Structural analysis of the Candida albicans mitochondrial DNA maintenance factor Gcf1p reveals a dynamic DNA-bridging mechanism.

The compaction of mitochondrial DNA (mtDNA) is regulated by architectural HMG-box proteins whose limited cross-species similarity suggests diverse underlying mechanisms. Viability of Candida albicans, a human antibiotic-resistant mucosal pathogen, is compromised by altering mtDNA regulators. Among them, there is the mtDNA maintenance factor Gcf1p, which differs in sequence and structure from its human and Saccharomyces cerevisiae counterparts, TFAM and Abf2p. Our crystallographic, biophysical, biochemical and computational analysis showed that Gcf1p forms dynamic protein/DNA multimers by a combined action of an N-terminal unstructured tail and a long helix. Furthermore, an HMG-box domain canonically binds the minor groove and dramatically bends the DNA while, unprecedentedly, a second HMG-box binds the major groove without imposing distortions. This architectural protein thus uses its multiple domains to bridge co-aligned DNA segments without altering the DNA topology, revealing a new mechanism of mtDNA condensation.

Label-Free Single Nanoparticle Identification and Characterization in Demanding Environment, Including Infectious Emergent Virus.

Unknown particle screening-including virus and nanoparticles-are keys in medicine, industry, and also in water pollutant determination. Here, RYtov MIcroscopy for Nanoparticles Identification (RYMINI) is introduced, a staining-free, non-invasive, and non-destructive optical approach that is merging holographic label-free 3D tracking with high-sensitivity quantitative phase imaging into a compact optical setup. Dedicated to the identification and then characterization of single nano-object in solution, it is compatible with highly demanding environments, such as level 3 biological laboratories, with high resilience to external source of mechanical and optical noise. Metrological characterization is performed at the level of each single particle on both absorbing and transparent particles as well as on immature and infectious HIV, SARS-CoV-2 and extracellular vesicles in solution. The capability of RYMINI to determine the nature, concentration, size, complex refractive index and mass of each single particle without knowledge or model of the particles' response is demonstrated. The system surpasses 90% accuracy for automatic identification between dielectric/metallic/biological nanoparticles and ≈80% for intraclass chemical determination of metallic and dielectric. It falls down to 50-70% for type determination inside the biological nanoparticle's class.

DNA specificities modulate the binding of human transcription factor A to mitochondrial DNA control region.

Human mitochondrial DNA (h-mtDNA) codes for 13 subunits of the oxidative phosphorylation pathway, the essential route that produces ATP. H-mtDNA transcription and replication depends on the transcription factor TFAM, which also maintains and compacts this genome. It is well-established that TFAM activates the mtDNA promoters LSP and HSP1 at the mtDNA control region where DNA regulatory elements cluster. Previous studies identified still uncharacterized, additional binding sites at the control region downstream from and slightly similar to LSP, namely sequences X and Y (Site-X and Site-Y) (Fisher et al., Cell 50, pp 247-258, 1987). Here, we explore TFAM binding at these two sites and compare them to LSP by multiple experimental and in silico methods. Our results show that TFAM binding is strongly modulated by the sequence-dependent properties of Site-X, Site-Y and LSP. The high binding versatility of Site-Y or the considerable stiffness of Site-X tune TFAM interactions. In addition, we show that increase in TFAM/DNA complex concentration induces multimerization, which at a very high concentration triggers disruption of preformed complexes. Therefore, our results suggest that mtDNA sequences induce non-uniform TFAM binding and, consequently, direct an uneven distribution of TFAM aggregation sites during the essential process of mtDNA compaction.

[Illuminating the nanoscopic world of viruses by fluorescence super-resolution microscopy]. / Apport de la microscopie de fluorescence super-résolue : dans l'intimité des virus !

