Replication of the coronavirus genome: A paradox among positive-strand RNA viruses.

Coronavirus (CoV) genomes consist of positive-sense single-stranded RNA and are among the largest viral RNAs known to date (∼30 kb). As a result, CoVs deploy sophisticated mechanisms to replicate these extraordinarily large genomes as well as to transcribe subgenomic messenger RNAs. Since 2003, with the emergence of three highly pathogenic CoVs (SARS-CoV, MERS-CoV, and SARS-CoV-2), significant progress has been made in the molecular characterization of the viral proteins and key mechanisms involved in CoV RNA genome replication. For example, to allow for the maintenance and integrity of their large RNA genomes, CoVs have acquired RNA proofreading 3'-5' exoribonuclease activity (in nonstructural protein nsp14). In order to replicate the large genome, the viral-RNA-dependent RNA polymerase (RdRp; in nsp12) is supplemented by a processivity factor (made of the viral complex nsp7/nsp8), making it the fastest known RdRp. Lastly, a viral structural protein, the nucleocapsid (N) protein, which is primarily involved in genome encapsidation, is required for efficient viral replication and transcription. Therefore, CoVs are a paradox among positive-strand RNA viruses in the sense that they use both a processivity factor and have proofreading activity reminiscent of DNA organisms in addition to structural proteins that mediate efficient RNA synthesis, commonly used by negative-strand RNA viruses. In this review, we present a historical perspective of these unsuspected discoveries and detail the current knowledge on the core replicative machinery deployed by CoVs.

The impact of airborne ultrafine particulate matter on human keratinocyte stem cells.

OBJECTIVE: Air pollution is today fully acknowledged to be a significant public health problem. Rapid urbanization exposed us to a variety of unhealthy ambient air pollutants at high concentrations. The emergence of airborne ultrafine particles has added an additional dimension to this already complex problem of air pollution. The skin has different functions, one of them being the protection against the deleterious effect of external agents. The aim of this study is to evaluate the impact of airborne ultrafine particles (UFP) pollution on skin aging and on keratinocyte differentiation. METHODS: Ex vivo human skin biopsies and cultured keratinocytes stem cells (KSC) were submitted to diesel exhaust-derived UFP. Reactive oxygen species (ROS) production was assessed with the MitoSOX™ probe. Keratinocyte stemness potential was evaluated by the immunodetection of keratin 15 (K15) and p63 (∆N isoforms). Effect of UFP on the epithelial niche maintenance was evaluated by immunodetection of Sox9. Reconstructed epidermis model was used to assess the impact of UFP on keratinocyte differentiation and aging. RESULTS: UFP exposure induced ROS production and disturbed K15, ∆Np63 and Sox9 expression in KSC or ex vivo skin. Finally, investigations on reconstructed epidermis revealed a phenotype marked by impaired keratinocyte differentiation. CONCLUSION: These results indicate that UFP pollution is a potent extrinsic factor of skin aging, affecting the keratinocyte stem cell potential and the skin renewal process.

OBJECTIF: La pollution de l'air est désormais pleinement reconnue comme un problème de santé publique important. L'urbanisation rapide nous a exposés à une variété de polluants atmosphériques ambiants malsains à des concentrations élevées. L'émergence de particules ultrafines en suspension dans l'air a ajouté une dimension supplémentaire à ce problème déjà complexe de la pollution de l'air. La peau exerce différentes fonctions, l'une d'elles étant la protection contre l'effet délétère d'agents extérieurs. L'objectif de cette étude est d'évaluer l'impact de la pollution par les particules ultrafines (UFP) aéroportées sur le vieillissement cutané et sur la différenciation des kératinocytes. MÉTHODES: Des biopsies de peau humaine ex vivo et des kératinocytes souches (KSC) en culture ont été mis en présence d'UFP provenant d'échappement de véhicule diesel. La production d'espèces réactives de l'oxygène (ROS) a été évaluée avec la sonde MitoSOX™. Le potentiel de souche des kératinocytes a été évalué par immunodétection de la kératine 15 (K15) et p63 (isoformes ∆N). L'effet des UFP sur la niche épithéliale a été évalué par immunodétection de Sox9. Un modèle d'épiderme reconstruit a été utilisé pour évaluer l'impact des UFP sur la différenciation et le vieillissement des kératinocytes. RÉSULTATS: L'exposition aux UFP a induit la production de ROS, a perturbé l'expression de K15, ∆Np63 et de Sox9 dans les KSC et dans la peau ex vivo. Enfin, des investigations sur des épidermes reconstruits ont révélé un phénotype marqué par une différenciation altérée des kératinocytes. CONCLUSION: Ces résultats indiquent que la pollution par les UFP est un facteur extrinsèque puissant du vieillissement cutané, affectant le potentiel des cellules souches de kératinocytes et le processus de renouvellement cutané.

