Monkeypox virus genomic accordion strategies.

The 2023 monkeypox (mpox) epidemic was caused by a subclade IIb descendant of a monkeypox virus (MPXV) lineage traced back to Nigeria in 1971. Person-to-person transmission appears higher than for clade I or subclade IIa MPXV, possibly caused by genomic changes in subclade IIb MPXV. Key genomic changes could occur in the genome's low-complexity regions (LCRs), which are challenging to sequence and are often dismissed as uninformative. Here, using a combination of highly sensitive techniques, we determine a high-quality MPXV genome sequence of a representative of the current epidemic with LCRs resolved at unprecedented accuracy. This reveals significant variation in short tandem repeats within LCRs. We demonstrate that LCR entropy in the MPXV genome is significantly higher than that of single-nucleotide polymorphisms (SNPs) and that LCRs are not randomly distributed. In silico analyses indicate that expression, translation, stability, or function of MPXV orthologous poxvirus genes (OPGs), including OPG153, OPG204, and OPG208, could be affected in a manner consistent with the established "genomic accordion" evolutionary strategies of orthopoxviruses. We posit that genomic studies focusing on phenotypic MPXV differences should consider LCR variability.

Comparison between the KARVI scale and the Child Development Evaluation test (EDI) as a screening tool for suspected neurodevelopmental delay.

BACKGROUND: Early detection of suspected neurodevelopmental delay allows for timely diagnosis and appropriate intervention, for which numerous screening tests have been developed. However, most are complex and impractical for health-care workers at the community level. This study aimed to validate the KARVI scale in the neurodevelopment assessment of children under 1 year of age. METHODS: We conducted an observational, longitudinal, comparative, inferential, and prospective study. Healthy children without risk factors for developing neurodevelopmental delay from 0 to 12 months of age were evaluated remotely using the Zoom® application. The Child Development Evaluation Test and the KARVI scale were applied once a month for four consecutive months. RESULTS: Fifty individuals were analyzed, with a predominance of males in 52%. Adequate percentages for a screening test were obtained in the first evaluation with a sensitivity of 70% (confidence interval [CI] 95% 34.75-93.33) and a specificity of 75% (CI 95% 58.8-87.31), and in the fourth evaluation with a sensitivity of 100% (CI 95% 29.4-100) and a specificity of 78.72% (CI 95% 64.34-89.3), being significant in both evaluations (p = 0.007 and p = 0.001, respectively). CONCLUSIONS: The KARVI scale has the elements to be an effective screening test for suspected neurodevelopmental delay, but more extensive studies are needed to obtain more reliable results.

INTRODUCCIÓN: La identificación temprana de retraso en el neurodesarrollo permite un diagnóstico oportuno y una intervención apropiada. Para ello, se han creado diversas pruebas de tamizaje; sin embargo, la mayoría son complejas y poco prácticas para el personal de la salud a nivel comunitario. El objetivo del estudio fue realizar la validación de la escala KARVI en la valoración del neurodesarrollo en niños menores de un año. MÉTODOS: Se realizó un estudio observacional, longitudinal, comparativo inferencial y prospectivo, en el cual se evaluaron, vía remota mediante la aplicación Zoom®, niños sanos de 0 a 12 meses de edad sin factores de riesgo para desarrollar retraso en el neurodesarrollo. Se aplicaron la prueba EDI (Evaluación del Desarrollo Infantil) y la escala KARVI una vez al mes por cuatro meses consecutivos. RESULTADOS: Se analizaron 50 individuos, con predominio del sexo masculino en el 52%. Se obtuvieron porcentajes adecuados para una prueba de tamizaje tanto en la primera evaluación, con sensibilidad de 70% (IC 95% 34.75-93.33) y especificidad de 75% (IC 95% 58.8-87.31), como en la cuarta, con sensibilidad de 100% (IC 95% 29.4-100) y especificidad de 78.72% (IC 95% 64.34-89.3), con significación estadística en ambas evaluaciones (p = 0.007 y p = 0.001, respectivamente). CONCLUSIONES: Se considera que la escala KARVI cuenta con los elementos para considerarla como una prueba de tamizaje efectiva para detectar retraso del neurodesarrollo, sin embargo. Sin requieren estudios más extensos para obtener resultados más confiables.

