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Murphy, E. A.

Publications and source records attributed to Murphy, E. A..

12 recordsLinked to original sources
bioRxiv2026

Preclinical evaluation of Brincidofovir in glioblastoma demonstrates improved long term-survival and cytomegalovirus-dependent and independent effects

Cytomegalovirus (CMV) has been implicated in glioblastoma (GBM) progression. Ongoing clinical trials are assessing therapeutic approaches targeting CMV in GBM but to date no new therapy has been approved outside the standard of care. Previous preclinical studies have highlighted the potential of the antiviral drug Cidofovir (CDV) in GBM; however, its clinical use is limited by dose-dependent nephrotoxicity and poor cellular uptake, necessitating high intravenous doses to achieve therapeutic activity. Brincidofovir (BCV), a lipid conjugate of CDV has been developed, which does not induce nephrotoxicity and has significantly greater cellular bioavailability. Here we examined the effects of BCV in a newly established CMV-driven GBM model (SB28) and in patient-derived tumor neurospheres. We show that BCV prolongs survival in vivo and exerts both CMV-dependent and independent antitumor effects. Mechanistically, BCV induces DNA damage and cell cycle dysregulation in GBM cells and inhibits proliferation of patient-derived neurospheres in a dose-dependent manner. These data identify BCV as a dual-action therapeutic that suppresses viral oncomodulation while directly targeting tumor cell viability.

cancer biology↗
bioRxiv2026

Human cytomegalovirus virion delivered UL14 is a viral tropism factor for epithelial cells

Infection with Human Cytomegalovirus (HCMV) can result in a significant burden of disease in those that are immunocompromised or immunonaive. HCMV encodes a repertoire of glycoproteins that facilitate its extensive viral tropism, some of which remain to be characterized. Currently, there is no effective vaccine or cure for HCMV, therefore emphasizing the need to identify viral proteins of critical function. UL14 was selected as an open reading frame of interest due to its high scoring on an in-silico prediction algorithm, as well as its conservation amongst CMVs. Our goal was to elucidate the function of this uncharacterized viral open reading frame. We hypothesized that UL14 functions in the establishment of infection in epithelial cells, due to its predicted structural similarity to UL141. This study demonstrates that HCMV UL14 is a glycosylated viral protein packaged with the virion. Importantly, the deletion of UL14 resulted in a significant reduction of viral growth in epithelial cells, whereas no growth defect was observed in fibroblasts. Mechanistically, we found this defect to be a result of post entry, pre-IE transcription in the establishment of infection, consistent with a defect endosomal escape. Taken together, our results suggest that UL14 functions in the establishment of infection in an epithelial cell-specific manner and may be a novel target for future vaccines or antiviral therapies. Author SummaryHCMV is found in a wide variety of human cells during the course of viral infection. As such, HCMV encodes several glycoprotein complexes that dictate tropism. In this work we report the identification of a novel glycoprotein, UL14, that is involved in establishing productive infections of epithelial cells, a common site of HCMV induced disease. We report that deletion of UL14 from the viral genome impacts its ability to infect ARPE19 cells at a stage indicative of viral events post viral entry but prior to viral transcriptional activation. Further, trans complementation of UL14 by expansion of mutant virus in cells expressing the viral glycoprotein, restore viral infectivity suggesting that UL14 mediates events early in viral infection. Importantly, the characterization of this viral envelope protein provides key insights into viral tropism and identifies a novel target for vaccine design and antiviral therapies.

microbiology↗
bioRxiv2024

Protein-S-Nitrosylation of Human Cytomegalovirus pp65 Reduces its Ability to Undermine cGAS.

