Zoonotic coronavirus (CoV) infections, such as those responsible for the current severe acute respiratory syndrome-CoV 2 (SARS-CoV-2) pandemic, cause grave international public health concern. In ...infected cells, the CoV RNA-synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (RO). Although double-membrane vesicles (DMVs) appear to be a pan-CoV RO element, studies to date describe an assortment of additional CoV-induced membrane structures. Despite much speculation, it remains unclear which RO element(s) accommodate viral RNA synthesis. Here we provide detailed 2D and 3D analyses of CoV ROs and show that diverse CoVs essentially induce the same membrane modifications, including the small open double-membrane spherules (DMSs) previously thought to be restricted to gamma- and delta-CoV infections and proposed as sites of replication. Metabolic labeling of newly synthesized viral RNA followed by quantitative electron microscopy (EM) autoradiography revealed abundant viral RNA synthesis associated with DMVs in cells infected with the beta-CoVs Middle East respiratory syndrome-CoV (MERS-CoV) and SARS-CoV and the gamma-CoV infectious bronchitis virus. RNA synthesis could not be linked to DMSs or any other cellular or virus-induced structure. Our results provide a unifying model of the CoV RO and clearly establish DMVs as the central hub for viral RNA synthesis and a potential drug target in CoV infection.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Viral RNA exported from the coronavirus replication organelle is likely to be mediated by a crown-shaped molecular pore.
A gateway to the cytosol
Coronaviruses transform host cell membranes into ...peculiar double-membrane vesicles that have long been thought to accommodate viral genome replication. However, because these compartments appeared to be completely sealed, it has remained unknown how the newly made viral RNA could be exported to the cytosol for translation and packaging into new virions. Wolff
et al.
used cryo–electron microscopy to identify a molecular pore that spans the double membrane (see the Perspective by Unchwaniwala and Ahlquist). Six copies of a large coronavirus transmembrane protein formed the core of this structure, which may constitute a viral RNA export channel and provide a target for future antiviral interventions.
Science
, this issue p.
1395
; see also p.
1306
Coronavirus genome replication is associated with virus-induced cytosolic double-membrane vesicles, which may provide a tailored microenvironment for viral RNA synthesis in the infected cell. However, it is unclear how newly synthesized genomes and messenger RNAs can travel from these sealed replication compartments to the cytosol to ensure their translation and the assembly of progeny virions. In this study, we used cellular cryo–electron microscopy to visualize a molecular pore complex that spans both membranes of the double-membrane vesicle and would allow export of RNA to the cytosol. A hexameric assembly of a large viral transmembrane protein was found to form the core of the crown-shaped complex. This coronavirus-specific structure likely plays a key role in coronavirus replication and thus constitutes a potential drug target.
The molecular mechanisms regulating antigen translocation into the cytosol for cross-presentation are under controversial debate, mainly because direct data is lacking. Here, we have provided direct ...evidence that the activity of the endoplasmic reticulum (ER) translocon protein Sec61 is essential for endosome-to-cytosol translocation. We generated a Sec61-specific intrabody, a crucial tool that trapped Sec61 in the ER and prevented its recruitment into endosomes without influencing Sec61 activity and antigen presentation in the ER. Expression of this ER intrabody inhibited antigen translocation and cross-presentation, demonstrating that endosomal Sec61 indeed mediates antigen transport across endosomal membranes. Moreover, we showed that the recruitment of Sec61 toward endosomes, and hence antigen translocation and cross-presentation, is dependent on dendritic cell activation by Toll-like receptor (TLR) ligands. These data shed light on a long-lasting question regarding antigen cross-presentation and point out a role of the ER-associated degradation machinery in compartments distinct from the ER.
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•Sec61 is recruited toward antigen-containing endosomes•An ER-retained intrabody to prevent Sec61 transport from the ER toward endosomes•Endosomal Sec61 is required for antigen translocation and cross-presentation•Recruitment of Sec61 toward antigen-containing endosomes via TRIF signaling
Presentation of extracellular antigens on MHC I molecules to activate cytotoxic T cells requires antigen translocation into the cytosol for proteasomal degradation. Burgdorf and colleagues demonstrate that such translocation is mediated by Sec61, a protein from the ER, which is translocated toward antigen-containing endosomes after stimulation of the dendritic cell with pro-inflammatory factors.
