Detection of enterovirus RNA in pancreas and lymphoid tissues of organ donors with type 1 diabetes.

Aims/hypothesis: The nPOD-Virus group collaboratively applied innovative technologies to detect and sequence viral RNA in pancreas and other tissues from organ donors with type 1 diabetes. These analyses involved the largest number of pancreas samples collected to date. Methods: We analysed pancreas, spleen, pancreatic lymph nodes, and duodenum samples from the following donor groups: a) donors with type 1 diabetes (n=71), with (n=35) or without (n=36) insulin-containing islets, (b) donors with single or double islet autoantibody positivity without diabetes (n=22) and c) autoantibody-negative donors without diabetes (control donors) (n=74). Five research laboratories participated in this collaborative effort using approaches for unbiased discovery of RNA viruses (two RNA-Seq platforms), targeted detection of Enterovirus A-D species using RT-PCR, and tests for virus growth in cell-culture. Results: Direct RNA-Seq did not detect virus signal in pancreas samples, whereas RT-PCR detected enterovirus RNA confirmed by sequencing in low amounts in pancreas samples in three of the five donor groups, namely donors with type 1 diabetes with insulin-containing islets, 16% (5/32) donors being positive, donors with single islet autoantibody positivity with 53% (8/15) donors being positive, and non-diabetic donors with 8% (4/49) being enterovirus RNA positive. Detection of enterovirus RNA was significantly more frequent in single islet autoantibody-positive donors compared to donors with type 1 diabetes with insulin-deficient islets (p-value <0.001) and control donors (p-value 0.004). In some donors, pancreatic lymph nodes were also positive. RT-PCR detected enterovirus RNA also in spleen of a small number of donors and virus enrichment in susceptible cell lines before RT-PCR resulted in much higher rate in spleen positivity, particularly in donors with type 1 diabetes. Interestingly, the enterovirus strains detected did not cause a typical lytic infection, possibly reflecting their persistence-prone nature. Conclusions/interpretation: This was the largest coordinated effort to examine the presence of enterovirus RNA in pancreas of organ donors with type 1 diabetes, using a multitude of assays. These findings are consistent with the notion that both the subjects with type 1 diabetes and those with islet autoantibodies may carry a low-grade enterovirus infection in the pancreas and lymphoid tissues. Research in context: What is already known about this subject?Enterovirus infections are among the prime candidates for environmental triggers of type 1 diabetes.Pancreas (and other tissue) samples of subjects with type 1 diabetes have not been extensively studied for the presence of enterovirus RNA.What is the key question?Can enterovirus RNA be detected in the pancreas and lymphoid tissues of individuals with and without type 1 diabetes?What are the new findings?Enterovirus RNA can be detected in low amounts in the pancreas and lymphoid tissues using selected enterovirus-specific methods.Detection of enterovirus RNA in the pancreas was most frequent in prediabetic subjects.Enterovirus RNA was found also in pancreatic lymph nodes and in spleen where it was more frequently detected in donors with type 1 diabetes compared to non-diabetic donors, with properties previously observed in persistent infections.How might this impact on clinical practice in the foreseeable future?The findings support the enterovirus - type 1 diabetes association and may have an effect on the primary and secondary prevention strategies towards the disease.

Distinct transcriptomic profiles in children prior to the appearance of type 1 diabetes-linked islet autoantibodies and following enterovirus infection.

Although the genetic basis and pathogenesis of type 1 diabetes have been studied extensively, how host responses to environmental factors might contribute to autoantibody development remains largely unknown. Here, we use longitudinal blood transcriptome sequencing data to characterize host responses in children within 12 months prior to the appearance of type 1 diabetes-linked islet autoantibodies, as well as matched control children. We report that children who present with insulin-specific autoantibodies first have distinct transcriptional profiles from those who develop GADA autoantibodies first. In particular, gene dosage-driven expression of GSTM1 is associated with GADA autoantibody positivity. Moreover, compared with controls, we observe increased monocyte and decreased B cell proportions 9-12 months prior to autoantibody positivity, especially in children who developed antibodies against insulin first. Lastly, we show that control children present transcriptional signatures consistent with robust immune responses to enterovirus infection, whereas children who later developed islet autoimmunity do not. These findings highlight distinct immune-related transcriptomic differences between case and control children prior to case progression to islet autoimmunity and uncover deficient antiviral response in children who later develop islet autoimmunity.

