Fatty acid synthase and stearoyl-CoA desaturase-1 are conserved druggable cofactors of Old World Alphavirus genome replication

International audienc

HIV-1 nucleocapsid and ESCRT-component Tsg101 interplay prevents HIV from turning into a DNA-containing virus

International audienceHIV-1, the agent of the AIDS pandemic, is an RNA virus that reverse transcribes its RNA genome (gRNA) into DNA, shortly after its entry into cells. Within cells, retroviral assembly requires thousands of structural Gag proteins and two copies of gRNA as well as cellular factors, which converge to the plasma membrane in a finely regulated timeline. In this process, the nucleocapsid domain of Gag (GagNC) ensures gRNA selection and packaging into virions. Subsequent budding and virus release require the recruitment of the cellular ESCRT machinery. Interestingly, mutating GagNC results into the release of DNA-containing viruses, by promoting reverse transcription (RTion) prior to virus release , through an unknown mechanism. Therefore, we explored the biogenesis of these DNA-containing particles, combining live-cell total internal-reflection fluorescent microscopy, electron microscopy, trans-complementation assays and biochemical characterization of viral particles. Our results reveal that DNA virus production is the consequence of budding defects associated with Gag aggregation at the plasma membrane and deficiency in the recruitment of Tsg101, a key ESCRT-I component. Indeed, targeting Tsg101 to virus assembly sites restores budding , restricts RTion and favors RNA packaging into viruses. Altogether, our results highlight the role of GagNC in the spatiotemporal control of RTion, via an ESCRT-I-dependent mechanism

Specific intracellular signature of SARS-CoV-2 infection using confocal Raman microscopy

International audienceAbstract SARS-CoV-2 infection remains spread worldwide and requires a better understanding of virus-host interactions. Here, we analyzed biochemical modifications due to SARS-CoV-2 infection in cells by confocal Raman microscopy. Obtained results were compared with the infection with another RNA virus, the measles virus. Our results have demonstrated a virus-specific Raman molecular signature, reflecting intracellular modification during each infection. Advanced data analysis has been used to distinguish non-infected versus infected cells for two RNA viruses. Further, classification between non-infected and SARS-CoV-2 and measles virus-infected cells yielded an accuracy of 98.9 and 97.2 respectively, with a significant increase of the essential amino-acid tryptophan in SARS-CoV-2-infected cells. These results present proof of concept for the application of Raman spectroscopy to study virus-host interaction and to identify factors that contribute to the efficient SARS-CoV-2 infection and may thus provide novel insights on viral pathogenesis, targets of therapeutic intervention and development of new COVID-19 biomarkers

Insight into the mechanism of action of EP-39, a bevirimat derivative that inhibits HIV-1 maturation

International audienceMaturation of human immunodeficiency virus type 1 (HIV-1) particles is a key step for viral infectivity. This process can be blocked using maturation inhibitors (MIs) that affect the cleavage of the capsid-spacer peptide 1 (CA-SP1) junction. Here, we investigated the mechanisms underlying the activity of EP-39, a bevirimat (BVM) derivative with better hydrosolubility. To this aim, we selected in vitro EP-39- and BVM-resistant mutants. We found that EP-39-resistant viruses have four mutations within the CA domain (CA-A194T, CA-T200N, CA-V230I, and CA-V230A) and one in the first residue of SP1 (SP1-A1V). We also identified six mutations that confer BVM resistance (CA-A194T, CA-L231F, CA-L231M, SP1-A1V, SP1-S5N and SP1-V7A). To characterize the EP-39 and BVM-resistant mutants, we studied EP-39 effects on mutant virus replication and performed a biochemical analysis with both MIs. We observed common and distinct characteristics, suggesting that, although EP-39 and BVM share the same chemical skeleton, they could interact in a different way with the Gag polyprotein precursor (Pr55Gag). Using an in silico approach, we observed that EP-39 and BVM present different predicted positions on the hexameric crystal structure of the CACTD-SP1 Gag fragment. To clearly understand the relationship between assembly and maturation, we investigated the impact of all identified mutations on virus assembly by expressing Pr55Gag mutants. Finally, using NMR, we have shown that the interaction of EP-39 with a peptide carrying the SP1-A1V mutation (CA-SP1(A1V)-NC) is almost suppressed in comparison with the wild type peptide. These results suggest that EP-39 and BVM could interact differently with the Pr55Gag lattice and that the mutation of the first SP1 residue induces a loss of interaction between Pr55Gag and EP-39