Unveiling the Multifaceted Roles of ISG15: From Immunomodulation to Therapeutic Frontiers.

The Interferon Stimulated Gene 15 (ISG15), a unique Ubiquitin-like (Ubl) modifier exclusive to vertebrates, plays a crucial role in the immune system. Primarily induced by interferon (IFN) type I, ISG15 functions through diverse mechanisms: (i) covalent protein modification (ISGylation); (ii) non-covalent intracellular action; and (iii) exerting extracellular cytokine activity. These various roles highlight its versatility in influencing numerous cellular pathways, encompassing DNA damage response, autophagy, antiviral response, and cancer-related processes, among others. The well-established antiviral effects of ISGylation contrast with its intriguing dual role in cancer, exhibiting both suppressive and promoting effects depending on the tumour type. The multifaceted functions of ISG15 extend beyond intracellular processes to extracellular cytokine signalling, influencing immune response, chemotaxis, and anti-tumour effects. Moreover, ISG15 emerges as a promising adjuvant in vaccine development, enhancing immune responses against viral antigens and demonstrating efficacy in cancer models. As a therapeutic target in cancer treatment, ISG15 exhibits a double-edged nature, promoting or suppressing oncogenesis depending on the tumour context. This review aims to contribute to future studies exploring the role of ISG15 in immune modulation and cancer therapy, potentially paving the way for the development of novel therapeutic interventions, vaccine development, and precision medicine.

Lipin-2 regulates the antiviral and anti-inflammatory responses to interferon.

Interferons (IFN) are crucial antiviral and immunomodulatory cytokines that exert their function through the regulation of a myriad of genes, many of which are not yet characterized. Here, we reveal that lipin-2, a phosphatidic acid phosphatase whose mutations produce an autoinflammatory syndrome known as Majeed syndrome in humans, is regulated by IFN in a STAT-1-dependent manner. Lipin-2 inhibits viral replication both in vitro and in vivo. Moreover, lipin-2 also acts as a regulator of inflammation in a viral context by reducing the signaling through TLR3 and the generation of ROS and release of mtDNA that ultimately activate the NLRP3 inflammasome. Inhibitors of mtDNA release from mitochondria restrict IL-1ß production in lipin-2-deficient animals in a model of viral infection. Finally, analyses of databases from COVID-19 patients show that LPIN2 expression levels negatively correlate with the severity of the disease. Overall, these results uncover novel regulatory mechanisms of the IFN response driven by lipin-2 and open new perspectives for the future management of patients with LPIN2 mutations.

Genomic and ultrastructural analysis of monkeypox virus in skin lesions and in human/animal infected cells reveals further morphofunctional insights into viral pathogenicity.

Monkeypox (MPOX) is a zoonotic disease that affects humans and other primates, resulting in a smallpox-like illness. It is caused by monkeypox virus (MPXV), which belongs to the Poxviridae family. Clinically manifested by a range of cutaneous and systemic findings, as well as variable disease severity phenotypes based on the genetic makeup of the virus, the cutaneous niche and respiratory mucosa are the epicenters of MPXV pathogenicity. Herein, we describe the ultrastructural features of MPXV infection in both human cultured cells and cutaneous clinical specimens collected during the 2022-2023 MPOX outbreak in New York City that were revealed through electron microscopy. We observed typical enveloped virions with brick-shaped morphologies that contained surface protrusions, consistent with the classic ultrastructural features of MPXV. In addition, we describe morpho-functional evidence that point to roles of distinct cellular organelles in viral assembly during clinical MPXV infection. Interestingly, in skin lesions, we found abundant melanosomes near viral assembly sites, particularly in the vicinity of mature virions, which provides further insight into virus-host interactions at the subcellular level that contribute to MPXV pathogenesis. These findings not only highlight the importance of electron microscopic studies for further investigation of this emerging pathogen but also in characterizing MPXV pathogenesis during human infection.

An MVA-based vector expressing cell-free ISG15 increases IFN-I production and improves HIV-1-specific CD8 T cell immune responses.