TDRD3 is an antiviral restriction factor that promotes IFN signaling with G3BP1.

Stress granules (SGs) are highly dynamic cytoplasmic foci that form in response to activation of the integrated stress response (ISR) that results in eIF2α phosphorylation and global translation shutdown. Stress granules, which are largely nucleated by G3BP1, serve as hubs for mRNA triage, but there is mounting evidence that they also perform cell signaling functions that are vital to cell survival, particularly during viral infection. We previously showed that SG formation leads to NFκB activation and JNK signaling and that this association may be due in part to G3BP1-dependent recruitment of PKR to SGs. Others have reported close associations between G3BP1 and various innate immune PRRs of the type 1 interferon signaling system, including RIG-I. We also reported SG assembly dynamics is dependent on the arginine-methylation status of G3BP1. Another protein that rapidly localizes to SGs, TDRD3, is a methyl reader protein that performs transcriptional activation and adaptor functions within the nucleus, but neither the mechanism nor its function in SGs is clear. Here, we present evidence that TDRD3 localizes to SGs partly based upon methylation potential of G3BP1. We also characterize granules that TDRD3 forms during overexpression and show that these granules can form in the absence of G3BP but also contain translation components found in canonical SGs. We also show for the first time that SGs recruit additional interferon effectors IRF3, IRF7, TBK1, and Sting, and provide evidence that TDRD3 may play a role in recruitment of these factors. We also present evidence that TDRD3 is a novel antiviral protein that is cleaved by enteroviral 2A proteinase. G3BP1 and TDRD3 knockdown in cells results in altered transcriptional regulation of numerous IFN effectors in complex modulatory patterns that are distinctive for G3BP1 and TDRD3. Overall, we describe a novel role of TDRD3 in innate immunity in which G3BP1 and TDRD3 may coordinate to play important roles in regulation of innate antiviral defenses.

Enteroviruses and Type 1 Diabetes: Multiple Mechanisms and Factors?

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by insulin deficiency and resultant hyperglycemia. Complex interactions of genetic and environmental factors trigger the onset of autoimmune mechanisms responsible for development of autoimmunity to ß cell antigens and subsequent development of T1D. A potential role of virus infections has long been hypothesized, and growing evidence continues to implicate enteroviruses as the most probable triggering viruses. Recent studies have strengthened the association between enteroviruses and development of autoimmunity in T1D patients, potentially through persistent infections. Enterovirus infections may contribute to different stages of disease development. We review data from both human cohort studies and experimental research exploring the potential roles and molecular mechanisms by which enterovirus infections can impact disease outcome.

Expansion of germline RPS20 mutation phenotype to include Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a ribosomopathy of variable expressivity and penetrance characterized by red cell aplasia, congenital anomalies, and predisposition to certain cancers, including early-onset colorectal cancer (CRC). DBA is primarily caused by a dominant mutation of a ribosomal protein (RP) gene, although approximately 20% of patients remain genetically uncharacterized despite exome sequencing and copy number analysis. Although somatic loss-of-function mutations in RP genes have been reported in sporadic cancers, with the exceptions of 5q-myelodysplastic syndrome (RPS14) and microsatellite unstable CRC (RPL22), these cancers are not enriched in DBA. Conversely, pathogenic variants in RPS20 were previously implicated in familial CRC; however, none of the reported individuals had classical DBA features. We describe two unrelated children with DBA lacking variants in known DBA genes who were found by exome sequencing to have de novo novel missense variants in RPS20. The variants affect the same amino acid but result in different substitutions and reduce the RPS20 protein level. Yeast models with mutation of the cognate residue resulted in defects in growth, ribosome biogenesis, and polysome formation. These findings expand the phenotypic spectrum of RPS20 mutation beyond familial CRC to include DBA, which itself is associated with increased risk of CRC.

Differential Detection of Encapsidated versus Unencapsidated Enterovirus RNA in Samples Containing Pancreatic Enzymes-Relevance for Diabetes Studies.

