Biology subjects

Goodfellow, I.

Publications and source records attributed to Goodfellow, I..

14 recordsLinked to original sources

bioRxiv2025

Development of an ex vivo model to study the zoonotic transmission of noroviruses

Human noroviruses are the most common cause of viral gastroenteritis worldwide, causing sporadic cases and outbreaks. Genetically related animal noroviruses infect wild and domestic animals including pigs and dogs. To investigate the potential of noroviruses to jump the species barrier, we used porcine, canine, and avian precision-cut intestinal slices. We show that human norovirus can bind to pig and dog intestinal tissue and be taken up with a similar efficiency as the porcine and canine noroviruses, respectively. In contrast, no binding or internalization of human, porcine, or canine noroviruses was detected in chicken tissue. We further showed that human norovirus can replicate to some degree in pig intestinal tissue, infecting single cells. In contrast, while animal noroviruses attached to human intestinal cells, intracellular uptake was limited. This suggests that human-to-animal transmission is more likely than animal-to-human transmission and that viral uptake likely presents a species barrier.

microbiology↗

bioRxiv2025

Reducing Supply Chain Dependencies for Viral Genomic Surveillance: Get by with a Little HELP from Commercial Enzymes already in your Lab Freezer

The COVID-19 pandemic exposed vulnerabilities in global laboratory supply chains, disrupting genomic surveillance efforts essential to epidemic response. To address this challenge, we developed ARTIC HELP (Homebrew Enzymes for Library Preparation), a practical, open-source adaptation of the widely adopted ARTIC nanopore sequencing protocol for viral genomic surveillance. We describe generic, cost-effective alternatives to all enzyme mixes used in tiling multiplex RT-PCR amplification of the virus genome, and the nanopore native barcoding workflow, including end-prep (EP), barcode ligation (BL), and adapter ligation (AL), making it broadly applicable to any laboratory. Through systematic evaluation, we identified a wild-type M-MLV reverse transcriptase and two types of proofreading DNA polymerases as effective alternatives when standard reagents are unavailable due to high cost or limited supply: B-family Pfu-based polymerases with a fused Sso7d DNA-binding domain, and blends combining A-family (Taq-based) and B-family (Pfu-based) polymerases. Validation on clinical samples of SARS-CoV-2 and Norovirus GII confirmed that the HELP workflow achieves genome coverage comparable to the ARTIC LoCost protocol. For SARS-CoV-2 samples (Ct [≤]28), the wild-type M-MLV RT combined with selected Pfu or A+B polymerases, along with optimised HELP mixes (EP, BL, AL), achieved genome coverage of 84.0-99.6%. For Norovirus GII (Ct [≤]32), the HELP workflow using one of the Pfu polymerases achieved genome coverage of >85% for six out of eight genotypes tested. Notably, several of the other polymerases tested showed reduced performance at higher Ct values. However, they still achieved strong coverage at Ct <24, supporting their use as emergency alternatives in rapid outbreak-response sequencing when viral input is high and RNA quality is sufficient. Our approach, ARTIC HELP, provides a framework which can be implemented to address supply chain disruptions, while maintaining robust genomic sequencing capabilities. A cost analysis highlights the well-known significant global disparities in reagent pricing, driven not by protocol differences but by import fees and supply barriers. Thus, our findings highlight the need for fairer global pricing models and support for local sourcing strategies like HELP, to promote equity in genomic research and ensure preparedness for future public health challenges.

genomics↗

bioRxiv2024

Variable rates of SARS-CoV-2 evolution in chronic infections

An important feature of the evolution of the SARS-CoV-2 virus has been the emergence of highly mutated novel variants, which are characterised by the gain of multiple mutations relative to viruses circulating in the general global population. Cases of chronic viral infection have been suggested as an explanation for this phenomenon, whereby an extended period of infection, with an increased rate of evolution, creates viruses with substantial genetic novelty. However, measuring a rate of evolution during chronic infection is made more difficult by the potential existence of compartmentalisation in the viral population, whereby the viruses in a host form distinct subpopulations. We here describe and apply a novel statistical method to study within-host virus evolution, identifying the minimum number of subpopulations required to explain sequence data observed from cases of chronic infection, and inferring rates for within-host viral evolution. Across nine cases of chronic SARS-CoV-2 infection in hospitalised patients we find that non-trivial population structure is relatively common, with four cases showing evidence of more than one viral population evolving independently within the host. We find cases of within-host evolution proceeding significantly faster, and significantly slower, than that of the global SARS-CoV-2 population, and of cases in which viral subpopulations in the same host have statistically distinguishable rates of evolution. Non-trivial population structure was associated with high rates of within-host evolution that were systematically underestimated by a more standard inference method.

