Rational design of highly potent broad-spectrum enterovirus inhibitors targeting the nonstructural protein 2C.

There is a great need for antiviral drugs to treat enterovirus (EV) and rhinovirus (RV) infections, which can be severe and occasionally life-threatening. The conserved nonstructural protein 2C, which is an AAA+ ATPase, is a promising target for drug development. Here, we present a structure-activity relationship study of a previously identified compound that targets the 2C protein of EV-A71 and several EV-B species members, but not poliovirus (PV) (EV-C species). This compound is structurally related to the Food and Drug Administration (FDA)-approved drug fluoxetine-which also targets 2C-but has favorable chemical properties. We identified several compounds with increased antiviral potency and broadened activity. Four compounds showed broad-spectrum EV and RV activity and inhibited contemporary strains of emerging EVs of public health concern, including EV-A71, coxsackievirus (CV)-A24v, and EV-D68. Importantly, unlike (S)-fluoxetine, these compounds are no longer neuroactive. By raising resistant EV-A71, CV-B3, and EV-D68 variants against one of these inhibitors, we identified novel 2C resistance mutations. Reverse engineering of these mutations revealed a conserved mechanism of resistance development. Resistant viruses first acquired a mutation in, or adjacent to, the α2 helix of 2C. This mutation disrupted compound binding and provided drug resistance, but this was at the cost of viral fitness. Additional mutations at distantly localized 2C residues were then acquired to increase resistance and/or to compensate for the loss of fitness. Using computational methods to identify solvent accessible tunnels near the α2 helix in the EV-A71 and PV 2C crystal structures, a conserved binding pocket of the inhibitors is proposed.

Characterization of the c10orf76-PI4KB complex and its necessity for Golgi PI4P levels and enterovirus replication.

The lipid kinase PI4KB, which generates phosphatidylinositol 4-phosphate (PI4P), is a key enzyme in regulating membrane transport and is also hijacked by multiple picornaviruses to mediate viral replication. PI4KB can interact with multiple protein binding partners, which are differentially manipulated by picornaviruses to facilitate replication. The protein c10orf76 is a PI4KB-associated protein that increases PI4P levels at the Golgi and is essential for the viral replication of specific enteroviruses. We used hydrogen-deuterium exchange mass spectrometry to characterize the c10orf76-PI4KB complex and reveal that binding is mediated by the kinase linker of PI4KB, with formation of the heterodimeric complex modulated by PKA-dependent phosphorylation. Complex-disrupting mutations demonstrate that PI4KB is required for membrane recruitment of c10orf76 to the Golgi, and that an intact c10orf76-PI4KB complex is required for the replication of c10orf76-dependent enteroviruses. Intriguingly, c10orf76 also contributed to proper Arf1 activation at the Golgi, providing a putative mechanism for the c10orf76-dependent increase in PI4P levels at the Golgi.

Convergent evolution in the mechanisms of ACBD3 recruitment to picornavirus replication sites.

Enteroviruses, members of the family of picornaviruses, are the most common viral infectious agents in humans causing a broad spectrum of diseases ranging from mild respiratory illnesses to life-threatening infections. To efficiently replicate within the host cell, enteroviruses hijack several host factors, such as ACBD3. ACBD3 facilitates replication of various enterovirus species, however, structural determinants of ACBD3 recruitment to the viral replication sites are poorly understood. Here, we present a structural characterization of the interaction between ACBD3 and the non-structural 3A proteins of four representative enteroviruses (poliovirus, enterovirus A71, enterovirus D68, and rhinovirus B14). In addition, we describe the details of the 3A-3A interaction causing the assembly of the ACBD3-3A heterotetramers and the interaction between the ACBD3-3A complex and the lipid bilayer. Using structure-guided identification of the point mutations disrupting these interactions, we demonstrate their roles in the intracellular localization of these proteins, recruitment of downstream effectors of ACBD3, and facilitation of enterovirus replication. These structures uncovered a striking convergence in the mechanisms of how enteroviruses and kobuviruses, members of a distinct group of picornaviruses that also rely on ACBD3, recruit ACBD3 and its downstream effectors to the sites of viral replication.

