Conformational transitions and allosteric modulation in a heteromeric glycine receptor.

Glycine Receptors (GlyRs) provide inhibitory neuronal input in the spinal cord and brainstem, which is critical for muscle coordination and sensory perception. Synaptic GlyRs are a heteromeric assembly of α and ß subunits. Here we present cryo-EM structures of full-length zebrafish α1ßBGlyR in the presence of an antagonist (strychnine), agonist (glycine), or agonist with a positive allosteric modulator (glycine/ivermectin). Each structure shows a distinct pore conformation with varying degrees of asymmetry. Molecular dynamic simulations found the structures were in a closed (strychnine) and desensitized states (glycine and glycine/ivermectin). Ivermectin binds at all five interfaces, but in a distinct binding pose at the ß-α interface. Subunit-specific features were sufficient to solve structures without a fiduciary marker and to confirm the 4α:1ß stoichiometry recently observed. We also report features of the extracellular and intracellular domains. Together, our results show distinct compositional and conformational properties of α1ßGlyR and provide a framework for further study of this physiologically important channel.

11C-methionine PET aids localization of microprolactinomas in patients with intolerance or resistance to dopamine agonist therapy.

PURPOSE: To assess the potential for 11C-methionine PET (Met-PET) coregistered with volumetric magnetic resonance imaging (Met-PET/MRCR) to inform clinical decision making in patients with poorly visualized or occult microprolactinomas and dopamine agonist intolerance or resistance. PATIENTS AND METHODS: Thirteen patients with pituitary microprolactinomas, and who were intolerant (n = 11) or resistant (n = 2) to dopamine agonist therapy, were referred to our specialist pituitary centre for Met-PET/MRCR between 2016 and 2020. All patients had persistent hyperprolactinemia and were being considered for surgical intervention, but standard clinical MRI had shown either no visible adenoma or equivocal appearances. RESULTS: In all 13 patients Met-PET/MRCR demonstrated a single focus of avid tracer uptake. This was localized either to the right or left side of the sella in 12 subjects. In one patient, who had previously undergone surgery for a left-sided adenoma, recurrent tumor was unexpectedly identified in the left cavernous sinus. Five patients underwent endoscopic transsphenoidal selective adenomectomy, with subsequent complete remission of hyperprolactinaemia and normalization of other pituitary function; three patients are awaiting surgery. In the patient with inoperable cavernous sinus disease PET-guided stereotactic radiosurgery (SRS) was performed with subsequent near-normalization of serum prolactin. Two patients elected for a further trial of medical therapy, while two declined surgery or radiotherapy and chose to remain off medical treatment. CONCLUSIONS: In patients with dopamine agonist intolerance or resistance, and indeterminate pituitary MRI, molecular (functional) imaging with Met-PET/MRCR can allow precise localization of a microprolactinoma to facilitate selective surgical adenomectomy or SRS.

Better, Faster, Cheaper: Recent Advances in Cryo-Electron Microscopy.

Cryo-electron microscopy (cryo-EM) continues its remarkable growth as a method for visualizing biological objects, which has been driven by advances across the entire pipeline. Developments in both single-particle analysis and in situ tomography have enabled more structures to be imaged and determined to better resolutions, at faster speeds, and with more scientists having improved access. This review highlights recent advances at each stageof the cryo-EM pipeline and provides examples of how these techniques have been used to investigate real-world problems, including antibody development against the SARS-CoV-2 spike during the recent COVID-19 pandemic.

Flavivirus maturation leads to the formation of an occupied lipid pocket in the surface glycoproteins.

Flaviviruses such as Dengue (DENV) or Zika virus (ZIKV) assemble into an immature form within the endoplasmatic reticulum (ER), and are then processed by furin protease in the trans-Golgi. To better grasp maturation, we carry out cryo-EM reconstructions of immature Spondweni virus (SPOV), a human flavivirus of the same serogroup as ZIKV. By employing asymmetric localised reconstruction we push the resolution to 3.8 Å, enabling us to refine an atomic model which includes the crucial furin protease recognition site and a conserved Histidine pH-sensor. For direct comparison, we also solve structures of the mature forms of SPONV and DENV to 2.6 Å and 3.1 Å, respectively. We identify an ordered lipid that is present in only the mature forms of ZIKV, SPOV, and DENV and can bind as a consequence of rearranging amphipathic stem-helices of E during maturation. We propose a structural role for the pocket and suggest it stabilizes mature E.