Balance training aims to improve balance and transfer acquired skills to real-life tasks. How older adults adapt gait to different conditions, and whether these adaptations are altered by balance ...training, remains unclear. We hypothesized that reorganization of modular control of muscle activity is a mechanism underlying adaptation of gait to training and environmental constraints. We investigated the transfer of standing balance training, shown to enhance unipedal balance control, to gait and adaptations in neuromuscular control of gait between normal and narrow-base walking in twenty-two older adults (72.6 ± 4.2 years). At baseline, after one, and after ten training sessions, kinematics and EMG of normal and narrow-base treadmill walking were measured. Gait parameters and temporal activation profiles of five muscle synergies were compared between time-points and gait conditions. Effects of balance training and an interaction between training and gait condition on step width were found, but not on synergies. After ten training sessions step width decreased in narrow-base walking, while step width variability decreased in both conditions. Trunk center of mass displacement and velocity, and the local divergence exponent, were lower in narrow-base compared to normal walking. Activation duration in narrow-base compared to normal walking was shorter for synergies associated with dominant leg weight acceptance and non-dominant leg stance, and longer for the synergy associated with non-dominant heel-strike. Time of peak activation associated with dominant leg stance occurred earlier in narrow-base compared to normal walking, while it was delayed in synergies associated with heel-strikes and non-dominant leg stance. The adaptations of synergies to narrow-base walking may be interpreted as related to more cautious weight transfer to the new stance leg and enhanced control over center of mass movement in the stance phase. The improvement of gait stability due to standing balance training is promising for less mobile older adults.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The photocurrent in conjugated polymer-fullerene blends is dominated by the dissociation efficiency of bound electron-hole pairs at the donor-acceptor interface. A model based on Onsager's theory of ...geminate charge recombination explains the observed field and temperature dependence of the photocurrent in PPV:PCBM blends. At room temperature only 60% of the generated bound electron-hole pairs are dissociated and contribute to the short-circuit current, which is a major loss mechanism in photovoltaic devices based on this material system.
Betacoronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV), are important pathogens causing potentially lethal infections in humans and animals. Coronavirus RNA synthesis is ...thought to be associated with replication organelles (ROs) consisting of modified endoplasmic reticulum (ER) membranes. These are transformed into double-membrane vesicles (DMVs) containing viral double-stranded RNA and into other membranous elements such as convoluted membranes, together forming a reticulovesicular network. Previous evidence suggested that the nonstructural proteins (nsp's) 3, 4, and 6 of the severe acute respiratory syndrome coronavirus (SARS-CoV), which contain transmembrane domains, would all be required for DMV formation. We have now expressed MERS-CoV replicase self-cleaving polyprotein fragments encompassing nsp3-4 or nsp3-6, as well as coexpressed nsp3 and nsp4 of either MERS-CoV or SARS-CoV, to characterize the membrane structures induced. Using electron tomography, we demonstrate that for both MERS-CoV and SARS-CoV coexpression of nsp3 and nsp4 is required and sufficient to induce DMVs. Coexpression of MERS-CoV nsp3 and nsp4 either as individual proteins or as a self-cleaving nsp3-4 precursor resulted in very similar DMVs, and in both setups we observed proliferation of zippered ER that appeared to wrap into nascent DMVs. Moreover, when inactivating nsp3-4 polyprotein cleavage by mutagenesis, we established that cleavage of the nsp3/nsp4 junction is essential for MERS-CoV DMV formation. Addition of the third MERS-CoV transmembrane protein, nsp6, did not noticeably affect DMV formation. These findings provide important insight into the biogenesis of coronavirus DMVs, establish strong similarities with other nidoviruses (specifically, the arteriviruses), and highlight possible general principles in viral DMV formation.
