Saudi Cultural Missions Theses & Dissertations
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Item Restricted The role of IL-17 in CD95L-driven nonapoptotic pathology during respiratory disease(University of Liverpool, 2024-07) ALAidarous, Sondus; Stewart, JamesCD95L can induce a pathological nonapoptotic pathway and promote Th17 migration when cleaved from the cell surface by proteases. The targeted inhibition of this nonapoptotic pathway by blocking the calcium-inducing domain to impede IL-17 migration could be of therapeutic benefit in inflammation, as seen in a murine lupus model. We aimed to investigate the CD95L nonapoptotic pathway and factors involved in the cleavage of CD95L in the context of SARS-CoV-2 infection and pulmonary fibrosis. Three approaches were used: in vitro studies, in vivo murine models and human clinical sample-based studies. In vitro, the Jurkat cell line was stimulated with PHA, LPS, recombinant MMP9 and mouse sera to optimise conditions for the expression and cleavage of CD95L. MMP9 was investigated as a putative cleavage factor of CD95L and was found at higher levels in naïve lung supernatant than in serum. In a SARS-CoV-2 murine model, the effect of inhibitory peptide treatment on mice intranasally infected with 104 PFU SARS-CoV-2 was assessed. IL-17 serum levels in the peptide-treated group were significantly lower than in the control group, yet this difference did not translate to disease progression, measured by viral load, weight loss, histopathology and survival. IL-17 and sCD95L levels were tested in SARS-CoV-2 patient samples from a biobank cohort and healthy control samples. There was no difference between the groups, and sCD95L was undetectable in the SARS-CoV-2 patient group. Our hypothesis on pulmonary fibrosis was tested through a model of bleomycin-induced fibrosis in BALB/c mice across 36 days. Animals were challenged with 4x105 of murine gammaherpesvirus (MHV) intranasally to aid fibrosis development on day 0. Bleomycin was administered twice at 40 mg/kg on day 8 and day 17. Inhibitory peptide treatment, control peptide treatment or mock treatment was given the day after bleomycin administration. Post-mortem, cardiac bleeds and lung and liver tissue were collected. IL-17, MMP9 and sCD95L were measured by ELISA. Right lungs were processed using Trizol for qPCR, and left lungs, along with livers, were prepared for histopathology. No difference was found between groups in terms of IL-17, MMP9 or sCD95L serum levels, gene expression of il-17 or ccr6, fibrosis scores based on Masson’s trichrome staining, and weight loss or survival rate. However, a significant correlation was seen between weight loss and the fibrosis score. In human pulmonary fibrosis serum samples, IL-17 levels were barely detectable and did not differ from those in healthy controls. Interestingly, sCD95L levels were significantly higher and MMP9 levels were significantly lower than in healthy controls. In summary, blocking the calcium-inducing domain could reduce IL-17 levels but did not affect disease progression in a SARS-CoV-2 model. Moreover, these findings could not be translated to other respiratory diseases, like pulmonary fibrosis.38 0Item Restricted Investigating the impact of coronavirus infections on the host respiratory microbiome(University of Liverpool, 2024-05) Alrezaihi, Abdulrahman; Hiscox, JulianCoronaviruses, including the recent SARS-CoV-2, present a complex challenge for public health due to their ability to cause illnesses ranging from asymptomatic infection to patients suffering severe symptoms and death. This thesis employed advanced sequencing methods to identify and study different coronaviruses, from seasonal varieties to MERS-CoV and SARS- CoV-2, and their interactions with the host microbiome in both human clinical samples and animal models. This was underpinned by Oxford Nanopore long-read sequencing combined with modifications of sequence-independent single primer amplification (SISPA). This research aims were to improve the ability to detect coronaviruses, particularly in samples with low viral loads, by modifying different enrichment methods that were designed to detect SARS-CoV-2, identify lineages, and simultaneously define the nasal microbiome. The study revealed changes in the