Modulation of Piezo1 influences human skin architecture and oxytocin expression.

OBJECTIVE: Throughout our existence, the skin senses and analyses the mechanical forces imposed by the environment. In response to these environmental forces, skin can deform itself and achieve a biological response. The subsequent cutaneous plasticity emerges from mechanical properties arising from the collective action of the skin cells, particularly keratinocytes, that govern the tensile strength via cell-to-cell adhesions and via cell-matrix adhesion structures. In addition to serving as force-bearing entities, keratinocytes respond to forces by activating signalling pathways to control their own fate and function. To detect and adapt to mechanical signals, keratinocytes possess a panel of sensory receptors and junctional intercellular structures. Mechanically activated ion channel Piezo1 has been described as a force sensor and as being involved in pleasant touch perception. In this study, relationships between Piezo1 modulation and oxytocin synthesis were investigated. METHODS: The expression of Piezo1 in the skin was studied and compared with the expression of TRPV1. Dooku1 antagonist and Jedi1 agonist were used to modulate Piezo1. The level of E-cadherin and oxytocin was monitored in ex vivo skin biopsies by immunodetection. RESULTS: Taken together, our results illustrate the major role of mechanosensitive ion channel Piezo1 in skin barrier integrity, and in peripheral oxytocin synthesis in the skin. CONCLUSION: In conclusion, this study highlights the relationships between pleasant touch, soft touch and local oxytocin synthesis.

OBJECTIF: Tout au long de notre existence, la peau détecte et analyse les forces mécaniques imposées par l'environnement. En réponse à ces forces environnementales, la peau peut se déformer et obtenir une réponse biologique. La plasticité cutanée qui s'ensuit émerge des propriétés mécaniques découlant de l'action collective des cellules cutanées, en particulier les kératinocytes, qui déterminent la résistance à la traction via les adhérences intercellulaires et les structures d'adhésion cellule-matrice. En plus de servir d'entités porteuses de force, les kératinocytes répondent aux forces en activant les voies de signalisation pour contrôler leur propre destin et leur propre fonction. Pour détecter et s'adapter aux signaux mécaniques, les kératinocytes possèdent un panel de récepteurs sensoriels et de structures intercellulaires jonctionnelles. Le canal ionique activé mécaniquement Piezo1 a été décrit comme un capteur de force et comme étant impliqué dans la perception d'un toucher agréable. Dans cette étude, les relations entre la modulation Piezo1 et la synthèse de l'ocytocine ont été étudiées. MÉTHODES: L'expression de Piezo1 dans la peau a été étudiée et comparée à l'expression de TRPV1. L'antagoniste Dooku1 et l'agoniste Jedi1 ont été utilisés pour moduler Piezo1. Le taux de cadhérine-E et d'ocytocine a été contrôlé dans des biopsies cutanées ex vivo par immunodétection. RÉSULTATS: Dans l'ensemble, nos résultats illustrent le rôle majeur du canal ionique mécanosensible Piezo1 dans l'intégrité de la barrière cutanée et dans la synthèse de l'ocytocine périphérique dans la peau. CONCLUSION: En conclusion, cette étude met en évidence les relations entre le toucher agréable, le toucher doux et la synthèse d'ocytocine locale.

The C-Terminal Domain of the Sudan Ebolavirus L Protein Is Essential for RNA Binding and Methylation.

