Publications

          BioRxiv

Abstract
Human sapoviruses (HuSaVs) and noroviruses are considered the leading cause of acute gastroenteritis worldwide. While extensive research has focused on noroviruses, our understanding of sapoviruses (SaVs) and their interactions with the host’s immune response remains limited. HuSaVs have been challenging to propagate in vitro, making the porcine sapovirus (PSaV) Cowden strain a valuable model for studying SaV pathogenesis. In this study we show, for the first time, that PSaV Cowden strain has mechanisms to evade the host’s innate immune response. The virus 3C-like protease (NS6) inhibits type I IFN production by targeting TBK1. Catalytically active NS6, both during ectopic expression and during PSaV infection, targets TBK1 which is then led for rapid degradation by the proteasome. Moreover, deletion of TBK1 from porcine cells led to a significant increase in PSaV titres, emphasizing its role in regulating PSaV infection. Additionally, we successfully established PSaV infection in IPEC-J2 cells, an enterocytic cell line originating from the jejunum of a neonatal piglet. Overall, this study provides novel insights into PSaV evasion strategies, opening the way for future investigations for SaV-host interactions, and enabling the use of a new cell line model for PSaV research.

          BioRxiv

Abstract
Two mutations occurred in SARS-CoV-2 early during the COVID-19 pandemic that have come to define circulating virus lineages: first a change in the spike protein (D614G) that defines the B.1 lineage and second, a double substitution in the nucleocapsid protein (R203K, G204R) that defines the B.1.1 lineage, which has subsequently given rise to three Variants of Concern: Alpha, Gamma and Omicron. While the latter mutations appear unremarkable at the protein level, there are dramatic implications at the nucleotide level: the GGG→AAC substitution generates a new Transcription Regulatory Sequence (TRS) motif, driving SARS-CoV-2 to express a novel subgenomic mRNA (sgmRNA) encoding a truncated C-terminal portion of nucleocapsid (N.iORF3), which is an inhibitor of type I interferon production. We find that N.iORF3 also emerged independently within the Iota variant, and further show that additional TRS motifs have convergently evolved to express novel sgmRNAs; notably upstream of Spike within the nsp16 coding region of ORF1b, which is expressed during human infection. Our findings demonstrate that SARS-CoV-2 is undergoing evolutionary changes at the functional RNA level in addition to the amino acid level, reminiscent of eukaryotic evolution. Greater attention to this aspect in the assessment of emerging strains of SARS-CoV-2 is warranted.

          BioRxiv

Abstract
A major limitation to applying quantitative LC-MS/MS proteomics to small samples, such as single cells, are the losses incured during sample cleanup. To relieve this limitation, we developed a Minimal ProteOmic sample Preparation (mPOP) method for culture-grown mammalian cells. mPOP obviates cleanup and thus eliminates cleanup-related losses while expediting sample preparation and simplifying its automation. Bulk SILAC samples processed by mPOP or by conventional urea-based methods indicated that mPOP results in complete cell lysis and accurate relative quantification. We integrated mPOP lysis with the Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS) sample preparation, and benchmarked the quantification of such samples on a Q-exactive instrument. The results demonstrate low noise and high technical reproducibility. Then, we FACS sorted single U-937, HEK-293, and mouse ES cells into 96-well plates and analyzed them by automated mPOP and SCoPE-MS. The quantified proteins enabled separating the single cells by cell-type and cell-division-cycle phase.

        Pubmed       medRxiv       Journal

Abstract
Background: There have been dramatic clinical improvements in cystic fibrosis (CF) patients commenced on the cystic fibrosis conductance regulator (CFTR) modulator elexacaftor/tezacaftor/ivacaftor (ETI). Sputum proteomics is a powerful research technique capable of identifying important airway disease mechanisms. Using this technique, we evaluated how ETI changes the sputum proteome in people with CF. Methods: Sputum samples from 21 CF subjects pre- and post- ETI, 6 CF controls ineligible for ETI, and 15 healthy controls were analysed by liquid chromatography mass spectrometry. Results: Post-ETI, mean FEV1% increased by 13.7% (SD 7.9). Principal component and hierarchical clustering analysis revealed that the post-ETI proteome shifted to an intermediate state that was distinct from pre-ETI and healthy controls, even for those achieving normal lung function. Functional analysis showed incomplete resolution of neutrophilic inflammation. The CF control sputum proteome did not alter. At the protein-level many more proteins increased in abundance than decreased following ETI therapy (80 vs 30; adjusted p value <0.05), including many that have anti-inflammatory properties. Of those proteins that reduced in abundance many were pro-inflammatory neutrophil-derived proteins. Several important respiratory proteases were unchanged. Conclusions: Sputum proteomics can provide insights into CF lung disease mechanisms and how they are modified by therapeutic intervention, in this case ETI. This study identifies imbalances in pro- and anti- inflammatory proteins in sputum that partially resolve with ETI even in those achieving normal spirometry values. This post-ETI intermediate state could contribute to ongoing airway damage and therefore its relevance to clinical outcomes needs to be established.

   Journal       bioRxiv

Abstract
The SARS-CoV-2 genome encodes a multitude of accessory proteins. Using comparative genomic approaches, an additional accessory protein, ORF3c, has been predicted to be encoded within the ORF3a sgmRNA. Expression of ORF3c during infection has been confirmed independently by ribosome profiling. Despite ORF3c also being present in the 2002–2003 SARS-CoV, its function has remained unexplored. Here we show that ORF3c localises to mitochondria, where it inhibits innate immunity by restricting IFN-β production, but not NF-κB activation or JAK-STAT signalling downstream of type I IFN stimulation. We find that ORF3c is inhibitory after stimulation with cytoplasmic RNA helicases RIG-I or MDA5 or adaptor protein MAVS, but not after TRIF, TBK1 or phospho-IRF3 stimulation. ORF3c co-immunoprecipitates with the antiviral proteins MAVS and PGAM5 and induces MAVS cleavage by caspase-3. Together, these data provide insight into an uncharacterised mechanism of innate immune evasion by this important human pathogen.

