Human cerebral organoids: cellular composition and subcellular morphological features.

Introduction: Human cerebral organoids (hCOs) derived from pluripotent stem cells are very promising for the study of neurodevelopment and the investigation of the healthy or diseased brain. To help establish hCOs as a powerful research model, it is essential to perform the morphological characterization of their cellular components in depth. Methods: In this study, we analyzed the cell types consisting of hCOs after culturing for 45 days using immunofluorescence and reverse transcriptase qualitative polymerase chain reaction (RT-qPCR) assays. We also analyzed their subcellular morphological characteristics by transmission electron microscopy (TEM). Results: Our results show the development of proliferative zones to be remarkably similar to those found in human brain development with cells having a polarized structure surrounding a central cavity with tight junctions and cilia. In addition, we describe the presence of immature and mature migrating neurons, astrocytes, oligodendrocyte precursor cells, and microglia-like cells. Discussion: The ultrastructural characterization presented in this study provides valuable information on the structural development and morphology of the hCO, and this information is of general interest for future research on the mechanisms that alter the cell structure or function of hCOs.

Internalization and toxicity of polystyrene nanoplastics on inmortalized human neural stem cells.

Global plastic production has increased exponentially in recent decades, and a significant part of it persists in the environment, where it degrades into microplastics and nanoplastics (MPs and NPs). These can enter in humans by ingestion, inhalation, and dermal routes, and there is scientific evidence that they are able to reach the systemic circulation and penetrate and accumulate in various tissues and organs. Neurodevelopmental toxicity of NPs is one of the most worrying effects, as they can cross the blood-brain barrier. In the following study, we analyzed, by transmission electron microscopy, the in vitro uptake of 30-nm polystyrene nanoplastics (PS-NPs) into human neural stem cells (NSCs), their accumulation and subcellular localization within the cell. Furthermore, we studied the effects of different concentrations of PS-NPs on cell death, proliferation, and cell differentiation using immunocytochemistry and quantitative real time PCR for specific markers. This study demonstrated that PS-NPs were able to enter the cell, probably by endocytosis, accumulate, and aggregated in human NSCs, without being detected in the nucleus, causing cell death by apoptosis and decreased cell proliferation. This study provides new insights into the interaction and effects of PS-NPs in human NSC and supports the scientific evidence for the involvement of nanoplastic in neurodevelopmental disorders.

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.

Role of Oxidative Stress on the Etiology and Pathophysiology of Amyotrophic Lateral Sclerosis (ALS) and Its Relation with the Enteric Nervous System.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons in the spinal cord, cerebral cortex, and medulla oblongata. Most patients present a clinical phenotype of classic ALS-with predominant atrophy, muscle weakness, and fasciculations-and survival of 3 to 5 years following diagnosis. In the present review, we performed a literature search to provide an update on the etiology and pathophysiological mechanisms involved in ALS. There are two types of ALS: the familial form with genetic involvement, and the sporadic form with a multifactorial origin. ALS pathophysiology is characterized by involvement of multiple processes, including oxidative stress, glutamate excitotoxicity, and neuroinflammation. Moreover, it is proposed that conditioning risk factors affect ALS development, such as susceptibility to neurodegeneration in motor neurons, the intensity of performed physical activity, and intestinal dysbiosis with involvement of the enteric nervous system, which supports the existing theories of disease generation. To improve patients' prognosis and survival, it is necessary to further deepen our understanding of the etiopathogenesis of ALS.

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.

Role of peptidoglycan recycling enzymes AmpD and AnmK in Acinetobacter baumannii virulence features.