The recent revolution in optical fluorescence microscopy, supported by the optimization of both spatial resolution and acquisition speed, led to the ability to visualize nano-scaled objects. Currently, the use of a new generation of super-resolution fluorescence microscopes coupled to improved fluorescent probes gives the possibility to study the replicative cycle of viruses in living cells, at the single-virus and molecule level. In this review, after a brief chronological description of these new approaches, we highlight several examples of super-resolution microscopies that have allowed to revisit our understanding of several human viruses and of host-pathogen interactions.

Illuminating the nanoscopic world of viruses by fluorescence super-resolution microscopy.

The recent revolution in optical fluorescence microscopy, supported by the optimization of both spatial resolution and acquisition speed, led to the ability to visualize nano-scaled objects. Currently, the use of a new generation of super-resolution fluorescence microscopes coupled to improved fluorescent probes gives the possibility to study the replicative cycle of viruses in living cells, at the single-virus and molecule level. In this review, after a brief chronological description of these new approaches, we highlight several examples of super-resolution microscopies that have allowed to revisit our understanding of several human viruses and of host-pathogen interactions.

DNA structure directs positioning of the mitochondrial genome packaging protein Abf2p.

The mitochondrial genome (mtDNA) is assembled into nucleo-protein structures termed nucleoids and maintained differently compared to nuclear DNA, the involved molecular basis remaining poorly understood. In yeast (Saccharomyces cerevisiae), mtDNA is a ∼80 kbp linear molecule and Abf2p, a double HMG-box protein, packages and maintains it. The protein binds DNA in a non-sequence-specific manner, but displays a distinct 'phased-binding' at specific DNA sequences containing poly-adenine tracts (A-tracts). We present here two crystal structures of Abf2p in complex with mtDNA-derived fragments bearing A-tracts. Each HMG-box of Abf2p induces a 90° bend in the contacted DNA, causing an overall U-turn. Together with previous data, this suggests that U-turn formation is the universal mechanism underlying mtDNA compaction induced by HMG-box proteins. Combining this structural information with mutational, biophysical and computational analyses, we reveal a unique DNA binding mechanism for Abf2p where a characteristic N-terminal flag and helix are crucial for mtDNA maintenance. Additionally, we provide the molecular basis for A-tract mediated exclusion of Abf2p binding. Due to high prevalence of A-tracts in yeast mtDNA, this has critical relevance for nucleoid architecture. Therefore, an unprecedented A-tract mediated protein positioning mechanism regulates DNA packaging proteins in the mitochondria, and in combination with DNA-bending and U-turn formation, governs mtDNA compaction.

Oligonucleotide-Lipid Conjugates Forming G-Quadruplex Structures Are Potent and Pangenotypic Hepatitis C Virus Entry Inhibitors In Vitro and Ex Vivo.

A hepatitis C virus (HCV) epidemic affecting HIV-infected men who have sex with men (MSM) is expanding worldwide. In spite of the improved cure rates obtained with the new direct-acting antiviral drug (DAA) combinations, the high rate of reinfection within this population calls urgently for novel preventive interventions. In this study, we determined in cell culture and ex vivo experiments with human colorectal tissue that lipoquads, G-quadruplex DNA structures fused to cholesterol, are efficient HCV pangenotypic entry and cell-to-cell transmission inhibitors. Thus, lipoquads may be promising candidates for the development of rectally applied gels to prevent HCV transmission.

Nucleocapsid Protein: A Desirable Target for Future Therapies Against HIV-1.

The currently available anti-HIV-1 therapeutics is highly beneficial to infected patients. However, clinical failures occur as a result of the ability of HIV-1 to rapidly mutate. One approach to overcome drug resistance is to target HIV-1 proteins that are highly conserved among phylogenetically distant viral strains and currently not targeted by available therapies. In this respect, the nucleocapsid (NC) protein, a zinc finger protein, is particularly attractive, as it is highly conserved and plays a central role in virus replication, mainly by interacting with nucleic acids. The compelling rationale for considering NC as a viable drug target is illustrated by the fact that point mutants of this protein lead to noninfectious viruses and by the inability to select viruses resistant to a first generation of anti-NC drugs. In our review, we discuss the most relevant properties and functions of NC, as well as recent developments of small molecules targeting NC. Zinc ejectors show strong antiviral activity, but are endowed with a low therapeutic index due to their lack of specificity, which has resulted in toxicity. Currently, they are mainly being investigated for use as topical microbicides. Greater specificity may be achieved by using non-covalent NC inhibitors (NCIs) targeting the hydrophobic platform at the top of the zinc fingers or key nucleic acid partners of NC. Within the last few years, innovative methodologies have been developed to identify NCIs. Though the antiviral activity of the identified NCIs needs still to be improved, these compounds strongly support the druggability of NC and pave the way for future structure-based design and optimization of efficient NCIs.

