the TGN had not been previously recognized as a site for 𝛼-secretase processing. Overall, the findings presented in this thesis have shown that efficient post-Golgi trafficking of newly synthesized APP and BACE1, which are regulated by distinct transport mechanisms, plays a critical role in the segregation of the two proteins. Dysregulation in the Golgi exit of either APP or BACE1 increases the residency time of these membrane cargoes in the TGN resulting in enhanced pathogenic processing of APP, most likely as a consequence of the loss of segregation and increased colocalization.
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ItemInvestigating acquired resistance to Pol I transcription inhibitors for the treatment of haematologic malignancies( 2018)Previous work from our group and others has demonstrated that CX-5461 (Senhwa Biosciences), a first-in-class small molecule inhibitor of RNA Polymerase I transcription of the ribosomal RNA genes, is effective at treating a range of different cancers both in vitro and in vivo, and is currently in clinical trials for haematologic and solid tumours. However, despite initial tumour clearance in response to CX-5461 treatment in preclinical murine models of cancer, mice eventually relapse with tumours that are resistant to further CX-5461 treatment. This thesis investigates the mechanisms via which the tumours can develop resistance to CX-5461 treatment and extrapolates this research to better understand: 1) how CX-5461 functions as an anti-tumour agent; 2) which pathways are required to mediate resistance to CX-5461; and 3) how resistance can be overcome with combination therapy. Using DNA exome sequencing, we found that Top2α is frequently mutated in tumours that have acquired resistance to CX-5461 treatment in vivo. Functional characterization of a Top2α mutant cell line demonstrated that Top2α expression and activity were reduced in these cells. Indeed, we found that knockdown of Top2α was sufficient to cause resistance to CX-5461. This implies that Top2α could provide a novel biomarker for CX-5461 response in clinical trials. Further investigation of the CX-5461 resistance mechanism uncovered that CX-5461 also acts as a Top2 inhibitor in addition to its ability to inhibit rDNA transcription. However, unlike common chemotherapeutic Top2 inhibitors which kill cells by causing genome-wide DNA damage thereby initiating a DNA damage response, CX-5461 treatment causes comparatively fewer DNA breaks enriched at the ribosomal DNA promoter loci. Thus, CX-5461 is able to kill tumour cells via the DNA damage response in the absence of extensive DNA damage thereby potentially limiting the cytotoxicity of drug treatment. Together, the work presented in this thesis identifies novel mechanisms of action and resistance to CX-5461. We propose that CX-5461 and other second-generation inhibitors of RNA Polymerase I and Top2α may provide a viable, less genotoxic alternative to classic Top2 inhibitors.
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ItemStructural basis of the interaction between the C-terminal domain of rabies virus phosphoprotein and human STAT1( 2018)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.