microbiome of patients with coronavirus infections, showing how the virus and the nasal microbiome interact. The data indicated that certain bacterial populations were more predominant in specific groups of patients with COVID-19 or MERS, potentially affecting the course of their illness. The investigation showed that dysbiosis, or microbial imbalance, was significant within coronavirus infections and occurred irrespective of the virus type (MERS-CoV or SARS-CoV-2) and mortality outcome. The investigation compared these patterns in human patients with those observed in a mouse model of SARS-CoV-2 that recapitulated severe disease. This study specifically identified differences in the microbiome between the upper and lower respiratory tracts. Examining the lower respiratory tract in humans is not common as this involves invasive sampling, and use of samples from intensive care patients facilitated this research. This comparison clarified how SARS- CoV-2 affected various parts of the respiratory system and showed that changes in viral load affected the microbiome composition within these distinct areas. Furthermore, the study defined how the microbiome changed throughout the infection, particularly in patients with severe illness who required intensive care.38 0Item Restricted Specific Nucleic Acid Detection Using a Nanoparticle Hybridization Assay(The Catholic University Of America, 2024-05-09) Aldakheel, Arwa A; Raub, Christopher B.; Bui, HieuThe Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus has become a global health crisis, leading to significant public health challenges. Since symptoms overlap with other respiratory infections, prompt and accurate diagnosis is essential to manage the disease. Two issues arise from using PCR-based diagnostic tests for SARS-CoV-2: false positives at high cycle numbers from early nonspecific amplification and the amplification of virus nucleic acids present in trace amounts in the collected specimen that are not necessarily associated with active infection. These issues, standard across all tests that use nucleic acid amplification, necessitate the development of alternative diagnostic approaches. This thesis presents a novel approach to developing a molecular biosensor for detecting SARS-CoV-2 RNA, leveraging gold nanoparticles (AuNPs) combined with two oligonucleotides. The biosensor relies on forming aggregates of oligo-AuNPs in the presence of targeted viral nucleic acid sequences. Initial testing involved conjugating AuNPs with two designed oligonucleotides, reverse complements of specific SARS-CoV-2 target sequences. Notably, nanoparticle aggregate detection was performed by darkfield videomicroscopy, detecting nanoparticle tracks and estimating the aggregates’ diffusion coefficients. Ancillary identification of nanoparticle aggregation occurred by detecting a ~6 nm shift in the surface plasmon resonance peak measured with nano spectrophotometry and a mobility shift detected by agarose gel electrophoresis. To maximize target hybridization and reduce aggregation of oligo-coated nanoparticles in the presence of off-target nucleic acid sequences, two oligomers chosen for nanoparticle conjugation were optimized for hybridization efficiency with the chosen target and for lack of self- and cross-hybridization through a parameter-limited Primer-BLAST search and thermodynamic and structure analysis using NUPACK. Experimental results demonstrated that oligomers hybridized with a specific DNA sequence that complements the RdRp RNA gene sequence from the published virus genome. After successfully conjugating the oligomers to 10 nm diameter gold nanoparticles, the nanoparticles aggregated in the presence of target DNA, confirmed by spectrophotometry, nanoparticle, tracking, and gel electrophoresis. Computational models of nanoparticle diffusion and nanoparticle aggregation as systems of first-order reactions confirmed findings from experiments: that larger and aggregated nanoparticles produced lower diffusion coefficients and that more targets in the reaction mixture produced larger aggregates. Integrating primers with gold nanoparticle-based biosensors highlights the potential for developing rapid and portable diagnostic tools for SARS-CoV-2 detection. In conclusion, this thesis proposes and provides