The human immunodeficiency virus (HIV), responsible of the Acquired Immune Deficiency Syndrome (AIDS), continues to be a major global public health issue with any cure or vaccine available. The Interferon-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that is induced by interferons and plays a critical role in the immune response. ISG15 is a modifier protein that covalently binds to its targets via a reversible bond, a process known as ISGylation, which is the best-characterized activity of this protein to date. However, ISG15 can also interact with intracellular proteins via non-covalent binding or act as a cytokine in the extracellular space after secretion. In previous studies we proved the adjuvant effect of ISG15 when delivered by a DNA-vector in heterologous prime-boost combination with a Modified Vaccinia virus Ankara (MVA)-based recombinant virus expressing HIV-1 antigens Env/Gag-Pol-Nef (MVA-B). Here we extended these results evaluating the adjuvant effect of ISG15 when expressed by an MVA vector. For this, we generated and characterized two novel MVA recombinants expressing different forms of ISG15, the wild-type ISG15GG (able to perform ISGylation) or the mutated ISG15AA (unable to perform ISGylation). In mice immunized with the heterologous DNA prime/MVA boost regimen, the expression of the mutant ISG15AA from MVA-Δ3-ISG15AA vector in combination with MVA-B induced an increase in the magnitude and quality of HIV-1-specific CD8 T cells as well as in the levels of IFN-I released, providing a better immunostimulatory activity than the wild-type ISG15GG. Our results confirm the importance of ISG15 as an immune adjuvant in the vaccine field and highlights its role as a potential relevant component in HIV-1 immunization protocols.

LIPRNAseq: a method to discover lipid interacting RNAs by sequencing.

BACKGROUND: Current biological research extensively describes the interactions of molecules such as RNA with other nucleic acids or proteins. However, the relatively recent discovery of nuclear phospholipids playing biologically relevant processes outside membranes, as well as, RNA-lipid interactions shows the need for new methods to explore the identity of these RNAs. METHODS AND RESULTS: In this study, we describe the method for LIPID-RNA isolation followed by sequencing and analysis of the RNA that has the ability to interact with the selected lipids. Here we utilized specific phospholipid coated beads for selective RNA binding. We tested RNA from organisms belonging to different realms (human, plant, and yeast), and tested their ability to bind a specific lipid. CONCLUSIONS: The results show several RNAs differentially enriched in the pull-down of phosphatidyl Inositol 4,5 bisphosphate coated beads. This method is helpful to screen lipid-binding RNA, which may have relevant biological functions. The method can be used with different lipids and comparison of pull-downs and can narrow the selection of RNAs that interact with a particular lipid for further studies.

ISG15 Is Required for the Dissemination of Vaccinia Virus Extracellular Virions.

Viruses have developed many different strategies to counteract immune responses, and Vaccinia virus (VACV) is one of a kind in this aspect. To ensure an efficient infection, VACV undergoes a complex morphogenetic process resulting in the production of two types of infective virions: intracellular mature virus (MV) and extracellular enveloped virus (EV), whose spread depends on different dissemination mechanisms. MVs disseminate after cell lysis, whereas EVs are released or propelled in actin tails from living cells. Here, we show that ISG15 participates in the control of VACV dissemination. Infection of Isg15-/- mouse embryonic fibroblasts with VACV International Health Department-J (IHD-J) strain resulted in decreased EV production, concomitant with reduced induction of actin tails and the abolition of comet-shaped plaque formation, compared to Isg15+/+ cells. Transmission electron microscopy revealed the accumulation of intracellular virus particles and a decrease in extracellular virus particles in the absence of interferon-stimulated gene 15 (ISG15), a finding consistent with altered virus egress. Immunoblot and quantitative proteomic analysis of sucrose gradient-purified virions from both genotypes reported differences in protein levels and composition of viral proteins present on virions, suggesting an ISG15-mediated control of viral proteome. Lastly, the generation of a recombinant IHD-J expressing V5-tagged ISG15 (IHD-J-ISG15) allowed us to identify several viral proteins as potential ISG15 targets, highlighting the proteins A34 and A36, which are essential for EV formation. Altogether, our results indicate that ISG15 is an important host factor in the regulation of VACV dissemination. IMPORTANCE Viral infections are a constant battle between the virus and the host. While the host's only goal is victory, the main purpose of the virus is to spread and conquer new territories at the expense of the host's resources. Along millions of years of incessant encounters, poxviruses have developed a unique strategy consisting in the production two specialized "troops": intracellular mature virions (MVs) and extracellular virions (EVs). MVs mediate transmission between hosts, and EVs ensure advance on the battlefield mediating the long-range dissemination. The mechanism by which the virus "decides" to shed from the primary site of infection and its significant impact in viral transmission is not yet fully established. Here, we demonstrate that this process is finely regulated by ISG15/ISGylation, an interferon-induced ubiquitin-like protein with broad antiviral activity. Studying the mechanism that viruses use during infection could result in new ways of understanding our perpetual war against disease and how we might win the next great battle.