   Pubmed       BioRxiv       Journal      Research Briefing (Journal)

Abstract
Major aims of single-cell proteomics include increasing the consistency, sensitivity, and depth of protein quantification, especially for proteins and modifications of biological interest. To simultaneously advance all of these aims, we developed prioritized Single Cell ProtEomics (pSCoPE). pSCoPE ensures duty-cycle time for analyzing prioritized peptides across all single cells (thus increasing data consistency) while analyzing identifiable peptides at full duty-cycle, thus increasing proteome depth. These strategies increased the quantified data points for challenging peptides and the overall proteome coverage about 2-fold. pSCoPE enabled quantifying proteome polarization in primary mouse macrophages and linking it to phenotypic variability in endocytic activity. Proteins annotated to phagosome maturation and proton transport showed concerted variation for both untreated and lipopolysaccharide-treated macrophages, indicating a conserved axis of polarization. pSCoPE further quantified proteolytic products, suggesting a gradient of cathepsin activities within a treatment condition. pSCoPE is easily accessible and likely to benefit many applications, especially mechanistic analysis seeking to focus on proteins of interest without sacrificing proteome coverage.

   Pubmed       Arxiv      Journal

Abstract
Analyzing proteins from single cells by tandem mass spectrometry (MS) has become technically feasible. While such analysis has the potential to accurately quantify thousands of proteins across thousands of single cells, the accuracy and reproducibility of the results may be undermined by numerous factors affecting experimental design, sample preparation, data acquisition, and data analysis. Establishing community guidelines and standardized metrics will enhance rigor, data quality, and alignment between laboratories. Here we propose best practices, quality controls, and data reporting guidelines to assist in the broad adoption of reliable quantitative workflows for single-cell proteomics.

Journal        Journal – feature

Abstract
Electrospray ionization (ESI), for which John Fenn was awarded a share of the 2002 Nobel Prize in chemistry, has revolutionized the use of mass spectrometry for biological and clinical applications. Developed in the late 1980s, building on the pioneering experiments by Malcom Dole’s lab, John Fenn et al. demonstrated the use of ESI for the ‘soft ionization’ of biologically important ‘molecular elephants’, allowing high molecular weight biopolymers (i.e., proteins) to be analysed by mass spectrometry. Here we describe the principles and mechanisms of ESI. Using the field of proteomics, we provide illustrations of how ESI has underpinned discoveries in this field for decades and is enabling emerging methods even today.

Pubmed       Journal       BioRxiv      Github       MassIVE     SCoPE2 Website      Figshare(1) (2) (3) (4)

Abstract
Many biological systems are composed of diverse single cells. This diversity necessitates functional and molecular single-cell analysis. Single-cell protein analysis has long relied on affinity reagents, but emerging mass-spectrometry methods (either label-free or multiplexed) have enabled quantifying >1,000 proteins per cell while simultaneously increasing the specificity of protein quantification. Here we describe the Single Cell ProtEomics (SCoPE2) protocol, which uses an isobaric carrier to enhance peptide sequence identification. Single cells are isolated by FACS or CellenONE into multiwell plates and lysed by Minimal ProteOmic sample Preparation (mPOP), and their peptides labeled by isobaric mass tags (TMT or TMTpro) for multiplexed analysis. SCoPE2 affords a cost-effective single-cell protein quantification that can be fully automated using widely available equipment and scaled to thousands of single cells. SCoPE2 uses inexpensive reagents and is applicable to any sample that can be processed to a single-cell suspension. The SCoPE2 workflow allows analyzing ~200 single cells per 24 h using only standard commercial equipment. We emphasize experimental steps and benchmarks required for achieving quantitative protein analysis.

*joint first authors

Pubmed      Journal       BioRxiv       Github       Protocols.io: TMTpro HUNTER       Protocols.io: Preparing annotated spectra from MaxQuant output in xiSpec       PRIDE (See published paper for accession numbers)

Abstract
SARS-CoV-2 is the causative agent behind the COVID-19 pandemic, responsible for over 170 million infections, and over 3.7 million deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify previously unknown cleavage sites in multiple viral proteins, including major antigens S and N: the main targets for vaccine and antibody testing efforts. We discover significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases. We show that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, show a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19.

          BioRxiv       Journal

Abstract

Background

QconCATs are quantitative concatamers for proteomic applications that yield stoichiometric quantities of sets of stable isotope-labelled internal standards. However, changing a QconCAT design, for example, to replace poorly performing peptide standards has been a protracted process.

Results

We report a new approach to the assembly and construction of QconCATs, based on synthetic biology precepts of biobricks, making use of loop assembly to construct larger entities from individual biobricks. The basic building block (a Qbrick) is a segment of DNA that encodes two or more quantification peptides for a single protein, readily held in a repository as a library resource. These Qbricks are then assembled in a one tube ligation reaction that enforces the order of assembly, to yield short QconCATs that are useable for small quantification products. However, the DNA context of the short construct also allows a second cycle of loop assembly such that five different short QconCATs can be assembled into a longer QconCAT in a second, single tube ligation. From a library of Qbricks, a bespoke QconCAT can be assembled quickly and efficiently in a form suitable for expression and labelling in vivo or in vitro.

Conclusions

We refer to this approach as the ALACAT strategy as it permits à la carte design of quantification standards. ALACAT methodology is a major gain in flexibility of QconCAT implementation as it supports rapid editing and improvement of QconCATs and permits, for example, substitution of one peptide by another.

          BioRxiv       Journal

Abstract
Vesivirus 2117 is an adventitious agent that has been responsible for lost productivity in biopharmaceutical production following contamination of Chinese hamster ovary cell cultures in commercial bioreactors. A member of the Caliciviridae, 2117 is classified within the vesivirus genus in a clade that includes canine and mink caliciviruses but is distinct from the vesicular exanthema of swine clade, which includes the extensively studied feline calicivirus (FCV). We have used cryogenic electron microscopy (cryo-EM) to determine the structure of the capsid of this small, icosahedral, positive-sense RNA containing virus. We show that the outer face of the dimeric capsomeres, which contains the receptor binding site and major immunodominant epitopes in all caliciviruses studied thus far, is quite different from that of FCV. This is a consequence of a 22 amino-acid insertion in the sequence of the FCV major capsid protein that forms a cantilevered arm, which plays an important role in both receptor engagement and undergoes structural rearrangements thought to be important for genome delivery to the cytosol. Our data highlight a potentially important difference in the attachment and entry pathways employed by the different clades of the vesivirus genus.