Constitutive proteins of lumpy skin disease virion assessed by next-generation proteomics

The Poxviridae (POXV) is a family of large, complex, enveloped virions enclosing single linear double-stranded DNA genome (128-450 kbp) (1, 2) that replicates entirelyInternational audienceLumpy skin disease (LSD) virus, a ruminant poxvirus of the Capripoxvirus genus, is the etiologic agent of an economically important cattle disease categorized as a notifiable disease by the World Organisation for Animal Health. The large and complex enveloped LSD virus (LSDV) particle encloses a double-stranded linear DNA genome of 151 kbp, comprising 156 predicted ORFs, together with a variety of proteins that are not yet identified. In parallel with a recent widespread expansion of LSD, many LSDV whole genomes have been sequenced, but knowledge on protein composition of the LSDV particle remains missing. In this study, LSD mature virions (MV) from strain KSGP-0240 were purified through a multistep ultracentrifugation process. The protein composition of LSD virions was then analyzed using label-free shotgun proteomics, based upon nano-liquid chromatography (LC) and tandem mass spectrometry. This procedure resulted in the identification of a total of 111 LSDV proteins. Considering that this first MV proteome extended beyond packaged proteins into the field of contami nants, an analytical methodology was developed and made it possible to select 66 viral proteins as candidates for packaging into MV. These viral proteins were analyzed comparatively with proteins previously demonstrated to be constitutive of the vaccinia virus MV particle. Offering for the first time a comprehensive proteomic analysis of an LSDV strain, this study contributes to our understanding of the structural features of infectious LSDV MV particles and paves the way for further systematic proteomic characterization of other LSDV strains. IMPORTANCE: Lumpy skin disease virus (LSDV) is the causative agent of an economically important cattle disease which is notifiable to the World Organisation for Animal Health. Over the past decades, the disease has spread at an alarming rate throughout the African continent, the Middle East, Eastern Europe, the Russian Federation, and many Asian countries. While multiple LDSV whole genomes have made further genetic comparative analyses possible, knowledge on the protein composition of the LSDV particle remains lacking. This study provides for the first time a comprehensive proteomic analysis of an infectious LSDV particle, prompting new efforts toward further proteomic LSDV strain characterization. Furthermore, this first incursion within the capripoxvirus proteome represents one of very few proteomic studies beyond the sole Orthopoxvirus genus, for which most of the proteomics studies have been performed. Providing new informa tion about other chordopoxviruses may contribute to shedding new light on protein composition within the Poxviridae family

F-actin nanostructures rearrangements and regulation are essential for SARS-CoV-2 particle production in host pulmonary cells

Summary: Our study focused on deciphering the role of F-actin and related regulatory factors during SARS-CoV-2 particle production and transmission in human pulmonary cells. Quantitative high-resolution microscopies revealed that the late phases of SARS-CoV-2 infection induce a strong rearrangement of F-actin nanostructures dependent on the viral M, E, and N structural proteins. Intracellular vesicles containing viral components are labeled with Rab7 and Lamp1 and are surrounded by F-actin ring-shaped structures, suggesting their role in viral trafficking toward the cell membrane for virus release. Furthermore, filopodia-like nanostructures were loaded with viruses, potentially facilitating their egress and transmission between lung cells. Gene expression analysis revealed the involvement of alpha-actinins under the regulation of the protein kinase N (PKN). The use of a PKN inhibitor efficiently reduces virus particle production, restoring endoplasmic reticulum and F-actin cellular shape. Our results highlight an important role of F-actin rearrangements during the productive phases of SARS-CoV-2 particles

PTHrP, calcitonin and calcitriol in a case of severe, protracted and refractory hypercalcemia due to a pancreatic neuroendocrine tumor

A patient with a primary neuroendocrine tumor of the pancreas, presented with severe hypercalcemia. This hypercalcemia of malignancy (HCM) failed to respond to intensive bisphosphonate treatment and needed continuous enhanced diuresis. Only after successful antitumor therapy did the hypercalcemia subside. Hypercalcemia was associated with increased concentrations of plasma PTHrP, calcitonin and 1,25-(OH)2D3. Bone mineral density was markedly increased. We demonstrated the presence of both PTHrP and calcitonin in the tumor at the mRNA and protein level, using RT-PCR, immunohistochemistry and Western blotting. The high levels of plasma PTHrP and the demonstrated predominant renal mechanism in this case of HCM are suspected to be the cause for its refractoriness to bone resorption inhibitors. Our findings furthermore suggest that the tumoral production of calcitonin and PTHrP might have contributed to the increased bone mineral storage of calcium and thus probably attenuated the development of frank hypercalcemia. © 2006 Elsevier Inc. All rights reserved.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development