Phylogenetic landscape of Monkeypox Virus (MPV) during the early outbreak in New York City, 2022.

Monkeypox (MPOX) is a zoonotic disease endemic to regions of Central/Western Africa. The geographic endemicity of MPV has expanded, broadening the human-monkeypox virus interface and its potential for spillover. Since May 2022, a large multi-country MPV outbreak with no proven links to endemic countries has originated in Europe and has rapidly expanded around the globe, setting off genomic surveillance efforts. Here, we conducted a genomic analysis of 23 MPV-infected patients from New York City during the early outbreak, assessing the phylogenetic relationship of these strains against publicly available MPV genomes. Additionally, we compared the genomic sequences of clinical isolates versus culture-passaged samples from a subset of samples. Phylogenetic analysis revealed that MPV genomes included in this study cluster within the B.1 lineage (Clade IIb), with some of the samples displaying further differentiation into five different sub-lineages of B.1. Mutational analysis revealed 55 non-synonymous polymorphisms throughout the genome, with some of these mutations located in critical regions required for viral multiplication, structural and assembly functions, as well as the target region for antiviral treatment. In addition, we identified a large majority of polymorphisms associated with GA > AA and TC > TT nucleotide replacements, suggesting the action of human APOBEC3 enzyme. A comparison between clinical isolates and cell culture-passaged samples failed to reveal any difference. Our results provide a first glance at the mutational landscape of early MPV-2022 (B.1) circulating strains in NYC.

Evaluation and validation of an RT-PCR assay for specific detection of monkeypox virus (MPXV).

Monkeypox virus (MPXV) is a zoonotic orthopoxvirus within the Poxviridae family. MPXV is endemic to Central and West Africa. However, the world is currently witnessing an international outbreak with no clear epidemiological links to travel or animal exposure and with ever-increasing numbers of reported cases worldwide. Here, we evaluated and validated a new, sensitive, and specific real-time PCR-assay for MPXV diagnosis in humans and compare the performance of this novel assay against a Food & Drug Administration-cleared pan-Orthopox RT-PCR assay. We determined specificity, sensitivity, and analytic performance of the PKamp™ Monkeypox Virus RT-PCR assay targeting the viral F3L-gene. In addition, we further evaluated MPXV-PCR-positive specimens by viral culture, electron microscopy, and viral inactivation assays. The limit of detection was established at 7.2 genome copies/reaction, and MPXV was successfully identified in 20 clinical specimens with 100% correlation against the reference method with 100% sensitivity and specificity. Our results demonstrated the validity of this rapid, robust, and reliable RT-PCR assay for specific and accurate diagnosis of MPXV infection in human specimens collected both as dry swabs and in viral transport media. This assay has been approved by NYS Department of Health for clinical use.

ISG15 Is a Novel Regulator of Lipid Metabolism during Vaccinia Virus Infection.