Pubmed       Journal      BioRxiv

Abstract

Background

Macrophages are innate immune cells with diverse functional and molecular phenotypes. This diversity is largely unexplored at the level of single-cell proteomes because of the limitations of quantitative single-cell protein analysis.

Results

To overcome this limitation, we develop SCoPE2, which substantially increases quantitative accuracy and throughput while lowering cost and hands-on time by introducing automated and miniaturized sample preparation. These advances enable us to analyze the emergence of cellular heterogeneity as homogeneous monocytes differentiate into macrophage-like cells in the absence of polarizing cytokines. SCoPE2 quantifies over 3042 proteins in 1490 single monocytes and macrophages in 10 days of instrument time, and the quantified proteins allow us to discern single cells by cell type. Furthermore, the data uncover a continuous gradient of proteome states for the macrophages, suggesting that macrophage heterogeneity may emerge in the absence of polarizing cytokines. Parallel measurements of transcripts by 10× Genomics suggest that our measurements sample 20-fold more protein copies than RNA copies per gene, and thus, SCoPE2 supports quantification with improved count statistics. This allowed exploring regulatory interactions, such as interactions between the tumor suppressor p53, its transcript, and the transcripts of genes regulated by p53.

Conclusions

Even in a homogeneous environment, macrophage proteomes are heterogeneous. This heterogeneity correlates to the inflammatory axis of classically and alternatively activated macrophages. Our methodology lays the foundation for automated and quantitative single-cell analysis of proteins by mass spectrometry and demonstrates the potential for inferring transcriptional and post-transcriptional regulation from variability across single cells.

Pubmed       Journal      BioRxiv

Abstract
Herpesviruses are ubiquitous in the human population and they extensively remodel the cellular environment during infection. Multiplexed quantitative proteomic analysis over a whole time-course of herpes simplex virus (HSV)-1 infection was used to characterize changes in the host-cell proteome and to probe the kinetics of viral protein production. Several host-cell proteins were targeted for rapid degradation by HSV-1, including the cellular trafficking factor GOPC. We identify that the poorly-characterized HSV-1 protein pUL56 binds directly to GOPC, stimulating its ubiquitination and proteasomal degradation. Plasma membrane profiling revealed that pUL56 mediates specific changes to the surface proteome of infected cells, including loss of IL18 receptor and Toll-like receptor 2, and delivery of Toll-like receptor 2 to the cell-surface requires GOPC. Our study highlights an unanticipated and efficient mechanism whereby a single virus protein targets a cellular trafficking factor to modify the abundance of multiple signaling molecules at the surface of infected cells.

   Pubmed       Journal       COVID-19-MSC Website

No Abstract

   Pubmed       Journal       BioRxiv

Abstract

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.

    Pubmed      BioRxiv      Wellcome Open Research

Abstract

Background: Norovirus, 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. Methods: Ifit1 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. Results: Here, 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. Conclusions: Ifit1 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.

Pubmed       Journal       PeerJ Preprints

Abstract
Peer-reviewed journal publication is the main means for academic researchers in the life sciences to create a permanent, public record of their work. These publications are also the de facto currency for career progress, with a strong link between journal brand recognition and perceived value. The current peer-review process can lead to long delays between submission and publication, with cycles of rejection, revision and resubmission causing redundant peer review. This situation creates unique challenges for early career researchers (ECRs), who rely heavily on timely publication of their work to gain recognition for their efforts. ECRs face changes in the academic landscape including the increased interdisciplinarity of life sciences research, expansion of the researcher population and consequent shifts in employer and funding demands. The publication of preprints, publicly available scientific manuscripts posted on dedicated preprint servers prior to journal managed peer-review, can play a key role in addressing these ECR challenges. Preprinting benefits include rapid dissemination of academic work, open access, establishing priority or concurrence, receiving feedback and facilitating collaborations. While there is a growing appreciation for and adoption of preprints, a minority of all articles in life sciences and medicine are preprinted. The current low rate of preprint submissions in life sciences and ECR concerns regarding preprinting needs to be addressed. We provide a perspective from an interdisciplinary group of early career researchers on the value of preprints and advocate the wide adoption of preprints to advance knowledge and facilitate career development.

  Pubmed       Journal

Abstract

Contrary to the textbook model, recent measurements demonstrated unexpected diversity in ribosomal composition that likely enables specialized translational functions. Methods based on liquid chromatography coupled to tandem mass-spectrometry (LC-MS/MS) enable direct quantification of ribosomal proteins with high specificity, accuracy, and throughput. LC-MS/MS can be ‘top-down’, analyzing intact proteins, or more commonly ‘bottom-up’, where proteins are digested to peptides prior to analysis. Changes to rRNA can be examined using either LC-MS/MS or sequencing-based approaches.

Note: this feature is a companion to the earlier ‘Ribosome Stoichiometry: From Form to Function’ article.

         Pubmed       Journal      BioRxiv

Abstract
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.

       Pubmed       Journal       PeerJ Preprints

Abstract
The existence of eukaryotic ribosomes with distinct ribosomal protein (RP) stoichiometry and regulatory roles in protein synthesis been speculated for over sixty years. Recent advances in mass spectrometry and high throughput analysis have begun to identify and characterize distinct ribosome stoichiometry in yeast or mammalian systems. In addition to RP stoichiometry, ribosomes play host to a vast array of protein modifications, effectively expanding the number of human RPs from 80 to many thousands of distinct proteoforms. Is it possible these proteoforms combine to function as a ‘ribosome code’ to tune protein synthesis? We outline the specific benefits that translational regulation by specialized ribosomes can offer and discuss the means and methodologies available to correlate and characterize RP stoichiometry with function. We highlight previous research with a focus on formulating hypotheses that can guide future experiments and crack the ‘ribosome code’.

