The recruitment of TRiC chaperonin in rotavirus viroplasms correlates with virus replication.

Rotavirus (RV) replication takes place in the viroplasms, cytosolic inclusions that allow the synthesis of virus genome segments and their encapsidation in the core shell, followed by the addition of the second layer of the virion. The viroplasms are composed of several viral proteins, including NSP5, which serves as the main building block. Microtubules, lipid droplets, and miRNA-7 are among the host components recruited in viroplasms. We investigated the interaction between RV proteins and host components of the viroplasms by performing a pull-down assay of lysates from RV-infected cells expressing NSP5-BiolD2. Subsequent tandem mass spectrometry identified all eight subunits of the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for folding at least 10% of the cytosolic proteins. Our confirmed findings reveal that TRiC is brought into viroplasms and wraps around newly formed double-layered particles. Chemical inhibition of TRiC and silencing of its subunits drastically reduced virus progeny production. Through direct RNA sequencing, we show that TRiC is critical for RV replication by controlling dsRNA genome segment synthesis, particularly negative-sense single-stranded RNA. Importantly, cryo-electron microscopy analysis shows that TRiC inhibition results in defective virus particles lacking genome segments and polymerase complex (VP1/VP3). Moreover, TRiC associates with VP2 and NSP5 but not with VP1. Also, VP2 is shown to be essential for recruiting TRiC in viroplasms and preserving their globular morphology. This study highlights the essential role of TRiC in viroplasm formation and in facilitating virion assembly during the RV life cycle. IMPORTANCE: The replication of rotavirus takes place in cytosolic inclusions termed viroplasms. In these inclusions, the distinct 11 double-stranded RNA genome segments are co-packaged to complete a genome in newly generated virus particles. In this study, we show for the first time that the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for the folding of at least 10% of the cytosolic proteins, is a component of viroplasms and is required for the synthesis of the viral negative-sense single-stranded RNA. Specifically, TRiC associates with NSP5 and VP2, the cofactor involved in RNA replication. Our study adds a new component to the current model of rotavirus replication, where TRiC is recruited to viroplasms to assist replication.

Recombinantly expressed virus-like particles (VLPs) of canine circovirus for development of an indirect ELISA.

Canine circovirus (CanineCV) is an emerging pathogen in domestic dogs, detected in multiple countries in association with varying clinical and pathological presentations including diarrhoea, vasculitis, granulomatous inflammation, and respiratory signs. Understanding the pathology of CanineCV is confounded by the fact that it has been detected in asymptomatic dogs as well as in diseased dogs concurrently infected with known pathogens. Recombinantly expressed self-assembling Virus-like particles (VLPs) lack viral genomic material but imitate the capsid surface conformations of wild type virion, allowing arrays of biological applications including subunit vaccine development and immunodiagnostics. In this study, full length CanineCV capsid gene was expressed in Escherichia coli followed by two-step purification process to yield soluble capsid protein in high concentration. Transmission electron microscopy (TEM) confirmed the capsid antigen self-assembled into 17-20 nm VLPs in glutathione S-transferase (GST) buffer, later utilised to develop an indirect enzyme-linked immunosorbent assay (iELISA). The respective sensitivity and specificity of the proposed iELISA were 94.10% and 88.40% compared with those obtained from Western blot. The mean OD450 value for western blot positive samples was 1.22 (range 0.12-3.39) and negative samples was 0.21 (range 0.07-0.41). An optimal OD450 cut-off of 0.35 was determined by ROC curve analysis. Median inter-assay and intra-assay validation revealed that the iELISA test results were reproducible with coefficients of variation 7.70 (range 5.6-11.9) and 4.21 (range 1.2-7.4). Our results demonstrated that VLP-based iELISA is a highly sensitive method for serological diagnosis of CanineCV infections in dogs, suitable for large-scale epidemiological studies.

Accumulation of endogenous free radicals is required to induce titan-like cell formation in Cryptococcus neoformans.

IMPORTANCE: Cryptococcus neoformans is an excellent model to investigate fungal pathogenesis. This yeast can produce "titan cells," which are cells of an abnormally larger size that contribute to the persistence of the yeast in the host. In this work, we have used a new approach to characterize them by identifying drugs that inhibit this process. We have used a repurposing off-patent drug library, combined with an automatic method to image and analyze fungal cell size. In this way, we have identified many compounds that inhibit this transition. Interestingly, several compounds were antioxidants, allowing us to confirm that endogenous ROS and mitochondrial changes are important for titan cell formation. This work provides new evidence of the mechanisms required for titanization. Furthermore, the future characterization of the inhibitory mechanisms of the identified compounds by the scientific community will contribute to better understand the role of titan cells in virulence.

