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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 an 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 into SaV-host interactions, and enabling the use of a new cell line model for PSaV research.
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Infecciones por Caliciviridae , Sapovirus , Animales , Línea Celular , Expresión Génica , Inmunidad Innata , Péptido Hidrolasas , Proteínas Serina-Treonina Quinasas , Sapovirus/genética , PorcinosRESUMEN
Vaccinia virus (VACV) is a large DNA virus that encodes scores of proteins that modulate the host immune response. VACV protein C4 is one such immunomodulator known to inhibit the activation of both the NF-κB signaling cascade and the DNA-PK-mediated DNA sensing pathway. Here, we show that the N-terminal region of C4, which neither inhibits NF-κB nor mediates interaction with DNA-PK, still contributes to virus virulence. Furthermore, this domain interacts directly and with high affinity to the C-terminal domain of filamin B (FLNB). FLNB is a large actin-binding protein that stabilizes the F-actin network and is implicated in other cellular processes. Deletion of FLNB from cells results in larger VACV plaques and increased infectious viral yield, indicating that FLNB restricts VACV spread. These data demonstrate that C4 has a new function that contributes to virulence and engages the cytoskeleton. Furthermore, we show that the cytoskeleton performs further previously uncharacterized functions during VACV infection. IMPORTANCE: Vaccinia virus (VACV), the vaccine against smallpox and monkeypox, encodes many proteins to counteract the host immune response. Investigating these proteins provides insights into viral immune evasion mechanisms and thereby indicates how to engineer safer and more immunogenic VACV-based vaccines. Here, we report that the N-terminal domain of VACV protein C4 interacts directly with the cytoskeletal protein filamin B (FLNB), and this domain of C4 contributes to virus virulence. Furthermore, VACV replicates and spreads better in cells lacking FLNB, thus demonstrating that FLNB has antiviral activity. VACV utilizes the cytoskeleton for movement within and between cells; however, previous studies show no involvement of C4 in VACV replication or spread. Thus, C4 associates with FLNB for a different reason, suggesting that the cytoskeleton has further uncharacterized roles during virus infection.
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Filaminas , Virus Vaccinia , Proteínas Virales , Humanos , Línea Celular , ADN/metabolismo , Filaminas/genética , Filaminas/metabolismo , FN-kappa B/metabolismo , Vaccinia/virología , Virus Vaccinia/patogenicidad , Virus Vaccinia/fisiología , Proteínas Virales/genética , Proteínas Virales/metabolismo , AnimalesRESUMEN
BACKGROUND: There have been dramatic clinical improvements in people with cystic fibrosis (PwCF) 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 PwCF. 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.
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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 localizes to mitochondria, where it inhibits innate immunity by restricting IFN-ß production, but not NF-κB activation or JAK-STAT signaling 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 uncharacterized mechanism of innate immune evasion by this important human pathogen.
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Major aims of single-cell proteomics include increasing the consistency, sensitivity and depth of protein quantification, especially for proteins and modifications of biological interest. Here, to simultaneously advance all these aims, we developed prioritized Single-Cell ProtEomics (pSCoPE). pSCoPE consistently analyzes thousands of prioritized peptides across all single cells (thus increasing data completeness) while maximizing instrument time spent analyzing identifiable peptides, thus increasing proteome depth. These strategies increased the sensitivity, data completeness and proteome coverage over twofold. The gains enabled quantifying protein variation in untreated and lipopolysaccharide-treated primary macrophages. Within each condition, proteins covaried within functional sets, including phagosome maturation and proton transport, similarly across both treatment conditions. This covariation is coupled to phenotypic variability in endocytic activity. pSCoPE also enabled quantifying proteolytic products, suggesting a gradient of cathepsin activities within a treatment condition. pSCoPE is freely available and widely applicable, especially for analyzing proteins of interest without sacrificing proteome coverage. Support for pSCoPE is available at http://scp.slavovlab.net/pSCoPE .
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Proteoma , Proteómica , Proteoma/análisis , Proteómica/métodos , Espectrometría de Masas , Péptidos/química , MacrófagosRESUMEN
Analyzing proteins from single cells by tandem mass spectrometry (MS) has recently 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. We expect that broadly accepted community guidelines and standardized metrics will enhance rigor, data quality and alignment between laboratories. Here we propose best practices, quality controls and data-reporting recommendations to assist in the broad adoption of reliable quantitative workflows for single-cell proteomics. Resources and discussion forums are available at https://single-cell.net/guidelines .