some data supporting a novel approach for detecting nucleic acids in solution without amplification: darkfield videomicroscopy with nanoparticle tracking and diffusion coefficient estimation. The steps needed to make this approach viable and the generalization of the assay beyond the detection of SARS-CoV-2 nucleic acids was discussed. The insights gained have implications for developing sensitive, rapid, and portable diagnostic platforms for various infectious diseases and detecting other types of nucleic acids.30 0Item Restricted Impact of SARS-CoV-2 Salivary Gland Infection on the Production of the Antifungal Peptide Histatin-5, Candida Colonization, and the oral Microbiota(University of Maryland, Baltimore, 2024) Alfaifi, Areej; Rizk, Mary Ann; Warner, Blake; Schneider, Abraham; Meiller, Timothy; Sultan, AhmedDespite the myriad oral manifestations during COVID-19 and presence of SARS-CoV-2 in saliva, the oral cavity remains an underappreciated site. However recent findings have shown that SARS-CoV-2 can replicate in salivary gland cells, leading to inflammation and tissue destruction. Saliva contains antimicrobial peptides considered integral components of innate immunity crucial for oral health, with the most notable being histatin-5. This peptide is exclusively produced in salivary glands and exhibits unique potent antifungal activity against Candida albicans. In this dissertation, we discovered that destruction of salivary gland by SARS-CoV-2 can compromise histatin-5 production, predisposing patients to oral candidiasis and dysbiosis in the oral microbiome. First, we utilized our novel customized immunoassay to measure salivary histatin-5 levels in a prospective study involving stratified COVID-19 cohorts. Our data indicates a trend showing a decrease in salivary histatin-5 and increase in Candida during COVID-19, persisting post-COVID-19 recovery, potentially contributing to the long COVID-19 syndrome. To provide lacking mechanistic insights into the pathophysiology of salivary gland dysfunction during COVID-19, we performed in situ hybridization coupled with immunofluorescence to co-localize SARS-C0V-2 and histatin-5, respectively, in salivary gland tissue from deceased COVID-19 patients. Our findings indicated diminished or absent histatin presence in salivary gland acini with proliferating SARS-CoV-2 providing the first direct evidence associating SARS-CoV-2 with histatin-5 production. Next, we conducted a comprehensive metagenomic analysis on clinical oral samples and identified potential COVID-19 associated pathologic dysbiotic shifts in the oral microbiome. Lastly, we conducted in-vitro experiments coupled with scanning electron microscopy and confocal imaging to identify the effect of histatin-5 on candida cells and biofilm concluding the antifungal effect of histatin-5 on candida albicans. This clinical study clearly shows the effect of SARS-CoV-2 on oral microbiota, highlighting the importance of understanding and managing the complex dynamics within the oral cavity of COVID-19 patients.15 0Item Restricted Deciphering the Immunological Landscape of SARS-CoV-2 Infection and Co-infection: Insights into Cytokines, Gene Expression, and Clinical Outcomes(University of Liverpool, 2024-06-05) Alosaimi, Samar; Stewart, JamesCoronavirus disease 2019 (COVID-19), originating from the novel human pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a severe ailment associated with widespread global morbidity and mortality. The immune response to SARS-CoV-2 infection involves the production and expression of numerous cytokines, playing a crucial role in determining the infection's outcome. This study aimed to explore the correlation between specific immune cytokines and the consequences of SARS-CoV-2 infection, as well as to identify key genes associated with the immune response against the virus. Transgenic mice expressing the human ACE2 receptor were chosen as they constitute a robust model for studying COVID- 19 infection, thoroughly mirroring the human infection. To complement animal modelling data, cytokine levels were measured in human serum samples. The role of IL-6 in the immune response was evaluated by measuring the potential impact of IL-6 blocking at different stages of the disease and using three variants of SARS-CoV-2 