Localized reconstruction in Scipion expedites the analysis of symmetry mismatches in cryo-EM data.

Technological advances in transmission electron microscopes and detectors have turned cryogenic electron microscopy (cryo-EM) into an essential tool for structural biology. A commonly used cryo-EM data analysis method, single particle analysis, averages hundreds of thousands of low-dose images of individual macromolecular complexes to determine a density map of the complex. The presence of symmetry in the complex is beneficial since each projection image can be assigned to multiple views of the complex. However, data processing that applies symmetry can average out asymmetric features and consequently data analysis methods are required to resolve asymmetric structural features. Scipion is a cryo-EM image processing framework that integrates functions from different image processing packages as plugins. To extend its functionality for handling symmetry mismatches, we present here a Scipion plugin termed LocalRec implementing the localized reconstruction method. When tested on an adenovirus data set, the plugin enables resolving the symmetry-mismatched trimeric fibre bound to the five-fold vertices of the capsid. Furthermore, it improves the structure determination of the icosahedral capsid by dealing with the defocus gradient across the particle. LocalRec is expected to be widely applicable in a range of cryo-EM investigations of flexible and symmetry mismatched complexes.

Multiple liquid crystalline geometries of highly compacted nucleic acid in a dsRNA virus.

Characterizing the genome of mature virions is pivotal to understanding the highly dynamic processes of virus assembly and infection. Owing to the different cellular fates of DNA and RNA, the life cycles of double-stranded (ds)DNA and dsRNA viruses are dissimilar. In terms of nucleic acid packing, dsDNA viruses, which lack genome segmentation and intra-capsid transcriptional machinery, predominantly display single-spooled genome organizations1-8. Because the release of dsRNA into the cytoplasm triggers host defence mechanisms9, dsRNA viruses retain their genomes within a core particle that contains the enzymes required for RNA replication and transcription10-12. The genomes of dsRNA viruses vary greatly in the degree of segmentation. In members of the Reoviridae family, genomes consist of 10-12 segments and exhibit a non-spooled arrangement mediated by RNA-dependent RNA polymerases11-14. However, whether this arrangement is a general feature of dsRNA viruses remains unknown. Here, using cryo-electron microscopy to resolve the dsRNA genome structure of the tri-segmented bacteriophage ɸ6 of the Cystoviridae family, we show that dsRNA viruses can adopt a dsDNA-like single-spooled genome organization. We find that in this group of viruses, RNA-dependent RNA polymerases do not direct genome ordering, and the dsRNA can adopt multiple conformations. We build a model that encompasses 90% of the genome, and use this to quantify variation in the packing density and to characterize the different liquid crystalline geometries that are exhibited by the tightly compacted nucleic acid. Our results demonstrate that the canonical model for the packing of dsDNA can be extended to dsRNA viruses.

Assembly of complex viruses exemplified by a halophilic euryarchaeal virus.

Many of the largest known viruses belong to the PRD1-adeno structural lineage characterised by conserved pseudo-hexameric capsomers composed of three copies of a single major capsid protein (MCP). Here, by high-resolution cryo-EM analysis, we show that a class of archaeal viruses possess hetero-hexameric MCPs which mimic the PRD1-adeno lineage trimer. These hetero-hexamers are built from heterodimers and utilise a jigsaw-puzzle system of pegs and holes, and underlying minor capsid proteins, to assemble the capsid laterally from the 5-fold vertices. At these vertices proteins engage inwards with the internal membrane vesicle whilst 2-fold symmetric horn-like structures protrude outwards. The horns are assembled from repeated globular domains attached to a central spine, presumably facilitating multimeric attachment to the cell receptor. Such viruses may represent precursors of the main PRD1-adeno lineage, similarly engaging cell-receptors via 5-fold spikes and using minor proteins to define particle size.

Dual Role of a Viral Polymerase in Viral Genome Replication and Particle Self-Assembly.