evolutionary biology↗

bioRxiv2022

TRIM7 restricts Coxsackievirus and norovirus infection by detecting the C-terminalglutamine generated by 3C protease processing

TRIM7 catalyses the ubiquitination of multiple substrates with unrelated biological functions. This cross-reactivity is at odds with the specificity usually displayed by enzymes, including ubiquitin ligases. Here we show that TRIM7s extreme substrate promiscuity is due to a highly unusual binding mechanism, in which the PRYSPRY domain captures any ligand with a C-terminal helix that terminates in a hydrophobic residue followed by a glutamine. Many of the non-structural proteins found in RNA viruses contain C-terminal glutamines as a result of polyprotein cleavage by 3C protease. This viral processing strategy generates novel substrates for TRIM7 and explains its ability to inhibit Coxsackie virus and norovirus replication. In addition to viral proteins, cellular proteins such as glycogenin have evolved C-termini that make them a TRIM7 substrate. The helix-{Phi}Q degron motif recognised by TRIM7 is reminiscent of the N-end degron system and is found in [~] 1% of cellular proteins. These features, together with TRIM7s restricted tissue expression and lack of immune regulation suggest that viral restriction may not be its physiological function.

biochemistry↗

bioRxiv2022

Genome wide screen of RNAi molecules against SARS-CoV-2 creates a broadly potent prophylaxis

Expanding the arsenal of prophylactic approaches against SARS-CoV-2 is of utmost importance, specifically those strategies that are resistant to antigenic drift in Spike. Here, we conducted a screen with over 16,000 RNAi triggers against the SARS-CoV-2 genome using a massively parallel assay to identify hyper-potent siRNAs. We selected 10 candidates for in vitro validation and found five siRNAs that exhibited hyper-potent activity with IC50<20pM and strong neutralisation in live virus experiments. We further enhanced the activity by combinatorial pairing of the siRNA candidates to develop siRNA cocktails and found that these cocktails are active against multiple types of variants of concern (VOC). We examined over 2,000 possible mutations to the siRNA target sites using saturation mutagenesis and identified broad protection against future variants. Finally, we demonstrated that intranasal administration of the siRNA cocktail effectively attenuates clinical signs and viral measures of disease in the Syrian hamster model. Our results pave the way to development of an additional layer of antiviral prophylaxis that is orthogonal to vaccines and monoclonal antibodies.

genomics↗

bioRxiv2020

Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using nanopore

Genome sequencing has been widely deployed to study the evolution of SARS-CoV-2 with more than 90,000 genome sequences uploaded to the GISAID database. We published a method for SARS-CoV-2 genome sequencing (https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w) online on January 22, 2020. This approach has rapidly become the most popular method for sequencing SARS-CoV-2 due to its simplicity and cost-effectiveness. Here we present improvements to the original protocol: i) an updated primer scheme with 22 additional primers to improve genome coverage, ii) a streamlined library preparation workflow which improves demultiplexing rate for up to 96 samples and reduces hands-on time by several hours and iii) cost savings which bring the reagent cost down to {pound}10 per sample making it practical for individual labs to sequence thousands of SARS-CoV-2 genomes to support national and international genomic epidemiology efforts.

genomics↗

bioRxiv2020

The short and long-range RNA-RNA Interactome of SARS-CoV-2

The Coronaviridae is a family of positive-strand RNA viruses that includes SARS-CoV-2, the etiologic agent of the COVID-19 pandemic. Bearing the largest single-stranded RNA genomes in nature, coronaviruses are critically dependent on long-distance RNA-RNA interactions to regulate the viral transcription and replication pathways. Here we experimentally mapped the in vivo RNA-RNA interactome of the full-length SARS-CoV-2 genome and subgenomic mRNAs. We uncovered a network of RNA-RNA interactions spanning tens of thousands of nucleotides. These interactions reveal that the viral genome and subgenomes adopt alternative topologies inside cells, and engage in different interactions with host RNAs. Notably, we discovered a long-range RNA-RNA interaction - the FSE-arch - that encircles the programmed ribosomal frameshifting element. The FSE-arch is conserved in the related MERS-CoV and is under purifying selection. Our findings illuminate RNA structure based mechanisms governing replication, discontinuous transcription, and translation of coronaviruses, and will aid future efforts to develop antiviral strategies.

molecular biology↗

bioRxiv2020

A thermostable, closed, SARS-CoV-2 spike protein trimer.