virus. Weight loss, viral loads in lungs and nasal turbinates, serum IL-6 level, and the histopathological analysis of the lungs were assessed. Anti-IL-6 antibody treatment demonstrated no effect on SARS-CoV-2 infected mice and may cause unfavourable outcomes when used early as a countermeasure in SARS-CoV-2 infection. Given the global impact and public health significance of the Influenza A virus (IAV), coupled with its capacity to infect a substantial number of individuals annually, its effect was studied with SAR-CoV-2 virus. The differentially expressed immune genes and the effects of a two-step infection involving IAV and SARS-CoV-2 were measured through samples obtained from a transgenic mouse model. These samples were collected at 6 dpi and 10 dpi and subjected to sequencing using the Illumina short-read length cDNA-PCR sequencing method. Co-infection caused more weight loss than SARS- CoV-2 single infection. The comparison between SARS-CoV-2 single infection and Co-infection revealed differentially expressed genes on both day 6 and day 10 post infection. Gene expression analysis indicated that influenza infection was primarily linked to the innate immune response, whereas SARS-CoV-2 infection was predominantly associated with the adaptive immune response. COVID-19 patients’ serum cytokines levels were assessed to find out whether there is a notable association between the severity of COVID-19 disease and the levels of specific cytokines. Patients who were asymptomatic had higher levels of IL-2Rα, IL-8, IL-18, IP-10, MCP-1, MIP-1β, and D-dimer compared to healthy subjects. Only MCP-1 changed between symptomatic patients and asymptomatic patients. Patients who were admitted to ICU and died had higher levels of IL-2Rα, IL-8, IP-10, MCP- 1, and CRP compared to those who were admitted to ICU and survived. Disease severity and elevated mortality observed in COVID-19 patients may result not only from SARS-CoV-2 virus infection but also from the immune response elicited by the infection.16 0Item Restricted Developing Novel Antiviral Agents: Targeting the N-Terminal Domain of SARS-CoV-2 Nucleocapsid Protein with Small Molecule Inhibitors(Virginia Commonwealth University, 2024-05-13) Alkhairi, Mona A.; Safo, Martin K.The COVID-19 pandemic, caused by SARS-CoV-2, persists globally with over 7 million deaths and 774 million infections. Urgent research is needed to understand virus behavior, especially considering the limited availability of approved medications. Despite vaccination efforts, the virus continues to pose a significant threat, highlighting the need for innovative approaches to combat it. The SARS-CoV-2 nucleocapsid protein (NP) emerges as a crucial target due to its role in viral replication and pathogenesis. The SARS-CoV-2 NP, essential for various stages of the viral life cycle, including genomic replication, virion assembly, and evasion of host immune defenses, comprises three critical domains: the N-terminal domain (NTD), C-terminal domain (CTD), and the central linker region (LKR). Notably, the NTD is characterized by a conserved electropositive pocket, which is crucial for viral RNA binding during packaging stages. This highlights the multifunctionality of the nucleocapsid protein and its potential as a therapeutic target due to its essential roles and conserved features across diverse pathogenic coronavirus species. Our collaborators previously initiated an intriguing drug repurposing screen, identifying certain β-lactam antibiotics as potential SARS-CoV-2 NP-NTD protein inhibitors in vitro. The current study employed ensemble of computational methodologies, biophysical, biochemical and X-ray crystallographic studies to discover novel chemotype hits against NP-NTD. Utilizing a combination of traditional molecular docking tools such as AutoDock Vina, alongside AI-enhanced techniques including Gnina and DiffDock for enhanced performance, eleven structurally diverse hit compounds predicted to target the SARS-CoV-2 NP-NTD were identified from the virtual screening (VS) studies. The hits include MY1, MY2, MY3, MY4, NP6, NP7, NP1, NP2, NP3, NP4 and NP5, which demonstrated favorable binding orientations and affinity scores. Additionally, one supplementary compound provided by Dr. Cen’s laboratory (denoted as CE) was assessed