The large (L) protein of Ebola virus is a key protein for virus replication. Its N-terminal region harbors the RNA-dependent RNA polymerase activity, and its C terminus contains a cap assembling line composed of a capping domain and a methyltransferase domain (MTase) followed by a C-terminal domain (CTD) of unknown function. The L protein MTase catalyzes methylation at the 2'-O and N-7 positions of the cap structures. In addition, the MTase of Ebola virus can induce cap-independent internal adenosine 2'-O-methylation. In this work, we investigated the CTD role in the regulation of the cap-dependent and cap-independent MTase activities of the L protein. We found that the CTD, which is enriched in basic amino acids, plays a key role in RNA binding and in turn regulates the different MTase activities. We demonstrated that the mutation of CTD residues modulates specifically the different MTase activities. Altogether, our results highlight the pivotal role of the L protein CTD in the control of viral RNA methylation, which is critical for Ebola virus replication and escape from the innate response in infected cells.IMPORTANCE Ebola virus infects human and nonhuman primates, causing severe infections that are often fatal. The epidemics, in West and Central Africa, emphasize the urgent need to develop antiviral therapies. The Ebola virus large protein (L), which is the central protein for viral RNA replication/transcription, harbors a methyltransferase domain followed by a C-terminal domain of unknown function. We show that the C-terminal domain regulates the L protein methyltransferase activities and consequently participates in viral replication and escape of the host innate immunity.

Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA.

Coronaviruses (CoVs) stand out among RNA viruses because of their unusually large genomes (∼30 kb) associated with low mutation rates. CoVs code for nsp14, a bifunctional enzyme carrying RNA cap guanine N7-methyltransferase (MTase) and 3'-5' exoribonuclease (ExoN) activities. ExoN excises nucleotide mismatches at the RNA 3'-end in vitro, and its inactivation in vivo jeopardizes viral genetic stability. Here, we demonstrate for severe acute respiratory syndrome (SARS)-CoV an RNA synthesis and proofreading pathway through association of nsp14 with the low-fidelity nsp12 viral RNA polymerase. Through this pathway, the antiviral compound ribavirin 5'-monophosphate is significantly incorporated but also readily excised from RNA, which may explain its limited efficacy in vivo. The crystal structure at 3.38 Šresolution of SARS-CoV nsp14 in complex with its cofactor nsp10 adds to the uniqueness of CoVs among RNA viruses: The MTase domain presents a new fold that differs sharply from the canonical Rossmann fold.

Understanding the Mechanism of the Broad-Spectrum Antiviral Activity of Favipiravir (T-705): Key Role of the F1 Motif of the Viral Polymerase.

Favipiravir (T-705) is a broad-spectrum antiviral agent that has been approved in Japan for the treatment of influenza virus infections. T-705 also inhibits the replication of various RNA viruses, including chikungunya virus (CHIKV). We demonstrated earlier that the K291R mutation in the F1 motif of the RNA-dependent RNA polymerase (RdRp) of CHIKV is responsible for low-level resistance to T-705. Interestingly, this lysine is highly conserved in the RdRp of positive-sense single-stranded RNA (+ssRNA) viruses. To obtain insights into the unique broad-spectrum antiviral activity of T-705, we explored the role of this lysine using another +ssRNA virus, namely, coxsackievirus B3 (CVB3). Introduction of the corresponding K-to-R substitution in the CVB3 RdRp (K159R) resulted in a nonviable virus. Replication competence of the K159R variant was restored by spontaneous acquisition of an A239G substitution in the RdRp. A mutagenesis analysis at position K159 identified the K159M variant as the only other viable variant which had also acquired the A239G substitution. The K159 substitutions markedly decreased the processivity of the purified viral RdRp, which was restored by the introduction of the A239G mutation. The K159R A239G and K159M A239G variants proved, surprisingly, more susceptible than the wild-type virus to T-705 and exhibited lower fidelity in polymerase assays. Furthermore, the K159R A239G variant was found to be highly attenuated in mice. We thus demonstrate that the conserved lysine in the F1 motif of the RdRp of +ssRNA viruses is involved in the broad-spectrum antiviral activity of T-705 and that it is a key amino acid for the proper functioning of the enzyme.IMPORTANCE In this study, we report the key role of a highly conserved lysine residue of the viral polymerase in the broad-spectrum antiviral activity of favipiravir (T-705) against positive-sense single-stranded RNA viruses. Substitutions of this conserved lysine have a major negative impact on the functionality of the RdRp. Furthermore, we show that this lysine is involved in the fidelity of the RdRp and that the RdRp fidelity influences the sensitivity of the virus for the antiviral efficacy of T-705. Consequently, these results provide insights into the mechanism of the antiviral activity of T-705 and may lay the basis for the design of novel chemical scaffolds that may be endowed with a more potent broad-spectrum antiviral activity than that of T-705.