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Benchmarking , Proteómica , Benchmarking/métodos , Proteómica/métodos , Reproducibilidad de los Resultados , Proteínas/análisis , Espectrometría de Masas en Tándem/métodos , Proteoma/análisisRESUMEN
The exact function(s) of the lagovirus non-structural protein p23 is unknown as robust cell culture systems for the Rabbit haemorrhagic disease virus (RHDV) and other lagoviruses have not been established. Instead, a range of in vitro and in silico models have been used to study p23, revealing that p23 oligomerizes, accumulates in the cytoplasm, and possesses a conserved C-terminal region with two amphipathic helices. Furthermore, the positional homologs of p23 in other caliciviruses have been shown to possess viroporin activity. Here, we report on the mechanistic details of p23 oligomerization. Site-directed mutagenesis revealed the importance of an N-terminal cysteine for dimerization. Furthermore, we identified cellular interactors of p23 using stable isotope labeling with amino acids in cell culture (SILAC)-based proteomics; heat shock proteins Hsp70 and 110 interact with p23 in transfected cells, suggesting that they 'chaperone' p23 proteins before their integration into cellular membranes. We investigated changes to the global transcriptome and proteome that occurred in infected rabbit liver tissue and observed changes to the misfolded protein response, calcium signaling, and the regulation of the endoplasmic reticulum (ER) network. Finally, flow cytometry studies indicate slightly elevated calcium concentrations in the cytoplasm of p23-transfected cells. Taken together, accumulating evidence suggests that p23 is a viroporin that might form calcium-conducting channels in the ER membranes.
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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.
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Péptidos/aislamiento & purificación , Proteoma/aislamiento & purificación , Proteómica/métodos , Análisis de la Célula Individual/métodos , Animales , Benchmarking , Cromatografía Liquida/métodos , Cromatografía Liquida/normas , Células HeLa , Humanos , Indicadores y Reactivos/química , Ratones , Oocitos/citología , Oocitos/metabolismo , Péptidos/química , Péptidos/clasificación , Cultivo Primario de Células , Proteoma/química , Proteoma/clasificación , Células RAW 264.7 , Análisis de la Célula Individual/normas , Espectrometría de Masas en Tándem/métodos , Espectrometría de Masas en Tándem/normas , Células U937RESUMEN
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.
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Proteínas , Proteómica , Biblioteca de Genes , Técnicas Genéticas , Péptidos , Proteínas/análisisRESUMEN
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.
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Antivirales/farmacología , COVID-19/metabolismo , Inhibidores de Proteasas/farmacología , SARS-CoV-2/efectos de los fármacos , Animales , Línea Celular , Dipéptidos/farmacología , Humanos , Mutación , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Quinasa de Cadena Ligera de Miosina/genética , Quinasa de Cadena Ligera de Miosina/metabolismo , Proteolisis , Proteómica , ARN Interferente Pequeño/farmacología , SARS-CoV-2/genética , Proteasas Virales/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismo , Tratamiento Farmacológico de COVID-19RESUMEN
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 virus (VESV) 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" that both plays an important role in 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. IMPORTANCE Vesivirus 2117 has caused significant losses in manufacturing of biopharmaceutical products following contamination of cell cultures used in their production. We report the structure of the vesivirus 2117 capsid, the shell that encloses the virus's genome. Comparison of this structure with that of a related vesivirus, feline calicivirus (FCV), highlighted potentially important differences related to virus attachment and entry. Our findings suggest that these two viruses may bind differently to receptors at the host cell surface. We also show that a region of the capsid protein of FCV that rearranges following receptor engagement is not present in vesivirus 2117. These structural changes in the FCV capsid have been shown to allow the assembly of a portal-like structure that is hypothesized to deliver the viral genome to the cell's interior. Our data suggest that the 2117 portal assembly may employ a different means of anchoring to the outer face of the capsid.
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Cápside/metabolismo , Vesivirus/metabolismo , Animales , Células CHO , Proteínas de la Cápside/genética , Línea Celular , Cricetinae , Cricetulus , Microscopía por Crioelectrón , Estructura Cuaternaria de Proteína/fisiología , Virión/metabolismo , Acoplamiento ViralRESUMEN
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.