Interferon-stimulated gene 15 (ISG15) is a 15-kDa ubiquitin-like modifier that binds to target proteins in a process termed ISGylation. ISG15, first described as an antiviral molecule against many viruses, participates in numerous cellular processes, from immune modulation to the regulation of genome stability. Interestingly, the role of ISG15 as a regulator of cell metabolism has recently gained strength. We previously described ISG15 as a regulator of mitochondrial functions in bone marrow-derived macrophages (BMDMs) in the context of Vaccinia virus (VACV) infection. Here, we demonstrate that ISG15 regulates lipid metabolism in BMDMs and that ISG15 is necessary to modulate the impact of VACV infection on lipid metabolism. We show that Isg15-/- BMDMs demonstrate alterations in the levels of several key proteins of lipid metabolism that result in differences in the lipid profile compared with Isg15+/+ (wild-type [WT]) BMDMs. Specifically, Isg15-/- BMDMs present reduced levels of neutral lipids, reflected by decreased lipid droplet number. These alterations are linked to increased levels of lipases and are independent of enhanced fatty acid oxidation (FAO). Moreover, we demonstrate that VACV causes a dysregulation in the proteomes of BMDMs and alterations in the lipid content of these cells, which appear exacerbated in Isg15-/- BMDMs. Such metabolic changes are likely caused by increased expression of the metabolic regulators peroxisome proliferator-activated receptor-γ (PPARγ) and PPARγ coactivator-1α (PGC-1α). In summary, our results highlight that ISG15 controls BMDM lipid metabolism during viral infections, suggesting that ISG15 is an important host factor to restrain VACV impact on cell metabolism. IMPORTANCE The functions of ISG15 are continuously expanding, and growing evidence supports its role as a relevant modulator of cell metabolism. In this work, we highlight how the absence of ISG15 impacts macrophage lipid metabolism in the context of viral infections and how poxviruses modulate metabolism to ensure successful replication. Our results open the door to new advances in the comprehension of macrophage immunometabolism and the interaction between VACV and the host.

Interferon-stimulated gene 15 pathway is a novel mediator of endothelial dysfunction and aneurysms development in angiotensin II infused mice through increased oxidative stress.

AIMS: Interferon-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that induces a reversible post-translational modification (ISGylation) and can also be secreted as a free form. ISG15 plays an essential role as host-defence response to microbial infection; however, its contribution to vascular damage associated with hypertension is unknown. METHODS AND RESULTS: Bioinformatics identified ISG15 as a mediator of hypertension-associated vascular damage. ISG15 expression positively correlated with systolic and diastolic blood pressure and carotid intima-media thickness in human peripheral blood mononuclear cells. Consistently, Isg15 expression was enhanced in aorta from hypertension models and in angiotensin II (AngII)-treated vascular cells and macrophages. Proteomics revealed differential expression of proteins implicated in cardiovascular function, extracellular matrix and remodelling, and vascular redox state in aorta from AngII-infused ISG15-/- mice. Moreover, ISG15-/- mice were protected against AngII-induced hypertension, vascular stiffness, elastin remodelling, endothelial dysfunction, and expression of inflammatory and oxidative stress markers. Conversely, mice with excessive ISGylation (USP18C61A) show enhanced AngII-induced hypertension, vascular fibrosis, inflammation and reactive oxygen species (ROS) generation along with elastin breaks, aortic dilation, and rupture. Accordingly, human and murine abdominal aortic aneurysms showed augmented ISG15 expression. Mechanistically, ISG15 induces vascular ROS production, while antioxidant treatment prevented ISG15-induced endothelial dysfunction and vascular remodelling. CONCLUSION: ISG15 is a novel mediator of vascular damage in hypertension through oxidative stress and inflammation.

Motif WFYY of human PrimPol is crucial to stabilize the incoming 3'-nucleotide during replication fork restart.

PrimPol is the second primase in human cells, the first with the ability to start DNA chains with dNTPs. PrimPol contributes to DNA damage tolerance by restarting DNA synthesis beyond stalling lesions, acting as a TLS primase. Multiple alignment of eukaryotic PrimPols allowed us to identify a highly conserved motif, WxxY near the invariant motif A, which contains two active site metal ligands in all members of the archeo-eukaryotic primase (AEP) superfamily. In vivo and in vitro analysis of single variants of the WFYY motif of human PrimPol demonstrated that the invariant Trp87 and Tyr90 residues are essential for both primase and polymerase activities, mainly due to their crucial role in binding incoming nucleotides. Accordingly, the human variant F88L, altering the WFYY motif, displayed reduced binding of incoming nucleotides, affecting its primase/polymerase activities especially during TLS reactions on UV-damaged DNA. Conversely, the Y89D mutation initially associated with High Myopia did not affect the ability to rescue stalled replication forks in human cells. Collectively, our data suggest that the WFYY motif has a fundamental role in stabilizing the incoming 3'-nucleotide, an essential requisite for both its primase and TLS abilities during replication fork restart.

