Biology subjects

Galloway, D. A.

Publications and source records attributed to Galloway, D. A..

8 recordsLinked to original sources

bioRxiv2025

E2F1-3 activate polyomavirus early transcription and viral replication

Merkel cell polyomavirus (MCPyV) is a DNA virus that establishes a persistent asymptomatic infection during childhood and can cause Merkel cell carcinoma (MCC) later in life. Its Large and Small Tumor antigens (LT, ST), splice variants of a common viral early transcript, drive viral replication and tumorigenesis by binding to and perturbing the function of host proteins. LT binds and inhibits RB1, deregulating E2F activity and host cell cycle control to permit viral replication during S phase. While the functions of LT and ST are relatively well characterized, how their expression is controlled is poorly defined. Here, we discovered that E2F1-3/DP1 dimers bind the MCPyV Non-Coding Control Region (NCCR) via a consensus E2 site close to the LT/ST transcriptional start site. Inhibiting E2F-NCCR binding, either by deleting the E2 site or treatment with a E2F small molecule inhibitor, downregulated LT/ST mRNA and protein expression in MCC cells and in 293A cells transfected with MCPyV. Our findings reveal an E2F/LT/RB1 positive feedback loop that appears to have evolved to support viral replication and is hijacked in MCC cells to promote cellular proliferation. Furthermore, we identified E2 sites in the NCCRs of PyVs closely related to MCPyV, including murine PyV, which mediate E2F/DP binding and potentiate viral early transcription. E2F/DP also bound weakly to the SV40 and BKPyV NCCRs, despite lacking an E2 site. Our findings challenge the prevailing model that PyV LT expression drives S phase entry and suggest, in contrast, that S phase entry stimulates PyV early transcription and replication. Significance statementPolyomaviruses (PyVs) express Large and Small Tumor antigens (LT, ST), splice variants of a common viral early transcript, that drive viral replication and tumorigenesis by binding and perturbing the function of host cells. LTs bind and inhibit RB1 via conserved LxCxE motifs, deregulating E2F activity and host cell cycle control to permit viral replication. Here, we discovered that E2F1-3 bind to the Non-Coding Control Regions (NCCRs) of many, but not all, PyVs to activate LT/ST transcription, revealing an E2F/LT/RB1 positive feedback loop that appears to have evolved to promote viral replication. Our findings challenge the existing paradigm that PyV LT expression drives S phase entry and suggest, in contrast, that S phase entry stimulates PyV LT/ST transcription and replication.

microbiology↗

bioRxiv2025

miR-146a is a Pleiotropic Regulator of Motor Neuron Degeneration

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons. Here, we have profiled motor neuron microRNAs (miRNAs) during motor neuron degeneration in vivo to gain a better understanding of ALS pathophysiology. We demonstrate that one miRNA, miR-146a, is downregulated in diseased motor neurons despite upregulation in bulk tissue. Genetic deletion of miR-146a significantly extended survival in SOD1G93A mice with heterozygous animals demonstrating the largest benefit. A corresponding reduction in spinal cord gliosis but not motor neuron loss was observed. Finally, we observed that a proportion of miR-146a knockout animals develop spontaneous paralysis, motor neuron loss and chronic neuroinflammation with advanced age. Together these findings demonstrate that a single miRNA influences multiple aspects of motor neuron disease and highlights the complex role for neuroinflammation in ALS pathogenesis.

neuroscience↗

bioRxiv2025

B cells specific for polyomavirus-derived oncoprotein are predictive of Merkel cell carcinoma progression