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Macrófagos/metabolismo , Proteómica , Transcriptoma , Línea Celular , Citocinas/genética , Proteínas en la Dieta , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Células HEK293 , Humanos , Activación de Macrófagos , Monocitos/metabolismo , Fenotipo , Proteoma/genéticaRESUMEN
Herpesviruses are ubiquitous in the human population and they extensively remodel the cellular environment during infection. Multiplexed quantitative proteomic analysis over the time course of herpes simplex virus 1 (HSV-1) infection was used to characterize changes in the host-cell proteome and the kinetics of viral protein production. Several host-cell proteins are targeted for rapid degradation by HSV-1, including the cellular trafficking factor Golgi-associated PDZ and coiled-coil motif-containing protein (GOPC). We show that the poorly characterized HSV-1 pUL56 directly binds GOPC, stimulating its ubiquitination and proteasomal degradation. Plasma membrane profiling reveals that pUL56 mediates specific changes to the cell-surface proteome of infected cells, including loss of interleukin-18 (IL18) receptor and Toll-like receptor 2 (TLR2), and that cell-surface expression of TLR2 is GOPC dependent. Our study provides significant resources for future investigation of HSV-host interactions and highlights an efficient mechanism whereby a single virus protein targets a cellular trafficking factor to modify the surface of infected cells.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de la Matriz de Golgi/metabolismo , Herpesvirus Humano 1/metabolismo , Proteómica/métodos , Células HEK293 , Humanos , TransfecciónAsunto(s)
Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/terapia , Espectrometría de Masas , Neumonía Viral/diagnóstico , Neumonía Viral/terapia , Betacoronavirus , Biomarcadores , COVID-19 , Biología Computacional , Humanos , Difusión de la Información , Pandemias , Pronóstico , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/químicaRESUMEN
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 ribosome recruitment. Our data uncovers a novel function for G3BP1 in the life cycle of positive sense RNA viruses and identifies the first host factor with pan-norovirus pro-viral activity.
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ADN Helicasas/metabolismo , Interacciones Huésped-Patógeno , Norovirus/crecimiento & desarrollo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Biosíntesis de Proteínas , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas Virales/biosíntesis , Animales , Infecciones por Caliciviridae , Línea Celular , Humanos , RatonesRESUMEN
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 (interferon induced proteins with tetratricopeptide repeats), 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 to 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.
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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. Today, ECRs face a changing 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. Although 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 need to be addressed. We provide a perspective from an interdisciplinary group of ECRs on the value of preprints and advocate their wide adoption to advance knowledge and facilitate career development.
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Revisión de la Investigación por Pares/métodos , Preimpresos como Asunto , Investigadores/psicología , Investigación Biomédica , Movilidad Laboral , Humanos , Publicaciones Periódicas como AsuntoRESUMEN
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. The regulation of protein synthesis by specialized ribosomes can be examined by multiple methods. These include the popular 'Ribo-Seq' method for analyzing ribosome density on a given mRNA, as well as LC-MS/MS approaches incorporating pulse-labelling with stable isotopes (SILAC) to monitor protein synthesis and degradation.
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ARN Ribosómico/química , Ribosomas/química , Cromatografía Liquida , Modelos Moleculares , Proteínas/química , Espectrometría de Masas en TándemRESUMEN
Norovirus infections are a major cause of acute viral gastroenteritis and a significant burden on global human health. A vital process for norovirus replication is the processing of the nonstructural polyprotein by a viral protease into the viral components required to form the viral replication complex. This cleavage occurs at different rates, resulting in the accumulation of stable precursor forms. Here, we characterized how precursor forms of the norovirus protease accumulate during infection. Using stable forms of the protease precursors, we demonstrated that all of them are proteolytically active in vitro, but that when expressed in cells, their activities are determined by both substrate and protease localization. Although all precursors could cleave a replication complex-associated substrate, only a subset of precursors lacking the NS4 protein were capable of efficiently cleaving a cytoplasmic substrate. By mapping the full range of protein-protein interactions among murine and human norovirus proteins with the LUMIER assay, we uncovered conserved interactions between replication complex members that modify the localization of a protease precursor subset. Finally, we demonstrate that fusion to the membrane-bound replication complex components permits efficient cleavage of a fused substrate when active polyprotein-derived protease is provided in trans These findings offer a model for how norovirus can regulate the timing of substrate cleavage throughout the replication cycle. Because the norovirus protease represents a key target in antiviral therapies, an improved understanding of its function and regulation, as well as identification of interactions among the other nonstructural proteins, offers new avenues for antiviral drug design.
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Norovirus/enzimología , Norovirus/metabolismo , Péptido Hidrolasas/metabolismo , Poliproteínas/metabolismo , Replicación Viral , Animales , Infecciones por Caliciviridae/metabolismo , Infecciones por Caliciviridae/virología , Línea Celular , Células HeLa , Humanos , Ratones , Norovirus/genética , Péptido Hidrolasas/genética , Unión Proteica , Replicación Viral/genéticaRESUMEN
The existence of eukaryotic ribosomes with distinct ribosomal protein (RP) stoichiometry and regulatory roles in protein synthesis has been speculated for over 60 years. Recent advances in mass spectrometry (MS) and high-throughput analysis have begun to identify and characterize distinct ribosome stoichiometry in yeast and mammalian systems. In addition to RP stoichiometry, ribosomes host a vast array of protein modifications, effectively expanding the number of human RPs from 80 to many thousands of distinct proteoforms. Is it possible that 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.