Enhancement of HIV-1 Env-Specific CD8 T Cell Responses Using Interferon-Stimulated Gene 15 as an Immune Adjuvant.

Induction of the endogenous innate immune system by interferon (IFN) triggers the expression of many proteins that serve like alarm bells in the body, activating an immune response. After a viral infection, one of the genes activated by IFN induction is the IFN-stimulated gene 15 (ISG15), which encodes a ubiquitin-like protein that undergoes a reversible posttranslational modification (ISGylation). ISG15 protein can also act unconjugated, intracellularly and secreted, acting as a cytokine. Although ISG15 has an essential role in host defense responses to microbial infection, its role as an immunomodulator in the vaccine field remains to be defined. In this investigation, we showed that ISG15 exerts an immunomodulatory role in human immunodeficiency virus (HIV) vaccines. In mice, after priming with a DNA-ISG15 vector mixed with a DNA expressing HIV-1 gp120 (DNA-gp120), followed by a booster with a modified vaccinia virus Ankara (MVA) vector expressing HIV-1 antigens, both wild-type ISG15-conjugated (ISG15-wt) and mutant unconjugated (ISG15-mut) proteins act as immune adjuvants by increasing the magnitude and quality of HIV-1-specific CD8 T cells, with ISG15-wt providing better immunostimulatory activity than ISG15-mut. The HIV-1 Env-specific CD8 T cell responses showed a predominant T effector memory (TEM) phenotype in all groups. Moreover, the amount of DNA-gp120 used to immunize mice could be reduced 5-fold after mixing with DNA-ISG15 without affecting the potency and the quality of the HIV-1 Env-specific immune responses. Our study clearly highlights the potential use of the IFN-induced ISG15 protein as immune adjuvant to enhance immune responses to HIV antigens, suggesting that this molecule might be exploitable for prophylactic and therapeutic vaccine approaches against pathogens.IMPORTANCE Our study described the potential role of ISG15 as an immunomodulatory molecule in the optimization of HIV/AIDS vaccine candidates. Using a DNA prime-MVA boost immunization protocol, our results indicated an increase in the potency and the quality of the HIV-1 Env-specific CD8 T cell response. These results highlight the adjuvant potency of ISG15 to elicit improved viral antigen presentation to the immune system, resulting in an enhanced HIV-1 vaccine immune response. The DNA-ISG15 vector could find applicability in the vaccine field in combination with other nucleic acid-based vector vaccines.

Habilidades motrices y su relación con las actividades y creencias parentales en preescolares; comparaciones por nivel socio-económico / Preschoolers' motor skills and their relation to parental activities and beliefs: comparisons by socio-economic status

El objetivo de esta investigación es analizar cómo se relacionan las habilidades motrices gruesas y finas en preescolares, la frecuencia de actividades motrices reportadas por los padres y las creencias sobre el desarrollo motor en diferente Nivel SocioEconómico (NSE). Participaron 75 padres de familia y sus respectivos hijos de NSE bajo y alto. La frecuencia con que realizan actividades motrices con sus hijos y sus creencias sobre el desarrollo motor se registraron por medio de un cuestionario; las habilidades motrices se evaluaron con las sub-escales de motricidad fina y gruesa del Inventario de Desarrollo Battelle (BDI-2). En los resultados los padres afirmaron otorgar mayor importancia al desarrollo de la motricidad fina que al desarrollo de la motricidad gruesa. Los niños de NSE bajo obtuvieron una puntuación más alta en motricidad gruesa que sus pares de NSE alto, sin embargo no se encontraron diferencias entre las puntuaciones de motricidad fina entre ambos grupos. Concluimos que el desarrollo de las habilidades motrices en preescolar no parece estar asociado a las creencias de los padres ni a la frecuencia con que suelen realizar actividades motrices con sus hijos; sin embargo existen diferencias por NSE en el desempeño de los menores