Merkel cell carcinomas typically arise from clonal integration of the Merkel cell polyomavirus. Immunogenic viral oncoproteins then lead to tumorigenesis. Oncoprotein-specific T cells are essential for anti-MCC immunity, but it is unclear whether B cells promote tumor control. Here, we analyzed the frequency and phenotype of viral oncoprotein-specific and total B cells in 47 blood samples and 19 unmatched tumors from MCC patients-- of which 8 out 19 progressed. The phenotype of blood B cells did not correlate with MCC patient outcomes. In contrast, all 11 patients with robust oncoprotein-specific antibody-secreting and/or germinal center B cells in tumors experienced long-term MCC control. In vitro, B cells engineered to be specific for viral oncoproteins increased the sensitivity of oncoprotein-specific CD4+ T cells by over 50-fold. Together, our findings suggest that cancer-specific B cells promote anti-tumor immunity via increased T cell responses and that cancer-specific B cell augmentation could be therapeutically relevant. Statement of SignificanceThe link between cancer-specific B cells in anti-tumor immunity and clinical outcomes remains poorly defined. Here, we show that tumor-associated B cells specific for a viral oncoprotein expressed in MCC patient tumors predict disease control with remarkable accuracy, establishing their potential as active participants in tumor immunity.

immunology↗

bioRxiv2025

HPV16 neutralizing monoclonal antibodies show evidence for common developmental pathways and public epitopes

Antibodies to human papillomavirus (HPV) primarily recognize surface exposed residues on five loops of the major capsid protein (L1) that vary significantly among HPV types. We determined which loops were required for neutralization for 70 HPV16 specific human monoclonal antibodies (mAbs) cloned from participants who received an HPV vaccine, and we describe molecular features of those antibodies. Chimeric HPV16 pseudovirus (cpsV), each having one surface loop bearing multiple amino acid substitutions, were used to determine neutralization specificity. The HPV16-FG-loop was the loop most frequently required for neutralization (44 of 70, 62.9%), however, all other surface loops were used for neutralization by multiple mAbs: HI (13, 18.6%), DE (15, 21.4%), EF (six, 8.6%), BC (four, 5.7%). Antibodies that required multiple loops were common (17, 24.3%). Three mAbs (4.3%) required sequences on the c-terminus of L1 and for another three mAbs the neutralization specificity could not be determined. Two types of mAbs appeared to be overrepresented: ten mAbs used VH 2-70 IGHV paired with VL {lambda}1-40, having characteristic mutations in complementarity determining region two (CDRL2). Cryogenic electron microscopy (Cryo-EM) revealed that two of these antibodies bound five Fabs per pentamer interacting with all five L1-surface loops. The other type of mAbs that appeared to be overrepresented were ten mAbs using VH 4-34, seven of which also used DH3-16*02 with conserved CDRH3 sequences. Cryo-EM for one of these mAbs, that required the FG-loop for neutralization, was shown to bind one Fab per pentamer at the apex, interacting with the DE- and FG-loops, with sequences of the Fab CDRH3 inserted between the DE- and FG-loops from two protomers. These two types of mAbs were found repeatedly in the four participants suggesting that these antibodies shared developmental pathways and bound to similar immunodominant epitopes on the virus. HighlightsMost human mAbs recognized L1 surface loops but three of 70 recognized sequences on the C-terminal arm of L1 Some antibodies induced by HPV vaccination follow shared developmental pathways. Human monoclonal antibodies using VH 2-70/VL {lambda}1-40 were found in all participants and bound with at a stoichiometry of five Fabs per capsomer. Human monoclonal antibodies using the diversity gene segment D3-16*02 were found in all participants and one Fab was shown to bind with a stoichiometry of one Fab per capsomer. In briefA panel of 70 HPV16 specific human monoclonal antibodies (mAbs), cloned from memory B cells or plasmablasts following HPV vaccination, was characterized by determining the surface loops of the major capsid protein (L1) required for neutralization and examined for shared molecular features. All five L1 loops were found to be used for neutralization by one or more antibodies, but the most frequent target of these antibodies was the FG loop followed by the HI and DE loops. Ten antibodies paired the heavy chain variable gene VH 2-70 with the light chain variable gene VL {lambda}1-40 and these antibodies had conserved mutations in the CDRL2 region of VL {lambda}1-40. Mutating the CDRL2 back to the predicted germline sequence significantly reduced neutralization. Cryo-EM analysis of two VH2-70/VL{lambda}1-40 mAbs showed five Fabs binding per L1 pentamer and a conserved epitope with Fabs interacting with all five variable loops across two adjacent protomers. Seven other mAbs had a heavy chain composed of the variable region VH4-34 with the diversity gene D3-16*02 resulting in the sequence motif WSGYR in the CDRH3. Mutation of that sequence to alanine ablated HPV16 neutralization activity. A cryo-EM structure of one of these antibodies showed one Fab binding the pentamer apex with the WSGYR motif inserting between three loops from two protomers. Antibodies with paired VH 2-70/VL {lambda}1-40 and the antibodies with CDRH3 containing the WSGYR sequence, were found in all four study participants suggesting that such antibodies may be commonly elicited following HPV vaccination.