Post-translational modifications (PTMs) are key regulators of various processes important for cell survival. These modifications are critical for dealing with stress conditions such as those observed in disease states and during infections with various pathogens. We previously reported that during infection of primary dermal fibroblasts, multiple Human Cytomegalovirus (HCMV) encoded proteins were post-translationally modified by the addition of a nitric oxide group to cysteine residues, a modification called protein-S-nitrosylation. For example, tegument protein pp71 is nitrosylated, diminishing its ability to inhibit STING, a protein necessary for DNA virus immune response. Herin, we report that an additional HCMV tegument protein, pp65, responsible for the inhibition of cGAS, is also modified by protein-S-nitrosylation on two cysteine residues. Utilizing site-directed mutagenesis to generate recombinant viruses that encode a pp65 that cannot be protein-S-nitrosylated, we evaluated the impact of this PTM on viral replication and how the virus impacts the cGAS/STING pathway. We report that the nitrosylation of pp65 negatively impacts its ability to block cGAS enzymatic functions. pp65 protein-S-nitrosylation mutants demonstrated a decrease in cGAS/STING induced IRF3 and TBK1 phosphorylation. Additionally, we observe a reduction in IFN-{beta}1 secretion in NuFF-1 cells expressing a nitrosylation-resistant pp65. We report that HCMV expressing a protein-S-nitrosylation deficient pp65 is resistant to the activation of cGAS in the infection of primary dermal fibroblasts. Our work suggests that nitrosylation of viral proteins may serve as a broadly neutralizing mechanism in HCMV infection. ImportancePost translational modifications (PTM) are utilized by host cells to limit an invading pathogens ability to establish a productive infection. A potent PTM called protein-S-nitrosylation has anti-bacterial and anti-viral properties. Increasing protein-S-nitrosylation with the addition of nitric oxide donor compounds, reduced HCMV replication in fibroblasts and epithelial cells 1. We previously reported that protein-S-nitrosylation of HCMV pp71 limits its ability to inhibit STING. Herein, we report that the protein-S-nitrosylation of HCMV pp65 impacts its ability to limit cGAS activity, an additional protein important in regulating interferon response. Therapeutically, patients provided nitric oxide by inhalation reduced viral replication in COVID-19, influenza and even impacted bacterial growth within patients lungs 2,3. It is thought an increase in free nitric oxide increases the frequency of nitrosylated proteins 4. Understanding how protein-S-nitrosylation regulates a common DNA virus like HCMV will provide insights into the development of broadly neautralizing therapeutics in drug resistant viral infections.

microbiology↗
bioRxiv2024

Human gut commensal Alistipes timonensis modulates the host lipidome and delivers anti-inflammatory outer membrane vesicles to suppress colitis in an Il10-deficient mouse model

Correlative studies have linked human gut microbes to specific health conditions. Alistipes is one such microbial genus negatively linked to inflammatory bowel disease (IBD). However, the protective role of Alistipes in IBD is understudied, and the underlying molecular mechanisms remain unknown. In this study, colonization of Il10-deficient mice with Alistipes timonensis DSM 27924 delays colitis development. Colonization does not significantly alter the gut microbiome composition, but instead shifts the host plasma lipidome, increasing phosphatidic acids while decreasing triglycerides. Outer membrane vesicles (OMVs) derived from Alistipes are detected in the plasma of colonized mice, carrying potentially immunomodulatory metabolites into the host circulatory system. Fractions of A. timonensis OMVs suppress LPS-induced Il6, Il1b, and Tnfa expression in vitro in murine macrophages. We detect putative bioactive lipids in the OMVs, including immunomodulatory sulfonolipids (SoLs) in the active fraction, which are also increased in the blood of colonized mice. Treating Il10-deficient mice with purified SoL B, a representative SoL, suppresses colitis development, suggesting its contribution to the anti-inflammatory phenotype observed with A. timonensis colonization. Thus, A. timonensis OMVs represent a potential mechanism for Alistipes-mediated delay of colitis in Il10-deficient mice via delivery of immunomodulatory lipids and modulation of the host plasma lipidome.

biochemistry↗
bioRxiv2024

The effective multiplicity of infection for HCMV depends on activity of the cellular 20S proteasome

