Structure and flexibility of the DNA polymerase holoenzyme of vaccinia virus.

The year 2022 was marked by the mpox outbreak caused by the human monkeypox virus (MPXV), which is approximately 98% identical to the vaccinia virus (VACV) at the sequence level with regard to the proteins involved in DNA replication. We present the production in the baculovirus-insect cell system of the VACV DNA polymerase holoenzyme, which consists of the E9 polymerase in combination with its co-factor, the A20-D4 heterodimer. This led to the 3.8 Å cryo-electron microscopy (cryo-EM) structure of the DNA-free form of the holoenzyme. The model of the holoenzyme was constructed from high-resolution structures of the components of the complex and the A20 structure predicted by AlphaFold 2. The structures do not change in the context of the holoenzyme compared to the previously determined crystal and NMR structures, but the E9 thumb domain became disordered. The E9-A20-D4 structure shows the same compact arrangement with D4 folded back on E9 as observed for the recently solved MPXV holoenzyme structures in the presence and the absence of bound DNA. A conserved interface between E9 and D4 is formed by a cluster of hydrophobic residues. Small-angle X-ray scattering data show that other, more open conformations of E9-A20-D4 without the E9-D4 contact exist in solution using the flexibility of two hinge regions in A20. Biolayer interferometry (BLI) showed that the E9-D4 interaction is indeed weak and transient in the absence of DNA although it is very important, as it has not been possible to obtain viable viruses carrying mutations of key residues within the E9-D4 interface.

APOBEC3F Is a Mutational Driver of the Human Monkeypox Virus Identified in the 2022 Outbreak.

BACKGROUND: On May 6, 2022, a powerful outbreak of monkeypox virus (MPXV) had been reported outside of Africa, with many continuing new cases being reported around the world. Analysis of mutations among the 2 different lineages present in the 2021 and 2022 outbreaks revealed the presence of G->A mutations occurring in the 5'GpA context, indicative of APOBEC3 cytidine deaminase activity. METHODS: By using a sensitive polymerase chain reaction (differential DNA denaturation PCR) method allowing differential amplification of AT-rich DNA, we analyzed the level of APOBEC3-induced MPXV editing in infected cells and in patients. RESULTS: We demonstrate that G->A hypermutated MPXV genomes can be recovered experimentally from APOBEC3 transfection followed by MPXV infection. Here, among the 7 human APOBEC3 cytidine deaminases (A3A-A3C, A3DE, A3F-A3H), only APOBEC3F was capable of extensively deaminating cytidine residues in MPXV genomes. Hyperedited genomes were also recovered in ∼42% of analyzed patients. Moreover, we demonstrate that substantial repair of these mutations occurs. Upon selection, corrected G->A mutations escaping drift loss contribute to the MPXV evolution observed in the current epidemic. CONCLUSIONS: Stochastic or transient overexpression of the APOBEC3F gene exposes the MPXV genome to a broad spectrum of mutations that may be modeling the mutational landscape after multiple cycles of viral replication.

Efficient Method for Generating Point Mutations in the Vaccinia Virus Genome Using CRISPR/Cas9.

The vaccinia virus (VACV) was previously used as a vaccine for smallpox eradication. Nowadays, recombinant VACVs are developed as vaccine platforms for infectious disease prevention and cancer treatment. The conventional method for genome editing of the VACV is based on homologous recombination, which is poorly efficient. Recently, the use of CRISPR/Cas9 technology was shown to greatly improve the speed and efficiency of the production of recombinant VACV expressing a heterologous gene. However, the ability to rapidly recover viruses bearing single nucleotide substitutions is still challenging. Notwithstanding, ongoing studies on the VACV and its interaction with the host cell could benefit from viral gene targeted mutagenesis. Here, we present a modified version of the CRISPR/Cas9 system for the rapid selection of mutant VACV carrying point mutations. For this purpose, we introduced a silent mutation into the donor gene (which will replace the wildtype gene) that serves a double function: it is located in the PAM (NGG) sequence, which is essential for Cas9 cleavage, and it alters a restriction site. This silent mutation, once introduced into the VACV genome, allows for rapid selection and screening of mutant viruses carrying a mutation of interest in the targeted gene. As a proof of concept, we produced several recombinant VACVs, with mutations in the E9L gene, upon which, phenotypic analysis was performed.

Fatal Cowpox Virus Infection in Human Fetus, France, 2017.