International audienceThe Q fever bacterium Coxiella burnetii replicates inside host cells within a large Coxiella-containing vacuole (CCV) whose biogenesis relies on the Dot/Icm-dependent secretion of bacterial effectors. Several membrane trafficking pathways contribute membranes, proteins, and lipids for CCV biogenesis. These include the endo-cytic and autophagy pathways, which are characterized by phos-phatidylinositol 3-phosphate [PI(3)P]-positive membranes. Here we show that the C. burnetii secreted effector Coxiella vacuolar protein B (CvpB) binds PI(3)P and phosphatidylserine (PS) on CCVs and early endosomal compartments and perturbs the activity of the phosphatidylinositol 5-kinase PIKfyve to manipulate PI(3)P metabolism. CvpB association to early endosome triggers vacuolation and clustering, leading to the channeling of large PI(3)P-positive membranes to CCVs for vacuole expansion. At CCVs, CvpB binding to early endosome-and autophagy-derived PI(3)P and the concom-itant inhibition of PIKfyve favor the association of the autophago-somal machinery to CCVs for optimal homotypic fusion of the Coxiella-containing compartments. The importance of manipulating PI(3)P metabolism is highlighted by mutations in cvpB resulting in a multivacuolar phenotype, rescuable by gene complementa-tion, indicative of a defect in CCV biogenesis. Using the insect model Galleria mellonella, we demonstrate the in vivo relevance of defective CCV biogenesis by highlighting an attenuated viru-lence phenotype associated with cvpB mutations

Low Selectivity Indices of Ivermectin and Macrocyclic Lactones on SARS-CoV-2 Replication In Vitro

Ivermectin was first approved for human use as an endectocide in the 1980s. It remains one of the most important global health medicines in history and has recently been shown to exert in vitro activity against SARS-CoV-2. However, the macrocyclic lactone family of compounds has not previously been evaluated for activity against SARS-CoV-2. The present study aims at comparing their anti-viral activity in relevant human pulmonary cell lines in vitro. Here, in vitro antiviral activity of the avermectins (ivermectin and selamectin) and milbemycins (moxidectin and milbemycin oxime) were assessed against a clinical isolate from a CHU Montpellier patient infected with SARS-CoV-2 in 2020. Ivermectin, like the other macrocyclic lactones moxidectin, milbemycin oxime and selamectin, reduced SARS-CoV-2 replication in vitro (EC50 of 2–5 μM). Immunofluorescence assays with ivermectin and moxidectin showed a reduction in the number of infected and polynuclear cells, suggesting a drug action on viral cell fusion. However, cellular toxicity of the avermectins and milbemycins during infection showed a very low selectivity index of <10. Thus, none of these agents appears suitable for human use for its anti-SARS-CoV-2 activity per se, due to low selectivity index

Functional Characterization of the N-Acetylmuramyl-l-Alanine Amidase, Ami1, from Mycobacterium abscessus

International audiencePeptidoglycan (PG) is made of a polymer of disaccharides organized as a three-dimensional mesh-like network connected together by peptidic cross-links. PG is a dynamic structure that is essential for resistance to environmental stressors. Remodeling of PG occurs throughout the bacterial life cycle, particularly during bacterial division and separation into daughter cells. Numerous autolysins with various substrate specificities participate in PG remodeling. Expression of these enzymes must be tightly regulated, as an excess of hydrolytic activity can be detrimental for the bacteria. In non-tuberculous mycobacteria such as Mycobacterium abscessus, the function of PG-modifying enzymes has been poorly investigated. In this study, we characterized the function of the PG amidase, Ami1 from M. abscessus. An ami1 deletion mutant was generated and the phenotypes of the mutant were evaluated with respect to susceptibility to antibiotics and virulence in human macrophages and zebrafish. The capacity of purified Ami1 to hydrolyze muramyl-dipeptide was demonstrated in vitro. In addition, the screening of a 9200 compounds library led to the selection of three compounds inhibiting Ami1 in vitro. We also report the structural characterization of Ami1 which, combined with in silico docking studies, allows us to propose a mode of action for these inhibitors