Understanding the effect of the nature of the nucleobase in the loops on the stability of the i-motif structure.

The nature and the length of loops connecting cytosine tracts in i-motif structures may affect their stability. In this work, the influence of the nature of the nucleobases located in two of the loops of an intramolecular i-motif is studied using spectroscopy, separation techniques, and multivariate data analysis. The insertion of bases other than thymine induces an additional acid-base equilibrium with pKa ∼ 4.5. The presence of two guanine bases in the loops, placed opposite to each other, decreases the thermal stability of the structure. In contrast, thymine and cytosine bases in these positions stabilize the structure.

Solution equilibria of cytosine- and guanine-rich sequences near the promoter region of the n-myc gene that contain stable hairpins within lateral loops.

BACKGROUND: Cytosine- and guanine-rich regions of DNA are capable of forming complex structures named i-motifs and G-quadruplexes, respectively. In the present study the solution equilibria at nearly physiological conditions of a 34-base long cytosine-rich sequence and its complementary guanine-rich strand corresponding to the first intron of the n-myc gene were studied. Both sequences, not yet studied, contain a 12-base tract capable of forming stable hairpins inside the i-motif and G-quadruplex structures, respectively. METHODS: Spectroscopic, mass spectrometry and separation techniques, as well as multivariate data analysis methods, were used to unravel the species and conformations present. RESULTS: The cytosine-rich sequence forms two i-motifs that differ in the protonation of bases located in the loops. A stable Watson-Crick hairpin is formed by the bases in the first loop, stabilizing the i-motif structure. The guanine-rich sequence adopts a parallel G-quadruplex structure that is stable throughout the pH range 3-7, despite the protonation of cytosine and adenine bases at lower pH values. The presence of G-quadruplex aggregates was confirmed using separation techniques. When mixed, G-quadruplex and i-motif coexist with the Watson-Crick duplex across a pH range from approximately 3.0 to 6.5. CONCLUSIONS: Two cytosine- and guanine-rich sequences in n-myc gene may form stable i-motif and G-quadruplex structures even in the presence of long loops. pH modulates the equilibria involving the intramolecular structures and the intermolecular Watson-Crick duplex. GENERAL SIGNIFICANCE: Watson-Crick hairpins located in the intramolecular G-quadruplexes and i-motifs in the promoter regions of oncogenes could play a role in stabilizing these structures.

Evaluation and activity of new porphyrin-peptide cage-type conjugates for the photoinactivation of Mycobacterium abscessus.

Mycobacterium abscessus is increasingly recognized as an emerging opportunistic pathogen causing severe lung diseases and cutaneous infections. However, treatment of M. abscessus infections remains particularly challenging, largely due to intrinsic resistance to a wide panel of antimicrobial agents. New therapeutic alternatives are urgently needed. Herein, we show that, upon limited irradiation with a blue-light source, newly developed porphyrin-peptide cage-type photosensitizers exert a strong bactericidal activity against smooth and rough variants of M. abscessus in planktonic cultures and in biofilms, at low concentrations. Atomic force microscopy unraveled important morphological alterations that include a wrinkled and irregular bacterial surface. The potential of these compounds for a photo-therapeutic use to treat M. abscessus skin infections requires further evaluations.IMPORTANCEMycobacterium abscessus causes persistent infections and is extremely difficult to eradicate. Despite intensive chemotherapy, treatment success rates remain very low. Thus, given the unsatisfactory performances of the current regimens, more effective therapeutic alternatives are needed. In this study, we evaluated the activity of newly described porphyrin-peptide cage-type conjugates in the context of photodynamic therapy. We show that upon light irradiation, these compounds were highly bactericidal against M. abscessus in vitro, thus qualifying these compounds for future studies dedicated to photo-therapeutic applications against M. abscessus skin infections.