One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities.

In addition to members causing milder human infections, the Coronaviridae family includes potentially lethal zoonotic agents causing severe acute respiratory syndrome (SARS) and the recently emerged Middle East respiratory syndrome. The ∼30-kb positive-stranded RNA genome of coronaviruses encodes a replication/transcription machinery that is unusually complex and composed of 16 nonstructural proteins (nsps). SARS-CoV nsp12, the canonical RNA-dependent RNA polymerase (RdRp), exhibits poorly processive RNA synthesis in vitro, at odds with the efficient replication of a very large RNA genome in vivo. Here, we report that SARS-CoV nsp7 and nsp8 activate and confer processivity to the RNA-synthesizing activity of nsp12. Using biochemical assays and reverse genetics, the importance of conserved nsp7 and nsp8 residues was probed. Whereas several nsp7 mutations affected virus replication to a limited extent, the replacement of two nsp8 residues (P183 and R190) essential for interaction with nsp12 and a third (K58) critical for the interaction of the polymerase complex with RNA were all lethal to the virus. Without a loss of processivity, the nsp7/nsp8/nsp12 complex can associate with nsp14, a bifunctional enzyme bearing 3'-5' exoribonuclease and RNA cap N7-guanine methyltransferase activities involved in replication fidelity and 5'-RNA capping, respectively. The identification of this tripartite polymerase complex that in turn associates with the nsp14 proofreading enzyme sheds light on how coronaviruses assemble an RNA-synthesizing machinery to replicate the largest known RNA genomes. This protein complex is a fascinating example of the functional integration of RNA polymerase, capping, and proofreading activities.

Coronavirus Nsp10, a critical co-factor for activation of multiple replicative enzymes.

The RNA-synthesizing machinery of the severe acute respiratory syndrome Coronavirus (SARS-CoV) is composed of 16 non-structural proteins (nsp1-16) encoded by ORF1a/1b. The 148-amino acid nsp10 subunit contains two zinc fingers and is known to interact with both nsp14 and nsp16, stimulating their respective 3'-5' exoribonuclease and 2'-O-methyltransferase activities. Using alanine-scanning mutagenesis, in cellulo bioluminescence resonance energy transfer experiments, and in vitro pulldown assays, we have now identified the key residues on the nsp10 surface that interact with nsp14. The functional consequences of mutations introduced at these positions were first evaluated biochemically by monitoring nsp14 exoribonuclease activity. Disruption of the nsp10-nsp14 interaction abrogated the nsp10-driven activation of the nsp14 exoribonuclease. We further showed that the nsp10 surface interacting with nsp14 overlaps with the surface involved in the nsp10-mediated activation of nsp16 2'-O-methyltransferase activity, suggesting that nsp10 is a major regulator of SARS-CoV replicase function. In line with this notion, reverse genetics experiments supported an essential role of the nsp10 surface that interacts with nsp14 in SARS-CoV replication, as several mutations that abolished the interaction in vitro yielded a replication-negative viral phenotype. In contrast, mutants in which the nsp10-nsp16 interaction was disturbed proved to be crippled but viable. These experiments imply that the nsp10 surface that interacts with nsp14 and nsp16 and possibly other subunits of the viral replication complex may be a target for the development of antiviral compounds against pathogenic coronaviruses.

RNA 3'-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease complex.

The replication/transcription complex of severe acute respiratory syndrome coronavirus is composed of at least 16 nonstructural proteins (nsp1-16) encoded by the ORF-1a/1b. This complex includes replication enzymes commonly found in positive-strand RNA viruses, but also a set of RNA-processing activities unique to some nidoviruses. The nsp14 protein carries both exoribonuclease (ExoN) and (guanine-N7)-methyltransferase (N7-MTase) activities. The nsp14 ExoN activity ensures a yet-uncharacterized function in the virus life cycle and must be regulated to avoid nonspecific RNA degradation. In this work, we show that the association of nsp10 with nsp14 stimulates >35-fold the ExoN activity of the latter while playing no effect on N7-MTase activity. Nsp10 mutants unable to interact with nsp14 are not proficient for ExoN activation. The nsp10/nsp14 complex hydrolyzes double-stranded RNA in a 3' to 5' direction as well as a single mismatched nucleotide at the 3'-end mimicking an erroneous replication product. In contrast, di-, tri-, and longer unpaired ribonucleotide stretches, as well as 3'-modified RNAs, resist nsp10/nsp14-mediated excision. In addition to the activation of nsp16-mediated 2'-O-MTase activity, nsp10 also activates nsp14 in an RNA processing function potentially connected to a replicative mismatch repair mechanism.