Lippia origanoides Essential Oil or Thymol in Combination with Fluconazole Produces Damage to Cells and Reverses the Azole-Resistant Phenotype of a Candida tropicalis Strain.

Candida tropicalis is one of the most pathogenic species within the genus. Increased antifungal resistance has been reported, which is in part due to the organism's ability to form biofilms. In natural products derived from plants, such as essential oils (EOs) or their major components, there is significant potential to develop new antifungals or to both enhance the efficacy and reduce the toxicity of conventional antifungals. This study aimed to evaluate the effect of combining an EO of Lippia origanoides or thymol with fluconazole on an azole-resistant C. tropicalis strain. Synergism was observed in the combination of fluconazole with the EO and with thymol, and minimal inhibitory concentrations for fluconazole decreased at least 32-fold. As a consequence of the synergistic interactions, mitochondrial membrane potential was reduced, and mitochondrial superoxide production increased. Alteration in nuclear morphology, cell surface, and ultrastructure was also observed. In conclusion, the synergistic interaction between L. origanoides EO or thymol with fluconazole reverted the azole-resistant C. tropicalis phenotype. These findings suggest that L. origanoides EO or thymol alone, or in combination with fluconazole, have the potential for development as antifungal therapies for this yeast, including resistant strains.

MicroRNA signature from extracellular vesicles of HCV/HIV co-infected individuals differs from HCV mono-infected.

Hepatitis C virus (HCV) coinfection with human immunodeficiency virus (HIV) has a detrimental impact on disease progression. Increasing evidence points to extracellular vesicles (EVs) as important players of the host-viral cross-talk. The microRNAs (miRNAs), as essential components of EVs cargo, are key regulators of normal cellular processes and also promote viral replication, viral pathogenesis, and disease progression. We aimed to characterize the plasma-derived EVs miRNA signature of chronic HCV infected and HIV coinfected patients to unravel the molecular mechanisms of coinfection. EVs were purified and characterized from 50 plasma samples (21 HCV mono- and 29 HCV/HIV co-infected). EV-derived small RNAs were isolated and analyzed by massive sequencing. Known and de novo miRNAs were identified with miRDeep2. Significant differentially expressed (SDE) miRNA identification was performed with generalized linear models and their putative dysregulated biological pathways were evaluated. Study groups were similar for most clinical and epidemiological characteristics. No differences were observed in EVs size or concentration between groups. Therefore, HCV/HIV co-infection condition did not affect the concentration or size of EVs but produced a disturbance in plasma-derived EVs miRNA cargo. Thus, a total of 149 miRNAs were identified (143 known and 6 de novo) leading to 37 SDE miRNAs of which 15 were upregulated and 22 downregulated in HCV/HIV co-infected patients. SDE miRNAs regulate genes involved in inflammation, fibrosis, and cancer, modulating different biological pathways related to HCV and HIV pathogenesis. These findings may help to develop new generation biomarkers and treatment strategies, in addition to elucidate the mechanisms underlying virus-host interaction. KEY MESSAGES: HCV and HCV/HIV displayed similar plasma-EV size and concentration. EVs- derived miRNA profile was characterized by NGS. 37 SDE miRNAs between HCV and HCV/HIV were observed. SDE miRNAs regulate genes involved in inflammation, fibrosis and cancer.

Rotavirus Particle Disassembly and Assembly In Vivo and In Vitro.

Rotaviruses (RVs) are non-enveloped multilayered dsRNA viruses that are major etiologic agents of diarrheal disease in humans and in the young in a large number of animal species. The viral particle is composed of three different protein layers that enclose the segmented dsRNA genome and the transcriptional complexes. Each layer defines a unique subparticle that is associated with a different phase of the replication cycle. Thus, while single- and double-layered particles are associated with the intracellular processes of selective packaging, genome replication, and transcription, the viral machinery necessary for entry is located in the third layer. This modular nature of its particle allows rotaviruses to control its replication cycle by the disassembly and assembly of its structural proteins. In this review, we examine the significant advances in structural, molecular, and cellular RV biology that have contributed during the last few years to illuminating the intricate details of the RV particle disassembly and assembly processes.