Using immunohistochemistry, enterovirus capsid proteins were demonstrated in pancreatic islets of patients with type 1 diabetes. Virus proteins are mainly located in beta cells, supporting the hypothesis that enterovirus infections may contribute to the pathogenesis of type 1 diabetes. In samples of pancreatic tissue, enterovirus RNA was also detected, but in extremely small quantities and in a smaller proportion of cases compared to the enteroviral protein. Difficulties in detecting viral RNA could be due to the very small number of infected cells, the possible activity of PCR inhibitors, and the presence-during persistent infection-of the viral genome in unencapsidated forms. The aim of this study was twofold: (a) to examine if enzymes or other compounds in pancreatic tissue could affect the molecular detection of encapsidated vs. unencapsidated enterovirus forms, and (b) to compare the sensitivity of RT-PCR methods used in different laboratories. Dilutions of encapsidated and unencapsidated virus were spiked into human pancreas homogenate and analyzed by RT-PCR. Incubation of pancreatic homogenate on wet ice for 20 h did not influence the detection of encapsidated virus. In contrast, a 15-min incubation on wet ice dramatically reduced detection of unencapsidated forms of virus. PCR inhibitors could not be found in pancreatic extract. The results show that components in the pancreas homogenate may selectively affect the detection of unencapsidated forms of enterovirus. This may lead to difficulties in diagnosing persisting enterovirus infection in the pancreas of patients with type 1 diabetes.

PACS1 is an HIV-1 cofactor that functions in Rev-mediated nuclear export of viral RNA.

HIV-1 is dependent upon cellular proteins to mediate the many processes required for viral replication. One such protein, PACS1, functions to localize Furin to the trans-Golgi network where Furin cleaves HIV-1 gp160 Envelope into gp41 and gp120. We show here that PACS1 also shuttles between the nucleus and cytoplasm, associates with the viral Rev protein and its cofactor CRM1, and contributes to nuclear export of viral transcripts. PACS1 appears specific to the Rev-CRM1 pathway and not other retroviral RNA export pathways. Over-expression of PACS1 increases nuclear export of unspliced viral RNA and significantly increases p24 expression in HIV-1-infected Jurkat CD4+ T cells. SiRNA depletion and over-expression experiments suggest that PACS1 may promote trafficking of HIV-1 GagPol RNA to a pathway distinct from that of translation on polyribosomes.

Metagenomics of the faecal virome indicate a cumulative effect of enterovirus and gluten amount on the risk of coeliac disease autoimmunity in genetically at risk children: the TEDDY study.

OBJECTIVE: Higher gluten intake, frequent gastrointestinal infections and adenovirus, enterovirus, rotavirus and reovirus have been proposed as environmental triggers for coeliac disease. However, it is not known whether an interaction exists between the ingested gluten amount and viral exposures in the development of coeliac disease. This study investigated whether distinct viral exposures alone or together with gluten increase the risk of coeliac disease autoimmunity (CDA) in genetically predisposed children. DESIGN: The Environmental Determinants of Diabetes in the Young study prospectively followed children carrying the HLA risk haplotypes DQ2 and/or DQ8 and constructed a nested case-control design. From this design, 83 CDA case-control pairs were identified. Median age of CDA was 31 months. Stool samples collected monthly up to the age of 2 years were analysed for virome composition by Illumina next-generation sequencing followed by comprehensive computational virus profiling. RESULTS: The cumulative number of stool enteroviral exposures between 1 and 2 years of age was associated with an increased risk for CDA. In addition, there was a significant interaction between cumulative stool enteroviral exposures and gluten consumption. The risk conferred by stool enteroviruses was increased in cases reporting higher gluten intake. CONCLUSIONS: Frequent exposure to enterovirus between 1 and 2 years of age was associated with increased risk of CDA. The increased risk conferred by the interaction between enteroviruses and higher gluten intake indicate a cumulative effect of these factors in the development of CDA.

Prospective virome analyses in young children at increased genetic risk for type 1 diabetes.