Pubmed      Wellcome Open Research

Abstract
Background: Human parainfluenza viruses (HPIVs) are significant causes of both upper and lower respiratory tract infections with type 3 (HPIV3) causing the most severe disease in the immunocompromised cohorts. The objective of this study was to analyse the epidemiological nature of a cluster of cases of HPIV3 in a pediatric oncology unit of a major teaching hospital.
Methods: In order to determine whether the activity observed represented a deviation from the norm, seasonal trends of HPIV3 in the surrounding geographical area as well as on the ward in question were analysed. The genetic link between cases was established by the phylogenetic analysis of the non-coding hypervariable region between the M (Matrix) and F (fusion) genes of HPIV3. The 15 cases involved and 15 unrelated cases were sequenced. Transmission routes were subsequently inferred and visualized using Konstanz Information Miner (KNIME) 3.3.2.
Results: Of the 15 cases identified, 14 were attributed to a point source outbreak. Two out of 14 outbreak cases were found to differ by a single mutation A182C. The outbreak strain was also seen in 1 out of 15 unrelated cases, indicating that it was introduced from the community. Transmission modeling was not able to link all the cases and establish a conclusive chain of transmission. No staff were tested during the outbreak period. No deaths occurred as a result of the outbreak.
Conclusion: on an oncology pediatric unit in a major teaching hospital. This raised concern about the possibility of a future more serious outbreak. Weaknesses in existing systems were identified and a new dedicated respiratory virus monitoring system introduced. Pediatric oncology units require sophisticated systems for early identification of potentially life-threatening viral outbreaks.

         Pubmed      Wellcome Open Research

Abstract
Background: Human parainfluenza viruses type 3 (HPIV3) are a prominent cause of respiratory infection with a significant impact in both pediatric and transplant patient cohorts.  Currently there is a paucity of whole genome sequence data that would allow for detailed epidemiological and phylogenetic analysis of circulating strains in the UK. Although it is known that HPIV3 peaks annually in the UK, to date there are no whole genome sequences of HPIV3 UK strains available.
Methods: Clinical strains were obtained from HPIV3 positive respiratory patient samples collected between 2011 and 2015.  These were then amplified using an amplicon based method, sequenced on the Illumina platform and assembled using a new robust bioinformatics pipeline. Phylogenetic analysis was carried out in the context of other epidemiological studies and whole genome sequence data currently available with stringent exclusion of significantly culture-adapted strains of HPIV3.
Results: In the current paper we have presented twenty full genome sequences of UK circulating strains of HPIV3 and a detailed phylogenetic analysis thereof.  We have analysed the variability along the HPIV3 genome and identified a short hypervariable region in the non-coding segment between the M (matrix) and F (fusion) genes. The epidemiological classifications obtained by using this region and whole genome data were then compared and found to be identical.
Conclusions: The majority of HPIV3 strains were observed at different geographical locations and with a wide temporal spread, reflecting the global distribution of HPIV3. Consistent with previous data, a particular subcluster or strain was not identified as specific to the UK, suggesting that a number of genetically diverse strains circulate at any one time. A small hypervariable region in the HPIV3 genome was identified and it was shown that, in the absence of full genome data, this region could be used for epidemiological surveillance of HPIV3.

Pubmed       Journal       BioRxiv      Data – PRIDE

Abstract
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.

Pubmed        Journal

Abstract
Human norovirus (HuNoV) is a major cause of nonbacterial gastroenteritis worldwide yet, despite their impact on society, vaccines and antivirals are currently lacking. A HuNoV replicon system has been widely applied to the evaluation of antiviral compounds and has thus accelerated the process of drug discovery against HuNoV infection. Rupintrivir, an irreversible inhibitor of the human rhinovirus 3C protease, has been reported to inhibit the replication of the Norwalk virus replicon via the inhibition of the norovirus protease. Here we report, for the first time, the generation of rupintrivir-resistant human Norwalk virus replicon in vitro. Sequence analysis revealed that these replicon cells contained amino acid substitutions of alanine 105 to valine (A105V) and isoleucine 109 to valine (I109V) in the viral protease NS6. The application of a cell-based fluorescence resonance energy transfer (FRET) assay for protease activity demonstrated that these substitutions were involved in the enhanced resistance to rupintrivir. Furthermore, we validated the effect of these mutations using the reverse genetics in murine norovirus (MNV), demonstrating that a recombinant MNV with a single I109V substitution in the protease also showed reduced susceptibility to rupintrivir. In summary, using a combination of different approaches, we have demonstrated that, under the correct conditions, mutations in the norovirus protease can rapidly occur that lead to the generation of resistant mutants.

  Pubmed        Journal

Abstract
Background
Human parainfluenza type 3 (HPIV3) is an important respiratory pathogen. Although a number of potential therapeutic candidates exist, there is currently no licensed therapy or vaccine. Ribavirin (RBV), favipiravir (FVP) and zanamivir (ZNV) are inhibitors with proven activity against influenza and with potential inhibitory activity against HPIV3 laboratory adapted strains in vitro.

Objectives
To evaluate RBV, FVP and ZNV as inhibitors of minimally passaged UK clinical strains of HPIV3 as well as a laboratory adapted strain MK9 in vitro.

Study Design
The inhibitory action of RBV, FVP and ZNV was evaluated against nine minimally passaged clinical strains and a laboratory adapted strain MK9 using plaque reduction and growth curve inhibition in a cell culture model.

Results
Clinical isolates were found to be at least as susceptible as the laboratory adapted strains to RBV and FVP and significantly more susceptible to ZNV. However the inhibitory concentrations achieved by ZNV against clinical strains remain prohibitively high in vivo.

Conclusions
RBV, FVP and ZNV were found to be effective inhibitors of HPIV3 in vitro. The lack of efficacy of RBV in vivo may be due to inability to reach required therapeutic levels. FVP, on the other hand, is a good potential therapeutic agent against HPIV3. Further studies using wild type clinical strains, as well as better formulation and delivery mechanisms may improve the utility of these three inhibitors.

  Pubmed        Journal        BioRxiv

Abstract
Eukaryotic cells use conserved multisubunit membrane tethering complexes, including CORVET and HOPS, to control the fusion of endomembranes. These complexes have been extensively studied in yeast, but to date there have been far fewer studies of metazoan CORVET and HOPS. Both of these complexes comprise six subunits: a common four-subunit core and two unique subunits. Once assembled, these complexes function to recognise specific endosomal membrane markers and facilitate SNARE-mediated membrane fusion. CORVET promotes the homotypic fusion of early endosomes, while HOPS promotes the fusion of lysosomes to late endosomes and autophagosomes. Many of the subunits of both CORVET and HOPS contain putative C-terminal zinc-finger domains. Here, the contribution of these domains to the assembly of the human CORVET and HOPS complexes has been examined. Using biochemical techniques, we demonstrate that the zinc-containing RING domains of human VPS18 and VPS41 interact directly to form a stable heterodimer. In cells, these RING domains are able to integrate into endogenous HOPS, showing that the VPS18 RING domain is required to recruit VPS41 to the core complex subunits. Importantly, this mechanism is not conserved throughout eukaryotes, as yeast Vps41 does not contain a C-terminal zinc-finger motif. The subunit analogous to VPS41 in human CORVET is VPS8, in which the RING domain has an additional C-terminal segment that is predicted to be disordered. Both the RING and disordered C-terminal domains are required for integration of VPS8 into endogenous CORVET complexes, suggesting that HOPS and CORVET recruit VPS41 and VPS8 via distinct molecular interactions.