Targeting of the hydrophobic metabolome by pathogens.

The hydrophobic molecules of the metabolome - also named the lipidome - constitute a major part of the entire metabolome. Novel technologies show the existence of a staggering number of individual lipid species, the biological functions of which are, with the exception of only a few lipid species, unknown. Much can be learned from pathogens that have evolved to take advantage of the complexity of the lipidome to escape the immune system of the host organism and to allow their survival and replication. Different types of pathogens target different lipids as shown in interaction maps, allowing visualization of differences between different types of pathogens. Bacterial and viral pathogens target predominantly structural and signaling lipids to alter the cellular phenotype of the host cell. Fungal and parasitic pathogens have complex lipidomes themselves and target predominantly the release of polyunsaturated fatty acids from the host cell lipidome, resulting in the generation of eicosanoids by either the host cell or the pathogen. Thus, whereas viruses and bacteria induce predominantly alterations in lipid metabolites at the host cell level, eukaryotic pathogens focus on interference with lipid metabolites affecting systemic inflammatory reactions that are part of the immune system. A better understanding of the interplay between host-pathogen interactions will not only help elucidate the fundamental role of lipid species in cellular physiology, but will also aid in the generation of novel therapeutic drugs.

Modulation of the Host Lipid Landscape to Promote RNA Virus Replication: The Picornavirus Encephalomyocarditis Virus Converges on the Pathway Used by Hepatitis C Virus.

Cardioviruses, including encephalomyocarditis virus (EMCV) and the human Saffold virus, are small non-enveloped viruses belonging to the Picornaviridae, a large family of positive-sense RNA [(+)RNA] viruses. All (+)RNA viruses remodel intracellular membranes into unique structures for viral genome replication. Accumulating evidence suggests that picornaviruses from different genera use different strategies to generate viral replication organelles (ROs). For instance, enteroviruses (e.g. poliovirus, coxsackievirus, rhinovirus) rely on the Golgi-localized phosphatidylinositol 4-kinase III beta (PI4KB), while cardioviruses replicate independently of the kinase. By which mechanisms cardioviruses develop their ROs is currently unknown. Here we show that cardioviruses manipulate another PI4K, namely the ER-localized phosphatidylinositol 4-kinase III alpha (PI4KA), to generate PI4P-enriched ROs. By siRNA-mediated knockdown and pharmacological inhibition, we demonstrate that PI4KA is an essential host factor for EMCV genome replication. We reveal that the EMCV nonstructural protein 3A interacts with and is responsible for PI4KA recruitment to viral ROs. The ensuing phosphatidylinositol 4-phosphate (PI4P) proved important for the recruitment of oxysterol-binding protein (OSBP), which delivers cholesterol to EMCV ROs in a PI4P-dependent manner. PI4P lipids and cholesterol are shown to be required for the global organization of the ROs and for viral genome replication. Consistently, inhibition of OSBP expression or function efficiently blocked EMCV RNA replication. In conclusion, we describe for the first time a cellular pathway involved in the biogenesis of cardiovirus ROs. Remarkably, the same pathway was reported to promote formation of the replication sites of hepatitis C virus, a member of the Flaviviridae family, but not other picornaviruses or flaviviruses. Thus, our results highlight the convergent recruitment by distantly related (+)RNA viruses of a host lipid-modifying pathway underlying formation of viral replication sites.

Tyrphostin AG1478 Inhibits Encephalomyocarditis Virus and Hepatitis C Virus by Targeting Phosphatidylinositol 4-Kinase IIIα.