The spike (S) protein of SARS-CoV-2 mediates receptor binding and cell entry and is the dominant target of the immune system. S exhibits substantial conformational flexibility. It transitions from closed to open conformations to expose its receptor binding site, and subsequently from prefusion to postfusion conformations to mediate fusion of viral and cellular membranes. S protein derivatives are components of vaccine candidates and diagnostic assays, as well as tools for research into the biology and immunology of SARS-CoV-2. Here we have designed mutations in S which allow production of thermostable, crosslinked, S protein trimers that are trapped in the closed, pre-fusion, state. We have determined the structures of crosslinked and non-crosslinked proteins, identifying two distinct closed conformations of the S trimer. We demonstrate that the designed, thermostable, closed S trimer can be used in serological assays. This protein has potential applications as a reagent for serology, virology and as an immunogen.

biochemistry↗

bioRxiv2019

Murine norovirus virulence factor 1 (VF1) protein contributes to viral fitness during persistent infection

BackgroundMurine norovirus (MNV) is widely used as a model for studying norovirus biology. While MNV isolates vary in their pathogenesis, infection of immunocompetent mice mostly results in persistent infection. The ability of a virus to establish a persistent infection is dependent on its ability to subvert or avoid the host immune response. Previously, we described the identification and characterization of virulence factor 1 (VF1) in MNV, and established that it acts as an innate immune antagonist. Here, we explore the role of VF1 during persistent MNV infection in an immunocompetent host.\n\nMethodsUsing reverse genetics, we generated VF1-knockout MNV-3 that contained a single or a triple termination codon inserted in the VF1 open reading frame.\n\nResultsVF1-knockout MNV-3 replicated to comparable levels to the wildtype virus in tissue culture. Comparative studies between MNV-3 and an acute MNV-1 strain show that MNV-3 VF1 exerts the same functions as MNV-1 VF1, but with reduced potency. Mice infected with VF1-knockout MNV-3 showed significantly reduced replication kinetics during the acute phase of the infection, but viral loads rapidly reached the levels seen in mice infected with wildtype virus after phenotypic restoration of VF1 expression. Infection with an MNV-3 mutant that had three termination codons inserted into VF1, in which reversion was suppressed, resulted in consistently lower replication throughout a three-month persistent infection in mice, suggesting a role for VF1 in viral fitness in vivo.\n\nConclusionOur results indicate that VF1 expressed by a persistent strain of MNV also functions to antagonise the innate response to infection. We found that VF1 is not essential for viral persistence, but instead contributes to viral fitness in mice. These data fit with the hypothesis that noroviruses utilise multiple mechanisms to avoid and/or control the host response to infection and that VF1 is just one component of this.

microbiology↗

bioRxiv2019

Noroviruses subvert the core stress granule component G3BP1 to promote viral VPg-dependent translation.

Knowledge of the host factors required for norovirus replication has been hindered by the challenges associated with culturing human noroviruses. We have combined proteomic analysis of the viral translation and replication complexes with a CRISPR screen, to identify host factors required for norovirus infection. The core stress granule component G3BP1 was identified as a host factor essential for efficient human and murine norovirus infection, demonstrating a conserved function across the Norovirus genus. Furthermore, we show that G3BP1 functions in the novel paradigm of viral VPg-dependent translation initiation, contributing to the assembly of translation complexes on the VPg-linked viral positive sense RNA genome by facilitating 40S recruitment. Our data suggest that G3BP1 functions by providing viral RNA a competitive advantage over capped cellular RNAs, uncovering a novel function for G3BP1 in the life cycle of positive sense RNA viruses and identifying the first host factor with pan-norovirus pro-viral activity.

microbiology↗

bioRxiv2019

A robust human norovirus replication model in zebrafish larvae

Human noroviruses (HuNoVs) are an important cause of epidemic and endemic acute gastroenteritis worldwide; annually about 700 million people develop a HuNoV infection resulting in [~]219,000 deaths and a societal cost estimated at 60 billion US dollars 1. The lack of robust small animal models has significantly hindered the understanding of norovirus biology and the development of effective therapeutics against HuNoV. Here we report that HuNoV GI and GII replicate to high titers in zebrafish (Danio rerio) larvae; replication peaks at day 2 post infection and is detectable for at least 6 days. HuNoV is detected in cells of the hematopoietic lineage, the intestine, liver and pancreas. Antiviral treatment reduces HuNoV replication by >2 log10, showing that this model is suited for antiviral studies. Downregulation of fucosyltransferase 8 (fut8) in the larvae reduces HuNoV replication, highlighting a common feature with infection in humans. Zebrafish larvae constitute a simple and robust replication model that will largely facilitate studies of HuNoV biology and the development of antiviral strategies.