Pubmed         Journal       Data – PRIDE

Abstract
Bluetongue virus (BTV) causes infections in wild and domesticated ruminants with high morbidity and mortality and is responsible for significant economic losses in both developing and developed countries. BTV serves as a model for the study of other members of the Orbivirus genus. Previously, the importance of casein kinase 2 for BTV replication was demonstrated. To identify intracellular signalling pathways and novel host-cell kinases involved during BTV infection, the phosphoproteome of BTV infected cells was analysed. Over 1000 phosphosites were identified using mass spectrometry, which were then used to determine the corresponding kinases involved during BTV infection. This analysis yielded protein kinase A (PKA) as a novel kinase activated during BTV infection. Subsequently, the importance of PKA for BTV infection was validated using a PKA inhibitor and activator. Our data confirmed that PKA was essential for efficient viral growth. Further, we showed that PKA is also required for infection of equid cells by African horse sickness virus, another member of the Orbivirus genus. Thus, despite their preference in specific host species, orbiviruses may utilize the same host signaling pathways during their replication.

Pubmed          Journal          Editorial          BioRxiv          Data (1) – PRIDE          Data (2) – PRIDE

Abstract
Noroviruses produce viral RNAs lacking a 5′ cap structure and instead use a virus-encoded viral protein genome-linked (VPg) protein covalently linked to viral RNA to interact with translation initiation factors and drive viral protein synthesis. Norovirus infection results in the induction of the innate response leading to interferon stimulated gene (ISG) transcription. However, the translation of the induced ISG mRNAs is suppressed. A SILAC-based mass spectrometry approach was employed to analyze changes to protein abundance in both whole cell and m7GTP-enriched samples to demonstrate that diminished host mRNA translation correlates with changes to the composition of the eukaryotic initiation factor complex. The suppression of host ISG translation correlates with the activity of the viral protease (NS6) and the activation of cellular caspases leading to the establishment of an apoptotic environment. These results indicate that noroviruses exploit the differences between viral VPg-dependent and cellular cap-dependent translation in order to diminish the host response to infection.

Pubmed          Journal

Abstract
Vesivirus 2117 is an adventitious agent that, in 2009, was identified as a contaminant of Chinese hamster ovary cells propagated in bioreactors at a pharmaceutical manufacturing plant belonging to Genzyme. The consequent interruption in supply of Fabrazyme and Cerezyme (drugs used to treat Fabry and Gaucher diseases, respectively) caused significant economic losses. Vesivirus 2117 is a member of the Caliciviridae, a family of small icosahedral viruses encoding a positive-sense RNA genome. We have used cryo-electron microscopy and three-dimensional image reconstruction to calculate a structure of vesivirus 2117 virus-like particles as well as feline calicivirus and a chimeric sapovirus. We present a structural comparison of several members of the Caliciviridae, showing that the distal P domain of vesivirus 2117 is morphologically distinct from that seen in other known vesivirus structures. Furthermore, at intermediate resolutions, we found a high level of structural similarity between vesivirus 2117 and Caliciviridae from other genera: sapovirus and rabbit hemorrhagic disease virus. Phylogenetic analysis confirms vesivirus 2117 as a vesivirus closely related to canine vesiviruses. We postulate that morphological differences in virion structure seen between vesivirus clades may reflect differences in receptor usage.

Pubmed          Journal

Abstract
The viral protease represents a key drug target for the development of antiviral therapeutics. Because many protease inhibitors mimic protease substrates, differences in substrate recognition between proteases may affect their sensitivity to a given inhibitor. Here we use a cell-based FRET sensor to investigate the activity of different norovirus proteases upon cleavage of various norovirus cleavage sites inserted into a linker region separating cyan fluorescent protein and yellow fluorescent protein. Using this system, we demonstrate that differences in substrate processing exist between proteases from human noroviruses (genogroups I (GI) and II) and the commonly used murine norovirus (MNV, genogroup V) model. These altered the cleavage efficiency of specific cleavage sites both within and between genogroups. The differences observed between these proteases may affect sensitivity to protease inhibitors and the suitability of MNV as a model system for testing such molecules against the human norovirus protease. Finally, we demonstrate that replacement of MNV polyprotein cleavage sites with the GI or GII equivalents, with the exception of the NS6-7 junction, leads to the production of infectious virus when the MNV NS6 protease, but not the GI or GII proteases, are present.

Pubmed          Journal

Abstract
Human noroviruses (HuNoVs) are a major cause of viral gastroenteritis, with an estimated 3 million cases per year in the United Kingdom. HuNoVs have recently been isolated from pet dogs in Europe (M. Summa, C.-H. von Bonsdorff, and L. Maunula, J Clin Virol 53:244-247, 2012, http://dx.doi.org/10.1016/j.jcv.2011.12.014), raising concerns about potential zoonotic infections. With 31% of United Kingdom households owning a dog, this could prove to be an important transmission route. To examine this risk, canine tissues were studied for their ability to bind to HuNoV in vitro. In addition, canine stool samples were analyzed for the presence of viral nucleic acid, and canine serum samples were tested for the presence of anti-HuNoV antibodies. The results showed that seven different genotypes of HuNoV virus-like particles (VLPs) can bind to canine gastrointestinal tissue, suggesting that infection is at least theoretically possible. Although HuNoV RNA was not identified in stool samples from 248 dogs, serological evidence of previous exposure to HuNoV was obtained in 43/325 canine serum samples. Remarkably, canine seroprevalence for different HuNoV genotypes mirrored the seroprevalence in the human population. Though entry and replication within cells have not been demonstrated, the canine serological data indicate that dogs produce an immune response to HuNoV, implying productive infection. In conclusion, this study reveals zoonotic implications for HuNoV, and to elucidate the significance of this finding, further epidemiological and molecular investigations will be essential.