in parallel. These hits were further evaluated for their in vitro activity using various biophysical and biochemical techniques including differential scanning fluorimetry (DSF), microscale thermophoresis (MST), fluorescence polarization (FP), and electrophoretic mobility shift assay (EMSA). DSF revealed native NTD had a baseline thermal melting temperature (Tm) of 43.82°C. The compounds NP3, NP6 and NP7 notably increased the Tm by 2.55°C, 2.47°C and 2.93°C respectively, indicating strong thermal stabilization over the native protein. In contrast, NP4 and NP5 only achieved marginal Tm increases. MST studies showed NP1, NP3, and NP7 exhibited the strongest affinity with low micromolar dissociation constants (KD) of 0.32 μM, 0.57 μM, and 0.87 μM, respectively, significantly outperforming the control compounds PJ34 and Suramin, with dissociation constants of 8.35 μM and 5.24 μM, respectively. Although NP2, NP6, and CE showed relatively weaker affinity, these compounds still demonstrated better binding affinities with dissociation constants of 4.1 μM, 2.50 μM, and 1.81 μM, respectively than the control compounds PJ34 and Suramin. These results substantiate the potential of these scaffolds as modulators of NTD activity. In FP competition assays, NP1 and NP3 exhibited the lowest half-maximal inhibitory concentrations (IC50) of 5.18 μM and 5.66 μM, respectively, indicating the highest potency at disrupting the NTD-ssRNA complex among the compounds, outperforming the positive controls PJ34 and Suramin, with IC50 of 21.72 μM and 17.03 μM, respectively. The compounds NP6, NP7, CE, and NP2 also showed significant IC50 values that ranged from 7.00 μM to 10.13 μM. EMSA studies confirmed the NTD-ssRNA complex disruptive abilities of the compounds, with NP1 and NP3 as the most potent with IC50 of 2.70 μM and 3.31 μM, respectively. These values compare to IC50 of 8.64 μM and 3.61 μM of the positive controls PJ34 and Suramin, respectively. NP7, CE, NP6, and NP2 also showed IC50 ranging from 4.31 μM to 7.61 μM. The use of full-length nucleocapsid protein also showed that NP1 and NP3 disrupted the NP-ssRNA binding with IC50 of 1.67 μM and 1.95 μM, which was better than Suramin with IC50 of 3.24 μM. These consistent results from both FP and EMSA highlight the superior effectiveness of NP1 and NP3 in disrupting nucleocapsid protein-ssRNA binding, showcasing their potential as particularly powerful antiviral agents. Extensive crystallization trials were conducted to elucidate the atomic structures of SARS-CoV-2 NP-NTD in complex with selected hit compounds, assessing over 8000 unique crystallization conditions. Ultimately, only a PJ34-bound structure could be determined, albeit with weak ligand density, likely due to tight crystal packing impeding binding site access. The crystal structure was determined to 2.2 Å by molecular replacement using the published apo NP-NTD (PDB 7CDZ) coordinates as a search model, and refined to R-factors of 0.193 (Rwork) and 0.234 (Rfree). The refined NP-NTD structure showed conserved intermolecular interactions with PJ34 at the RNA binding pocket as observed in the previously reported HCoV-OC43 NP-NTD-PJ34 complex (PDB 4KXJ). This multi-faceted drug discovery endeavor, combining computational screening and in vitro assays resulted in successful identification of novel compounds inhibiting the SARS-CoV-2 nucleocapsid N-terminal domain. Biophysical and biochemical studies established compounds NP1 and NP3 as superior hits with low micromolar binding affinities, as well as low micromolar potency superior to standard inhibitors at disrupting both isolated N-NTD-RNA and full-length nucleocapsid-RNA complex formation. Though crystallographic efforts encountered challenges, important validation was achieved through a resolved crystal structure of PJ34 in complex with NP-NTD. Future effort will be to obtain co-crystals of NP-NTD with our compounds to allow for targeted structure modification to improve on the potency of the compounds.6 0Item Restricted Developing Antiviral Drugs for COVID-19 and Hepatitis C: Targeting Key Viral Proteases(Virginia Commonwealth University, 2024-05-14) AlAwadh, Mohammed; Safo, Martin K.Viruses are submicroscopic infectious agents causing immense global disease burdens. Propagation of viral particles