Validation of flavivirus infectious clones carrying fluorescent markers for antiviral drug screening and replication studies.

Flaviviruses have emerged as major arthropod-transmitted pathogens and represent an increasing public health problem worldwide. High-throughput screening can be facilitated using viruses that easily express detectable marker proteins. Therefore, developing molecular tools, such as reporter-carrying versions of flaviviruses, for studying viral replication and screening antiviral compounds represents a top priority. However, the engineering of flaviviruses carrying either fluorescent or luminescent reporters remains challenging due to the genetic instability caused by marker insertion; therefore, new approaches to overcome these limitations are needed. Here, we describe reverse genetic methods that include the design and validation of infectious clones of Zika, Kunjin, and Dengue viruses harboring different reporter genes for infection, rescue, imaging, and morphology using super-resolution microscopy. It was observed that different flavivirus constructs with identical designs displayed strikingly different genetic stabilities, and corresponding virions resembled wild-type virus particles in shape and size. A successful strategy was assessed to increase the stability of rescued reporter virus and permit antiviral drug screening based on quantitative automated fluorescence microscopy and replication studies.

Dynamic Profile of S-Layer Proteins Controls Surface Properties of Emetic Bacillus cereus AH187 Strain.

Many prokaryotes are covered by a two-dimensional array of proteinaceous subunits. This surface layers (S-layer) is incompletely characterized for many microorganisms. Here, we studied Bacillus cereus AH187. A genome analysis identified two genes encoding the S-layer proteins SL2 and EA1, which we experimentally confirmed to encode the two protein components of the S-layer covering the surface of B. cereus. Shotgun proteomics analysis indicated that SL2 is the major component of the B. cereus S-layer at the beginning of exponential growth, whereas EA1 becomes more abundant than SL2 during later stages of stationary growth. Microscopy analysis revealed the spatial organization of SL2 and EA1 at the surface of B. cereus to depend on their temporal-dynamics during growth. Our results also show that a mutant strain lacking functional SL2 and EA1 proteins has distinct surface properties compared to its parental strain, in terms of stiffness and hydrophilicity during the stationary growth phase. Surface properties, self-aggregation capacity, and bacterial adhesion were observed to correlate. We conclude that the dynamics of SL2 and EA1 expression is a key determinant of the surface properties of B. cereus AH187, and that the S-layer could contribute to B. cereus survival in starvation conditions.

The Syp1/FCHo2 protein induces septin filament bundling through its intrinsically disordered domain.

The septin collar of budding yeast is an ordered array of septin filaments that serves a scaffolding function for the cytokinetic machinery at the bud neck and compartmentalizes the membrane between mother and daughter cell. How septin architecture is aided by septin-binding proteins is largely unknown. Syp1 is an endocytic protein that was implicated in the timely recruitment of septins to the newly forming collar through an unknown mechanism. Using advanced microscopy and in vitro reconstitution assays, we show that Syp1 is able to align laterally and tightly pack septin filaments, thereby forming flat bundles or sheets. This property is shared by the Syp1 mammalian counterpart FCHo2, thus emphasizing conserved protein functions. Interestingly, the septin-bundling activity of Syp1 resides mainly in its intrinsically disordered region. Our data uncover the mechanism through which Syp1 promotes septin collar assembly and offer another example of functional diversity of unstructured protein domains.

Optimized production and fluorescent labeling of SARS-CoV-2 virus-like particles.