Viruses are implicated in autoimmune destruction of pancreatic islet ß cells, which results in insulin deficiency and type 1 diabetes (T1D)1-4. Certain enteroviruses can infect ß cells in vitro5, have been detected in the pancreatic islets of patients with T1D6 and have shown an association with T1D in meta-analyses4. However, establishing consistency in findings across studies has proven difficult. Obstacles to convincingly linking RNA viruses to islet autoimmunity may be attributed to rapid viral mutation rates, the cyclical periodicity of viruses7 and the selection of variants with altered pathogenicity and ability to spread in populations. ß cells strongly express cell-surface coxsackie and adenovirus receptor (CXADR) genes, which can facilitate enterovirus infection8. Studies of human pancreata and cultured islets have shown significant variation in enteroviral virulence to ß cells between serotypes and within the same serotype9,10. In this large-scale study of known eukaryotic DNA and RNA viruses in stools from children, we evaluated fecally shed viruses in relation to islet autoimmunity and T1D. This study showed that prolonged enterovirus B rather than independent, short-duration enterovirus B infections may be involved in the development of islet autoimmunity, but not T1D, in some young children. Furthermore, we found that fewer early-life human mastadenovirus C infections, as well as CXADR rs6517774, independently correlated with islet autoimmunity.

Rationale for enteroviral vaccination and antiviral therapies in human type 1 diabetes.

In type 1 diabetes, pancreatic beta cells are destroyed by chronic autoimmune responses. The disease develops in genetically susceptible individuals, but a role for environmental factors has been postulated. Viral infections have long been considered as candidates for environmental triggers but, given the lack of evidence for an acute, widespread, cytopathic effect in the pancreas in type 1 diabetes or for a closely related temporal association of diabetes onset with such infections, a role for viruses in type 1 diabetes remains unproven. Moreover, viruses have rarely been isolated from the pancreas of individuals with type 1 diabetes, mainly (but not solely) due to the inaccessibility of the organ. Here, we review past and recent literature to evaluate the proposals that chronic, recurrent and, possibly, persistent enteroviral infections occur in pancreatic beta cells in type 1 diabetes. We also explore whether these infections may be sustained by different virus strains over time and whether multiple viral hits can occur during the natural history of type 1 diabetes. We emphasise that only a minority of beta cells appear to be infected at any given time and that enteroviruses may become replication defective, which could explain why they have been isolated from the pancreas only rarely. We argue that enteroviral infection of beta cells largely depends on the host innate and adaptive immune responses, including innate responses mounted by beta cells. Thus, we propose that viruses could play a role in type 1 diabetes on multiple levels, including in the triggering and chronic stimulation of autoimmunity and in the generation of inflammation and the promotion of beta cell dysfunction and stress, each of which might then contribute to autoimmunity, as part of a vicious circle. We conclude that studies into the effects of vaccinations and/or antiviral drugs (some of which are currently on-going) is the only means by which the role of viruses in type 1 diabetes can be finally proven or disproven.

Temporal development of the gut microbiome in early childhood from the TEDDY study.

The development of the microbiome from infancy to childhood is dependent on a range of factors, with microbial-immune crosstalk during this time thought to be involved in the pathobiology of later life diseases1-9 such as persistent islet autoimmunity and type 1 diabetes10-12. However, to our knowledge, no studies have performed extensive characterization of the microbiome in early life in a large, multi-centre population. Here we analyse longitudinal stool samples from 903 children between 3 and 46 months of age by 16S rRNA gene sequencing (n = 12,005) and metagenomic sequencing (n = 10,867), as part of the The Environmental Determinants of Diabetes in the Young (TEDDY) study. We show that the developing gut microbiome undergoes three distinct phases of microbiome progression: a developmental phase (months 3-14), a transitional phase (months 15-30), and a stable phase (months 31-46). Receipt of breast milk, either exclusive or partial, was the most significant factor associated with the microbiome structure. Breastfeeding was associated with higher levels of Bifidobacterium species (B. breve and B. bifidum), and the cessation of breast milk resulted in faster maturation of the gut microbiome, as marked by the phylum Firmicutes. Birth mode was also significantly associated with the microbiome during the developmental phase, driven by higher levels of Bacteroides species (particularly B. fragilis) in infants delivered vaginally. Bacteroides was also associated with increased gut diversity and faster maturation, regardless of the birth mode. Environmental factors including geographical location and household exposures (such as siblings and furry pets) also represented important covariates. A nested case-control analysis revealed subtle associations between microbial taxonomy and the development of islet autoimmunity or type 1 diabetes. These data determine the structural and functional assembly of the microbiome in early life and provide a foundation for targeted mechanistic investigation into the consequences of microbial-immune crosstalk for long-term health.