relies on proteolytic cleavage of polyprotein precursors by host or virally-encoded proteases to liberate functional components necessary for replication and infection cycles. These processing events present vulnerable intervention points for antiviral targeting. This thesis focused on two indispensable viral proteases - the SARS-CoV-2 main protease and the NS3 protease domain from hepatitis C virus. The first project centered on the discovery of small molecule inhibitors against the SARS-CoV-2 main protease (Mpro). As a cysteine protease, Mpro plays an indispensable role in processing the virally-encoded replicase polyproteins through specific cleavages to liberate functional non-structural proteins that regulate virion maturation and assembly pathways. Owing to such critical involvement, Mpro offered an attractive target for coronavirus pathogenesis intervention. Its near-identical architecture with the SARS-CoV strain enabled rapid knowledge transfer for drug design using prior scaffolds. Therefore, an ensemble small molecule discovery platform consolidating computational screening, synthetic chemistry, enzymology and biophysical characterization was constructed to systematically retrieve inhibitors against this important drug target. Three virtual screening protocols using complementary in silico techniques – ligand-based 3D pharmacophore searches, protein structure-centric molecular docking, and artificial intelligence models employed deep neural networks. This triaged computational workflow efficiently narrowed a search space of millions to selectively cherry pick prospective hit candidates. In parallel, quantitative structure-activity examinations of a small, focused library of 168 synthetically derived α-ketoamide compounds revealed a reactive Michael acceptor warhead amenable for covalently targeting the key catalytic cysteine residue. Downstream characterization in a tiered cascade of biochemical and biophysical techniques validated the tandem computational-experimental screening approach. Fluorescence resonance energy transfer (FRET) enzyme assays confirmed dose-dependent SARS CoV-2 Mpro inhibition for 10 ligands – 7 from virtual screening pipelines and 3 α-ketoamide derivatives – with low micromolar half maximal inhibitory concentrations between 1.7-55 μM. Direct binding quantification via label-free biophysical methods like microscale thermophoresis and isothermal titration calorimetry supplemented functional data. The tightest-binder, compound MA4, achieved a binding affinity of around 5 μM. Attempts to co-crystallize Mpro with ligands for atomic perspectives encountered technical limitations likely owing to poor aqueous solubility, nevertheless yielding 1.8 Å resolution apo-enzyme insight into plasticity elements lining the substrate binding cleft. Microsecond timescale explicit-solvent molecular dynamics simulations tracked long-term dynamic stabilities of inhibitor-bound complexes, corroborated through rigorously computed binding free energy predictions. Lastly, objective hit enrichment and success rate metrics evaluated relative virtual screening performances, demonstrating superior early retrieval rates for the deep learning technique that leveraged biochemical data patterns. The second collaborative project expanded targeting scope beyond conventionally exploited catalytic sites to explore an allosteric regulatory protein-protein interface on the hepatitis C NS3 protease domain. NS3 requires binding of a co-factor NS4A peptide to achieve sufficient catalytic activity essential for mediating downstream viral polyprotein processing events linked to replication competency. NS4A triggers key structural rearrangements in otherwise natively disordered NS3 that enable organization of the catalytic triad into a configuration competent for catalyzing substrates. This activation paradigm presented possibilities for blocking the interaction site with engineered variants retaining affinity but subtly distorting functional geometries through strategic mutations. Results validated this, revealing a designed nanomolar-binding NS4A variant with a single cyclohexylglycine substitution that associated with NS3 but eliminated enzyme activity. Microscale thermophoresis quantifications revealed PEP15 associated with the NS3 protease domain