SARS-CoV-2 is an RNA enveloped virus responsible for the COVID-19 pandemic that conducted in 6 million deaths worldwide so far. SARS-CoV-2 particles are mainly composed of the 4 main structural proteins M, N, E and S to form 100 nm diameter viral particles. Based on productive assays, we propose an optimal transfected plasmid ratio mimicking the viral RNA ratio in infected cells. This allows SARS-CoV-2 Virus-Like Particle (VLPs) formation composed of the viral structural proteins M, N, E and mature S. Furthermore, fluorescent or photoconvertible VLPs were generated by adding a fluorescent protein tag on N or M mixing with unlabeled viral proteins and characterized by western blots, atomic force microscopy coupled to fluorescence and immuno-spotting. Thanks to live fluorescence and super-resolution microscopies, we quantified VLPs size and concentration. SARS-CoV-2 VLPs present a diameter of 110 and 140 nm respectively for MNE-VLPs and MNES-VLPs with a concentration of 10e12 VLP/ml. In this condition, we were able to establish the incorporation of the Spike in the fluorescent VLPs. Finally, the Spike functionality was assessed by monitoring fluorescent MNES-VLPs docking and internalization in human pulmonary cells expressing or not the receptor hACE2. Results show a preferential maturation of S on N(GFP) labeled VLPs and an hACE2-dependent VLP internalization and a potential fusion in host cells. This work provides new insights on the use of non-fluorescent and fluorescent VLPs to study and visualize the SARS-CoV-2 viral life cycle in a safe environment (BSL-2 instead of BSL-3). Moreover, optimized SARS-CoV-2 VLP production can be further adapted to vaccine design strategies.

The FDA-approved drug Auranofin has a dual inhibitory effect on SARS-CoV-2 entry and NF-κB signaling.

Patients with severe COVID-19 show an altered immune response that fails to control the viral spread and suffer from exacerbated inflammatory response, which eventually can lead to death. A major challenge is to develop an effective treatment for COVID-19. NF-κB is a major player in innate immunity and inflammatory process. By a high-throughput screening approach, we identified FDA-approved compounds that inhibit the NF-κB pathway and thus dampen inflammation. Among these, we show that Auranofin prevents post-translational modifications of NF-κB effectors and their recruitment into activating complexes in response to SARS-CoV-2 infection or cytokine stimulation. In addition, we demonstrate that Auranofin counteracts several steps of SARS-CoV-2 infection. First, it inhibits a raft-dependent endocytic pathway involved in SARS-CoV-2 entry into host cells; Second, Auranofin alters the ACE2 mobility at the plasma membrane. Overall, Auranofin should prevent SARS-CoV-2 infection and inflammatory damages, offering new opportunities as a repurposable drug candidate to treat COVID-19.

Atomic force microscopy analysis of native infectious and inactivated SARS-CoV-2 virions.

SARS-CoV-2 is an enveloped virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic. Here, single viruses were analyzed by atomic force microscopy (AFM) operating directly in a level 3 biosafety (BSL3) facility, which appeared as a fast and powerful method to assess at the nanoscale level and in 3D infectious virus morphology in its native conformation, or upon inactivation treatments. AFM imaging reveals structurally intact infectious and inactivated SARS-CoV-2 upon low concentration of formaldehyde treatment. This protocol combining AFM and plaque assays allows the preparation of intact inactivated SARS-CoV-2 particles for safe use of samples out of level 3 laboratory to accelerate researches against the COVID-19 pandemic. Overall, we illustrate how adapted BSL3-AFM is a remarkable toolbox for rapid and direct virus analysis based on nanoscale morphology.

Comparative Genome-Wide Transcriptome Analysis of Brucella suis and Brucella microti Under Acid Stress at pH 4.5: Cold Shock Protein CspA and Dps Are Associated With Acid Resistance of B. microti.

