Circularization of flavivirus genomic RNA inhibits de novo translation initiation.

Members of the Flaviviridae family, including dengue virus (DENV) and yellow fever virus, cause serious disease in humans, whilst maternal infection with Zika virus (ZIKV) can induce microcephaly in newborns. Following infection, flaviviral RNA genomes are translated to produce the viral replication machinery but must then serve as a template for the transcription of new genomes. However, the ribosome and viral polymerase proceed in opposite directions along the RNA, risking collisions and abortive replication. Whilst generally linear, flavivirus genomes can adopt a circular conformation facilitated by long-range RNA-RNA interactions, shown to be essential for replication. Using an in vitro reconstitution approach, we demonstrate that circularization inhibits de novo translation initiation on ZIKV and DENV RNA, whilst the linear conformation is translation-competent. Our results provide a mechanism to clear the viral RNA of ribosomes in order to promote efficient replication and, therefore, define opposing roles for linear and circular conformations of the flavivirus genome

A conserved structure within the HIV gag open reading frame that controls translation initiation directly recruits the 40S subunit and eIF3

Translation initiation on HIV genomic RNA relies on both cap and Internal Ribosome Entry Site (IRES) dependant mechanisms that are regulated throughout the cell cycle. During a unique phenomenon, the virus recruits initiation complexes through RNA structures located within Gag coding sequence, downstream of the initiation codon. We analyzed initiation complexes paused on the HIV-2 gag IRES and revealed that they contain all the canonical initiation factors except eIF4E and eIF1. We report that eIF3 and the small ribosomal subunit bind HIV RNA within gag open reading frame. We thus propose a novel two step model whereby the initial event is the formation of a ternary eIF3/40S/IRES complex. In a second step, dependent on most of the canonical initiation factors, the complex is rearranged to transfer the ribosome on the initiation codons. The absolute requirement of this large structure for HIV translation defines a new function for a coding region. Moreover, the level of information compaction within this viral genome reveals an additional level of evolutionary constraint on the coding sequence. The conservation of this IRES and its properties in rapidly evolving viruses suggest an important role in the virus life cycle and highlight an attractive new therapeutic target

Conserved functional domains and a novel tertiary interaction near the pseudoknot drive translational activity of hepatitis C virus and hepatitis C virus-like internal ribosome entry sites

The translational activity of the hepatitis C virus (HCV) internal ribosome entry site (IRES) and other HCV-like IRES RNAs depends on structured RNA elements in domains II and III, which serve to recruit the ribosomal 40S subunit, eukaryotic initiation factor (eIF) 3 and the ternary eIF2/Met-tRNAiMet/GTP complex and subsequently domain II assists subunit joining. Porcine teschovirus-1 talfan (PTV-1) is a member of the Picornaviridae family, with a predicted HCV-like secondary structure, but only stem-loops IIId and IIIe in the 40S-binding domain display significant sequence conservation with the HCV IRES. Here, we use chemical probing to show that interaction sites with the 40S subunit and eIF3 are conserved between HCV and HCV-like IRESs. In addition, we reveal the functional role of a strictly conserved co-variation between a purine–purine mismatch near the pseudoknot (A–A/G) and the loop sequence of domain IIIe (GAU/CA). These nucleotides are involved in a tertiary interaction, which serves to stabilize the pseudoknot structure and correlates with translational efficiency in both the PTV-1 and HCV IRES. Our data demonstrate conservation of functional domains in HCV and HCV-like IRESs including a more complex structure surrounding the pseudoknot than previously assumed

High-density functional-RNA arrays as a versatile platform for studying RNA-based interactions

We are just beginning to unravel the myriad of interactions in which non-coding RNAs participate. The intricate RNA interactome is the foundation of many biological processes, including bacterial virulence and human disease, and represents unexploited resources for the development of potential therapeutic interventions. However, identifying specific associations of a given RNA from the multitude of possible binding partners within the cell requires robust high-throughput systems for their rapid screening. Here, we present the first demonstration of functional-RNA arrays as a novel platform technology designed for the study of such interactions using immobilized, active RNAs. We have generated high-density RNA arrays by an innovative method involving surface-capture of in vitro transcribed RNAs. This approach has significant advantages over existing technologies, particularly in its versatility in regards to binding partner character. Indeed, proof-of-principle application of RNA arrays to both RNA-small molecule and RNA-RNA pairings is demonstrated, highlighting their potential as a platform technology for mapping RNA-based networks and for pharmaceutical screening. Furthermore, the simplicity of the method supports greater user-accessibility over currently available technologies. We anticipate that functional-RNA arrays will find broad utility in the expanding field of RNA characterization

Norovirus infection results in eIF2α independent host translation shut-off and remodels the G3BP1 interactome evading stress granule formation.