target with remarkably high, low nanomolar binding affinity exhibiting a dissociation constant (KD) of 22.23 ± 0.297 nM. This was approximately two orders of magnitude stronger binding compared to the native NS4A cofactor peptide, which achieved a KD of 2.595 ± 0.0015 μM in the same assay configuration. The exceptionally improved affinity despite a single residue substitution substantiates the significant energetics contributions of the engineered glycine mutation and validates the allosteric targeting rationale underlying the inhibitor design. Differential scanning fluorimetry indicated unexpected reductions in thermal stability relative to native complex or isolated protein controls. Metadynamics simulations provided insights into the unexpected biophysical findings by modeling dynamics and stability of the PEP15-NS3 complex. The trajectories revealed favorable occupying of the deep hydrophobic environment lining the NS3 allosteric pocket by the engineered glycine substitution. Notably, the modelling also captured shifting of the key SER139 hydroxyl moiety away from the organized catalytic triad geometric center. Displacement of this nucleophilic residue plausibly misaligns other proximal components due to intricate hydrogen bonding networks. Structural rearrangement of active site elements likely contributes to the abolished enzymatic activity despite high affinity binding of the strategic PEP15 peptide.33 0Item Restricted Investigation of severe coronavirus infection with the host(University of Liverpool, 2023-07) Alruwaili, Muhannad Falah J; Hiscox, Julian A.Over the last two decades, three zoonotic viruses, SARS-CoV, MERS-CoV, and SARS-CoV-2 have emerged and posed significant threats to human health. After the release of the viral genome in the cytoplasm, 16 NSPs are cleaved and released from the ORF1a and ORF1ab essential for forming the replication-transcription complex to replicate the viral genome. Virus replication relies on the interaction with host proteins for the synthesis of the viral genome, and this process could introduce mutations into the viral genome, but the interactome profile and functionality are undetermined. Also, no developed tool for studying mutations, especially recombination, for the MERS-CoV and SARS-CoV-2 allows for sequencing long amplicons. Here, this thesis demonstrated that an amplicon-sequencing approach, named Rapid Sequencing Long Amplicons (RSLAs), combined with Oxford Nanopore technology can generate data from long amplicons from MERS-CoV and SARS-CoV- 2 genomes in clinical samples suitable for indel and recombination studies. Also, this thesis showed that NSP9 and NSP12, the main components of RTC, interacted with host proteins essential for virus replication. The functioning of SARS-CoV-2 NSP9 was dependent on eEF1A1 and MTHFD1, and NSP12 binding to TRiC/CCT complex was critical to the viral replication and NSP12 stability. Furthermore, NSP12 variants exhibited distinct associations with components of a phosphatase complex, including PP2A and STRN3. The virus that carried NSP12L323 demonstrated reduced susceptibility to disruption in PP2A. These findings allow the scientific communities to investigate mutational profiles of coronavirus for a better understanding of any potential evolution of coronavirus in the future using RSLA. They also showed the dependency of SARS-CoV-2 replication on host proteins revealing new targets for therapeutic interventions.15 0Item Restricted Biofunctionalization of Micro/Nanoparticles for Neutralizing SARS-CoV-2 Surrogate and Targeting ACE2-expressing Lung Cells(Saudi Digital Library, 2024) Alkhaldi, Soha; Peng, Ching-AnThe SARS-CoV-2 virus, which led to the COVID-19 pandemic, first emerged in China in the end of 2019 and swiftly propagated to affect the entire world. COVID-19 has caused millions of illnesses and deaths worldwide. The illness from virus infection may lead to acute respiratory distress syndrome (ARDS), necessitating hospitalization in intensive care units and the use of mechanical ventilators for some individuals. Efforts to mitigate the effects of the pandemic have depended heavily on measures like vaccination drives and public health strategies such as the use of masks and maintaining physical distance to curb the virus's transmission. The