Encephalomyocarditis virus (EMCV), like hepatitis C virus (HCV), requires phosphatidylinositol 4-kinase IIIα (PI4KA) for genome replication. Here, we demonstrate that tyrphostin AG1478, a known epidermal growth factor receptor (EGFR) inhibitor, also inhibits PI4KA activity, both in vitro and in cells. AG1478 impaired replication of EMCV and HCV but not that of an EMCV mutant previously shown to escape PI4KA inhibition. This work uncovers novel cellular and antiviral properties of AG1478, a compound previously regarded only as a cancer chemotherapy agent.

Cholesterol shuttling is important for RNA replication of coxsackievirus B3 and encephalomyocarditis virus.

Picornaviruses are a family of positive-strand RNA viruses that includes important human and animal pathogens. Upon infection, picornaviruses induce an extensive remodelling of host cell membranes into replication organelles (ROs), which is critical for replication. Membrane lipids and lipid remodelling processes are at the base of RO formation, yet their involvement remains largely obscure. Recently, phosphatidylinositol-4-phosphate was the first lipid discovered to be important for the replication of a number of picornaviruses. Here, we investigate the role of the lipid cholesterol in picornavirus replication. We show that two picornaviruses from distinct genera that rely on different host factors for replication, namely the enterovirus coxsackievirus B3 (CVB3) and the cardiovirus encephalomyocarditis virus (EMCV), both recruited cholesterol to their ROs. Although CVB3 and EMCV both required cholesterol for efficient genome replication, the viruses appeared to rely on different cellular cholesterol pools. Treatments that altered the distribution of endosomal cholesterol inhibited replication of both CVB3 and EMCV, showing the importance of endosomal cholesterol shuttling for the replication of these viruses. Summarizing, we here demonstrate the importance of cholesterol homeostasis for efficient replication of CVB3 and EMCV.

Recruitment of PI4KIIIß to coxsackievirus B3 replication organelles is independent of ACBD3, GBF1, and Arf1.

UNLABELLED: Members of the Enterovirus (poliovirus [PV], coxsackieviruses, and human rhinoviruses) and Kobuvirus (Aichi virus) genera in the Picornaviridae family rely on PI4KIIIß (phosphatidylinositol-4-kinase IIIß) for efficient replication. The small membrane-anchored enteroviral protein 3A recruits PI4KIIIß to replication organelles, yet the underlying mechanism has remained elusive. Recently, it was shown that kobuviruses recruit PI4KIIIß through interaction with ACBD3 (acyl coenzyme A [acyl-CoA]-binding protein domain 3), a novel interaction partner of PI4KIIIß. Therefore, we investigated a possible role for ACBD3 in recruiting PI4KIIIß to enterovirus replication organelles. Although ACBD3 interacted directly with coxsackievirus B3 (CVB3) 3A, its depletion from cells by RNA interference did not affect PI4KIIIß recruitment to replication organelles and did not impair CVB3 RNA replication. Enterovirus 3A was previously also proposed to recruit PI4KIIIß via GBF1/Arf1, based on the known interaction of 3A with GBF1, an important regulator of secretory pathway transport and a guanine nucleotide exchange factor (GEF) of Arf1. However, our results demonstrate that inhibition of GBF1 or Arf1 either by pharmacological inhibition or depletion with small interfering RNA (siRNA) treatment did not affect the ability of 3A to recruit PI4KIIIß. Furthermore, we show that a 3A mutant that no longer binds GBF1 was capable of recruiting PI4KIIIß, even in ACBD3-depleted cells. Together, our findings indicate that unlike originally envisaged, coxsackievirus recruits PI4KIIIß to replication organelles independently of ACBD3 and GBF1/Arf1. IMPORTANCE: A hallmark of enteroviral infection is the generation of new membranous structures to support viral RNA replication. The functionality of these "replication organelles" depends on the concerted actions of both viral nonstructural proteins and co-opted host factors. It is thus essential to understand how these structures are formed and which cellular components are key players in this process. GBF1/Arf1 and ACBD3 have been proposed to contribute to the recruitment of the essential lipid-modifying enzyme PI4KIIIß to enterovirus replication organelles. Here we show that the enterovirus CVB3 recruits PI4KIIIß by a mechanism independent of both GBF1/Arf1 and ACBD3. This study shows that the strategy employed by coxsackievirus to recruit PI4KIIIß to replication organelles is far more complex than initially anticipated.