Viral infections impose major stress on the host cell. In response, stress pathways can rapidly deploy defence mechanisms by shutting off the protein synthesis machinery and triggering the accumulation of mRNAs into stress granules to limit the use of energy and nutrients. Because this threatens viral gene expression, viruses need to evade these pathways to propagate. Human norovirus is responsible for gastroenteritis outbreaks worldwide. Here we examined how norovirus interacts with the eIF2α signaling axis controlling translation and stress granules. While norovirus infection represses host cell translation, our mechanistic analyses revealed that eIF2α signaling mediated by the stress kinase GCN2 is uncoupled from translational stalling. Moreover, infection results in a redistribution of the RNA-binding protein G3BP1 to replication complexes and remodelling of its interacting partners, allowing the avoidance from canonical stress granules. These results define novel strategies by which norovirus undergo efficient replication whilst avoiding the host stress response and manipulating the G3BP1 interactome

Dentists' use of validated child dental anxiety measures in clinical practice: a mixed methods study

Background: Assessing anxiety is an important part of the assessment of a child presenting for dental treatment; however, the use of dental anxiety scales in practice is not well-documented. Aims: To introduce child dental anxiety scales, and to monitor the extent to which dentists used them; to explore the experience and views of dentists regarding anxiety assessment. Design: A mixed-methods design was employed. Method: A protocol for child anxiety assessment was introduced to paediatric dentists in Eastman Dental Hospital. After 6 months, 100 patient files were audited to examine compliance with the protocol. Fourteen dentists were interviewed to explore their experience and views regarding anxiety assessment. Results: Only five patients were assessed using the scales. Thematic analysis of the dentist interviews revealed three themes: ‘Clinical observations and experience: The gold standard’; ‘Scales as an estimate or adjunct’; and ‘Shortcomings and barriers to using scales’. Conclusions: The dentists in our study did not use anxiety scales, instead they rely on their own experience/judgement. Therefore, scales should be recommended as an adjunct to judgement. Brief scales are recommended as clinicians lack time and expertise in administering anxiety questionnaires. Advantages of using scales and hands-on experience could be incorporated more in undergraduate training

A national cross-sectional survey of dental anxiety in the French adult population

<p>Abstract</p> <p>Background</p> <p>Dental anxiety is a public health problem but no epidemiological study has been undertaken in France to evaluate its prevalence. The aim of this study was to estimate the prevalence, severity and associations of dental anxiety in a sample of the French adult population.</p> <p>Methods</p> <p>A convenience sample of 2725 adults (mean age = 47 years, SD16, minimum = 16, maximum = 101 years), representative of the French population with regard to age and urban distribution, completed a French version of the Corah Dental Anxiety scale (DAS) and a questionnaire relating to their dental appointments.</p> <p>Results</p> <p>Moderate dental anxiety (14≥DAS≥13) was revealed for 172 persons (6.2%), while 195 (7.3%) had severe dental anxiety (DAS≥15), giving an overall prevalence of dental anxiety of 13.5%. Prevalence was lower proportionally with age (P < 0.001) and was higher in French overseas territories and in the countryside (P < 0.01). Farmers and low skilled workers were significantly more anxious than executives and shopkeepers (P < 0.001). Anxiety was associated with avoidance of care (p < 0.001) and lack of regular dental appointments (p < 0.001).</p> <p>Conclusion</p> <p>Dental anxiety in France appears to concern a similar proportion of the population as in other industrialised European, Australasian or North American countries. Recommendations for prevention and management of dental anxiety are made with reference to dental education and health care services in France.</p

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

Knowledge of the host factors required for norovirus replication has been hindered by the challenges associated with culturing human noroviruses. We have combined proteomic analysis of the viral translation and replication complexes with a CRISPR screen, to identify host factors required for norovirus infection. The core stress granule component G3BP1 was identified as a host factor essential for efficient human and murine norovirus infection, demonstrating a conserved function across the Norovirus genus. Furthermore, we show that G3BP1 functions in the novel paradigm of viral VPg-dependent translation initiation, contributing to the assembly of translation complexes on the VPg-linked viral positive sense RNA genome by facilitating 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

Rescue of Advanced Pompe Disease in Mice with Hepatic Expression of Secretable Acid α-Glucosidase.

Pompe disease is a neuromuscular disorder caused by disease-associated variants in the gene encoding for the lysosomal enzyme acid α-glucosidase (GAA), which converts lysosomal glycogen to glucose. We previously reported full rescue of Pompe disease in symptomatic 4-month-old Gaa knockout (Gaa-/-) mice by adeno-associated virus (AAV) vector-mediated liver gene transfer of an engineered secretable form of GAA (secGAA). Here, we showed that hepatic expression of secGAA rescues the phenotype of 4-month-old Gaa-/- mice at vector doses at which the native form of GAA has little to no therapeutic effect. Based on these results, we then treated severely affected 9-month-old Gaa-/- mice with an AAV vector expressing secGAA and followed the animals for 9 months thereafter. AAV-treated Gaa-/- mice showed complete reversal of the Pompe phenotype, with rescue of glycogen accumulation in most tissues, including the central nervous system, and normalization of muscle strength. Transcriptomic profiling of skeletal muscle showed rescue of most altered pathways, including those involved in mitochondrial defects, a finding supported by structural and biochemical analyses, which also showed restoration of lysosomal function. Together, these results provide insight into the reversibility of advanced Pompe disease in the Gaa-/- mouse model via liver gene transfer of secGAA.This work was supported by Genethon, the French Muscular Dystro-phy Association (AFM), and Spark Therapeutics. It was also sup-ported by the European Union’s Research and Innovation Programunder grant agreement number 667751 (to F.M.), the EuropeanResearch Council Consolidator Grant under grant agreement number617432 (to F.M.), and Marie Skłodowska-Curie Actions-IndividualFellowship (MSCA-IF) grant agreement number 797144 (to U.C.)S