Development of 3D gingival in vitro models using primary gingival cells.

Since March 2013, animal testing for toxicity evaluation of cosmetic ingredients is banned in Europe. This directive applies to all personal care ingredients including oral ingredients. Gingival in vitro 3D models are commercially available. However, it is essential to develop "in house model" to modulate several parameters to study oral diseases, determine the toxicity of ingredients, test biocompatibility, and evaluate different formulations of cosmetic ingredients. Our expertise in tissue engineering allowed us to reconstruct human oral tissues from normal human gingival cells (fibroblasts and keratinocytes). Indeed, isolation from surgical leftover was performed to culture these gingival cells. These cells keep their endogenous capacity to proliferate allowing reconstruction of equivalent tissue close to in vivo tissue. Reconstruction of gingival epithelium, chorion equivalent, and the combination of these two tissues (full thickness) using primary gingival cells displayed all characteristics of an in vivo gingival model.

Synergistic effect of Panax ginseng, Polygonatum cyrtonema, Epiphyllum oxypetalum, Nelumbo nucifera and Osmanthus fragrans extracts on skin aging regulation. From in silico predictions to in vitro outcome.

Intrinsic and extrinsic aging affect the health of human skin. Extracellular matrix protein degradation, DNA damage and oxidative stress are known to disturb skin architecture and skin homeostasis leading to skin aging. Traditional Chinese Medicine (TCM) delivers a large amount of knowledge regarding the phytotherapeutic power of diverse plants. Panax ginseng, Polygonatum cyrtonema, Epiphyllum oxypetalum, Nelumbo nucifera and Osmanthus fragrans are five plants used in TCM for their protective effect. In this study, several combinations of these TCM plants were explored: first, an in silico analysis was performed to predict their potential to target biological activities in the skin and then, some predictions were verified with in vitro studies to underline the synergistic effect of plant extracts. The results showed a stronger anti-aging activity for the combination with the five plants compared to the combination with Panax ginseng, Polygonatum cyrtonema, Epiphyllum oxypetalum and, compared to Panax ginseng alone.

Crystal structure and functional analysis of the SARS-coronavirus RNA cap 2'-O-methyltransferase nsp10/nsp16 complex.

Cellular and viral S-adenosylmethionine-dependent methyltransferases are involved in many regulated processes such as metabolism, detoxification, signal transduction, chromatin remodeling, nucleic acid processing, and mRNA capping. The Severe Acute Respiratory Syndrome coronavirus nsp16 protein is a S-adenosylmethionine-dependent (nucleoside-2'-O)-methyltransferase only active in the presence of its activating partner nsp10. We report the nsp10/nsp16 complex structure at 2.0 Šresolution, which shows nsp10 bound to nsp16 through a ∼930 Ų surface area in nsp10. Functional assays identify key residues involved in nsp10/nsp16 association, and in RNA binding or catalysis, the latter likely through a SN2-like mechanism. We present two other crystal structures, the inhibitor Sinefungin bound in the S-adenosylmethionine binding pocket and the tighter complex nsp10(Y96F)/nsp16, providing the first structural insight into the regulation of RNA capping enzymes in +RNA viruses.

Structures and exoribonuclease activity fonctions in arenavirus and coronavirus.

RNA viruses encode dedicated protein machinery required through the viral life cycle. Some enzymatic activities are generally associated with RNA viruses such as RNA- or DNA-dependent RNA polymerases, RNA helicases or proteases. Some viral enzyme activities are however unique to some viral families. This is the case of two 3'-5' exoribonuclease activities identified in arenavirus and coronavirus proteomes. Arenaviruses have a segmented ambisense single stranded RNA genome of negative polarity while coronaviruses have a positive single-stranded genomic RNA. Although both enzymes belong to the same exo(ribo)nuclease superfamily, available data indicate that they are involved in very different pathways. Indeed, the exoribonuclease activity carried by the arenavirus nucleoprotein seems to counteract the innate immunity antiviral response while the exoribonuclease activity carried by the coronavirus nsp14 protein is likely involved in a unique RNA repair mechanism. In this review, we present our current knowledge about these two viral enzymes and their functions in the viral life cycle.