The purpose of the present study is to analyze the relationship between preschoolers' gross and fine motor skills, parent reported frequency of motor activities, and parental beliefs about motor development in different socio-economic contexts. Seventy-five parents and their children from low and high socio-economic status participated in the study. The frequency with which parents engaged in motor activities with their children and their beliefs about motor development were assessed using a questionnaire; children's motor skills were assessed using the fine and gross motor skill subscales from the Battelle Developmental Inventory (BDI-2). Results show that parents report that they consider the development of fine- as more important than gross motor skills. Children from Low SES performed better on the gross motor skill assessment than their higher SES counterparts, however, performance did not differ by SES on the fine motor skill assessment. We conclude that the development of motor skills in preschool does not appear to be associated to parental beliefs or to parent reported frequency of motor activities; however, we did find differences by SES on children's performance

Mechanism of DNA primer synthesis by human PrimPol.

PrimPol is the second primase discovered in eukaryotic cells, whose function is to restart the stalled replication forks during both mitochondrial and nuclear DNA replication. This chapter revises our current knowledge about the mechanism of synthesis of DNA primers by human PrimPol, and the importance of its distinctive Zn-finger domain (ZnFD). After PrimPol forms a binary complex with ssDNA, the formation of the pre-ternary complex strictly requires the presence of Mn2+ ions to stabilize the interaction of the incoming deoxynucleotide at the 3'-site. The capacity to bind both ssDNA template and 3'-deoxynucleotide was shown to reside in the AEP core of PrimPol, with ZnFD being dispensable at these two early steps of the primase reaction. Sugar selection favoring dNTPs versus NTPs at the 3' site is mediated by a specific tyrosine (Tyr100) acting as a steric gate. Besides, a specific glutamate residue (Glu116) conforming a singular A motif (DxE) promotes the use of Mn2+ to stabilize the pre-ternary complex. Mirroring the function of the PriL subunit of dimeric AEP primases, the ZnFD of PrimPol is crucial to stabilize the initiating 5'-nucleotide, specifically interacting with the gamma-phosphate. Such an interaction is crucial to optimize dimer formation and the subsequent translocation events leading to the processive synthesis of a mature DNA primer. Finally, the capacity of PrimPol to tolerate lesions is discussed in the context of its DNA primase function, and its potential as a TLS primase.

The invariant glutamate of human PrimPol DxE motif is critical for its Mn2+-dependent distinctive activities.

PrimPol is a human primase/polymerase specialized in downstream repriming of stalled forks during both nuclear and mitochondrial DNA replication. Like most primases and polymerases, PrimPol requires divalent metal cations, as Mg2+ or Mn2+, used as cofactors for catalysis. However, little is known about the consequences of using these two metal cofactors in combination, which would be the most physiological scenario during PrimPol-mediated reactions, and the individual contribution of the putative carboxylate residues (Asp114, Glu116 and Asp280) acting as metal ligands. By site-directed mutagenesis in human PrimPol, we confirmed the catalytic relevance of these three carboxylates, and identified Glu116 as a relevant enhancer of distinctive PrimPol reactions, which are highly dependent on Mn2+. Herein, we evidenced that PrimPol Glu116 contributes to error-prone tolerance of 8oxodG more markedly when both Mg2+ and Mn2+ ions are present. Moreover, Glu116 was important for TLS events mediated by primer/template realignments, and crucial to achieving an optimal primase activity, processes in which Mn2+ is largely preferred. EMSA analysis of PrimPol:ssDNA:dNTP pre-ternary complex indicated a critical role of each metal ligand, and a significant impairment when Glu116 was changed to a more conventional aspartate. These data suggest that PrimPol active site requires a specific motif A (DxE) to favor the use of Mn2+ ions in order to achieve optimal incoming nucleotide stabilization, especially required during primer synthesis.