Distinct role of subcomplexes of the COPI coat in the regulation of ArfGAP2 activity.

COPI vesicles serve for transport of proteins and membrane lipids in the early secretory pathway. Their coat protein (coatomer) is a heptameric complex that is recruited to the Golgi by the small GTPase Arf1. Although recruited en bloc, coatomer can be viewed as a stable assembly of an adaptin-like tetrameric subcomplex (CM4) and a trimeric 'cage' subcomplex (CM3). Following recruitment, coatomer stimulates ArfGAP-dependent GTP hydrolysis on Arf1. Here, we employed recombinant coatomer subcomplexes to study the role of coatomer components in the regulation of ArfGAP2, an ArfGAP whose activity is strictly coatomer-dependent. Within CM4, we define a novel hydrophobic pocket for ArfGAP2 interaction on the appendage domain of γ1-COP. The CM4 subcomplex (but not CM3) is recruited to membranes through Arf1 and can subsequently recruit ArfGAP2. Neither CM3 nor CM4 in itself is effective in stimulating ArfGAP2 activity, but stimulation is regained when both subcomplexes are present. Our findings point to a distinct role of each of the two coatomer subcomplexes in the regulation of ArfGAP2-dependent GTP hydrolysis on Arf1, where the CM4 subcomplex functions in GAP recruitment, while, similarly to the COPII system, the cage-like CM3 subcomplex stimulates the catalytic reaction.

Phenotypic profiling of the human genome reveals gene products involved in plasma membrane targeting of SRC kinases.

SRC proteins are non-receptor tyrosine kinases that play key roles in regulating signal transduction by a diverse set of cell surface receptors. They contain N-terminal SH4 domains that are modified by fatty acylation and are functioning as membrane anchors. Acylated SH4 domains are both necessary and sufficient to mediate specific targeting of SRC kinases to the inner leaflet of plasma membranes. Intracellular transport of SRC kinases to the plasma membrane depends on microdomains into which SRC kinases partition upon palmitoylation. In the present study, we established a live-cell imaging screening system to identify gene products involved in plasma membrane targeting of SRC kinases. Based on siRNA arrays and a human model cell line expressing two kinds of SH4 reporter molecules, we conducted a genome-wide analysis of SH4-dependent protein targeting using an automated microscopy platform. We identified and validated 54 gene products whose down-regulation causes intracellular retention of SH4 reporter molecules. To detect and quantify this phenotype, we developed a software-based image analysis tool. Among the identified gene products, we found factors involved in lipid metabolism, intracellular transport, and cellular signaling processes. Furthermore, we identified proteins that are either associated with SRC kinases or are related to various known functions of SRC kinases such as other kinases and phosphatases potentially involved in SRC-mediated signal transduction. Finally, we identified gene products whose function is less defined or entirely unknown. Our findings provide a major resource for future studies unraveling the molecular mechanisms that underlie proper targeting of SRC kinases to the inner leaflet of plasma membranes.

Recombinant heptameric coatomer complexes: novel tools to study isoform-specific functions.

COPI (coat protein I)-coated vesicles are implicated in various transport steps within the early secretory pathway. The major structural component of the COPI coat is the heptameric complex coatomer (CM). Recently, four isoforms of CM were discovered that may help explain various transport steps in which the complex has been reported to be involved. Biochemical studies of COPI vesicles currently use CM purified from animal tissue or cultured cells, a mixture of the isoforms, impeding functional and structural studies of individual complexes. Here we report the cloning into single baculoviruses of all CM subunits including their isoforms and their combination for expression of heptameric CM isoforms in insect cells. We show that all four isoforms of recombinant CM are fully functional in an in vitro COPI vesicle biogenesis assay. These novel tools enable functional and structural studies on CM isoforms and their subcomplexes and allow studying mutants of CM.

