Biology subjects

Hankinson, J.

Publications and source records attributed to Hankinson, J..

8 recordsLinked to original sources

bioRxiv2026

The dynamics and strategy of RNA replication in astroviruses

Astroviruses are positive-sense single-stranded RNA viruses that cause significant disease across avian and mammalian hosts, yet their replication mechanisms remain poorly understood. The replication of astrovirus RNA occurs via a double-stranded RNA intermediate that is used as a template for the synthesis of new positive-sense RNA, which is covalently linked to the virus-encoded protein VPg. These viruses also produce a capsid-encoding subgenomic (sg) RNA that is 3'-coterminal with the genomic RNA. The mechanisms by which the astrovirus sgRNA is produced and regulated during infection have not yet been characterized. Using high throughput sequencing of RNA from cells infected with each of five different astrovirus strains, we demonstrate that the presence of a (-)sgRNA is a conserved feature of infection, supporting a premature termination model of subgenomic RNA production. A pronounced pile-up in the mapping positions of the 3' ends of negative-sense RNA reads marks the precise 3' terminus of the (-)sgRNA. We investigate the relative abundance and dynamics of positive and negative RNA species during virus replication and virion packaging, and perform a mutational analysis of conserved residues in the genomic and subgenomic 5' termini. Together, this work elucidates the dynamics of genomic and subgenomic RNA synthesis during astrovirus infection. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/700307v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1d71111org.highwire.dtl.DTLVardef@a1a024org.highwire.dtl.DTLVardef@118d126org.highwire.dtl.DTLVardef@4b3c4e_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Viral protease-mediated polyprotein processing in human astroviruses

Positive-sense RNA viruses often encode large polyproteins that are proteolytically processed by viral and host proteases into functional replication proteins. Astroviruses infect intestinal and neuronal cells across diverse human and animal hosts and also rely on polyprotein cleavage for replication. In this study, we mapped the cleavage sites of the nonstructural polyproteins of classical human astrovirus 1 (HAstV1) and neurotropic astrovirus strain MLB2 using complementary N-terminomics of infected cells and analyses of untagged overexpressed polyproteins. Notably, we identified two adjacent cleavage sites at the N-terminus of HAstV1 and MLB2 proteases, as well as a similar dual cleavage site at the C-terminus of the MLB2 protease. We also demonstrated processing of the hypervariable region and VPg in both astrovirus strains. This allowed us to define the boundaries of individual protein products and identify conserved and divergent processing features between classical and non-classical astroviruses. Additionally, we characterized several polyprotein precursors and evaluated the replication properties of cleavage-deficient mutant replicons, revealing the critical role of polyprotein processing for functional replication complex formation. Understanding the dynamics of polyprotein processing is essential for interpreting the stages of viral infection and identifying new drug targets and antiviral strategies.

microbiology↗

bioRxiv2025

ZNFX1, an immunoregulatory RNA helicase and E3 ubiquitin ligase, assembles into pleiomorphic polymers

ZNFX1 is an SF1-family RNA helicase essential for innate immunity. Patients with ZNFX1 mutations experience recurrent infections, yet the underlying mechanism remains unclear. We determined cryo-EM structures of ZNFX1 in apo and RNA-bound forms, revealing auto-inhibition of the helicase through a regulatory insertion occluding the RNA-binding groove. ZNFX1 also functions as a bi-catalytic E3 ubiquitin ligase, containing an RZ-finger homologous to RNF213 and a previously unidentified Miz-like domain that catalyze ubiquitylation independently or cooperatively, with activity enhanced by ubiquitin chains. Patient mutations demonstrate that the helicase, E3 ligase, and rigid zinc-finger stalk connecting them are required for function. ZNFX1 can assemble into structured, pleiomorphic polymers via multivalent protein-protein interactions, revealing a mechanism that may facilitate RNA sequestration in stress granules and antiviral activity. These findings establish ZNFX1 as a multifunctional enzyme in innate immunity that couples RNA sensing to ubiquitin signaling and assembles into higher-order structures for signal amplification.

biochemistry↗

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↗

bioRxiv2025

Flexibility and modulation of translation initiation in enterovirus genomes

Enteroviruses comprise a large group of mammalian pathogens that often utilize two open reading frames (ORFs) to encode their proteins: the upstream protein (UP) and the main polyprotein. In some enteroviruses, in addition to the canonical upstream AUG (uAUG), there is another AUG that may represent an alternative upstream initiation site. An analysis of enterovirus sequences containing additional upstream AUGs identified several clusters, including strains of pathogenic Enterovirus alphacoxsackie and E. coxsackiepol. Using ribosome profiling on coxsackievirus CVA-13 (E. coxsackiepol), we demonstrate that both upstream AUG codons can be used for translation initiation in infected cells. Moreover, we confirm translation from both upstream AUGs using a reporter system. Mutating the additional upstream AUG in the context of CVA-13 did not result in phenotypic changes in immortalized cell lines. However, the wild-type virus outcompeted this mutant in human intestinal organoids and differentiated neuronal systems, representing an advantage in physiologically relevant infection sites. Mutation of the stop codon of the shorter upstream ORF led to dysregulated translation of the other ORFs in the reporter system, suggesting a potential role for the additional uORF in modulating the expression level of the other ORFs. These findings demonstrate the remarkable plasticity of enterovirus IRES-mediated initiation and the competitive advantage of double-upstream-AUG-containing viruses in terminally differentiated intestinal organoids and neuronal systems.