A cancer-associated point mutation disables the steric gate of human PrimPol.

PrimPol is a human primase/polymerase specialized in re-starting stalled forks by repriming beyond lesions such as pyrimidine dimers, and replication-perturbing structures including G-quadruplexes and R-loops. Unlike most conventional primases, PrimPol proficiently discriminates against ribonucleotides (NTPs), being able to start synthesis using deoxynucleotides (dNTPs), yet the structural basis and physiological implications for this discrimination are not understood. In silico analyses based on the three-dimensional structure of human PrimPol and related enzymes enabled us to predict a single residue, Tyr100, as the main effector of sugar discrimination in human PrimPol and a change of Tyr100 to histidine to boost the efficiency of NTP incorporation. We show here that the Y100H mutation profoundly stimulates NTP incorporation by human PrimPol, with an efficiency similar to that for dNTP incorporation during both primase and polymerase reactions in vitro. As expected from the higher cellular concentration of NTPs relative to dNTPs, Y100H expression in mouse embryonic fibroblasts and U2OS osteosarcoma cells caused enhanced resistance to hydroxyurea, which decreases the dNTP pool levels in S-phase. Remarkably, the Y100H PrimPol mutation has been identified in cancer, suggesting that this mutation could be selected to promote survival at early stages of tumorigenesis, which is characterized by depleted dNTP pools.

ISG15, a Small Molecule with Huge Implications: Regulation of Mitochondrial Homeostasis.

Viruses are responsible for the majority of infectious diseases, from the common cold to HIV/AIDS or hemorrhagic fevers, the latter with devastating effects on the human population. Accordingly, the development of efficient antiviral therapies is a major goal and a challenge for the scientific community, as we are still far from understanding the molecular mechanisms that operate after virus infection. Interferon-stimulated gene 15 (ISG15) plays an important antiviral role during viral infection. ISG15 catalyzes a ubiquitin-like post-translational modification termed ISGylation, involving the conjugation of ISG15 molecules to de novo synthesized viral or cellular proteins, which regulates their stability and function. Numerous biomedically relevant viruses are targets of ISG15, as well as proteins involved in antiviral immunity. Beyond their role as cellular powerhouses, mitochondria are multifunctional organelles that act as signaling hubs in antiviral responses. In this review, we give an overview of the biological consequences of ISGylation for virus infection and host defense. We also compare several published proteomic studies to identify and classify potential mitochondrial ISGylation targets. Finally, based on our recent observations, we discuss the essential functions of mitochondria in the antiviral response and examine the role of ISG15 in the regulation of mitochondrial processes, specifically OXPHOS and mitophagy.

ISG15 governs mitochondrial function in macrophages following vaccinia virus infection.

The interferon (IFN)-stimulated gene 15 (ISG15) encodes one of the most abundant proteins induced by interferon, and its expression is associated with antiviral immunity. To identify protein components implicated in IFN and ISG15 signaling, we compared the proteomes of ISG15-/- and ISG15+/+ bone marrow derived macrophages (BMDM) after vaccinia virus (VACV) infection. The results of this analysis revealed that mitochondrial dysfunction and oxidative phosphorylation (OXPHOS) were pathways altered in ISG15-/- BMDM treated with IFN. Mitochondrial respiration, Adenosine triphosphate (ATP) and reactive oxygen species (ROS) production was higher in ISG15+/+ BMDM than in ISG15-/- BMDM following IFN treatment, indicating the involvement of ISG15-dependent mechanisms. An additional consequence of ISG15 depletion was a significant change in macrophage polarization. Although infected ISG15-/- macrophages showed a robust proinflammatory cytokine expression pattern typical of an M1 phenotype, a clear blockade of nitric oxide (NO) production and arginase-1 activation was detected. Accordingly, following IFN treatment, NO release was higher in ISG15+/+ macrophages than in ISG15-/- macrophages concomitant with a decrease in viral titer. Thus, ISG15-/- macrophages were permissive for VACV replication following IFN treatment. In conclusion, our results demonstrate that ISG15 governs the dynamic functionality of mitochondria, specifically, OXPHOS and mitophagy, broadening its physiological role as an antiviral agent.