Human cytomegalovirus (HCMV) is a betaherpesvirus capable of infecting numerous cell types and persisting throughout an infected individuals life. Disease usually occurs in individuals with compromised or underdeveloped immune systems. Several antivirals exist but have limitations relating to toxicity and resistance. HCMV replication involves upregulation of host proteasomal activities which play important roles in the temporal stages of replication. Here, we defined the impact on replication kinetics of the proteasome inhibitor, bortezomib. We demonstrate that bortezomib significantly reduces levels of viral genomes and infectious virions produced from a population of cells. Inhibition reduced expression of viral proteins that are influenced by genome synthesis. When added prior to 24 hpi, we observe decreases in PCNA and Cdk1 while increases in p21 whose regulations contribute to efficient replication. This response synergized with an antiviral, maribavir. Since some replication occurred, we tested the hypothesis that a subset of infected cells might break through inhibition. Initially, we simulated bortezomib activities using a mechanistic computational model of late-lytic replication. Upon reducing MOI in-silico, we observed near identical simulated results compared to experimental data. Next, we analyzed replication using live-cell imaging. This revealed treated cultures do contain a population of cells with fully developed late-stage cytoplasmic assembly compartments but at significantly lower numbers. We refer to this as the effective MOI. Overall, our studies support a hypothesis in which 20S proteasome inhibition disrupts HCMV replication by reducing the MOI to an effective MOI, defined by a fraction of infected cells capable of progressing to fulminant infection. IMPORTANCEHCMV infection and reactivation continues to contribute to morbidity and mortality around the world. Antiviral compounds are available but have limitations. Here, we have defined the impact of the proteasome inhibitor bortezomib on HCMV replication. Proteasomal activities play a critical role in temporal changes required for replication. We demonstrate that disrupting these activities inhibits viral replication while likely supporting increased antiviral activity of the anti-HCMV agent, maribavir. Using a combination of live cell imaging and computational tools, we discover that a subset of infected cells progresses to fulminant infection which we define as the effective MOI, and this subset would otherwise be missed when analyzing the average of the population.

microbiology↗
bioRxiv2024

Targeting the host transcription factor HSF1 prevents human cytomegalovirus replication in vitro and in vivo

FDA-approved antivirals against HCMV have several limitations, including only targeting the later stages of the viral replication cycle, adverse side effects, and the emergence of drug-resistant strains. Antivirals targeting host factors specifically activated within infected cells and necessary for viral replication could address the current drawbacks of anti-HCMV standard-of-care drugs. In this study, we found HCMV infection stimulated the activation of the stress response transcription factor heat shock transcription factor 1 (HSF1). HCMV entry into fibroblasts rapidly increased HSF1 activity and subsequent relocalization from the cytoplasm to the nucleus, which was maintained throughout viral replication and in contrast to the transient burst of activity induced by canonical heat shock. Prophylactic pharmacological inhibition or genetic depletion of HSF1 prior to HCMV infection attenuated the expression of all classes of viral genes, including immediate early (IE) genes, and virus production, suggesting HSF1 promotes the earliest stages of the viral replication cycle. Therapeutic treatment with SISU-102, an HSF1 inhibitor tool compound, after IE expression also reduced the levels of L proteins and progeny production, suggesting HSF1 regulates multiple steps along the HCMV replication cycle. Leveraging a newly developed human skin xenograft transplant murine model, we found prophylactic treatment with SISU-102 significantly attenuated viral replication in transplanted human skin xenografts as well as viral dissemination to distal sites. These data demonstrate HCMV infection rapidly activates and relocalizes HSF1 to the nucleus to promote viral replication, which can be exploited as a host-directed antiviral strategy. One Sentence SummaryInhibiting of HSF1 as a host-directed antiviral therapy attenuates HCMV replication in vitro and in vivo.

microbiology↗
bioRxiv2024

Evolutionary genomics of the emergence of brown algae as key components of coastal ecosystems

Brown seaweeds are keystone species of coastal ecosystems, often forming extensive underwater forests, that are under considerable threat from climate change. Despite their ecological and evolutionary importance, this phylogenetic group, which is very distantly related to animals and land plants, is still poorly characterised at the genome level. Here we analyse 60 new genomes that include species from all the major brown algal orders. Comparative analysis of these genomes indicated the occurrence of several major events coinciding approximately with the emergence of the brown algal lineage. These included marked gain of new orthologous gene families, enhanced protein domain rearrangement, horizontal gene transfer events and the acquisition of novel signalling molecules and metabolic pathways. The latter include enzymes implicated in processes emblematic of the brown algae such as biosynthesis of the alginate-based extracellular matrix, and halogen and phlorotannin biosynthesis. These early genomic innovations enabled the adaptation of brown algae to their intertidal habitats. The subsequent diversification of the brown algal orders tended to involve loss of gene families, and genomic features were identified that correlated with the emergence of differences in life cycle strategy, flagellar structure and halogen metabolism. Analysis of microevolutionary patterns within the genus Ectocarpus indicated that deep gene flow between species may be an important factor in genome evolution on more recent timescales. Finally, we show that integration of large viral genomes has had a significant impact on brown algal genome content and propose that this process has persisted throughout the evolutionary history of the lineage.

genomics↗
bioRxiv2024

Herpes Simplex Virus-1 targets the 2-3cGAMP importer SLC19A1 as an antiviral countermeasure.