Cowpox virus (CPXV) has an animal reservoir and is typically transmitted to humans by contact with infected animals. In 2017, CPXV infection of a pregnant woman in France led to the death of her fetus. Fetal death after maternal orthopoxvirus (smallpox) vaccination has been reported; however, this patient had not been vaccinated. Investigation of the patient's domestic animals failed to demonstrate prevalence of CPXV infection among them. The patient's diagnosis was confirmed by identifying CPXV DNA in all fetal and maternal biopsy samples and infectious CPXV in biopsy but not plasma samples. This case of fetal death highlights the risk for complications of orthopoxvirus infection during pregnancy. Among orthopoxviruses, fetal infection has been reported for variola virus and vaccinia virus; our findings suggest that CPXV poses the same threats for infection complications as vaccinia virus.

Mesenchymal stromal cells confer chemoresistance to myeloid leukemia blasts through Side Population functionality and ABC transporter activation.

Targeting chemoresistant malignant cells is one of the current major challenges in oncology. Therefore, it is mandatory to refine the characteristics of these cells to monitor their survival and develop adapted therapies. This is of particular interest in acute myeloid leukemia (AML), for which the 5-year survival rate only reaches 30%, regardless of the prognosis. The role of the microenvironment is increasingly reported to be a key regulator for blast survival. In this context, we demonstrate that contact with mesenchymal stromal cells promotes a better survival of blasts in culture in the presence of anthracycline through the activation of ABC transporters. Stroma-dependent ABC transporter activation leads to the induction of a Side Population (SP) phenotype in a subpopulation of primary leukemia blasts through alpha (α)4 engagement. The stroma-promoting effect is reversible and is observed with stromal cells isolated from either healthy donors or leukemia patients. Blasts expressing an SP phenotype are mostly quiescent and are chemoresistant in vitro and in vivo in patient-derived xenograft mouse models. At the transcriptomic level, blasts from the SP are specifically enriched in the drug metabolism program. This detoxification signature engaged in contact with mesenchymal stromal cells represents promising ways to target stroma-induced chemoresistance of AML cells.

Macrophage-derived oncostatin M contributes to human and mouse neurogenic heterotopic ossifications.

Neurogenic heterotopic ossification (NHO) is the formation of ectopic bone generally in muscles surrounding joints following spinal cord or brain injury. We investigated the mechanisms of NHO formation in 64 patients and a mouse model of spinal cord injury-induced NHO. We show that marrow from human NHOs contains hematopoietic stem cell (HSC) niches, in which mesenchymal stromal cells (MSCs) and endothelial cells provide an environment supporting HSC maintenance, proliferation, and differentiation. The transcriptomic signature of MSCs from NHOs shows a neuronal imprinting associated with a molecular network required for HSC support. We demonstrate that oncostatin M (OSM) produced by activated macrophages promotes osteoblastic differentiation and mineralization of human muscle-derived stromal cells surrounding NHOs. The key role of OSM was confirmed using an experimental model of NHO in mice defective for the OSM receptor (OSMR). Our results provide strong evidence that macrophages contribute to NHO formation through the osteogenic action of OSM on muscle cells within an inflammatory context and suggest that OSM/OSMR could be a suitable therapeutic target. Altogether, the evidence of HSCs in ectopic bones growing at the expense of soft tissue in spinal cord/brain-injured patients indicates that inflammation and muscle contribute to HSC regulation by the brain-bone-blood triad.

Effect of Mesenchymal Stromal Cells on T Cells in a Septic Context: Immunosuppression or Immunostimulation?

Sepsis is a complex process, including a first wave of damage partially due to the body's response to pathogens, followed by a phase of immune cell dysfunction. The efficacy of a pharmacological approach facing a rapidly evolving system implies a perfect timing of administration-this difficulty could explain the recent failure of clinical trials. Mesenchymal stromal cells (MSCs) are usually defined as immunosuppressive and their beneficial effects in preclinical models of acute sepsis have been shown to rely partly on such ability. If nonregulated, this phenotype could be harmful in the immunosuppressed context arising hours after sepsis onset. However, MSCs being environment sensitive, we hypothesized that they could reverse their immunosuppressive properties when confronted with suffering immune cells. Our objective was to evaluate the effect of human MSCs on activated human lymphocytes in an in vitro endotoxemia model. Peripheral blood mononuclear cells (PBMCs) underwent a 24-h lipopolysaccharide (LPS) intoxication and were stimulated with phytohemagglutinin (PHA) in contact with MSCs. MSCs induced a differential effect on lymphocytes depending on PBMC intoxication with LPS. Unintoxicated lymphocytes were highly proliferative with PHA and were inhibited by MSCs, whereas LPS-intoxicated lymphocytes showed a low proliferation rate, but were supported by MSCs, even when monocytes were depleted. These data, highlighting MSC plasticity in their immunomodulatory activity, pave the way for further studies investigating the mechanisms of mutual interactions between MSCs and immune cells in sepsis. Thus, MSCs might be able to fight against both early sepsis-induced hyperinflammatory response and later time points of immune dysfunction.