Genetic and phenotypic stability of poliovirus shed from infants who received novel type 2 or Sabin type 2 oral poliovirus vaccines in Panama: an analysis of two clinical trials.

BACKGROUND: Sabin strains used in oral poliovirus vaccines (OPV) can revert to virulence and, in rare instances, cause disease or generate vaccine-derived strains leading to outbreaks in areas of low immunisation coverage. A novel OPV2 (nOPV2) was designed to stabilise the viral genome against reversion and reduce recombination events that might lead to virulent strains. In this study, we evaluated the genetic and phenotypic stability of shed poliovirus following administration of one dose of monovalent OPV2 (mOPV2) or nOPV2 to infants aged 18-22 weeks. METHODS: In two similarly designed clinical trials (NCT02521974 and NCT03554798) conducted in Panama, infants aged 18-22-weeks, after immunisation with three doses of bivalent OPV (types 1 and 3) and one dose of inactivated poliovirus vaccine, were administered one or two doses of mOPV2 or nOPV2. In this analysis of two clinical trials, faecally shed polioviruses following one dose of mOPV2 or nOPV2 were isolated from stools meeting predetermined criteria related to sample timing and viral presence and quantity and assessed for nucleotide polymorphisms using next-generation sequencing. A transgenic mouse neurovirulence test was adapted to assess the effect of the possible phenotypic reversion of shed mOPV2 and nOPV2 with a logistic regression model. FINDINGS: Of the 91 eligible samples, 86 were able to be sequenced, with 72 evaluated in the transgenic mouse assay. Sabin-2 poliovirus reverts rapidly at nucleotide 481, the primary attenuation site in domain V of the 5' untranslated region of the genome. There was no evidence of neurovirulence-increasing polymorphisms in domain V of shed nOPV2. Reversion of shed Sabin-2 virus corresponded with unadjusted paralysis rates of 47·6% at the 4 log10 50% cell culture infectious dose (CCID50) and 76·7% at the 5 log10 CCID50 inoculum levels, with rates of 2·8% for 4 log10 CCID50 and 11·8% for 5 log10 CCID50 observed for shed nOPV2 samples. The estimated adjusted odds ratio at 4·5 log10 of 0·007 (95% CI 0·002-0·023; p<0·0001) indicates significantly reduced odds of mouse paralysis from virus obtained from nOPV2 recipients compared with mOPV2 recipients. INTERPRETATION: The data indicate increased genetic stability of domain V of nOPV2 relative to mOPV2, with significantly lower neurovirulence of shed nOPV2 virus compared with shed mOPV2. While this vaccine is currently being deployed under an emergency use listing, the data on the genetic stability of nOPV2 will support further regulatory and policy decision-making regarding use of nOPV2 in outbreak responses. FUNDING: Bill & Melinda Gates Foundation.

Evaluation and validation of next-generation sequencing to support lot release for a novel type 2 oral poliovirus vaccine.

A novel, genetically-stabilized type 2 oral polio vaccine (nOPV2), developed to assist in the global polio eradication program, was recently the first-ever vaccine granted Emergency Use Listing by the WHO. Lot release tests for this vaccine included-for the first time to our knowledge-the assessment of genetic heterogeneity using next-generation sequencing (NGS). NGS ensures that the genetically-modified regions of the vaccine virus genome remain as designed and that levels of polymorphisms which may impact safety or efficacy are controlled during routine production. The variants present in nOPV2 lots were first assessed for temperature sensitivity and neurovirulence using molecular clones to inform which polymorphisms warranted formal evaluation during lot release. The novel use of NGS as a lot release test required formal validation of the method. Analysis of an nOPV2 lot spiked with the parental Sabin-2 strain enabled performance characteristics of the method to be assessed simultaneously at over 40 positions in the genome. These characteristics included repeatability and intermediate precision of polymorphism measurement, linearity of both spike-induced and nOPV2 lot-specific polymorphisms, and the limit-of-detection of spike-induced polymorphisms. The performance characteristics of the method met pre-defined criteria for 34 spike-induced polymorphic sites and 8 polymorphisms associated with the nOPV2 preparation; these sites collectively spanned most of the viral genome. Finally, the co-location of variants of interest on genomes was evaluated, with implications for the interpretation of NGS discussed.