Acinetobacter baumannii is an important causative agent of hospital acquired infections. In addition to acquired resistance to many currently-available antibiotics, it is intrinsically resistant to fosfomycin. It has previously been shown that AmpD and AnmK contribute to intrinsic fosfomycin resistance in A. baumannii due to their involvement in the peptidoglycan recycling pathway. However, the role that these two enzymes play in the fitness and virulence of A. baumannii has not been studied. The aim of this study was to characterize several virulence-related phenotypic traits in A. baumannii mutants lacking AmpD and AnmK. Specifically, cell morphology, peptidoglycan thickness, membrane permeability, growth under iron-limiting conditions, fitness, resistance to disinfectants and antimicrobial agents, twitching motility and biofilm formation of the mutant strains A. baumannii ATCC 17978 ΔampD::Kan and ΔanmK::Kan were compared to the wild type strain. Our results demonstrate that bacterial growth and fitness of both mutants were compromised, especially in the ΔampD::Kan mutant. In addition, biofilm formation was decreased by up to 69%, whereas twitching movement was reduced by about 80% in both mutants. These results demonstrate that, in addition to increased susceptibility to fosfomycin, alteration of the peptidoglycan recycling pathway affects multiple aspects related to virulence. Inhibition of these enzymes could be explored as a strategy to develop novel treatments for A. baumannii in the future. Furthermore, this study establishes a link between intrinsic fosfomycin resistance mechanisms and bacterial fitness and virulence traits.

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.

A New Approach to the Identification of Palliative Care Needs and Advanced Chronic Patients among Nursing Home Residents.

BACKGROUND: Proper planning of Palliative Care in nursing homes requires advanced knowledge of the care needs that residents show. The aim of the study was to evaluate Palliative Needs and other conditions such as fragility, complexity, and prognosis and also to suggest new indicators for the establishment of the resident's advanced chronic condition. METHODS: Cross-sectional study conducted in 149 nursing homes Complex Chronic residents evaluated by trained professionals. Palliative Care Needs, assessed by the NECPAL ICO-CCOMS© tool, and fragility, case and palliative complexity and prognosis were evaluate through a comprehensive assessment. Descriptive analyses and association measures were performed setting the statistical significance at 0.05. RESULTS: More than 50% of the residents had positive Surprise Question and other Palliative Needs and were classified as Advanced Chronic Patients. Distress and/or Severe Adaptative Disorder was the most frequent need shown by the residents and significant differences in levels of frailty and other characteristics, were found between the Positive and the Negative Surprise Question Groups. Statistically significant correlations were also found between aspects of both groups. CONCLUSIONS: Nursing homes residents show Palliative Needs regardless of the response to the Surprise Question of the NECPAL tool. Other characteristics such as presence of an intermediate level of frailty are suggested as a new perspective to identify advanced chronic patients among nursing homes residents.

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.

Detection of SARS-CoV-2 in a cat owned by a COVID-19-affected patient in Spain.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, is considered a zoonotic pathogen mainly transmitted human to human. Few reports indicate that pets may be exposed to the virus. The present report describes a cat suffering from severe respiratory distress and thrombocytopenia living with a family with several members affected by COVID-19. Clinical signs of the cat prompted humanitarian euthanasia and a detailed postmortem investigation to assess whether a COVID-19-like disease was causing the condition. Necropsy results showed the animal suffered from feline hypertrophic cardiomyopathy and severe pulmonary edema and thrombosis. SARS-CoV-2 RNA was only detected in nasal swab, nasal turbinates, and mesenteric lymph node, but no evidence of histopathological lesions compatible with a viral infection were detected. The cat seroconverted against SARS-CoV-2, further evidencing a productive infection in this animal. We conclude that the animal had a subclinical SARS-CoV-2 infection concomitant to an unrelated cardiomyopathy that led to euthanasia.

Changes in Mediterranean dietary pattern of university students: a comparative study between Spain and Algeria / Cambios en el patrón dietético mediterráneo de estudiantes universitarios: un estudio comparativo entre España y Argelia

AIM: Assess and compare the diet quality of university students living in two different Mediterranean countries (Algeria and Spain). METHODS: 303 university students from two different Mediterranean areas (Algeria and Spain) were enrolled in this cross-sectional study. The data collection of students' diet and other personal variables was based on a self-reported method; a nutritional evaluation was carried out using the DIAL(R) nutritional software. RESULTS: There were found significant differences in the diets, not only because of gender but also nationality, probably due to inherent cultural and socioeconomic characteristics of each country resulting in variants of Mediterranean diet. CONCLUSIONS: Nutrition education programs based on the promotion of Mediterranean lifestyle must be aimed at all ages, but especially in university stage