Co-culture of iNeurons with primary human skin cells provides a reliable model to examine intercellular communication.

OBJECTIVE: The skin is a sensory organ, densely innervated with various types of sensory nerve endings, capable of discriminating touch, environmental sensations, proprioception, and physical affection. Neurons communication with skin cells confer to the tissue the ability to undergo adaptive modifications during response to environmental changes or wound healing after injury. Thought for a long time to be dedicated to the central nervous system, the glutamatergic neuromodulation is increasingly described in peripheral tissues. Glutamate receptors and transporters have been identified in the skin. There is a strong interest in understanding the communication between keratinocytes and neurons, as the close contacts with intra-epidermal nerve fibers is a favorable site for efficient communication. To date, various coculture models have been described. However, these models were based on non-human or immortalized cell line. Even the use of induced pluripotent stem cells (iPSCs) is posing limitations because of epigenetic variations during the reprogramming process. METHODS: In this study, we performed small molecule-driven direct conversion of human skin primary fibroblasts into induced neurons (iNeurons). RESULTS: The resulting iNeurons were mature, showed pan-neuronal markers, and exhibited a glutamatergic subtype and C-type fibers characteristics. Autologous coculture of iNeurons with human primary keratinocytes, fibroblasts, and melanocytes was performed and remained healthy for many days, making possible to study the establishment of intercellular interactions. CONCLUSION: Here, we report that iNeurons and primary skin cells established contacts, with neurite ensheathment by keratinocytes, and demonstrated that iNeurons cocultured with primary skin cells provide a reliable model to examine intercellular communication.

Structure and genetic analysis of the arterivirus nonstructural protein 7alpha.

Arterivirus replicase polyproteins are cleaved into at least 13 mature nonstructural proteins (nsps), and in particular the nsp5-to-nsp8 region is subject to a complex processing cascade. The function of the largest subunit from this region, nsp7, which is further cleaved into nsp7α and nsp7ß, is unknown. Using nuclear magnetic resonance (NMR) spectroscopy, we determined the solution structure of nsp7α of equine arteritis virus, revealing an interesting unique fold for this protein but thereby providing little clue to its possible functions. Nevertheless, structure-based reverse genetics studies established the importance of nsp7/nsp7α for viral RNA synthesis, thus providing a basis for future studies.

In vitro reconstitution of SARS-coronavirus mRNA cap methylation.

SARS-coronavirus (SARS-CoV) genome expression depends on the synthesis of a set of mRNAs, which presumably are capped at their 5' end and direct the synthesis of all viral proteins in the infected cell. Sixteen viral non-structural proteins (nsp1 to nsp16) constitute an unusually large replicase complex, which includes two methyltransferases putatively involved in viral mRNA cap formation. The S-adenosyl-L-methionine (AdoMet)-dependent (guanine-N7)-methyltransferase (N7-MTase) activity was recently attributed to nsp14, whereas nsp16 has been predicted to be the AdoMet-dependent (nucleoside-2'O)-methyltransferase. Here, we have reconstituted complete SARS-CoV mRNA cap methylation in vitro. We show that mRNA cap methylation requires a third viral protein, nsp10, which acts as an essential trigger to complete RNA cap-1 formation. The obligate sequence of methylation events is initiated by nsp14, which first methylates capped RNA transcripts to generate cap-0 (7Me)GpppA-RNAs. The latter are then selectively 2'O-methylated by the 2'O-MTase nsp16 in complex with its activator nsp10 to give rise to cap-1 (7Me)GpppA(2'OMe)-RNAs. Furthermore, sensitive in vitro inhibition assays of both activities show that aurintricarboxylic acid, active in SARS-CoV infected cells, targets both MTases with IC(50) values in the micromolar range, providing a validated basis for anti-coronavirus drug design.

[Capping strategies in RNA viruses]. / Stratégies de formation de la structure coiffe chez les virus à ARN.