Equilibrium Dynamics of a Biomolecular Complex Analyzed at Single-amino Acid Resolution by Cryo-electron Microscopy.

The biological function of macromolecular complexes depends not only on large-scale transitions between conformations, but also on small-scale conformational fluctuations at equilibrium. Information on the equilibrium dynamics of biomolecular complexes could, in principle, be obtained from local resolution (LR) data in cryo-electron microscopy (cryo-EM) maps. However, this possibility had not been validated by comparing, for a same biomolecular complex, LR data with quantitative information on equilibrium dynamics obtained by an established solution technique. In this study we determined the cryo-EM structure of the minute virus of mice (MVM) capsid as a model biomolecular complex. The LR values obtained correlated with crystallographic B factors and with hydrogen/deuterium exchange (HDX) rates obtained by mass spectrometry (HDX-MS), a gold standard for determining equilibrium dynamics in solution. This result validated a LR-based cryo-EM approach to investigate, with high spatial resolution, the equilibrium dynamics of biomolecular complexes. As an application of this approach, we determined the cryo-EM structure of two mutant MVM capsids and compared their equilibrium dynamics with that of the wild-type MVM capsid. The results supported a previously suggested linkage between mechanical stiffening and impaired equilibrium dynamics of a virus particle. Cryo-EM is emerging as a powerful approach for simultaneously acquiring information on the atomic structure and local equilibrium dynamics of biomolecular complexes.

Identification and Characterization of Epithelial Cell-Derived Dense Bodies Produced upon Cytomegalovirus Infection.

Dense bodies (DB) are complex, noninfectious particles produced during CMVinfection containing envelope and tegument proteins that may be ideal candidates as vaccines. Although DB were previously described in fibroblasts, no evidence of DB formation has been shown after propagating CMV in epithelial cells. In the present study, both fibroblast MRC-5 and epithelial ARPE-19 cells were used to study DB production during CMV infection. We demonstrate the formation of epithelial cell-derived DB, mostly located as cytoplasmic inclusions in the perinuclear area of the infected cell. DB were gradient-purified, and the nature of the viral particles was confirmed using CMV-specific immunelabeling. Epithelial cell-derived DB had higher density and more homogeneous size (200-300 nm) compared to fibroblast-derived DB (100-600 nm).In agreement with previous results characterizing DB from CMV-infected fibroblasts, the pp65 tegument protein was predominant in the epithelial cell-derived DB. Our results also suggest that epithelial cells had more CMV capsids in the cytoplasm and had spherical bodies compatible with nucleus condensation (pyknosis) in cells undergoing apoptosis that were not detected in MRC-5 infected cells at the tested time post-infection. Our results demonstrate the formation of DB in CMV-infected ARPE-19 epithelial cells that may be suitable candidate to develop a multiprotein vaccine with antigenic properties similar to that of the virions while not including the viral genome.

Cryo-EM structures show the mechanistic basis of pan-peptidase inhibition by human α2-macroglobulin.

Human α2-macroglobulin (hα2M) is a multidomain protein with a plethora of essential functions, including transport of signaling molecules and endopeptidase inhibition in innate immunity. Here, we dissected the molecular mechanism of the inhibitory function of the ∼720-kDa hα2M tetramer through eight cryo­electron microscopy (cryo-EM) structures of complexes from human plasma. In the native complex, the hα2M subunits are organized in two flexible modules in expanded conformation, which enclose a highly porous cavity in which the proteolytic activity of circulating plasma proteins is tested. Cleavage of bait regions exposed inside the cavity triggers rearrangement to a compact conformation, which closes openings and entraps the prey proteinase. After the expanded-to-compact transition, which occurs independently in the four subunits, the reactive thioester bond triggers covalent linking of the proteinase, and the receptor-binding domain is exposed on the tetramer surface for receptor-mediated clearance from circulation. These results depict the molecular mechanism of a unique suicidal inhibitory trap.

A Small Non-Coding RNA Modulates Expression of Pilus-1 Type in Streptococcus pneumoniae.