OBJETIVO: evaluar y comparar la calidad de la dieta de los estudiantes universitarios que viven en dos países mediterráneos diferentes (Argelia y España). MÉTODO: 303 estudiantes universitarios de dos áreas mediterráneas diferentes (Argelia y España) se inscribieron en este estudio transversal. La recopilación de datos de la dieta de los estudiantes y otras variables personales se recogío mediante en un cuestionario autoinformado; Se realizó una evaluación nutricional utilizando el software nutricional DIAL(R). RESULTADOS: Se encontraron diferencias significativas en las dietas, no solo debido al género sino también a la nacionalidad, probablemente debido a las distintas características culturales y socioeconómicas de cada país que se reflejan en algunas variantes de la dieta mediterránea. CONCLUSIÓN: los programas de educación nutricional basados en la promoción del estilo de vida mediterráneo deben estar dirigidos a todas las edades, pero especialmente en la etapa universitaria

Chimeric Pneumoviridae fusion proteins as immunogens to induce cross-neutralizing antibody responses.

Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV), two members of the Pneumoviridae family, account for the majority of severe lower respiratory tract infections worldwide in very young children. They are also a frequent cause of morbidity and mortality in the elderly and immunocompromised adults. High levels of neutralizing antibodies, mostly directed against the viral fusion (F) glycoprotein, correlate with protection against either hRSV or hMPV However, no cross-neutralization is observed in polyclonal antibody responses raised after virus infection or immunization with purified F proteins. Based on crystal structures of hRSV F and hMPV F, we designed chimeric F proteins in which certain residues of well-characterized antigenic sites were swapped between the two antigens. The antigenic changes were monitored by ELISA with virus-specific monoclonal antibodies. Inoculation of mice with these chimeras induced polyclonal cross-neutralizing antibody responses, and mice were protected against challenge with the virus used for grafting of the heterologous antigenic site. These results provide a proof of principle for chimeric fusion proteins as single immunogens that can induce cross-neutralizing antibody and protective responses against more than one human pneumovirus.

Structural insights into the assembly and regulation of distinct viral capsid complexes.

The assembly and regulation of viral capsid proteins into highly ordered macromolecular complexes is essential for viral replication. Here, we utilize crystal structures of the capsid protein from the smallest and simplest known viruses capable of autonomously replicating in animal cells, circoviruses, to establish structural and mechanistic insights into capsid morphogenesis and regulation. The beak and feather disease virus, like many circoviruses, encode only two genes: a capsid protein and a replication initiation protein. The capsid protein forms distinct macromolecular assemblies during replication and here we elucidate these structures at high resolution, showing that these complexes reverse the exposure of the N-terminal arginine rich domain responsible for DNA binding and nuclear localization. We show that assembly of these complexes is regulated by single-stranded DNA (ssDNA), and provide a structural basis of capsid assembly around single-stranded DNA, highlighting novel binding interfaces distinct from the highly positively charged N-terminal ARM domain.

Engineering, Structure and Immunogenicity of the Human Metapneumovirus F Protein in the Postfusion Conformation.

Human metapneumovirus (hMPV) is a paramyxovirus that is a common cause of bronchiolitis and pneumonia in children less than five years of age. The hMPV fusion (F) glycoprotein is the primary target of neutralizing antibodies and is thus a critical vaccine antigen. To facilitate structure-based vaccine design, we stabilized the ectodomain of the hMPV F protein in the postfusion conformation and determined its structure to a resolution of 3.3 Å by X-ray crystallography. The structure resembles an elongated cone and is very similar to the postfusion F protein from the related human respiratory syncytial virus (hRSV). In contrast, significant differences were apparent with the postfusion F proteins from other paramyxoviruses, such as human parainfluenza type 3 (hPIV3) and Newcastle disease virus (NDV). The high similarity of hMPV and hRSV postfusion F in two antigenic sites targeted by neutralizing antibodies prompted us to test for antibody cross-reactivity. The widely used monoclonal antibody 101F, which binds to antigenic site IV of hRSV F, was found to cross-react with hMPV postfusion F and neutralize both hRSV and hMPV. Despite the cross-reactivity of 101F and the reported cross-reactivity of two other antibodies, 54G10 and MPE8, we found no detectable cross-reactivity in the polyclonal antibody responses raised in mice against the postfusion forms of either hMPV or hRSV F. The postfusion-stabilized hMPV F protein did, however, elicit high titers of hMPV-neutralizing activity, suggesting that it could serve as an effective subunit vaccine. Structural insights from these studies should be useful for designing novel immunogens able to induce wider cross-reactive antibody responses.