emergence of SARS-CoV-2 variants has raised concerns due to their increased ability to spread, their potential to lead to more severe illness, and their reduced responsiveness to existing treatments and vaccines. Preventing viral nanoparticles from entering susceptible lung cells is an approach to curb the transmission of SARS-CoV-2 and any future related coronaviruses. In this study, two strategies were harnessed to achieve the goal – (i) blocking SARS- CoV-2 spike proteins on the outer surface of virions, and (ii) blocking recombinant human angiotensin- converting enzyme 2 (hACE2) receptors on the targeting lung cells. Micro/nanoparticles (biotinylated fluorescent polystyrene particles and perfluorooctyl bromide (PFOB)-based oxygen nanoemulsions) tethered with the recombinant hACE2 were designed and fabricated for the neutralization of spike protein pseudotyped lentivirus (employed as SARS-CoV-2 surrogate). For blocking the hACE2 receptors on the target cells, biotinylated fluorescent polystyrene particles tethered with the recombinant receptor-binding domain (RBD) to saturate hACE2 receptors, thereby preventing the SARS-CoV-2 surrogate from infecting cells. Furthermore, since mesenchymal stem cell-derived extracellular vesicles (MSC-derived EVs) have been proven to possess therapeutic potential for repairing lung injuries, an innovative method for the separation of MSC-derived EVs was developed using chitin magnetic microparticles bound with chitin-binding domain (CBD) fusion with self- cleaving intein tag and lactadherin C1C2, which has a high affinity to phosphatidylserine (PS) exposed on EV membranes. As a result, purified MSC-derived EVs tethered with RBD of SARS-CoV-2 spike protein and loaded with small interfering RNA (siRNA) green fluorescent protein (GFP) could specifically target hACE2 receptors on GFP-expressing A549 lung adenocarcinoma cells and dramatically diminish the level of GFP expressed in the cells.27 0Item Restricted Comparative interactomic analysis of MERS-CoV & SARS-CoV-2 proteins in human, bat, and camel cells(Saudi Digital Library, 2023-12-05) Alaa, Abed; Davidson, AndrewThe Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are Betacoronaviruses capable of causing fatal human infections. Both viruses are believed to have emerged from bats via an intermediate host (camels for MERS-CoV, unknown for SARS-CoV-2) into the human population. MERSCoV and SARS-CoV-2 are enveloped positive-sense RNA viruses which encode four structural proteins (envelope (E), nucleocapsid, spike, and membrane (M)). The E and M proteins are involved in virus assembly, budding, envelope formation, and pathogenesis. Finding cellular protein interactors for these viral proteins, conserved across species, will increase our understanding of the coronavirus lifecycle and identify targets for antiviral development. Initial validation of the results for 11 cellular proteins interacting with the MERS-CoV E and/or M proteins by co-immunoprecipitation (co-IP) /Western blot analysis and immunofluorescence co-localisation, confirmed 10 of the interactions. Three cell lines (human HEK293, bat Pteropus alecto PaKiT and Camelus dromedarius Dubca) were used for transient expression of the MERS-CoV and SARS-CoV-2 E and M proteins (FLAG epitope-tagged) followed by co-IP and high-throughput mass spectrometrybased interactomic analysis. Bioinformatic analysis revealed E/M protein interactions with ER, Golgi, mitochondrial and nuclear proteins. There were 32 high-confidence cellular interaction proteins conserved amongst the different cell lines and viruses (p < 0.05, > 0 log 2 fold change compared to the controls). To determine the importance of the 11 cellular proteins interacting with the MERS-CoV E and/or M proteins and 32 cellular proteins conserved across species, in the virus lifecycle, functional validation was done by siRNA depletion in human cells, followed by infection with SARS-CoV-2. An interesting four cellular proteins were shown to be important for SARS-CoV- 2 replication. These interesting proteins include (CERS2, LPCAT1, UBA52, and TM9SF2) that when it was knocked down, succeeded in reducing the SARS-CoV-2 replication. This can be followed by working to find if these proteins can be safely targeted by a drug to be reduced in cells.14 0