A comprehensive overview of the vertebrate p24 family: identification of a novel tissue-specifically expressed member.

The members of the p24 protein family have an important but unclear role in transport processes in the early secretory pathway. The p24 family consists of four subfamilies (alpha, beta, gamma, and delta), whereby the exact composition of the family varies among species. Despite more than 15 years of p24 research, the vertebrate p24 family is still surprisingly ill characterized. Here, we describe the human, mouse, Xenopus, and zebrafish orthologues of 10 p24 family members and a new member that we term p24gamma(5). Of these eleven p24 family members, nine are conserved throughout the vertebrate lineage, whereas two (p24gamma(4) and p24delta(2)) occur in some but not all vertebrates. We further show that all p24 proteins are widely expressed in mouse, except for p24alpha(1) and p24gamma(5) that display restricted expression patterns. Thus, we present for the first time a comprehensive overview of the phylogeny and expression of the vertebrate p24 protein family.

The p24 family and selective transport processes at the ER-Golgi interface.

The secretory pathway is of vital importance for eukaryotic cells and has a pivotal role in the synthesis, sorting, processing and secretion of a large variety of bioactive molecules involved in intercellular communication. One of the key processes in the secretory pathway concerns the transport of cargo proteins from the ER (endoplasmic reticulum) to the Golgi. Type-I transmembrane proteins of approximately 24 kDa are abundantly present in the membranes of the early secretory pathway, and bind the COPI and COPII coat complexes that cover vesicles travelling between the membranes. These p24 proteins are thought to play an important role in the selective transport processes at the ER-Golgi interface, although their exact functioning is still obscure. One model proposes that p24 proteins couple cargo selection in the lumen with vesicle coat recruitment in the cytosol. Alternatively, p24 proteins may furnish subcompartments of the secretory pathway with the correct subsets of machinery proteins. Here we review the current knowledge of the p24 proteins and the various roles proposed for the p24 family members.

Functional diversity among p24 subfamily members.

BACKGROUND INFORMATION: The p24 protein family plays an important but unclear role at the ER (endoplasmic reticulum)-Golgi interface. A p24 member from each subfamily (p24alpha(3), beta(1), gamma(3) and delta(2)) is upregulated with the prohormone POMC (pro-opiomelanocortin) when Xenopus laevis intermediate pituitary melanotrope cells are physiologically activated. Here we explored the role of p24 by generating and analysing Xenopus with melanotrope cell-specific transgene expression of p24beta(1) or p24gamma(3), two of the p24 proteins coexpressed with POMC, and compared the results with those previously reported for the two other coexpressed p24s (p24alpha(3) and p24delta(2)). RESULTS: The transgene expression of p24beta(1) or p24gamma(3) did not affect the endogenous p24 proteins or affected only endogenous p24gamma(3) respectively, whereas in transgenics expressing p24alpha(3) and p24delta(2), the levels of all endogenous p24 proteins were strongly decreased. Nevertheless, as for p24alpha(3) but albeit to a lesser extent, in the p24beta(1)-transgenic melanotrope cells the rate of cargo cleavage was reduced, probably reflecting reduced cargo transport from the ER, and POMC glycosylation and sulfation in the Golgi were not affected. The p24gamma(3)-transgenic cells displayed features of both the p24alpha(3)-transgenics (reduced cargo cleavage, normal POMC sulfation) and the p24delta(2)-transgenics (affected POMC glycosylation). CONCLUSIONS: Our results show that the four upregulated proteins p24alpha(3), beta(1), gamma(3) and delta(2) have non-redundant roles in the early secretory pathway, and suggest that each p24 subfamily member provides a proper ER/Golgi subcompartmental microenvironment, together allowing correct secretory protein transport and processing.