Pubmed          Journal

Abstract
Protein synthesis is a tightly controlled process responding to several stimuli, including viral infection. As obligate intracellular parasites, viruses depend on the translation machinery of the host and can manipulate it by affecting the availability and function of specific eukaryotic initiation factors (eIFs). Human norovirus is a member of the Caliciviridae family and is responsible for gastroenteritis outbreaks. Previous studies on feline calicivirus and murine norovirus 1 (MNV1) demonstrated that the viral protein, genome-linked (VPg), acts to direct translation by hijacking the host protein synthesis machinery. Here we report that MNV1 infection modulates the MAPK pathway to activate eIF4E phosphorylation. Our results show that the activation of p38 and Mnk during MNV1 infection is important for MNV1 replication. Furthermore, phosphorylated eIF4E relocates to the polysomes, and this contributes to changes in the translational state of specific host mRNAs. We propose that global translational control of the host by eIF4E phosphorylation is a key component of the host-pathogen interaction.

Pubmed          Journal

Abstract
Viruses have evolved a variety of mechanisms to usurp the host cell translation machinery to enable translation of the viral genome in the presence of high levels of cellular mRNAs. Noroviruses, a major cause of gastroenteritis in man, have evolved a mechanism that relies on the interaction of translation initiation factors with the virus-encoded VPg protein covalently linked to the 5′ end of the viral RNA. To further characterize this novel mechanism of translation initiation, we have used proteomics to identify the components of the norovirus translation initiation factor complex. This approach revealed that VPg binds directly to the eIF4F complex, with a high affinity interaction occurring between VPg and eIF4G. Mutational analyses indicated that the C-terminal region of VPg is important for the VPg-eIF4G interaction; viruses with mutations that alter or disrupt this interaction are debilitated or non-viable. Our results shed new light on the unusual mechanisms of protein-directed translation initiation.

Pubmed          Journal

Abstract
Murine norovirus (MNV) is a positive-sense, plus-stranded RNA virus in the Caliciviridae family. It is the most common pathogen in biomedical research colonies. MNV is also related to the human noroviruses, which cause the majority of nonbacterial gastroenteritis worldwide. Like the human noroviruses, MNV is an enteric virus that replicates in the intestine and is transmitted by the fecal-oral route. MNV replicates in murine macrophages and dendritic cells in cells in culture and in the murine host. This virus is often used to study mechanisms in norovirus biology, because human noroviruses are refractory to growth in cell culture. MNV combines the availability of a cell culture and reverse genetics system with the ability to study infection in the native host. Herein, we describe a panel of techniques that are commonly used to study MNV biology.

Pubmed          Journal

Abstract
Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

Pubmed           Journal

Abstract
Noroviruses are associated with intestinal disease in humans, cows, pigs, mice, and, more recently, dogs. In 2007, the first canine norovirus (CNV) was identified and characterized in Italy. Subsequent studies have identified CNV in stools of dogs from Portugal, Greece, and the United States. To investigate the prevalence of CNV in the UK dog population, 228 canine stool samples were screened for CNV by qPCR, and 396 serum samples were screened for anti-CNV antibodies. qPCR of RNA extracted from canine stool samples did not reveal any CNV-positive samples, based on samples collected from diarrhoeic and control dogs in 2012-2013. CNV virus-like particles to three different CNV strains were produced using recombinant baculoviruses and a seroprevalence screen undertaken. Anti-CNV antibodies were identified at significant levels in canine serum; 38.1% of samples collected between 1999-2001 and 60.1% of samples collected in 2012-2013 were seropositive. The increase in seroprevalence over time (p less than 0.001) suggests that the CNV strains screened for are becoming more widespread. Variation in seroprevalence to different CNV strains was also identified. Two-thirds of the dogs were seropositive to a single strain, whereas the remaining third were seropositive to two or three of the strains analysed. This study has provided the first evidence that CNV is present in the UK, with seroprevalence identified to multiple circulating strains. This warrants further study and increased awareness of this recently discovered canine virus.

Pubmed           Journal

Abstract
Noroviruses are now recognized as the major cause of acute gastroenteritis in the developed world, yet our ability to prevent and control infection is limited. Recent work has highlighted that, while typically an acute infection in the population, immunocompromised patients often experience long-term infections that may last many years. This cohort of patients and those regularly exposed to infectious material, for example, care workers and others, would benefit greatly from the development of a vaccine or antiviral therapy. While a licensed vaccine or antiviral has yet to be developed, work over the past 10 years in this area has intensified and trials with a vaccine candidate have proven promising. Numerous antiviral targets and small molecule inhibitors that have efficacy in cell culture have now been identified; however, further studies in this area are required in order to make these suitable for clinical use.

Pubmed          Journal

Abstract
The coronavirus nucleocapsid (N) protein plays a multifunctional role in the virus life cycle, from regulation of replication and transcription and genome packaging to modulation of host cell processes. These functions are likely to be facilitated by interactions with host cell proteins. The potential interactome of the infectious bronchitis virus (IBV) N protein was mapped using stable isotope labeling with amino acids in cell culture (SILAC) coupled to a green fluorescent protein-nanotrap pulldown methodology and liquid chromatography-tandem mass spectrometry. The addition of the SILAC label allowed discrimination of proteins that were likely to specifically bind to the N protein over background binding. Overall, 142 cellular proteins were selected as potentially binding to the N protein, many as part of larger possible complexes. These included ribosomal proteins, nucleolar proteins, translation initiation factors, helicases, and hnRNPs. The association of selected cellular proteins with IBV N protein was confirmed by immunoblotting, cosedimentation, and confocal microscopy. Further, the localization of selected proteins in IBV-infected cells as well as their activity during virus infection was assessed by small interfering RNA-mediated depletion, demonstrating the functional importance of cellular proteins in the biology of IBV. This interactome not only confirms previous observations made with other coronavirus and IBV N proteins with both overexpressed proteins and infectious virus but also provides novel data that can be exploited to understand the interaction between the virus and the host cell.