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide, and about 30% of the pneumococcal clinical isolates show type I pili-like structures. These long proteinaceous polymers extending from the bacterial surface are encoded by pilus islet 1 and play major roles in adhesion and host colonization. Pili expression is bistable and is controlled by the transcriptional activator RlrA. In this work, we demonstrate that the previously identified small noncoding RNA srn135 also participates in pilus regulation. Our findings show that srn135 is generated upon processing of the 5'-UTR region of rrgA messenger and its deletion prevents the synthesis of RrgA, the main pili adhesin. Moreover, overexpression of srn135 increases the expression of all pili genes and rises the percentage of piliated bacteria within a clonal population. This regulation is mediated by the stabilization of rlrA mRNA since higher levels of srn135 increase its half-life to 165%. Our findings suggest that srn135 has a dual role in pilus expression acting both in cis- (on the RrgA levels) and in trans- (modulating the levels of RlrA) and contributes to the delicate balance between pili expressing and non-expressing bacteria.

Structural Perspectives of Beak and Feather Disease Virus and Porcine Circovirus Proteins.

Circoviruses represent a rapidly expanding group of viruses that infect both vertebrate and invertebrate hosts. Members are responsible for diseases of veterinary and economic importance, including postweaning multisystemic wasting syndrome in pigs, and beak and feather disease (BFD) in birds. These viruses are associated with lymphoid depletion and immunosuppressive conditions in infected animals leading to systemic illness. Circoviruses are small nonenveloped DNA viruses containing a single-stranded circular genome, encoding two major proteins: the capsid-associated protein (Cap), comprising the entirety of the viral capsid, and the replication-associated protein (Rep). Cap is the only protein component of the virion and plays crucial roles throughout the virus replication cycle, including viral attachment, cell entry, genome uncoating, and packaging of newly formed viral particles. Rep mediates recognition of replication origin motifs in the viral genome sequence and is responsible for endonuclease activity enabling nicking of the circular DNA and initiation of rolling-circle replication (RCR). Porcine circovirus 2 (PCV2) was the first circovirus capsid structure to be solved at atomic resolution using X-ray crystallography. The structure revealed an assembly comprising 60 monomeric subunits to form virus-like particles. Each Cap monomer harbors a canonical viral jelly roll domain composed of two, four-stranded antiparallel ß-sheets. Crystal structures of two distinct macromolecular assemblies from BFD virus Cap were also resolved at high resolution. In these structures, the exposure of the N-terminal arginine-rich motif, responsible for DNA binding and nuclear localization is reversed. Additional structural investigations have also elucidated a PCV2 type-specific neutralizing epitope, and interaction between the PCV2 capsid and polymers such as heparin. In this review, we provide a snapshot of the structural and functional aspects of circovirus proteins.

Four-Dimensional Characterization of the Babesia divergens Asexual Life Cycle, from the Trophozoite to the Multiparasite Stage.

Babesia is an apicomplexan parasite of significance that causes the disease known as babesiosis in domestic and wild animals and in humans worldwide. Babesia infects vertebrate hosts and reproduces asexually by a form of binary fission within erythrocytes/red blood cells (RBCs), yielding a complex pleomorphic population of intraerythrocytic parasites. Seven of them, clearly visible in human RBCs infected with Babesia divergens, are considered the main forms and named single, double, and quadruple trophozoites, paired and double paired pyriforms, tetrad or Maltese Cross, and multiparasite stage. However, these main intraerythrocytic forms coexist with RBCs infected with transient parasite combinations of unclear origin and development. In fact, little is understood about how Babesia builds this complex population during its asexual life cycle. By combining cryo-soft X-ray tomography and video microscopy, main and transitory parasites were characterized in a native whole cellular context and at nanometric resolution. The architecture and kinetics of the parasite population was observed in detail and provide additional data to the previous B. divergens asexual life cycle model that was built on light microscopy. Importantly, the process of multiplication by binary fission, involving budding, was visualized in live parasites for the first time, revealing that fundamental changes in cell shape and continuous rounds of multiplication occur as the parasites go through their asexual multiplication cycle. A four-dimensional asexual life cycle model was built highlighting the origin of several transient morphological forms that, surprisingly, intersperse in a chronological order between one main stage and the next in the cycle.IMPORTANCE Babesiosis is a disease caused by intraerythrocytic Babesia parasites, which possess many clinical features that are similar to those of malaria. This worldwide disease is increasing in frequency and geographical range and has a significant impact on human and animal health. Babesia divergens is one of the species responsible for human and cattle babesiosis causing death unless treated promptly. When B. divergens infects its vertebrate hosts, it reproduces asexually within red blood cells. During its asexual life cycle, B. divergens builds a population of numerous intraerythrocytic (IE) parasites of difficult interpretation. This complex population is largely unexplored, and we have therefore combined three- and four-dimensional imaging techniques to elucidate the origin, architecture, and kinetics of IE parasites. Unveiling the nature of these parasites has provided a vision of the B. divergens asexual cycle in unprecedented detail and is a key step to develop control strategies against babesiosis.