immunology↗

bioRxiv2025

Prior epigenetic status predicts future susceptibility to seizures in mice

Wide variation of responses to identical stimuli presented to genetically inbred mice suggests the hypothesis that stochastic epigenetic variation during neurodevelopment can mediate such phenotypic differences. However , this hypothesis is largely untested since capturing pre-existing molecular states requires non-destructive, longitudinal recording. Therefore, we tested the potential of Calling Cards (CC) to record transient neuronal enhancer activity during postnatal development, and thereby associate epigenetic variation with a subsequent phenotypic presentation - degree of seizure response to the pro-convulsant pentylenetetrazol. We show that recorded differences in epigenetics at 243 loci predict a severe vs. mild response, and that these are enriched near genes associated with human epilepsy. We also validated pharmacologically a seizure -modifying role for two novel genes, Htr1f and Let7c. This proof-of-principle supports using CC broadly to discover predisposition loci for other neuropsychiatric traits and behaviors. Finally, as, human disease is also influenced by non-inherited factors, similar epigenetic predispositions are possible in humans.

genomics↗

bioRxiv2025

Delta-catenin is required for cell proliferation in virus positive Merkel cell carcinoma cell lines but not in human fibroblasts

Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine skin cancer often driven by the integration of Merkel cell polyomavirus (MCPyV) into the host genome and the persistent expression of its viral oncoproteins, small tumor (ST) antigen and truncated large tumor (t-LT) antigen. While human fibroblasts support MCPyV replication, the cell of origin for MCC remains unknown. We hypothesized that MCPyV initially replicates in fibroblasts but, in rare cases, infects Merkel cell progenitors, contributing to MCC development. Using TurboID mass spectrometry, we identified {delta}-catenin as a novel ST interactor in fibroblasts. However, while ST binds {delta}-catenin in fibroblasts, this interaction is absent in virus-positive (VP)-MCC cell lines. Despite this, {delta}-catenin is essential for VP-MCC, but not for fibroblast, cell proliferation. We found that fibroblasts predominantly express {delta}-catenin isoform 1, whereas VP-MCC cells mainly express isoform 3. Overexpression of isoform 1 in VP-MCC failed to restore ST binding. {delta}-catenin promotes VP-MCC proliferation by regulating cell cycle gene expression through its interaction with Kaiso, a transcriptional repressor. Additionally, we found that LSD1 (KDM1A) regulates {delta}-catenin isoform 3 expression by modulating ESRP1, a {delta}-catenin splicing factor. Our findings reveal novel host factors involved in MCPyV infection and MCC tumorigenesis, suggesting that the host cell supporting viral replication and the MCC cell of origin may be distinct cell types. ImportanceMerkel cell polyomavirus (MCPyV), the only known human oncogenic polyomavirus, is the primary cause of Merkel cell carcinoma (MCC), a rare and aggressive type of skin cancer. MCC is driven by two viral proteins: small T (ST) and large T (LT). While the virus can replicate in skin fibroblasts, it is still unknown which type of skin cell becomes cancerous. We found that ST binds to a host protein, {delta}-catenin in fibroblasts, potentially playing a role in the virus lifecycle, but this interaction is missing in the cancer cells. Our study provides evidence that the cells in which the virus replicates and causes cancer are different.

cancer biology↗

bioRxiv2024

Polyomavirus ALTOs, but not MTs, downregulate viral early gene expression by activating the NF-κB pathway