microbiology↗

bioRxiv2024

The astrovirus N-terminal nonstructural protein anchors replication complexes to the perinuclear ER membranes

An essential aspect of viral replication is the anchoring of the replication complex (RC) to cellular membranes. Positive-sense RNA viruses employ diverse strategies, including co-translational membrane targeting through signal peptides and co-opting cellular membrane trafficking components. Often, N-terminal nonstructural proteins play a crucial role in linking the RC to membranes, facilitating the early association of the replication machinery. Astroviruses utilize a polyprotein strategy to synthesize nonstructural proteins, relying on subsequent processing to form replication-competent complexes. In this study, we provide evidence for the perinuclear ER membrane association of RCs in five distinct human astrovirus strains. Using tagged recombinant classical human astrovirus 1 and neurotropic MLB2 strains, we establish that the N-terminal domain guides the ER membrane association. Through mutational analysis of the N-terminal domain in replicon and reverse genetics systems, we identified di-arginine motifs responsible for the perinuclear ER retention and formation of functional RCs. Our findings highlight the intricate virus-ER interaction mechanism employed by astroviruses, potentially leading to the development of novel antiviral intervention strategies. Author SummaryHuman astroviruses are a significant cause of acute gastroenteritis, accounting for up to 9% of cases in young children. Immunocompromised individuals and infants experience more critical symptoms, such as severe and persistent diarrhea, as well as sporadic systemic and even fatal diseases. To date, no drugs have been developed to protect against astrovirus infection. Our study provides the first evidence that the integrity of the N-terminal domain of nsP1a is essential for establishing early replication. Central to this process, the di-arginine motifs in the N-terminal domain are responsible for ER retention, the formation of functional replication complexes, and viral replication. Therefore, selectively targeting N-terminal domain-mediated ER retention could be a promising therapeutic strategy to effectively control astrovirus infection.

microbiology↗

bioRxiv2020

An active chromatin interactome elucidates the biological mechanisms underlying genetic risk factors of dermatological conditions in disease relevant cell lines

Chromatin looping between regulatory elements and gene promoters presents a potential mechanism whereby disease risk variants affect their target genes. Here we use H3K27ac HiChIP, a method for assaying the active chromatin interactome in two cell lines: keratinocytes and skin derived CD8+ T cells. We integrate public datasets for a lymphoblastoid cell line and primary CD4+ T cells and identify gene targets at risk loci for skin-related disorders. Interacting genes enrich for pathways of known importance in each trait, such as cytokine response (psoriatic arthritis, psoriasis) and replicative senescence (melanoma). We show examples of how our analysis can inform changes in the current understanding of multiple psoriasis associated risk loci. For example, the variant rs10794648, which is generally assigned to IFNLR1, was linked to GRHL3 in our dataset, a gene essential in skin repair and development. Our findings, therefore, indicate a renewed importance of skin related factors in the risk of disease. O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/973271v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@99cd40org.highwire.dtl.DTLVardef@1725b6dorg.highwire.dtl.DTLVardef@d05d21org.highwire.dtl.DTLVardef@1924d07_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract In this article we take disease associated variants from 5 dermatological conditions and use cell type specific datasets to map genes that could be affected by these variants, providing insight into disease mechanisms. C_FIG

genomics↗

bioRxiv2019

Chromatin-based techniques map DNA interaction landscapes in psoriasis susceptibility loci and highlight KLF4 as a target gene in 9q31

Genome-wide association studies (GWAS) have uncovered many genetic risk loci for psoriasis, yet many remain uncharacterised in terms of the causal gene and their biological mechanism in disease. Here, we use a disease-focused Capture Hi-C experiment to link psoriasis-associated variants with their target genes in psoriasis-relevant cell lines (HaCaT keratinocytes and My-La CD8+ T cells). We confirm previously assigned genes, suggest novel candidates and provide evidence for complexity at psoriasis GWAS loci. In the 9q31 risk locus we combine further epigenomic evidence to demonstrate how the psoriasis association forms a functional interaction with the distant (>500 kb) KLF4 gene. We use CRISPR activation coupled with RNA-seq to demonstrate how activation of psoriasis-associated enhancers upregulates KLF4 in HaCaT cells. Our study design provides a robust pipeline for following up on GWAS disease-associated variants, paving the way for functional translation of genetic findings into clinical benefit.

genomics↗