Synthesis and antiviral effect of novel fluoxetine analogues as enterovirus 2C inhibitors.

Enteroviruses (EV) are a group of positive-strand RNA (+RNA) viruses that include many important human pathogens (e.g. poliovirus, coxsackievirus, echovirus, numbered enteroviruses and rhinoviruses). Fluoxetine was identified in drug repurposing screens as potent inhibitor of enterovirus B and enterovirus D replication. In this paper we are reporting the synthesis and the antiviral effect of a series of fluoxetine analogues. The results obtained offer a preliminary insight into the structure-activity relationship of its chemical scaffold and confirm the importance of the chiral configuration. We identified a racemic fluoxetine analogue, 2b, which showed a similar antiviral activity compared to (S)-fluoxetine. Investigating the stereochemistry of 2b revealed that the S-enantiomer exerts potent antiviral activity and increased the antiviral spectrum compared to the racemic mixture of 2b. In line with the observed antiviral effect, the S-enantiomer displayed a dose-dependent shift in the melting temperature in thermal shift assays, indicative for direct binding to the recombinant 2C protein.

Dynamic remodelling of the human host cell proteome and phosphoproteome upon enterovirus infection.

The group of enteroviruses contains many important pathogens for humans, including poliovirus, coxsackievirus, rhinovirus, as well as newly emerging global health threats such as EV-A71 and EV-D68. Here, we describe an unbiased, system-wide and time-resolved analysis of the proteome and phosphoproteome of human cells infected with coxsackievirus B3. Of the ~3,200 proteins quantified throughout the time course, a large amount (~25%) shows a significant change, with the majority being downregulated. We find ~85% of the detected phosphosites to be significantly regulated, implying that most changes occur at the post-translational level. Kinase-motif analysis reveals temporal activation patterns of certain protein kinases, with several CDKs/MAPKs immediately active upon the infection, and basophilic kinases, ATM, and ATR engaging later. Through bioinformatics analysis and dedicated experiments, we identify mTORC1 signalling as a major regulation network during enterovirus infection. We demonstrate that inhibition of mTORC1 activates TFEB, which increases expression of lysosomal and autophagosomal genes, and that TFEB activation facilitates the release of virions in extracellular vesicles via secretory autophagy. Our study provides a rich framework for a system-level understanding of enterovirus-induced perturbations at the protein and signalling pathway levels, forming a base for the development of pharmacological inhibitors to treat enterovirus infections.

Incomplete posttranslational prohormone modifications in hyperactive neuroendocrine cells.

BACKGROUND: In black-background-adapted Xenopus laevis, the intermediate pituitary melanotrope cells are hyperactive, producing large amounts of their major secretory cargo proopiomelanocortin (POMC, representing ~80% of all newly synthesised proteins), whereas in white-adapted frogs these cells are only basally active. Here we explored in the hyperactive and basally active melanotrope cells the capacity for posttranslational POMC processing events in the secretory pathway. RESULTS: We found that the hyperactive cells produced mainly non-complex N-glycosylated POMC, whereas in the basally active cells POMC was mostly complex N-glycosylated. Furthermore, the relative level of POMC sulphation was ~5.5-fold lower in the hyperactive than in the basally active cells. When the cargo load in the secretory pathway of the hyperactive cells was pharmacologically reduced, the relative amount of complex glycosylated POMC markedly increased. CONCLUSION: Collectively, our data show that the secretory pathway in hyperactive neuroendocrine secretory cells lacks the capacity to fully comply with the high demands for complex glycosylation and sulphation of the overload of secretory cargo. Thus, a hyperactive secretory cell may run short in providing an output of correctly modified biological signals.