Maximal viral information recovery from sequence data using VirMAP.

Accurate classification of the human virome is critical to a full understanding of the role viruses play in health and disease. This implies the need for sensitive, specific, and practical pipelines that return precise outputs while still enabling case-specific post hoc analysis. Viral taxonomic characterization from metagenomic data suffers from high background noise and signal crosstalk that confounds current methods. Here we develop VirMAP that overcomes these limitations using techniques that merge nucleotide and protein information to taxonomically classify viral reconstructions independent of genome coverage or read overlap. We validate VirMAP using published data sets and viral mock communities containing RNA and DNA viruses and bacteriophages. VirMAP offers opportunities to enhance metagenomic studies seeking to define virome-host interactions, improve biosurveillance capabilities, and strengthen molecular epidemiology reporting.

FGFR1-Activated Translation of WNT Pathway Components with Structured 5' UTRs Is Vulnerable to Inhibition of EIF4A-Dependent Translation Initiation.

Cooperativity between WNT and FGF signaling is well documented in embryonic development and cancer progression, but the molecular mechanisms underlying this cross-talk remain elusive. In this study, we interrogated the dynamics of RNA levels, ribosome occupancy, and protein expression as a function of inducible FGF signaling in mouse mammary glands with constitutive WNT hyperactivation. Multiomics correlation analysis revealed a substantial discrepancy between RNA and ribosome occupancy levels versus protein levels. However, this discrepancy decreased as cells became premalignant and dynamically responded to FGF signaling, implicating the importance of stringent gene regulation in nontransformed cells. Analysis of individual genes demonstrated that acute FGF hyperactivation increased translation of many stem cell self-renewal regulators, including WNT signaling components, and decreased translation of genes regulating cellular senescence. WNT pathway components translationally upregulated by FGF signaling had long and structured 5' UTRs with a high frequency of polypurine sequences, several of which harbored (CGG)4 motifs that can fold into either stable G-quadruplexes or other stable secondary structures. The FGF-mediated increase in translation of WNT pathway components was compromised by silvestrol, an inhibitor of EIF4A that clamps EIF4A to polypurine sequences to block 43S scanning and inhibits its RNA-unwinding activity important for translation initiation. Moreover, silvestrol treatment significantly delayed FGF-WNT-driven tumorigenesis. Taken together, these results suggest that FGF signaling selectively enhances translation of structured mRNAs, particularly WNT signaling components, and highlight their vulnerability to inhibitors that target the RNA helicase EIF4A.Significance: The RNA helicase EIF4A may serve as a therapeutic target for breast cancers that require FGF and WNT signaling. Cancer Res; 78(15); 4229-40. ©2018 AACR.

Histone arginine demethylase JMJD6 is linked to stress granule assembly through demethylation of the stress granule-nucleating protein G3BP1.

Stress granules (SG) are membrane-less organelles that are condensates of stalled translation initiation complexes and mRNAs. SG formation is a cytoprotective response to environmental stress and results from protein interactions involving regions of low amino acid complexity and poorly defined post-translational modifications of SG components. Many RNA-binding proteins are methylated, and we previously demonstrated that the potent SG-nucleating protein G3BP1 is methylated by protein arginine methyltransferase 1 and 5 (PRMT1 and PRMT5). G3BP1 methylation represses SG formation and is reversible. Here we functionally link JMJD6 (Jumonji C domain-containing protein 6) to G3BP1 demethylation. Our findings reveal that JMJD6 is a novel SG component that interacts with G3BP1 complexes, and its expression reduces G3BP1 monomethylation and asymmetric dimethylation at three Arg residues. Knockdown of JMJD6 repressed SG formation and G3BP1 demethylation, but SG formation and G3BP1 demethylation were rescued with catalytically active but not mutant JMJD6. These results suggest that JMJD6 functions directly or indirectly as an arginine demethylase of G3BP1 that promotes SG formation.

Casein Kinase 2 Is Linked to Stress Granule Dynamics through Phosphorylation of the Stress Granule Nucleating Protein G3BP1.