Pubmed          Journal

Abstract
Viruses continue to pose some of the greatest threats to human and animal health, and food security worldwide. Therefore, new approaches are required to increase our understanding of virus-host cell interactions and subsequently design more effective therapeutic countermeasures. Quantitative proteomics based on stable isotope labeling by amino acids in cell culture (SILAC), coupled to LC-MS/MS and bioinformatic analysis, is providing an excellent resource for studying host cell proteomes and can readily be applied for the study of virus infection. Here, we review this approach and discuss how virus-host cell interactions can best be studied, what is realistically feasible, and the potential limitations. For example, sub-cellular fractionation can reduce sample complexity for LC-MS/MS, increase data return and provide information regarding protein trafficking between different cellular compartments. The key to successful quantitative proteomics combines good experimental design and appropriate sample preparation with statistical analysis and validation of the MS data through the use of independent techniques and functional analysis. The annotation of the human genome and the increasing availability of biological reagents such as antibodies, provide the optimum parameters for studying viruses that infect humans, in human cell lines. SILAC-based quantitative proteomics can also be used to study the interactome of viral proteins with the host cell. Coupling proteomic studies with global transcriptomic and RNA depletion experiments will provide great insights into the complexity of the infection process, and potentially reveal new antiviral targets.

Pubmed          Journal

Abstract
Human respiratory syncytial virus (HRSV) is a major cause of pediatric lower respiratory tract disease to which there is no vaccine or efficacious chemotherapeutic strategy. Although RNA synthesis and virus assembly occur in the cytoplasm, HRSV is known to induce nuclear responses in the host cell as replication alters global gene expression. Quantitative proteomics was used to take an unbiased overview of the protein changes in transformed human alveolar basal epithelial cells infected with HRSV. Underpinning this was the use of stable isotope labeling with amino acids in cell culture coupled to LC-MS/MS, which allowed the direct and simultaneous identification and quantification of both cellular and viral proteins. To reduce sample complexity and increase data return on potential protein localization, cells were fractionated into nuclear and cytoplasmic extracts. This resulted in the identification of 1,140 cellular proteins and six viral proteins. The proteomics data were analyzed using Ingenuity Pathways Analysis to identify defined canonical pathways and functional groupings. Selected data were validated using Western blot, direct and indirect immunofluorescence confocal microscopy, and functional assays. The study served to validate and expand upon known HRSV-host cell interactions, including those associated with the antiviral response and alterations in subnuclear structures such as the nucleolus and ND10 (promyelocytic leukemia bodies). In addition, novel changes were observed in mitochondrial proteins and functions, cell cycle regulatory molecules, nuclear pore complex proteins and nucleocytoplasmic trafficking proteins. These data shed light into how the cell is potentially altered to create conditions more favorable for infection. Additionally, the study highlights the application and advantage of stable isotope labeling with amino acids in cell culture coupled to LC-MS/MS for the analysis of virus-host interactions.

Pubmed          Journal

Abstract
Influenza A virus (IAV) is a major human pathogen whose genotypic diversity results in unpredictable pandemics and epidemics. Interaction with the cell nucleus is essential to IAV infection, allowing recruitment of cellular components to facilitate virus replication. Viral proteins are also targeted to the nucleolus, a subnuclear structure involved in ribosomal biogenesis, RNA maturation, stress response, and control of cell growth, but the functional consequences of this are unclear. We took an unbiased approach to studying IAV-nucleolar interactions by using stable isotope labeling with amino acids in cell culture (SILAC) in conjunction with LC-MS/MS to quantify changes in the nucleolar proteome following infection with A/PR/8/34 (H1N1) and A/Udorn/72 (H3N2) strains of the virus. Only a minority of nucleolar proteins showed significant changes in abundance after infection; these alterations were mostly different between the two strains but could be validated by confocal microscopy of infected cells. Many of the affected proteins comprised functional groupings, including components of ribonuclease P, RNA polymerase I, the MLL1 histone methyltransferase complex, as well as nuclear paraspeckles and the RNA editing apparatus. This, as well as comparison with other viruses that cause changes in the nucleolar proteome, suggests that IAV targets specific nucleolar pathways.

Pubmed          Journal

Abstract
The nucleolus is a dynamic subnuclear compartment involved in ribosome subunit biogenesis, regulation of cell stress and modulation of cellular growth and the cell cycle, among other functions. The nucleolus is composed of complex protein/protein and protein/RNA interactions. It is a target of virus infection with many viral proteins being shown to localize to the nucleolus during infection. Perturbations to the structure of the nucleolus and its proteome have been predicted to play a role in both cellular and infectious disease. Stable isotope labeling with amino acids in cell culture coupled to LC-MS/MS with bioinformatic analysis using Ingenuity Pathway Analysis was used to investigate whether the nucleolar proteome altered in virus-infected cells. In this study, the avian nucleolar proteome was defined in the absence and presence of virus, in this case the positive strand RNA virus, avian coronavirus infectious bronchitis virus. Data sets, potential protein changes and the functional consequences of virus infection were validated using independent assays. These demonstrated that specific rather than generic changes occurred in the nucleolar proteome in infectious bronchitis virus-infected cells.

Pubmed          Journal

Abstract
Virus-host interactions involve complex interplay between viral and host factors, rendering them an ideal target for proteomic analysis. Here we detail a high throughput quantitative proteomics analysis of Vero cells infected with the coronavirus infectious bronchitis virus (IBV), a positive strand RNA virus that replicates in the cytoplasm. Stable isotope labeling with amino acids in cell culture (SILAC) was used in conjunction with LC-MS/MS to identify and quantify 1830 cellular and two viral proteins from IBV-infected cells. Fractionation of cells into cytoplasmic, nuclear, and nucleolar extracts was used to reduce sample complexity and provide information on the trafficking of proteins between the different compartments. Each fraction showed a proportion of proteins exhibiting >or=2-fold changes in abundance. Ingenuity Pathway Analysis revealed that proteins that changed in response to infection could be grouped into different functional categories. These included proteins regulated by NF-kappaB- and AP-1-dependent pathways and proteins involved in the cytoskeleton and molecular motors. A luciferase-based reporter gene assay was used to validate the up-regulation of AP-1- and NF-kappaB-dependent transcription in IBV-infected cells and confirmed using immunofluorescence. Immunofluorescence was used to validate changes in the subcellular localization of vimentin and myosin VI in IBV-infected cells. The proteomics analysis also confirmed the presence of the viral nucleocapsid protein as localizing in the cytoplasm, nucleus, and nucleolus and the viral membrane protein in the cytoplasmic fraction. This research is the first application of SILAC to study total host cell proteome changes in response to positive sense RNA virus infection and illustrates the versatility of this technique as applied to infectious disease research.

