Biology subjects

Croker, B. A.

Publications and source records attributed to Croker, B. A..

8 recordsLinked to original sources

bioRxiv2025

Genome-Wide Interrogation of SARS-CoV-2 RNA-Protein Interactions Uncovers Hidden Regulatory Sites

The global impact of the COVID-19 pandemic underscores the critical need for a comprehensive understanding of SARS-CoV-2 replication mechanisms. While the central roles of the RNA dependent RNA polymerase (NSP12), primase protein (NSP8), and nucleocapsid protein (N) in the virus life cycle are extensively studied, the precise nature of their interactions with the full-length viral RNA genome remain incompletely characterized. In this study, we sought to address this knowledge gap by employing enhanced crosslinking and immunoprecipitation (eCLIP) to map the binding sites of NSP8, NSP12, and N proteins across the SARS-CoV-2 genome at early stages of viral RNA and protein synthesis and late stages of virion assembly. Our findings revealed interactions of NSP8 and NSP12 to the 5 and 3 untranslated regions (UTRs) of both positive and negative sense RNA, regions known to regulate viral replication, transcription, and translation. We identified a surprising and essential NSP12 binding site within the RNA sequence encoding the conserved Y1 domain of NSP3, which regulates RNA abundance upstream of the site. Additionally, we found that N protein interacts with the 5 UTR and influences translation efficiency. Finally, we report a novel regulatory function of N protein in modulating ribosomal frameshifting proximal to the frameshift element, a crucial process for maintaining viral protein stoichiometry. Our results provide a detailed molecular map of SARS-CoV-2 protein-RNA interactions, revealing potential therapeutic targets for attenuating viral fitness and informing the development of next-generation antiviral strategies.

molecular biology↗

bioRxiv2024

Comparative single-cell lineage bias in human and murine hematopoietic stem cells

The commitment of hematopoietic stem cells (HSC) to myeloid, erythroid, and lymphoid lineages is influenced by microenvironmental cues, and governed by cell-intrinsic and epigenetic characteristics that are unique to the HSC population. To investigate the nature of lineage commitment bias in human HSC, mitochondrial single cell (sc) ATAC-Sequencing (mt-scATAC-Seq) was used to identify somatic mutations in mitochondrial DNA to act as natural genetic barcodes for tracking the ex vivo differentiation potential of HSC to mature cells. Clonal lineages of human CD34+ cells and their mature progeny were normally distributed across the hematopoietic lineage tree without evidence of significant skewing. To investigate commitment bias in vivo, mice were transplanted with limited numbers of long-term HSC (LT-HSC). Variation in the ratio of myeloid and lymphoid cells between donors, although suggestive of a skewed output, was not altered by increasing numbers of LT-HSC. These data suggest that the variation in myeloid and lymphoid engraftment is a stochastic process influenced by the irradiated recipient niche and not a cell-intrinsic lineage bias of LT-HSC.

genomics↗

bioRxiv2023

The lung employs an intrinsic surfactant-mediated inflammatory response for viral defense

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) causes an acute respiratory distress syndrome (ARDS) that resembles surfactant deficient RDS. Using a novel multi-cell type, human induced pluripotent stem cell (hiPSC)-derived lung organoid (LO) system, validated against primary lung cells, we found that inflammatory cytokine/chemokine production and interferon (IFN) responses are dynamically regulated autonomously within the lung following SARS-CoV-2 infection, an intrinsic defense mechanism mediated by surfactant proteins (SP). Single cell RNA sequencing revealed broad infectability of most lung cell types through canonical (ACE2) and non-canonical (endocytotic) viral entry routes. SARS-CoV-2 triggers rapid apoptosis, impairing viral dissemination. In the absence of surfactant protein B (SP-B), resistance to infection was impaired and cytokine/chemokine production and IFN responses were modulated. Exogenous surfactant, recombinant SP-B, or genomic correction of the SP-B deletion restored resistance to SARS-CoV-2 and improved viability.

immunology↗

bioRxiv2022

Conformational Flexibility in Neutralization of SARS-CoV-2 by Naturally Elicited Anti-SARS-CoV-2 Antibodies