Stress granules (SGs) are large macromolecular aggregates that contain translation initiation complexes and mRNAs. Stress granule formation coincides with translational repression, and stress granules actively signal to mediate cell fate decisions by signaling to the translation apparatus to (i) maintain translational repression, (ii) mount various transcriptional responses, including innate immunity, and (iii) repress apoptosis. Previous work showed that G3BP1 is phosphorylated at serine 149, which regulates G3BP1 oligomerization, stress granule assembly, and RNase activity intrinsic to G3BP1. However, the kinase that phosphorylates G3BP1 was not identified, leaving a key step in stress granule regulation uncharacterized. Here, using chemical inhibition, genetic depletion, and overexpression experiments, we show that casein kinase 2 (CK2) promotes stress granule dynamics. These results link CK2 activity with SG disassembly. We also show that casein kinase 2 phosphorylates G3BP1 at serine 149 in vitro and in cells. These data support a role for casein kinase 2 in regulation of protein synthesis by downregulating stress granule formation through G3BP1.

Arginine Demethylation of G3BP1 Promotes Stress Granule Assembly.

Stress granules (SGs) are cytoplasmic condensates of stalled messenger ribonucleoprotein complexes (mRNPs) that form when eukaryotic cells encounter environmental stress. RNA-binding proteins are enriched for arginine methylation and facilitate SG assembly through interactions involving regions of low amino acid complexity. How methylation of specific RNA-binding proteins regulates RNA granule assembly has not been characterized. Here, we examined the potent SG-nucleating protein Ras-GAP SH3-binding protein 1 (G3BP1), and found that G3BP1 is differentially methylated on specific arginine residues by protein arginine methyltransferase (PRMT) 1 and PRMT5 in its RGG domain. Several genetic and biochemical interventions that increased methylation repressed SG assembly, whereas interventions that decreased methylation promoted SG assembly. Arsenite stress quickly and reversibly decreased asymmetric arginine methylation on G3BP1. These data indicate that arginine methylation in the RGG domain prevents large SG assembly and rapid demethylation is a novel signal that regulates SG formation.

Enterovirus Control of Translation and RNA Granule Stress Responses.

Enteroviruses such as poliovirus (PV) and coxsackievirus B3 (CVB3) have evolved several parallel strategies to regulate cellular gene expression and stress responses to ensure efficient expression of the viral genome. Enteroviruses utilize their encoded proteinases to take over the cellular translation apparatus and direct ribosomes to viral mRNAs. In addition, viral proteinases are used to control and repress the two main types of cytoplasmic RNA granules, stress granules (SGs) and processing bodies (P-bodies, PBs), which are stress-responsive dynamic structures involved in repression of gene expression. This review discusses these processes and the current understanding of the underlying mechanisms with respect to enterovirus infections. In addition, the review discusses accumulating data suggesting linkage exists between RNA granule formation and innate immune sensing and activation.

Multiple Poliovirus Proteins Repress Cytoplasmic RNA Granules.

We have previously shown that poliovirus (PV) infection induces stress granule (SG) formation early in infection and then inhibits the formation of SG and disperses processing bodies (PBs) by the mid-phase of infection. Loss of SG was linked to cleavage of G3BP1 by viral 3C proteinase (3C(pro)), however dispersal of PBs was not strongly linked to cleavage of specific factors by viral proteinases, suggesting other viral proteins may play roles in inhibition of SG or PB formation. Here we have screened all viral proteins for roles in inducing or inhibiting the formation of RNA granules by creating fusions with mCherry and expressing them individually in cells. Expression of viral proteins separately revealed that the capsid region P1, 2A(pro), 3A, 3C(pro), the protease precursor 3CD and 3D polymerase all affect RNA granules to varying extents, whereas 2BC does not. 2A(pro), which cleaves eIF4GI, induced SGs as expected, and entered novel foci containing the SG nucleating protein G3BP1. Of the two forms of G3BP, only G3BP1 is cleaved by a virus proteinase, 3C(pro), whereas G3BP2 is not cleaved by 3C(pro) or 2A(pro). Surprisingly, 3CD, which contains proteinase activity, differentially repressed PBs but not SGs. Further, both 2A(pro) and 3C(pro) expression dispersed PBs, however molecular targets were different since PB dispersal due to 2A(pro) and heat shock protein (Hsp)90 inhibition but not 3C(pro), could be rescued by application of oxidative stress to cells. The data indicate that PV repression of SGs and PBs is multifactorial, though protease function is dominant.