Most viruses use the mRNA-cap dependent cellular translation machinery to translate their mRNAs into proteins. The addition of a cap structure at the 5' end of mRNA is therefore an essential step for the replication of many virus families. Additionally, the cap protects the viral RNA from degradation by cellular nucleases and prevents viral RNA recognition by innate immunity mechanisms. Viral RNAs acquire their cap structure either by using cellular capping enzymes, by stealing the cap of cellular mRNA in a process named "cap snatching", or using virus-encoded capping enzymes. Many viral enzymes involved in this process have recently been structurally and functionally characterized. These studies have revealed original cap synthesis mechanisms and pave the way towards the development of specific inhibitors bearing antiviral drug potential.

Les enzymes de la réplication/transcription chez les coronavirus.

The discovery of a new coronavirus (CoV) as the causative agent of the severe acute respiratory syndrome (SARS) pandemic outbreak in 2003 has stimulated a number of studies on the molecular biology of SARS-CoV and related viruses. This research has provided significant new insight into functions and activities of the CoV replication-transcription complex, a multi-protein complex that directs coordinated processes of both continuous and discontinuous RNA synthesis to replicate and transcribe the large CoV genome, a single-stranded, positive-sense RNA of ∼30 kilobases. In this review, we summarize current understanding of the expression and functions of key replicative enzymes, such as RNA polymerases, ribonucleases, methyltransferases and other replicase gene encoded proteins involved in genome expression, virus-host interactions and other processes. Collectively, these recent studies reveal fascinating details of a huge enzymatic machinery unique in the RNA virus world.

miR-203 represses keratinocyte stemness by targeting survivin.

OBJECTIVE: The epidermis possesses the capacity to replace dying cells and to heal wounds, thanks to resident stem cells, which have self-renewal properties. In skin physiology, miRNAs have been shown to be involved in many processes, including skin and hair morphogenesis. Recently, differentiation of epidermal stem cells was shown to be promoted by the miR-203. The miR-203 is upregulated during epidermal differentiation and is of interest because of significant targets. METHODS: By utilizing a bioinformatic tool, we identified a target site for miR-203 in the survivin mRNA. Silencing miR-203 was managed with the use of antagomir; the silencing of survivin was performed with a siRNA. Survivin expression was determined by qPCR or immunofluorescence in cultured cells, and by immunohistochemistry in skin sections. Involucrin expression was used as marker of keratinocyte differentiation. A rice extract with previously demonstrated anti-aging properties was evaluated on miR-203 modulation. RESULTS: In this study, we identified a miR-203/survivin axis, important for epidermal homeostasis. We report that differentiation of keratinocyte is dependent on the level of miR-203 expression and that inhibition of miR-203 can increase the expression of survivin, an epidermal marker of stemness. CONCLUSION: In summary, our findings suggest that miR-203 target 3'UTR region of survivin mRNA and directly represses survivin expression in the epidermis. The rice extract was identified as modulator of miR-203 and pointed out as a promising microRNA-based strategy in treating skin changes occurring with aging.

Structure Prediction and Analysis of Hepatitis E Virus Non-Structural Proteins from the Replication and Transcription Machinery by AlphaFold2.

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in humans globally. Considered for a long while a public health issue only in developing countries, the HEV infection is now a global public health concern. Most human infections are caused by the HEV genotypes 1, 2, 3 and 4 (HEV-1 to HEV-4). Although HEV-3 and HEV-4 can evolve to chronicity in immunocompromised patients, HEV-1 and HEV-2 lead to self-limited infections. HEV has a positive-sense single-stranded RNA genome of ~7.2 kb that is translated into a large pORF1 replicative polyprotein, essential for the viral RNA genome replication and transcription. Unfortunately, the composition and structure of these replicases are still unknown. The recent release of the powerful machine-learning protein structure prediction software AlphaFold2 (AF2) allows us to accurately predict the structure of proteins and their complexes. Here, we used AF2 with the replicase encoded by the polyprotein pORF1 of the human-infecting HEV-3. The boundaries and structures reveal five domains or nonstructural proteins (nsPs): the methyltransferase, Zn-binding domain, macro, helicase, and RNA-dependent RNA polymerase, reliably predicted. Their substrate-binding sites are similar to those observed experimentally for other related viral proteins. Precisely knowing enzyme boundaries and structures is highly valuable to recombinantly produce stable and active proteins and perform structural, functional and inhibition studies.