To establish a successful infection, herpes simplex virus-1 (HSV-1), a virus with high seropositivity in the human population, must undermine host innate and intrinsic immune defense mechanisms, including the stimulator of interferon genes (STING) pathway. Recently it was discovered that not only de novo produced intracellular 2-3cGAMP, but also extracellular 2-3cGAMP activates the STING pathway by being transported across the cell membrane via the folate transporter, SLC19A1, the first identified extracellular antiporter of this signaling molecule. We hypothesized that the import of exogenous 2-3cGAMP functions to establish an antiviral state like that seen with the paracrine antiviral activities of interferon. Further, to establish a successful infection, HSV-1 must undermine this induction of the STING pathway by inhibiting the biological functions of SLC19A1. Herein, we report that treatment of the monocytic cell line, THP-1 cells, epithelial cells (ARPE-19) and SH-SY5Y neuronal cell line with exogenous 2-3cGAMP induces interferon production and establishes an antiviral state. Using either pharmaceutical inhibition or genetic knockout of SLC19A1 blocks the 2-3cGAMP-induced antiviral state. Additionally, HSV-1 infection results in the reduction of SLC19A1 transcription, translation, and importantly, the rapid removal of SLC19A1 from the cell surface of infected cells. Our data indicate SLC19A1 functions as a newly identified antiviral mediator for extracellular 2-3cGAMP which is undermined by HSV-1. This work presents novel and important findings about how HSV-1 manipulates the hosts immune environment for viral replication and discovers details about an antiviral mechanism which information could aid in the development of better antiviral drugs in the future. ImportanceHSV-1 has evolved multiple mechanisms to neutralize of the hosts innate and intrinsic defense pathways, such as the STING pathway. Here, we identified an antiviral response in which extracellular 2-3cGAMP triggers IFN production via its transporter SLC19A1. Moreover, we report that HSV-1 blocks the functions of this transporter thereby impeding the antiviral response, suggesting exogenous 2-3cGAMP can act as an immunomodulatory molecule in uninfected cells to activate the STING pathway, and priming an antiviral state, similar to that seen in interferon responses. The details of this mechanism highlight important details about HSV-1 infections. This work presents novel findings about how HSV-1 manipulates the hosts immune environment for viral replication and reveals details about a novel antiviral mechanism. These findings expand our understanding of how viral infections undermine host responses and may help in the development of better broad based antiviral drugs in the future.

microbiology↗
bioRxiv2020

Tumor irradiation combined with vascular-targeted photodynamic therapy enhances anti-tumor effects in preclinical prostate cancer.

RationaleThere is an important clinical need to improve the treatment of high risk localized and locally advanced prostate cancer (PCa), and to reduce the side effects of these treatments. We hypothesized that multi-modality therapy combining radiotherapy and vascular-targeted photodynamic therapy (VTP) could PCa tumour control compared against monotherapy with each of these treatments alone. This could provide proof-of-concept to take to the clinic. VTP is a focal therapy for localized PCa, which rapidly destroys targeted tumors through vascular disruption. Tumor vasculature is characterized by vessel immaturity, increased permeability, aberrant branching and inefficient flow. Fractionated radiotherapy (FRT) alters the tumor microenvironment and promotes transient vascular normalization. ObjectiveWe investigated whether sequential delivery of FRT followed by VTP 7 days later improves PCa tumor control compared to monotherapy with FRT or VTP alone. FindingsFRT induced vascular normalization changes in PCa flank tumor allografts, improving vascular function as demonstrated using dynamic contrast enhanced magnetic resonance imaging. FRT followed by VTP significantly delayed tumor growth in flank PCa allograft pre-clinical models, compared with monotherapy with FRT or VTP alone, and improved overall survival. ConclusionTaken together, these results suggest that combining FRT and VTP could become a promising multimodal clinical strategy in PCa therapy. This provides proof-of-concept for this multi-modality therapy approach to take forward to early phase clinical trials.