Double-stranded RNA (dsRNA) viruses package several RNA-dependent RNA polymerases (RdRp) together with their dsRNA genome into an icosahedral protein capsid known as the polymerase complex. This structure is highly conserved among dsRNA viruses but is not found in any other virus group. RdRp subunits typically interact directly with the main capsid proteins, close to the 5-fold symmetric axes, and perform viral genome replication and transcription within the icosahedral protein shell. In this study, we utilized Pseudomonas phage Φ6, a well-established virus self-assembly model, to probe the potential roles of the RdRp in dsRNA virus assembly. We demonstrated that Φ6 RdRp accelerates the polymerase complex self-assembly process and contributes to its conformational stability and integrity. We highlight the role of specific amino acid residues on the surface of the RdRp in its incorporation during the self-assembly reaction. Substitutions of these residues reduce RdRp incorporation into the polymerase complex during the self-assembly reaction. Furthermore, we determined that the overall transcription efficiency of the Φ6 polymerase complex increased when the number of RdRp subunits exceeded the number of genome segments. These results suggest a mechanism for RdRp recruitment in the polymerase complex and highlight its novel role in virion assembly, in addition to the canonical RNA transcription and replication functions.IMPORTANCE Double-stranded RNA viruses infect a wide spectrum of hosts, including animals, plants, fungi, and bacteria. Yet genome replication mechanisms of these viruses are conserved. During the infection cycle, a proteinaceous capsid, the polymerase complex, is formed. An essential component of this capsid is the viral RNA polymerase that replicates and transcribes the enclosed viral genome. The polymerase complex structure is well characterized for many double-stranded RNA viruses. However, much less is known about the hierarchical molecular interactions that take place in building up such complexes. Using the bacteriophage Φ6 self-assembly system, we obtained novel insights into the processes that mediate polymerase subunit incorporation into the polymerase complex for generation of functional structures. The results presented pave the way for the exploitation and engineering of viral self-assembly processes for biomedical and synthetic biology applications. An understanding of viral assembly processes at the molecular level may also facilitate the development of antivirals that target viral capsid assembly.

Chimeric O1K foot-and-mouth disease virus with SAT2 outer capsid as an FMD vaccine candidate.

Foot-and-mouth disease virus (FMDV) is highly contagious and infects cloven-hoofed domestic livestock leading to foot-and-mouth disease (FMD). FMD outbreaks have severe economic impact due to production losses and associated control measures. FMDV is found as seven distinct serotypes, but there are numerous subtypes within each serotype, and effective vaccines must match the subtypes circulating in the field. In addition, the O and Southern African Territories (SAT) serotypes, are relatively more thermolabile and their viral capsids readily dissociate into non-immunogenic pentameric subunits, which can compromise the effectiveness of FMD vaccines. Here we report the construction of a chimeric clone between the SAT2 and O serotypes, designed to have SAT2 antigenicity. Characterisation of the chimeric virus showed growth kinetics equal to that of the wild type SAT2 virus with better thermostability, attributable to changes in the VP4 structural protein. Sequence and structural analyses confirmed that no changes from SAT2 were present elsewhere in the capsid as a consequence of the VP4 changes. Following exposure to an elevated temperature the thermostable SAT2-O1K chimera induced higher neutralizing-antibody titres in comparison to wild type SAT2 virus.

Beyond structures of highly symmetric purified viral capsids by cryo-EM.

Cryogenic transmission electron microscopy (cryo-EM) is widely used to determine high-resolution structures of symmetric virus capsids. The method holds promise for extending studies beyond purified capsids and their symmetric protein shells. The non-symmetric genome component has been addressed in dsRNA cypoviruses and ssRNA bacteriophages Qß and MS2. The structure of human herpes simplex virus type 1 capsids has been determined within intact virions to resolve capsid-tegument interactions. Electron tomography under cryogenic conditions (cryo-ET), has allowed resolving an early membrane fusion intermediate of Rift Valley fever virus. Antibody-affinity based sample grids allow capturing of virions directly from cell cultures or even clinical samples. These and other emerging methods will support studies to address viral entry, assembly and neutralization processes at increasingly high resolutions and native conditions.