microbiology↗

bioRxiv2019

Epigenetic suppression of interferon lambda receptor expression leads to an enhanced HuNoV replication in vitro

Human norovirus (HuNoV) is the main cause of acute gastroenteritis worldwide yet no therapeutics are currently available. Here, we utilize a human norovirus replicon in epithelial human gastric tumor (HGT) cells to identify host factors involved in promoting or inhibiting HuNoV replication. We observed that an IFN-cured population of replicon-harboring HGT cells (HGT-cured) was enhanced in their ability to replicate transfected HuNoV RNA compared to parental HGT cells, suggesting that differential gene expression in HGT-cured cells created an environment favouring the replication of viral RNA. Microarray analysis was used to identify genes differentially regulated in HGT-NV and HGT-cured compared to parental HGT cells. We found that the IFN lambda receptor alpha (IFNLR1) expression was highly reduced in HGT-NV and HGT-cured cells. All three cell lines responded to exogenous IFN-{beta} by inducing interferon stimulated genes (ISGs), however, HGT-NV and HGT-cured failed to respond to exogenous IFN-{lambda}. Inhibition of DNA methyltransferase activity with 5-aza-2-deoxycytidine partially reactivated IFNLR1 expression in HGT-NV and IFN-cured cells suggesting that host adaptation occurred via epigenetic reprogramming. In line with this observation, ectopic expression of the IFN-{lambda} receptor alpha rescued HGT-NV and HGT-cured cells response to IFN-{lambda}. We conclude that type III IFN is important in inhibiting HuNoV replication in vitro and that the loss of IFNLR1 enhances replication of HuNoV. To the best of our knowledge, this study unravels for the first time epigenetic reprograming of the interferon lambda receptor as a new mechanism of cellular adaptation during long-term RNA virus replication and shows that an endogenous level of interferon lambda signalling is able to control human norovirus replication.

microbiology↗

bioRxiv2018

Norovirus infection results in assembly of virus-specific G3BP1 granules and evasion of eIF2α signaling.

During viral infection, the accumulation of RNA replication intermediates or viral proteins imposes major stress on the host cell. In response, cellular stress pathways can rapidly impose defence mechanisms by shutting off the protein synthesis machinery, which viruses depend on, and triggering the accumulation of mRNAs into stress granules to limit the use of energy and nutrients. Because this threatens viral gene expression, viruses need to evade these pathways to propagate. Human norovirus is responsible for gastroenteritis outbreaks worldwide. Previously we showed that murine norovirus (MNV) regulates the activity of eukaryotic initiation factors (eIFs). Here we examined how MNV interacts with the eIF2a signaling axis controlling translation and stress granules accumulation. We show that while MNV infection represses host cell translation, it results in the assembly of virus-specific granules rather than stress granules. Further mechanistic analyses revealed that eIF2a signaling is uncoupled from translational stalling. Moreover the interaction of the RNA-binding protein G3BP1 with viral factors together with a redistribution of its cellular interacting partners could explain norovirus evasion of stress granules assembly. These results identify novel strategies by which norovirus ensure efficient replication propagation by manipulating the host stress response.

microbiology↗

bioRxiv2017

Spatial and temporal control of norovirus protease activity is determined by polyprotein processing and intermolecular interactions within the viral replication complex.

Norovirus infections are a major cause of acute viral gastroenteritis and a significant burden to human health globally. A vital process for norovirus replication is the processing of the nonstructural polyprotein, by an internal protease, into the necessary viral components required to form the viral replication complex. This cleavage occurs at different rates resulting in the accumulation of stable precursor forms. In this report, we characterized how precursor forms of the norovirus protease accumulate during infection. Using stable forms of the protease precursors we demonstrated that these are all proteolytically active in vitro, but that when expressed in cells, activity is determined by both substrate and protease localization. Whilst all precursors could cleave a replication complex-associated substrate, only a subset of precursors lacking NS4 were capable of efficiently cleaving a cytoplasmic substrate. For the first time, the full range of protein-protein interactions between murine and human norovirus proteins were mapped by LUMIER assay, with conserved interactions between replication complex members, modifying the localization of a subset of precursors. Finally, we demonstrate that re-targeting of a poorly cleaved artificial cytoplasmic substrate to the replication complex is sufficient to permit efficient cleavage in the context of norovirus infection. This offers a model for how norovirus can regulate the timing of substrate cleavage throughout the replication cycle. The norovirus protease represents a key target in the search for effective antiviral treatments for norovirus infection. An improved understanding of protease function and regulation, as well as identification of interactions between the other non-structural proteins, offers new avenues for antiviral drug design.

microbiology↗