As new variants of SARS-CoV-2 continue to emerge, it is important to assess the neutralizing capabilities of naturally elicited antibodies against SARS-CoV-2. In the present study, we evaluated the activity of nine anti-SARS-CoV-2 monoclonal antibodies (mAbs), previously isolated from convalescent donors infected with the Wuhan-Hu-1 strain, against the SARS-CoV-2 variants of concern (VOC) Alpha, Beta, Gamma, Delta and Omicron. By testing an array of mutated spike receptor binding domain (RBD) proteins, cell-expressed spike proteins from VOCs, and neutralization of SARS-CoV-2 VOCs as pseudoviuses, or as the authentic viruses in culture, we show that mAbs directed against the ACE2 binding site (ACE2bs) are far more sensitive to viral evolution compared to anti-RBD non-ACE2bs mAbs, two of which kept their potency against all VOCs tested. At the second part of our study, we reveal the neutralization mechanisms at high molecular resolution of two anti-SARS-CoV-2 neutralizing mAbs by structural characterization. We solved the structures of the Delta-neutralizing ACE2bs mAb TAU-2303 with the SARS-CoV-2 spike trimer and RBD at 4.5 [A] and 2.42 [A], respectively, revealing a similar mode of binding to that between the RBD and the ACE2 receptor. Furthermore, we provide five additional structures (at resolutions of 5.54 [A], 7.76 [A], 6.47 [A], 3.45 [A], and 7.32 [A]) of a second antibody, non-ACE2bs mAb TAU-2212, complexed with the SARS-CoV-2 spike trimer. TAU-2212 binds an exclusively quaternary epitope, and exhibits a unique, flexible mode of neutralization that involves transitioning between five different conformations, with both arms of the antibody recruited for cross linking intra- and inter-spike RBD subunits. Our study provides new mechanistic insights about how antibodies neutralize SARS-CoV-2 and its emerging variants and provides insight about the likelihood of reinfections.

immunology↗

bioRxiv2021

The Prolyl-tRNA Synthetase Inhibitor Halofuginone Inhibits SARS-CoV-2 Infection

Summary ParagraphWe identify the prolyl-tRNA synthetase (PRS) inhibitor halofuginone1, a compound in clinical trials for anti-fibrotic and anti-inflammatory applications2, as a potent inhibitor of SARS-CoV-2 infection and replication. The interaction of SARS-CoV-2 spike protein with cell surface heparan sulfate (HS) promotes viral entry3. We find that halofuginone reduces HS biosynthesis, thereby reducing spike protein binding, SARS-CoV-2 pseudotyped virus, and authentic SARS-CoV-2 infection. Halofuginone also potently suppresses SARS-CoV-2 replication post-entry and is 1,000-fold more potent than Remdesivir4. Inhibition of HS biosynthesis and SARS-CoV-2 infection depends on specific inhibition of PRS, possibly due to translational suppression of proline-rich proteins. We find that pp1a and pp1ab polyproteins of SARS-CoV-2, as well as several HS proteoglycans, are proline-rich, which may make them particularly vulnerable to halofuginones translational suppression. Halofuginone is orally bioavailable, has been evaluated in a phase I clinical trial in humans and distributes to SARS-CoV-2 target organs, including the lung, making it a near-term clinical trial candidate for the treatment of COVID-19.

microbiology↗

bioRxiv2020

Multi-Clonal Live SARS-CoV-2 In Vitro Neutralization by Antibodies Isolated from Severe COVID-19 Convalescent Donors

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe and not mild infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of viral inhibition. B cell receptor (BCR) sequencing revealed two VH genes, VH3-38 and VH3-53, that were enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against live SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and RBD mutagenesis, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.

immunology↗

bioRxiv2019

NOD1 mediates non-canonical inflammasome processing of interleukin-18 in epithelial cells to Helicobacter pylori infection.

The interleukin-1 family members, IL-1{beta} and IL-18, are processed into their biologically active forms by multi-protein complexes, known as inflammasomes. Although the inflammasome pathways that mediate IL-1{beta} processing in myeloid cells have been extensively studied, those involved in IL-18 processing, particularly in non-myeloid cells, are still poorly understood. Here, we have identified the cytosolic sensor NOD1 as a key regulator of IL-18 processing in epithelial cells responding to Helicobacter pylori infection. Importantly, NOD1 processing of IL-18 occurs independently of the canonical inflammasome proteins, NLRP3 and ASC. Instead, NOD1 interacts directly with caspase-1 via homotypic binding of caspase-activation recruitment domains. We show that IL-18 is important in maintaining tissue homeostasis and protecting against pre-neoplastic changes due to gastric H. pylori infection. These findings reveal an unanticipated role for NOD1 in a new type of inflammasome that regulates epithelial cell production of bioactive IL-18 with tissue protective functions.

immunology↗

bioRxiv2019

β-glucan dependent shuttling of conidia from neutrophils to macrophages occurs during fungal infection establishment

The initial host response to fungal pathogen invasion is critical to infection establishment and outcome. However, the diversity of leukocyte-pathogen interactions is only recently being appreciated. We describe a new form of interleukocyte conidial exchange called \"shuttling\". In Talaromyces marneffei and Aspergillus fumigatus zebrafish in vivo infections, live imaging demonstrated conidia initially phagocytosed by neutrophils were transferred to macrophages. Shuttling is unidirectional, not a chance event, involves alterations of phagocyte mobility, inter-cellular tethering, and phagosome transfer. Shuttling kinetics were fungal species-specific, implicating a fungal determinant. {beta}-glucan serves as a fungal-derived signal sufficient for shuttling. Murine phagocytes also shuttled in vitro. The impact of shuttling for microbiological outcomes of in vivo infections is difficult to specifically assess experimentally, but for these two pathogens, shuttling augments initial conidial redistribution away from fungicidal neutrophils into the favourable macrophage intracellular niche. Shuttling is a frequent host/pathogen interaction contributing to fungal infection establishment patterns.

cell biology↗