Rules of engagement between αvß6 integrin and foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) mediates cell entry by attachment to an integrin receptor, generally αvß6, via a conserved arginine-glycine-aspartic acid (RGD) motif in the exposed, antigenic, GH loop of capsid protein VP1. Infection can also occur in tissue culture adapted virus in the absence of integrin via acquired basic mutations interacting with heparin sulphate (HS); this virus is attenuated in natural infections. HS interaction has been visualized at a conserved site in two serotypes suggesting a propensity for sulfated-sugar binding. Here we determined the interaction between αvß6 and two tissue culture adapted FMDV strains by cryo-electron microscopy. In the preferred mode of engagement, the fully open form of the integrin, hitherto unseen at high resolution, attaches to an extended GH loop via interactions with the RGD motif plus downstream hydrophobic residues. In addition, an N-linked sugar of the integrin attaches to the previously identified HS binding site, suggesting a functional role.

Double-stranded RNA virus outer shell assembly by bona fide domain-swapping.

Correct outer protein shell assembly is a prerequisite for virion infectivity in many multi-shelled dsRNA viruses. In the prototypic dsRNA bacteriophage φ6, the assembly reaction is promoted by calcium ions but its biomechanics remain poorly understood. Here, we describe the near-atomic resolution structure of the φ6 double-shelled particle. The outer T=13 shell protein P8 consists of two alpha-helical domains joined by a linker, which allows the trimer to adopt either a closed or an open conformation. The trimers in an open conformation swap domains with each other. Our observations allow us to propose a mechanistic model for calcium concentration regulated outer shell assembly. Furthermore, the structure provides a prime exemplar of bona fide domain-swapping. This leads us to extend the theory of domain-swapping from the level of monomeric subunits and multimers to closed spherical shells, and to hypothesize a mechanism by which closed protein shells may arise in evolution.

Localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes.

Electron cryomicroscopy can yield near-atomic resolution structures of highly ordered macromolecular complexes. Often however some subunits bind in a flexible manner, have different symmetry from the rest of the complex, or are present in sub-stoichiometric amounts, limiting the attainable resolution. Here we report a general method for the localized three-dimensional reconstruction of such subunits. After determining the particle orientations, local areas corresponding to the subunits can be extracted and treated as single particles. We demonstrate the method using three examples including a flexible assembly and complexes harbouring subunits with either partial occupancy or mismatched symmetry. Most notably, the method allows accurate fitting of the monomeric RNA-dependent RNA polymerase bound at the threefold axis of symmetry inside a viral capsid, revealing for the first time its exact orientation and interactions with the capsid proteins. Localized reconstruction is expected to provide novel biological insights in a range of challenging biological systems.

Rapid infusion of streptokinase standard dose in acute myocardial infarction is followed by a higher rate of coronary reperfusion than standard protocol.

There are several opinions asserting that the accelerated t-PA is more efficient than the standard protocol of streptokinase (SK) administration in acute myocardial infarction (AMI). One hundred patients admitted within the first 6 hrs after the onset of the symptoms revealing AMI were divided in two subgroups, as follows: subgroup A (50 patients) in whom a dose of 1.5 M.U. SK was infused in 20 min (accelerated protocol) and subgroup B (50 patients) in whom the same dose was infused in 60 min (standard protocol). In order to assess the efficiency of thrombolytic therapy (TT), we used three noninvasive criteria: the rapid resolution of the chest pain, the rapid decreasing of the ST segment elevation by more than 50% from the initial value, and the rapid increasing of enzymes revealing necrosis. Using the above-mentioned criteria, we considered that coronary reperfusion appeared in 40 patients from subgroup A (80%) and in 29 patients from subgroup B (58%). The speed of coronary reperfusion was 40 +/- 26 min in patients with accelerated SK and this time was significantly shorter than the time of 60 +/- 24 min registered in the control group. No major hemorrhagic events appeared in both subgroups. Although hypotension appeared more frequently in subgroup A, this minor complication was well supported by our patients. The rapid infusion of the standard dose of SK was followed by a higher rate and speed of coronary reperfusion as compared to the standard protocol.