Polyomaviruses are small, circular dsDNA viruses that can cause cancer. Alternative splicing of polyomavirus early transcripts generates large and small tumor antigens (LT, ST) that play essential roles in viral replication and tumorigenesis. Some polyomaviruses also express middle tumor antigens (MTs) or Alternate LT ORFs (ALTOs), which are evolutionarily related but have distinct gene structures. MTs are a splice variant of the early transcript whereas ALTOs are overprinted on the second exon of the LT transcript in an alternate reading frame and are translated via an alternative start codon. Merkel cell polyomavirus (MCPyV), the only human polyomavirus that causes cancer, encodes an ALTO but its role in the viral lifecycle and tumorigenesis has remained elusive. Here, we show MCPyV ALTO acts as a tumor suppressor and is silenced in Merkel cell carcinoma (MCC). Rescuing ALTO in MCC cells induces growth arrest and activates NF-{kappa}B signaling. ALTO activates NF-{kappa}B by binding SQSTM1 and TRAF2&3 via two N-Terminal Activating Regions (NTAR1+2), resembling Epstein-Barr virus (EBV) Latent Membrane Protein 1 (LMP1).. Following activation, NF-{kappa}B dimers bind the MCPyV non-coding control region (NCCR) and downregulate early transcription. Beyond MCPyV, NTAR motifs are conserved in other polyomavirus ALTOs, which activate NF-{kappa}B signaling, but are lacking in MTs that do not. Furthermore, polyomavirus ALTOs downregulate their respective viral early transcription in an NF-{kappa}B and NTAR dependent manner. Our findings suggest that ALTOs evolved to suppress viral replication and promote viral latency and that MCPyV ALTO must be silenced for MCC to develop.

microbiology↗

bioRxiv2018

High-Risk Human Papillomavirus Oncogenes Disrupt the Fanconi Anemia DNA repair Pathway by Impairing Localization and De-ubiquitination of FancD2

Persistent expression of high-risk HPV oncogenes is necessary for the development of anogenital and oropharyngeal cancers. Here, we show that E6/E7 expressing cells are hypersensitive to DNA crosslinking agent cisplatin and have defects in repairing DNA interstrand crosslinks (ICL). Importantly, we elucidate how E6/E7 attenuate the Fanconi anemia (FA) DNA crosslink repair pathway. Though E6/E7 activated the pathway by increasing FancD2 monoubiquitination and foci formation, they inhibited the completion of the repair by multiple mechanisms. E6/E7 impaired FancD2 colocalization with double-strand breaks (DSB), which subsequently hindered the recruitment of downstream protein Rad51 to DSB in E6 cells. Further, E6 expression caused delayed FancD2 de-ubiquitination, an important process for effective ICL repair. Delayed FancD2 de-ubiquitination was attributed to the increased chromatin retention of FancD2 hindering USP1 de-ubiquitinating activity, and persistently activated ATR/CHK-1/pS565 FancI signaling. E6 mediated p53 degradation did not hamper the cell cycle specific process of FancD2 modifications but abrogated repair by disrupting FancD2 de-ubiquitination. Further, E6 reduced the expression and foci formation of Palb2, which is a repair protein downstream of FancD2. These findings uncover unique mechanisms by which HPV oncogenes contribute to genomic instability and the response to cisplatin therapies.\n\nAUTHOR SUMMARYHigh-risk human papillomavirus (HPV) causes nearly all cervical and many other anogenital cancers, and oropharyngeal cancers. As cisplatin is the most commonly used drug for cervical and HPV-associated oropharyngeal cancers, it is important to understand how HPV oncogenes disrupt the Fanconi anemia (FA) pathway involved primarily in the repair of cisplatin-induced DNA crosslinks. However, the mechanism by which HPV E6 and E7 attenuate the FA pathway is poorly understood. We demonstrate that E6/E7 expression disrupts crosslink repair and increase cisplatin sensitivity, and attenuate the FA pathway through multiple unique mechanisms. First, E6/E7 causes accumulation of FancD2, a central component of the FA pathway, at the sites away from DNA damage. This results in reduced recruitment of Rad51, another repair protein involved in the pathway. Second, E6 causes delayed FancD2 de-ubiquitination, an important process for effective repair. Third, E6 expressing cells decreases the expression and foci formation of Palb2 repair protein. Together, this work elucidates the mechanisms by which HPV attenuates the repair of DNA crosslinks increasing cisplatin cytotoxicity and efficacy in treating HPV-positive cancers.

cancer biology↗