Cryo-electron Microscopy Structure, Assembly, and Mechanics Show Morphogenesis and Evolution of Human Picobirnavirus.

Despite their diversity, most double-stranded-RNA (dsRNA) viruses share a specialized T=1 capsid built from dimers of a single protein that provides a platform for genome transcription and replication. This ubiquitous capsid remains structurally undisturbed throughout the viral cycle, isolating the genome to avoid triggering host defense mechanisms. Human picobirnavirus (hPBV) is a dsRNA virus frequently associated with gastroenteritis, although its pathogenicity is yet undefined. Here, we report the cryo-electron microscopy (cryo-EM) structure of hPBV at 2.6-Å resolution. The capsid protein (CP) is arranged in a single-shelled, ∼380-Å-diameter T=1 capsid with a rough outer surface similar to that of dsRNA mycoviruses. The hPBV capsid is built of 60 quasisymmetric CP dimers (A and B) stabilized by domain swapping, and only the CP-A N-terminal basic region interacts with the packaged nucleic acids. hPBV CP has an α-helical domain with a fold similar to that of fungal partitivirus CP, with many domain insertions in its C-terminal half. In contrast to dsRNA mycoviruses, hPBV has an extracellular life cycle phase like complex reoviruses, which indicates that its own CP probably participates in cell entry. Using an in vitro reversible assembly/disassembly system of hPBV, we isolated tetramers as possible assembly intermediates. We used atomic force microscopy to characterize the biophysical properties of hPBV capsids with different cargos (host nucleic acids or proteins) and found that the CP N-terminal segment not only is involved in nucleic acid interaction/packaging but also modulates the mechanical behavior of the capsid in conjunction with the cargo.IMPORTANCE Despite intensive study, human virus sampling is still sparse, especially for viruses that cause mild or asymptomatic disease. Human picobirnavirus (hPBV) is a double-stranded-RNA virus, broadly dispersed in the human population, but its pathogenicity is uncertain. Here, we report the hPBV structure derived from cryo-electron microscopy (cryo-EM) and reconstruction methods using three capsid protein variants (of different lengths and N-terminal amino acid compositions) that assemble as virus-like particles with distinct properties. The hPBV near-atomic structure reveals a quasisymmetric dimer as the structural subunit and tetramers as possible assembly intermediates that coassemble with nucleic acids. Our structural studies and atomic force microscopy analyses indicate that hPBV capsids are potentially excellent nanocages for gene therapy and targeted drug delivery in humans.

Cryo-electron microscopy for the study of virus assembly.

Although viruses are extremely diverse in shape and size, evolution has led to a limited number of viral classes or lineages, which is probably linked to the assembly constraints of a viable capsid. Viral assembly mechanisms are restricted to two general pathways, (i) co-assembly of capsid proteins and single-stranded nucleic acids and (ii) a sequential mechanism in which scaffolding-mediated capsid precursor assembly is followed by genome packaging. Cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), which are revolutionizing structural biology, are central to determining the high-resolution structures of many viral assemblies as well as those of assembly intermediates. This wealth of cryo-EM data has also led to the development and redesign of virus-based platforms for biomedical and biotechnological applications. In this Review, we will discuss recent viral assembly analyses by cryo-EM and cryo-ET showing how natural assembly mechanisms are used to encapsulate heterologous cargos including chemicals, enzymes, and/or nucleic acids for a variety of nanotechnological applications.

Structural Insights into Rotavirus Entry.

To initiate infection, non-enveloped viruses must recognize a target cell and penetrate the cell membrane by pore formation or membrane lysis. Rotaviruses are non-enveloped dsRNA viruses that infect the mature intestinal epithelium. They are major etiologic agents of diarrheal disease in human infants, as well as in young individuals of various avian and mammalian species. Rotavirus entry into the cell is a complex multistep process initiated by the interaction of the tip of the viral spike with glycan ligands at the cell surface, and driven by conformational changes of the proteins present in the outer protein capsid, the viral machinery for entry. This review feeds on the abundant structural information produced for rotavirus during the past 30 years and focuses on the structure and the dynamics of the rotavirus entry machinery. We survey the current models for rotavirus entry into cells.

Structural nanotechnology: three-dimensional cryo-EM and its use in the development of nanoplatforms for in vitro catalysis.