cancer biology↗
bioRxiv2020

Protein S-nitrosylation of Human Cytomegalovirus pp71 inhibits its ability to limit STING antiviral responses

Human Cytomegalovirus (HCMV) is a ubiquitous pathogen that has co-evolved with its host and in doing so, is highly efficient in undermining antiviral responses that limit successful infections. As a result, HCMV infections are highly problematic in individuals with weakened or underdeveloped immune systems including transplant recipients and newborns. Understanding how HCMV controls the microenvironment of an infected cell so as to favor productive replication is of critical importance. To this end, we took an unbiased proteomics approach to identify the highly reversible, stress induced, post-translational modification (PTM), protein S-nitrosylation, on viral proteins to determine the biological impact on viral replication. We identified protein S-nitrosylation of 13 viral proteins during infection of highly permissive fibroblasts. One of these proteins, pp71, is critical for efficient viral replication, as it undermines host antiviral responses, including STING activation. By exploiting site-directed mutagenesis of the specific amino acids we identified in pp71 as protein S-nitrosylated, we found this pp71 PTM diminishes its ability to undermine antiviral responses induced by the STING pathway. Our results suggest a model in which protein S-nitrosylation may function as a host response to viral infection that limits viral spread. IMPORTANCEIn order for a pathogen to establish a successful infection, it must undermine the host cell responses inhibitory to the pathogen. As such, herpesviruses encode multiple viral proteins that antagonize each host antiviral response, thereby allowing for efficient viral replication. Human Cytomegalovirus encodes several factors that limit host countermeasures to infection, including pp71. We identified a previously unreported modification of pp71 residues within the protein are protein S-nitrosylated. Using site-directed mutagenesis, we mutated the specific sites of this modification thereby blocking this pp71 post-translational modification. In contexts where pp71 is not protein S-nitrosylated, host antiviral response was inhibited. The net result of this post-translational modification is to render a viral protein with diminished abilities to block host responses to infection. This novel work supports a model in which protein S-nitrosylation may be an additional mechanism in which a cell inhibits a pathogen during the course of infection.

microbiology↗
bioRxiv2018

2,4-Diaminothienopyrimidines, a new class of anthelmintic with activity against adult and egg stages of whipworm

The human whipworm Trichuris trichiura is a parasite that infects around 500 million people globally, with consequences including damage to physical growth and educational performance. Current drugs such as mebendazole have a notable lack of efficacy against whipworm, compared to other soil-transmitted helminths. Mass drug administration programs are therefore unlikely to achieve eradication and new treatments for trichuriasis are desperately needed. All current drug control strategies focus on post-infection eradication, targeting the parasite in vivo. Here we propose developing novel anthelmintics which target the egg stage of the parasite in the soil as an adjunct environmental strategy. As evidence in support of such an approach we describe the actions of a new class of anthelmintic compounds, the 2,4-diaminothieno[3,2-d]pyrimidines (DATPs). This compound class has found broad utility in medicinal chemistry, but has not previously been described as having anthelmintic activity. Importantly, these compounds show efficacy against not only the adult parasite, but also both the embryonated and unembryonated egg stages and thereby may enable a break in the parasite lifecycle.\n\nAuthor SummaryThe human whipworm, Trichuris trichiura, infects around 500 million people globally, impacting on their physical growth and educational performance. There are currently huge mass drug administration (MDA) programs aiming to control whipworm, along with the other major soil transmitted helminths, Ascaris and hookworm. However single doses of albendazole and mebendazole, which are used in MDA, have particularly poor effectiveness against whipworm, with cure rates less than 40%. This means that MDA may not be able to control and eliminate whipworm infection, and risks the spread of resistance to albendazole and mebendazole in the parasite population.\n\nWe are attempting to develop new treatments for parasitic worm infection, particularly focused on whipworm. We report the identification of a class of compounds, diaminothienopyrimidines (DATPs), which have not previously been described as anthelmintics. These compounds are effective against adult stages of whipworm, and also block the development of the model nematode C. elegans.\n\nOur DATP compounds reduce the ability of treated eggs to successfully establish infection in a mouse model of human whipworm. These results support a potential environmental spray to control whipworm by targeting the infectious egg stage in environmental hotspots.

pharmacology and toxicology↗