Biology subjects

Sweeney, T. R.

Publications and source records attributed to Sweeney, T. R..

6 recordsLinked to original sources

bioRxiv2025

Single-cell phenotypic analysis and multiplet detection through incorporation of microscopy data into cellenONE-based single-cell proteomic data analysis

The reliability of single-cell proteomics (SCP) is intrinsically linked to the fidelity of cell isolation; however, the identification of co-isolated cells (doublets or multiplets) remains a persistent challenge for single-cell (sc-) omics. While single-cell transcriptomics has established probabilistic frameworks for doublet detection, these methods are ill-suited for the sparser throughput of SCP datasets. This work presents scpImaging, a novel computational pipeline that repurposes the latent microscopy data routinely generated during cellenONE-based sample preparation to provide deterministic quality control (QC) and high-content phenotypic profiling. We demonstrate that standard proteomic quality metrics for SCP (e.g., peptide count, signal intensity) paradoxically favour retaining doublets. In contrast, scpImaging applies machine-learning-based cell segmentation (Cellpose-SAM) to identify and exclude these artefacts with high precision. Furthermore, the framework integrates morphological metrics generated using the open-source CellProfiler, with proteomic abundance data, enabling joint analysis that links cell shape and texture to molecular cell state. Provided as an open-source R package, scpImaging offers a scalable, automated solution for enhancing data integrity and adding a phenotypic dimension to SCP experiments without increasing experimental cost or complexity.

systems biology↗

bioRxiv2025

IFIT3 controls IFIT1 accumulation and specificity preventing self mRNA targeting during the innate immune response.

IFIT1 is among the highest expressed proteins following virus detection and interferon (IFN) induction and binds the 5-end of non-self viral cap0-mRNAs lacking 2-O-methylation, blocking their translation. IFIT1 hetero-complexes with IFIT3, which enhances IFIT1 cap0-binding. Here, we demonstrate that IFIT3 is a master regulator of IFIT1, dictating its activity and stability. When expressed at high levels in the absence of IFIT3, IFIT1 can inhibit Semliki Forest virus replication, but can also inhibit translation of certain other self IFN-stimulated genes (ISGs), including the important innate immune proteins ISG15 and IFITM1 as exemplars. However, IFIT1:IFIT3 complexing rescues ISG15 and IFITM1 from IFIT1 translation inhibition. We demonstrate that IFIT1 is degraded by the proteasome in the absence of IFIT3, but that direct binding to IFIT3 protects IFIT1 from degradation, ensuring IFIT1 accumulation only occurs along with IFIT3, demonstrating a mechanism to control the fine balance between antiviral activity and self-targeting in the face of unrelenting viral evolution.

immunology↗

bioRxiv2025

Determinant host differences in ZIKV sfRNA accumulation.

Zika virus (Orthoflavivirus zikaese) is part of the Orthoflavivirus genus that contains several viral species posing major and expanding burdens to public health worldwide. Upon infection, orthoflaviviruses produce viral RNA fragments named sfRNAs (short flaviviral RNA), generated from viral genomic RNA incompletely degraded by Xrn1/Pcm, the major cellular 5-3 exonuclease. Several conserved elements in the orthoflavivirus 3 untranslated region (UTR) are responsible for XrnI-resistance, named xrRNA. The ubiquitous prevalence of xrRNA/sfRNA in flaviviruses indicate an important role of these structures in the viral life cycle, although their function is not completely understood. Here, we used in vitro reconstitution and infection models to examine the role of ZIKV xrRNA2 in mammalian and insect cells. SHAPE RNA structure probing revealed that disruption of ZIKV xrRNA2 does not affect xrRNA1 or dumbbell structure elsewhere in the 3UTR despite disrupting all sfRNA accumulation in mammalian cells. In vitro RNA degradation assays showed that xrRNA1 efficiently stalled human or mosquito Xrn1 independent of xrRNA2 integrity. Reversion in mammalian cells occurred at early passages in protein kinase R (PKR)-/- or IFN-receptor1 (IFAR1)-/-/PKR-/- knock out (KO) or double KO mammalian A549 cells, respectively, while wild-type virus outcompeted the xrRNA2 defective virus in RNAi-deficient mosquito cells. Together, our data reveals novel details of the mechanism of sfRNA accumulation in mammalian and insect cells We demonstrate a differential sensitivity to the absence of a functional xrRNA2 structure in mammalian and insect cells, with reversion to an active xrRNA2 occurring more rapidly in mammalian cells. Together, our work reveals new details on the requirements for ZIKV sfRNA accumulation in mammalian and mosquito cells.