The organization of enzymes into different subcellular compartments is essential for correct cell function. Protein-based cages are a relatively recently discovered subclass of structurally dynamic cellular compartments that can be mimicked in the laboratory to encapsulate enzymes. These synthetic structures can then be used to improve our understanding of natural protein-based cages, or as nanoreactors in industrial catalysis, metabolic engineering, and medicine. Since the function of natural protein-based cages is related to their three-dimensional structure, it is important to determine this at the highest possible resolution if viable nanoreactors are to be engineered. Cryo-electron microscopy (cryo-EM) is ideal for undertaking such analyses within a feasible time frame and at near-native conditions. This review describes how three-dimensional cryo-EM is used in this field and discusses its advantages. An overview is also given of the nanoreactors produced so far, their structure, function, and applications.

Antiviral activity of aged green tea extract in model food systems and under gastric conditions.

Aged-green tea extract (GTE) is known to reduce the infectivity of hepatitis A virus (HAV) and murine norovirus (MNV), a human norovirus surrogate, in vitro and in washing solutions. Initially, the effect of aged-GTE was evaluated on virus like particles (VLPs) of human norovirus (HuNoV) genogroup I (GI) by a porcine gastric mucine (PGM)-enzyme-linked immunosorbent assay (ELISA) and transmission electron microscopy (TEM), and on HuNoV GI suspensions by an in situ capture-RT-qPCR method, suggesting that HuNoVs are very sensitive to aged-GTE treatment at 37 °C. Moreover, the potential application of aged-GTE was evaluated using model foods and simulated gastric conditions. Then, aged-GTE samples prepared in orange juice, apple juice, horchata, and milk, respectively, were individually mixed with each virus and incubated overnight at 37 °C. Aged-GTE at 5 mg/ml in apple juice reduced MNV infectivity to undetectable levels and from 1.0 to 1.8 log in milk, horchata and orange juice. Aged-GTE at 5 mg/ml in orange juice, apple juice, horchata and milk reduced HAV infectivity by 1.2, 2.1, 1.5, and 1.7 log, respectively. Additionally, aged-GTE at 5 mg/ml in simulated intestinal fluid reduced MNV titers to undetectable levels and reduced HAV infectivity by ca. 2.0 log. The results show a potential for aged-GTE as a suitable natural option for preventive strategies for foodborne viral diseases.

Kinetics of the invasion and egress processes of Babesia divergens, observed by time-lapse video microscopy.

Based on confocal fluorescence and bright field video microscopy, we present detailed observations on the processes of invasion and egress of erythrocytes by the apicomplexan parasite Babesia divergens. Time-lapse images reveal numerous unexpected findings associated with the dynamics of B. divergens and its ability to manipulate the erythrocyte during both processes in its asexual cycle under in vitro conditions. Despite the speed at which these processes occur and the small size of the parasite, we capture infective merozoites moving vigorously and causing striking deformations in the erythrocyte's plasma membrane during an active invasion. We also observed intraerythrocytic dynamic stages as paired pyriforms, double paired pyriforms, tetrads, unattached pyriform sister cells and multiple parasite stages resulting in the release of large numbers of merozoites over a short period. Of considerable interest is that time-lapse images reveal a novel mechanism of egress used by B. divergens to exit the human erythrocyte. The release occurs when B. divergens parasites establish contacts with the plasma membrane of the erythrocyte from within, before exiting the cell. Visualization and analysis of the images enabled us to obtain useful information and broaden our knowledge of complex and crucial events involved with parasitisation of human erythrocytes by B. divergens.

Biophysical properties of single rotavirus particles account for the functions of protein shells in a multilayered virus.

The functions performed by the concentric shells of multilayered dsRNA viruses require specific protein interactions that can be directly explored through their mechanical properties. We studied the stiffness, breaking force, critical strain and mechanical fatigue of individual Triple, Double and Single layered rotavirus (RV) particles. Our results, in combination with Finite Element simulations, demonstrate that the mechanics of the external layer provides the resistance needed to counteract the stringent conditions of extracellular media. Our experiments, in combination with electrostatic analyses, reveal a strong interaction between the two outer layers and how it is suppressed by the removal of calcium ions, a key step for transcription initiation. The intermediate layer presents weak hydrophobic interactions with the inner layer that allow the assembly and favor the conformational dynamics needed for transcription. Our work shows how the biophysical properties of the three shells are finely tuned to produce an infective RV virion.