Transcriptome analysis of bone marrow mesenchymal stromal cells from patients with primary myelofibrosis.

Primary myelofibrosis (PMF) is a clonal myeloproliferative neoplasm whose severity and treatment complexity are attributed to the presence of bone marrow (BM) fibrosis and alterations of stroma impairing the production of normal blood cells. Despite the recently discovered mutations including the JAK2V617F mutation in about half of patients, the primitive event responsible for the clonal proliferation is still unknown. In the highly inflammatory context of PMF, the presence of fibrosis associated with a neoangiogenesis and an osteosclerosis concomitant to the myeloproliferation and to the increase number of circulating hematopoietic progenitors suggests that the crosstalk between hematopoietic and stromal cells is deregulated in the PMF BM microenvironmental niches. Within these niches, mesenchymal stromal cells (BM-MSC) play a hematopoietic supportive role in the production of growth factors and extracellular matrix which regulate the proliferation, differentiation, adhesion and migration of hematopoietic stem/progenitor cells. A transcriptome analysis of BM-MSC in PMF patients will help to characterize their molecular alterations and to understand their involvement in the hematopoietic stem/progenitor cell deregulation that features PMF.

Heterogeneous functions of perinatal mesenchymal stromal cells require a preselection before their banking for clinical use.

Perinatal sources of mesenchymal stromal cells (MSCs) have raised growing interest because they are readily and widely available with minimal ethical/legal issues and can easily be stored for allogeneic settings. In addition, perinatal tissues are known to be important in mediating the fetomaternal tolerance of pregnancy, which confer upon perinatal-MSCs (P-MSCs) a particular interest in immunomodulation. It has been recently shown that it is possible to deeply modify the secreted factor profiles of MSCs with different cytokine stimuli such as interferon gamma or tumor necrosis factor alpha to license MSCs for a better immunosuppresive potential. Therefore, we aimed to compare adult bone marrow-MSCs with MSCs from perinatal tissues (cord blood, umbilical cord, amnion, and chorion) on their in vitro immunological and stromacytic efficiencies under different priming conditions. Our results showed that P-MSCs had a potential to modulate the in vitro immune response and be useful for hematopoietic progenitor cell ex vivo expansion. However, we showed contrasted effects of cytokine priming embedded in an important between-donor variability. In conclusion, our study highlights the importance to elaborate predicitive in vitro tests to screen between-donor variability of perinatal tissues for banking allogeneic standardized MSCs.

CXCR7 participates in CXCL12-induced CD34+ cell cycling through ß-arrestin-dependent Akt activation.

In addition to its well-known effect on migration and homing of hematopoietic stem/progenitor cells (HSPCs), CXCL12 chemokine also exhibits a cell cycle and survival-promoting factor for human CD34(+) HSPCs. CXCR4 was suggested to be responsible for CXCL12-induced biological effects until the recent discovery of its second receptor, CXCR7. Until now, the participation of CXCR7 in CXCL12-induced HSPC cycling and survival is unknown. We show here that CXCL12 was capable of binding CXCR7 despite its scarce expression at CD34(+) cell surface. Blocking CXCR7 inhibited CXCL12-induced Akt activation as well as the percentage of CD34(+) cells in cycle, colony formation, and survival, demonstrating its participation in CXCL12-induced functional effects in HSPCs. At steady state, CXCR7 and ß-arrestin2 co-localized near the plasma membrane of CD34(+) cells. After CXCL12 treatment, ß-arrestin2 translocated to the nucleus, and this required both CXCR7 and CXCR4. Silencing ß-arrestin expression decreased CXCL12-induced Akt activation in CD34(+) cells. Our results demonstrate for the first time the role of CXCR7, complementary to that played by CXCR4, in the control of HSPC cycling, survival, and colony formation induced by CXCL12. We also provide evidence for the involvement of ß-arrestins as signaling hubs downstream of both CXCL12 receptors in primary human HSPCs.

Emerging therapy for improving wound repair of severe radiation burns using local bone marrow-derived stem cell administrations.