Brucellae are facultative intracellular coccobacilli causing brucellosis, one of the most widespread bacterial zoonosis affecting wildlife animals, livestock and humans. The genus Brucella comprises classical and atypical species, such as Brucella suis and Brucella microti, respectively. The latter is characterized by increased metabolic activity, fast growth rates, and extreme acid resistance at pH 2.5, suggesting an advantage for environmental survival. In addition, B. microti is more acid-tolerant than B. suis at the intermediate pH of 4.5. This acid-resistant phenotype of B. microti may have major implications for fitness in soil, food products and macrophages. Our study focused on the identification and characterization of acid resistance determinants of B. suis and B. microti in Gerhardt's minimal medium at pH 4.5 and 7.0 for 20 min and 2 h by comparative RNA-Seq-based transcriptome analysis, validated by RT-qPCR. Results yielded a common core response in both species with a total of 150 differentially expressed genes, and acidic pH-dependent genes regulated specifically in each species. The identified core response mechanisms comprise proton neutralization or extrusion from the cytosol, participating in maintaining physiological intracellular pH values. Differential expression of 441 genes revealed species-specific mechanisms in B. microti with rapid physiological adaptation to acid stress, anticipating potential damage to cellular components and critical energy conditions. Acid stress-induced genes encoding cold shock protein CspA, pseudogene in B. suis, and stress protein Dps were associated with survival of B. microti at pH 4.5. B. suis response with 284 specifically regulated genes suggested increased acid stress-mediated protein misfolding or damaging, triggering the set-up of repair strategies countering the consequences rather than the origin of acid stress and leading to subsequent loss of viability. In conclusion, our work supports the hypothesis that increased acid stress resistance of B. microti is based on selective pressure for the maintenance of functionality of critical genes, and on specific differential gene expression, resulting in rapid adaptation.

The HIV-1 Nucleocapsid Regulates Its Own Condensation by Phase-Separated Activity-Enhancing Sequestration of the Viral Protease during Maturation.

A growing number of studies indicate that mRNAs and long ncRNAs can affect protein populations by assembling dynamic ribonucleoprotein (RNP) granules. These phase-separated molecular 'sponges', stabilized by quinary (transient and weak) interactions, control proteins involved in numerous biological functions. Retroviruses such as HIV-1 form by self-assembly when their genomic RNA (gRNA) traps Gag and GagPol polyprotein precursors. Infectivity requires extracellular budding of the particle followed by maturation, an ordered processing of ∼2400 Gag and ∼120 GagPol by the viral protease (PR). This leads to a condensed gRNA-NCp7 nucleocapsid and a CAp24-self-assembled capsid surrounding the RNP. The choreography by which all of these components dynamically interact during virus maturation is one of the missing milestones to fully depict the HIV life cycle. Here, we describe how HIV-1 has evolved a dynamic RNP granule with successive weak-strong-moderate quinary NC-gRNA networks during the sequential processing of the GagNC domain. We also reveal two palindromic RNA-binding triads on NC, KxxFxxQ and QxxFxxK, that provide quinary NC-gRNA interactions. Consequently, the nucleocapsid complex appears properly aggregated for capsid reassembly and reverse transcription, mandatory processes for viral infectivity. We show that PR is sequestered within this RNP and drives its maturation/condensation within minutes, this process being most effective at the end of budding. We anticipate such findings will stimulate further investigations of quinary interactions and emergent mechanisms in crowded environments throughout the wide and growing array of RNP granules.

Features, processing states, and heterologous protein interactions in the modulation of the retroviral nucleocapsid protein function.

Retroviral nucleocapsid (NC) is central to viral replication. Nucleic acid chaperoning is a key function for NC through the action of its conserved basic amino acids and zinc-finger structures. NC manipulates genomic RNA from its packaging in the producer cell to reverse transcription into the infected host cell. This chaperone function, in conjunction with NC's aggregating properties, is up-modulated by successive NC processing events, from the Gag precursor to the fully mature protein, resulting in the condensation of the nucleocapsid within the capsid shell. Reverse transcription also depends on NC processing, whereas this process provokes NC dissociation from double-stranded DNA, leading to a preintegration complex (PIC), competent for host chromosomal integration. In addition NC interacts with cellular proteins, some of which are involved in viral budding, and also with several viral proteins. All of these properties are reviewed here, focusing on HIV-1 as a paradigmatic reference and highlighting the plasticity of the nucleocapsid architecture.