Pubmed          Journal

Abstract
The nucleolus is a dynamic structure that has roles in various processes, from ribosome biogenesis to regulation of the cell cycle and the cellular stress response. Such functions are frequently mediated by the sequestration or release of nucleolar proteins. Our understanding of protein targeting to the nucleolus is much less complete than our knowledge of membrane-spanning translocation systems–such as those involved in nuclear targeting–and the experimental evidence reveals that few parallels exist with these better-characterized systems. Here, we discuss the current understanding of nucleolar targeting, explore the types of sequence that control the localization of a protein to the nucleolus, and speculate that certain subsets of nucleolar proteins might act as hub proteins that are able to bind to multiple protein targets. In parallel to other subnuclear structures, such as PML bodies, the proteins that are involved in the formation and maintenance of the nucleolus are inexorably linked to nucleolar trafficking.

Pubmed          Journal

Abstract
Human adenovirus type 5 infection causes the disruption of structures in the cell nucleus termed promyelocytic leukaemia (PML) protein nuclear domains or ND10, which contain the PML protein as a critical component. This disruption is achieved through the action of the viral E4 Orf3 protein, which forms track-like nuclear structures that associate with the PML protein. This association is mediated by a direct interaction of Orf3 with a specific PML isoform, PMLII. We show here that the Orf3 interaction properties of PMLII are conferred by a 40 aa residue segment of the unique C-terminal domain of the protein. This segment was sufficient to confer interaction on a heterologous protein. The analysis was informed by prior application of a bioinformatic tool for the prediction of potential protein interaction sites within unstructured protein sequences (predictors of naturally disordered region analysis; PONDR). This tool predicted three potential molecular recognition elements (MoRE) within the C-terminal domain of PMLII, one of which was found to form the core of the Orf3 interaction site, thus demonstrating the utility of this approach. The sequence of the mapped Orf3-binding site on PML protein was found to be relatively poorly conserved across other species; however, the overall organization of MoREs within unstructured sequence was retained, suggesting the potential for conservation of functional interactions.

Pubmed          Journal

Abstract
Localisation of both viral and cellular proteins to the nucleolus is determined by a variety of factors including nucleolar localisation signals (NoLSs), but how these signals operate is not clearly understood. The nucleolar trafficking of wild type viral proteins and chimeric proteins, which contain altered NoLSs, were compared to investigate the role of NoLSs in dynamic nucleolar trafficking. Three viral proteins from diverse viruses were selected which localised to the nucleolus; the coronavirus infectious bronchitis virus nucleocapsid (N) protein, the herpesvirus saimiri ORF57 protein and the HIV-1 Rev protein. The chimeric proteins were N protein and ORF57 protein which had their own NoLS replaced with those from ORF57 and Rev proteins, respectively. By analysing the sub-cellular localisation and trafficking of these viral proteins and their chimeras within and between nucleoli using confocal microscopy and photo-bleaching we show that NoLSs are responsible for different nucleolar localisations and trafficking rates.

Journal       PeerJ Preprints

Abstract
Peer-reviewed journal publication is the main means for academic researchers in the life sciences to create a permanent, public record of their work. These publications are also the de facto currency for career progress, with a strong link between journal brand recognition and perceived value. The current peer-review process can lead to long delays between submission and publication, with cycles of rejection, revision and resubmission causing redundant peer review. This situation creates unique challenges for early career researchers (ECRs), who rely heavily on timely publication of their work to gain recognition for their efforts. ECRs face changes in the academic landscape including the increased interdisciplinarity of life sciences research, expansion of the researcher population and consequent shifts in employer and funding demands. The publication of preprints, publicly available scientific manuscripts posted on dedicated preprint servers prior to journal managed peer-review, can play a key role in addressing these ECR challenges. Preprinting benefits include rapid dissemination of academic work, open access, establishing priority or concurrence, receiving feedback and facilitating collaborations. While there is a growing appreciation for and adoption of preprints, a minority of all articles in life sciences and medicine are preprinted. The current low rate of preprint submissions in life sciences and ECR concerns regarding preprinting needs to be addressed. We provide a perspective from an interdisciplinary group of early career researchers on the value of preprints and advocate the wide adoption of preprints to advance knowledge and facilitate career development.

          Nature            List of Co-authors (SI)

Abstract
Following our PeerJ preprint (Maintaining confidence in the reporting of scientific outputs), I along with 8 co-authors published a correspondence in Nature. This appeared alongside two companion pieces – Preprints: safeguard rigour together, and Preprints: help not hinder journalism led by others in the open science and preprint comminity. We argue that suppressing preprints is harmful, and that if standard journalistic practices are followed the reporting of preprints poses no greater risk than reporting of peer reviewed articles.

          PeerJ Preprints

Abstract
The timely and accurate dissemination of scientific discoveries is of utmost importance so that scientific knowledge can be advanced and applied to benefit the public. Scientists communicate amongst themselves at conferences, via journal articles, and, increasingly in the life sciences, in preprint manuscripts which have not been subject to peer review. Journalists translate new research into a language the public can understand, relying on both work presented in scientific forums and interviews with experts. Critically, scientists and journalists both share the ethical principle that publications should be rigorously sourced and fact-checked, with errors subject to publicized corrections. Here we respond to concerns raised about the impact of reporting on results that have not passed through peer review, calling for improved dialogue between scientists and journalists to maintain public trust in research and arguing that imposing limits is against the public interest.

          The Conversation

Abstract
In a second article for The Conversation I talk about what are viruses really? Are they the particles we recognise from images in the media or actually the infected cells themselves? Even uninfected cells contain viruses (endogenous retroviruses) which are essential for life, and in infected cells there is vastly more ‘you’ involved in making new virus particles than comes from the virus itself. This article was subsequently taken up by a number of media outlets including IFLscience, phys.org, and the World Economic Forum.

          The Conversation

With just a tablespoon of norovirus you can infect a huge number of people (billions!). Image courtesy of Matt Cotton (@mlcotton13)

Abstract
Its peak norovirus season and hospitals are experiencing ward closures again. In this article I talk about how and why such an apparently mild virus is capable of causing this level of disruption in hospitals and what is being done to try and help avoid this in future. This article was originally written for The Conversation and was subsequently taken up by a variety of media outlets including IFLscience, the Independent, and the ‘i’ newspaper.