The RNA replication of positive stranded RNA viruses of eukaryotes is thought to take place at cytoplasmic membranous replication organelles (ROs). Double-membrane vesicles are a prominent type of viral ROs. They are induced by coronaviruses, such as SARS-CoV and MERS-CoV, as well as by a number of other important pathogens, yet little is known about their biogenesis. In this study, we explored the viral protein requirements for the formation of MERS-CoV- and SARS-CoV-induced DMVs and established that coexpression of two of the three transmembrane subunits of the coronavirus replicase polyprotein, nonstructural proteins (nsp's) 3 and 4, is required and sufficient to induce DMV formation. Moreover, release of nsp3 and nsp4 from the polyprotein by proteolytic maturation is essential for this process. These findings provide a strong basis for further research on the biogenesis and functionality of coronavirus ROs and may point to more general principles of viral DMV formation.
Complement activation by antibodies bound to pathogens, tumors, and self antigens is a critical feature of natural immune defense, a number of disease processes, and immunotherapies. How antibodies ...activate the complement cascade, however, is poorly understood. We found that specific noncovalent interactions between Fc segments of immunoglobulin G (IgG) antibodies resulted in the formation of ordered antibody hexamers after antigen binding on cells. These hexamers recruited and activated C1, the first component of complement, thereby triggering the complement cascade. The interactions between neighboring Fc segments could be manipulated to block, reconstitute, and enhance complement activation and killing of target cells, using all four human IgG subclasses. We offer a general model for understanding antibody-mediated complement activation and the design of antibody therapeutics with enhanced efficacy.
Prior studies suggested that SAGA and TFIID are alternative factors that promote RNA polymerase II transcription, with about 10% of genes in S. cerevisiae dependent on SAGA. We reassessed the role of ...SAGA by mapping its genome-wide location and role in global transcription in budding yeast. We find that SAGA maps to the UAS elements of most genes, overlapping with Mediator binding and irrespective of previous designations of SAGA- or TFIID-dominated genes. Disruption of SAGA through mutation or rapid subunit depletion reduces transcription from nearly all genes, measured by newly synthesized RNA. We also find that the acetyltransferase Gcn5 synergizes with Spt3 to promote global transcription and that Spt3 functions to stimulate TBP recruitment at all tested genes. Our data demonstrate that SAGA acts as a general cofactor required for essentially all RNA polymerase II transcription and is not consistent with the previous classification of SAGA- and TFIID-dominated genes.
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•SAGA is recruited to the regulatory regions of both SAGA- and TFIID-dominated genes•Inactivation of SAGA leads to a global decrease of RNA polymerase II transcription•SAGA is required for stable TBP recruitment at promoters•Gcn5 acetyltransferase synergizes with Spt3 (TBP-binding) to promote transcription
Baptista et al. show that SAGA, a transcriptional coactivator conserved in all eukaryotes, is involved in overall RNA polymerase II transcription in budding yeast. Using ChEC-seq, SAGA was shown to be recruited to both TATA-containing and TATA-less genes. In agreement, inactivation of SAGA leads to dramatic effects on nascent transcription.
All cells release a multitude of nanoscale extracellular vesicles (nEVs) into circulation, offering immense potential for new diagnostic strategies. Yet, clinical translation for nEVs remains a ...challenge due to their vast heterogeneity, our insufficient ability to isolate subpopulations, and the low frequency of disease-associated nEVs in biofluids. The growing field of nanoplasmonics is poised to address many of these challenges. Innovative materials engineering approaches based on exploiting nanoplasmonic phenomena, i.e., the unique interaction of light with nanoscale metallic materials, can achieve unrivaled sensitivity, offering real-time analysis and new modes of medical and biological imaging. We begin with an introduction into the basic structure and function of nEVs before critically reviewing recent studies utilizing nanoplasmonic platforms to detect and characterize nEVs. For the major techniques considered, surface plasmon resonance (SPR), localized SPR, and surface enhanced Raman spectroscopy (SERS), we introduce and summarize the background theory before reviewing the studies applied to nEVs. Along the way, we consider notable aspects, limitations, and considerations needed to apply plasmonic technologies to nEV detection and analysis.
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