microbiology↗

bioRxiv2025

Structural and mechanistic insights into translation initiation on the enterovirus Type 1 IRES

Enteroviruses are a diverse group of pathogens that cause over one billion human infections annually. Upon cell entry, translation of the viral genome is directed by an internal ribosome entry site (IRES) within the 5' untranslated region. Despite early identification of the Type 1 poliovirus IRES, the structural and mechanistic basis for its activity remains poorly understood due to its size, flexibility and dependence on multiple host cell factors. Here, we reconstitute human translation initiation on a model poliovirus IRES and examine 48S complexes by cryo-electron microscopy. Our structures reveal how IRES domain IVc contacts ribosomal proteins uS19 and uS13, whilst a conserved GNRA tetraloop engages with the initiator tRNA during start-codon recognition. Disruption of these interfaces impairs IRES-dependent translation and viral replication. Together, our results provide new structural and mechanistic insights into initiation on the Type 1 IRES and reveal conserved RNA-RNA interactions critical for enterovirus translation.

molecular biology↗

bioRxiv2019

Ifit1 regulates norovirus infection and enhances the interferon response in murine macrophage-like cells.

BackgroundNorovirus, also known as the winter vomiting bug, is the predominant cause of non-bacterial gastroenteritis worldwide. Disease control is predicated on a robust innate immune response during the early stages of infection. Double-stranded RNA intermediates generated during viral genome replication are recognised by host innate immune sensors in the cytoplasm, activating the strongly antiviral interferon gene programme. Ifit proteins, which are highly expressed during the interferon response, have been shown to directly inhibit viral protein synthesis as well as regulate innate immune signalling pathways. Ifit1 is well-characterised to inhibit viral translation by sequestration of eukaryotic initiation factors or by directly binding to the 5 terminus of foreign RNA, particularly those with non-self cap structures. However, noroviruses have a viral protein, VPg, covalently linked to the 5 end of the genomic RNA, which acts as a cap substitute to recruit the translation initiation machinery.\n\nMethodsIfit1 knockout RAW264.7 murine macrophage-like cells were generated using CRISPR-Cas9 gene editing. These cells were analysed for their ability to support murine norovirus infection, determined by virus yield, and respond to different immune stimuli, assayed by quantitative PCR. The effect of Ifit proteins on norovirus translation was also tested in vitro.\n\nResultsHere, we show that VPg-dependent translation is completely refractory Ifit1-mediated translation inhibition in vitro and Ifit1 cannot bind the 5 end of VPg-linked RNA. Nevertheless, knockout of Ifit1 promoted viral replication in murine norovirus infected cells. We then demonstrate that Ifit1 promoted interferon-beta expression following transfection of synthetic double-stranded RNA but had little effect on toll-like receptor 3 and 4 signalling.\n\nConclusionsIfit1 is an antiviral factor during norovirus infection but cannot directly inhibit viral translation. Instead, Ifit1 stimulates the antiviral state following cytoplasmic RNA sensing, contributing to restriction of norovirus replication.

immunology↗

bioRxiv2018

IFIT3 and IFIT2/3 promote IFIT1-mediated translation inhibition by enhancing binding to non-self RNA.

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed during the cell-intrinsic immune response to viral infection. IFIT1 inhibits translation by binding directly to the 5' end of foreign RNAs, particularly those with non-self cap structures, precluding the recruitment of the cap-binding eukaryotic translation initiation factor 4F and subsequent 40S recruitment. Interaction of different IFIT family members is well described, but little is known of the molecular basis of IFIT association or its impact on function. Here, we reconstituted different complexes of IFIT1, IFIT2 and IFIT3 in vitro, which enabled us to reveal critical aspects of IFIT complex assembly. IFIT1 interacts rapidly and strongly with IFIT3 forming a stable heterotetramer. IFIT2 and IFIT3 homodimers dissociate to form a more stable heterodimer that associates with IFIT1, forming an IFIT1:IFIT2:IFIT3 trimer. Site-directed mutagenesis revealed a C-terminal YxxxL motif in IFIT1 that mediates its association with IFIT3. Using various reporter mRNAs, we demonstrate for the first time that IFIT3 stabilises IFIT1 binding to cap0-mRNA and enhances its translation inhibition activity. Disrupting the binding interface between IFIT1 and IFIT3 abrogated this enhancement. This work reveals molecular aspects of IFIT assembly and provides an important missing link between IFIT interaction and function.

immunology↗