The therapeutic management of severe radiation burns remains a challenging issue today. Conventional surgical treatment including excision, skin autograft, or flap often fails to prevent unpredictable and uncontrolled extension of the radiation-induced necrotic process. In a recent very severe accidental radiation burn, we demonstrated the efficiency of a new therapeutic approach combining surgery and local cellular therapy using autologous mesenchymal stem cells (MSC), and we confirmed the crucial place of the dose assessment in this medical management. The patient presented a very significant radiation lesion located on the arm, which was first treated by several surgical procedures: iterative excisions, skin graft, latissimus muscle dorsi flap, and forearm radial flap. This conventional surgical therapy was unfortunately inefficient, leading to the use of an innovative cell therapy strategy. Autologous MSC were obtained from three bone marrow collections and were expanded according to a clinical-grade protocol using platelet-derived growth factors. A total of five local MSC administrations were performed in combination with skin autograft. After iterative local MSC administrations, the clinical evolution was favorable and no recurrence of radiation inflammatory waves occurred during the patient's 8-month follow-up. The benefit of this local cell therapy could be linked to the "drug cell" activity of MSC by modulating the radiation inflammatory processes, as suggested by the decrease in the C-reactive protein level observed after each MSC administration. The success of this combined treatment leads to new prospects in the medical management of severe radiation burns and more widely in the improvement of wound repair.

A cross-talk between stromal cell-derived factor-1 and transforming growth factor-beta controls the quiescence/cycling switch of CD34(+) progenitors through FoxO3 and mammalian target of rapamycin.

Cell cycle regulation plays a fundamental role in stem cell biology. A balance between quiescence and proliferation of hematopoietic stem cells in interaction with the microenvironment is critical for sustaining long-term hematopoiesis and for protection against stress. We analyzed the molecular mechanisms by which stromal cell-derived factor-1 (SDF-1) exhibited a cell cycle-promoting effect and interacted with transforming growth factor-beta (TGF-beta), which has negative effects on cell cycle orchestration of human hematopoietic CD34(+) progenitor cells. We demonstrated that a low concentration of SDF-1 modulated the expression of key cell cycle regulators such as cyclins, cyclin-dependent kinase inhibitors, and TGF-beta target genes, confirming its cell cycle-promoting effect. We showed that a cross-talk between SDF-1- and TGF-beta-related signaling pathways involving phosphatidylinositol 3-kinase (PI3K)/Akt phosphorylation participated in the control of CD34(+) cell cycling. We demonstrated a pivotal role of two downstream effectors of the PI3K/Akt pathway, FoxO3a and mammalian target of rapamycin, as connectors in the SDF-1-/TGF-beta-induced control of the cycling/quiescence switch and proposed a model integrating a dialogue between the two molecules in cell cycle progression. Our data shed new light on the signaling pathways involved in SDF-1 cell cycle-promoting activity and suggest that the balance between SDF-1- and TGF-beta-activated pathways is critical for the regulation of hematopoietic progenitor cell cycle status.

Mesenchymal stem cell abnormalities in patients with multiple myeloma.

Osteolytic bone lesions are common in patients with multiple myeloma (MM), a clonal plasma cell disorder, and result from increased osteoclastic bone resorption and decreased osteoblastic bone formation. Because mesenchymal stem cells (MSCs) are committed towards cells of the osteoblast lineage, we compared the in vitro characteristics of MSCs from the bone marrow of 18 MM patients (MM-MSCs) and eight normal donors (ND-MSCs). MM-MSCs displayed deficient growth that could be explained in part by the reduced expression of several growth factor receptors on the surface of MM-MSCs compared with ND-MSCs. Receptor downregulation was observed on RT-PCR analysis. A major finding was an approximately fivefold higher expression of osteoblast inhibitor DKK1 at transcript and protein levels in MM-MSCs than ND-MSCs. These data suggest that defective osteoblast function in patients with advanced MM may be related not only to factors released by tumor myeloma cells but also to MSC abnormalities.

Phenotypic and functional characteristics of CD34+ cells are related to their anatomical environment: is their versatility a prerequisite for their bio-availability?

Human CD34+ hematopoietic progenitors (HP) are mainly resident in adult bone marrow (BM). However, their recent revelation in nonhematopoietic tissues implies their circulation through peripheral blood (PB). The intimate mechanisms of this physiological process are not yet understood. Our results showed that steady-state CD34+ HP exhibit a differential phenotypic profile according to their BM versus PB localization. We demonstrated that this phenotype could be modulated by incubation in the presence of their counterpart mononuclear cells (MNC) through cell interactions and cytokine production. Such a modulation mainly concerns migration-mediated cytokine and chemokine receptors as well as some adhesion molecules and partly results from MNC specificity. These phenotypic profiles are associated with distinct cell-cycle position, cloning efficiency, and migration capacity of CD34+ cells from the different anatomical sources. We therefore propose a definition for a circulating versus resident CD34+ cell profile, which mostly depends on their cellular environment. We suggest that blood would represent a supply of cells for which phenotypic and functional characteristics would be a prerequisite for their bio-availability.