Ultrastructure of the Babesia divergens free merozoite.

The invasive form of the apicomplexan parasite Babesia divergens, the free merozoite, invades the erythrocytes of host vertebrates, leading to significant pathology. Although invasion is an active process critical for parasite survival, it is not yet entirely understood. Using techniques to isolate the viable free merozoite, as well as electron microscopy, we undertook a detailed morphological study and explored the sub-cellular structure of the invasive B. divergens free merozoite after it had left the host cell. We examined characteristic apicomplexan features such as the apicoplast, the inner and discontinuous double membrane complex, and the apical complex; some aspects of erythrocyte entry by B. divergens were also defined by electron microscopy. This study adds to our understanding of B. divergens free merozoites and their invasion of human erythrocytes.

Influence of Respiratory Syncytial Virus F Glycoprotein Conformation on Induction of Protective Immune Responses.

UNLABELLED: Human respiratory syncytial virus (hRSV) vaccine development has received new impetus from structure-based studies of its main protective antigen, the fusion (F) glycoprotein. Three soluble forms of F have been described: monomeric, trimeric prefusion, and trimeric postfusion. Most human neutralizing antibodies recognize epitopes found exclusively in prefusion F. Although prefusion F induces higher levels of neutralizing antibodies than does postfusion F, postfusion F can also induce protection against virus challenge in animals. However, the immunogenicity and protective efficacy of the three forms of F have not hitherto been directly compared. Hence, BALB/c mice were immunized with a single dose of the three proteins adjuvanted with CpG and challenged 4 weeks later with virus. Serum antibodies, lung virus titers, weight loss, and pulmonary pathology were evaluated after challenge. Whereas small amounts of postfusion F were sufficient to protect mice, larger amounts of monomeric and prefusion F proteins were required for protection. However, postfusion and monomeric F proteins were associated with more pathology after challenge than was prefusion F. Antibodies induced by all doses of prefusion F, in contrast to other F protein forms, reacted predominantly with the prefusion F conformation. At high doses, prefusion F also induced the highest titers of neutralizing antibodies, and all mice were protected, yet at low doses of the immunogen, these antibodies neutralized virus poorly, and mice were not protected. These findings should be considered when developing new hRSV vaccine candidates. IMPORTANCE: Protection against hRSV infection is afforded mainly by neutralizing antibodies, which recognize mostly epitopes found exclusively in the viral fusion (F) glycoprotein trimer, folded in its prefusion conformation, i.e., before activation for membrane fusion. Although prefusion F is able to induce high levels of neutralizing antibodies, highly stable postfusion F (found after membrane fusion) is also able to induce neutralizing antibodies and protect against infection. In addition, a monomeric form of hRSV F that shares epitopes with prefusion F was recently reported. Since each of the indicated forms of hRSV F may have advantages and disadvantages for the development of safe and efficacious subunit vaccines, a direct comparison of the immunogenic properties and protective efficacies of the different forms of hRSV F was made in a mouse model. The results obtained show important differences between the noted immunogens that should be borne in mind when considering the development of hRSV vaccines.

Cell Wall Changes in Amphotericin B-Resistant Strains from Candida tropicalis and Relationship with the Immune Responses Elicited by the Host.

We have morphologically characterizedCandida tropicalisisolates resistant to amphotericin B (AmB). These isolates present an enlarged cell wall compared to isolates of regular susceptibility. This correlated with higher levels of ß-1,3-glucan in the cell wall but not with detectable changes in chitin content. In line with this, AmB-resistant strains showed reduced susceptibility to Congo red. Moreover, mitogen-activated protein kinases (MAPKs) involved in cell integrity were already activated during regular growth in these strains. Finally, we investigated the response elicited by human blood cells and found that AmB-resistant strains induced a stronger proinflammatory response than susceptible strains. In agreement, AmB-resistant strains also induced stronger melanization ofGalleria mellonellalarvae, indicating that the effect of alterations of the cell wall on the immune response is conserved in different types of hosts. Our results suggest that resistance to AmB is associated with pleiotropic mechanisms that might have important consequences, not only for the efficacy of the treatment but also for the immune response elicited by the host.

Intracellular expression of Tat alters mitochondrial functions in T cells: a potential mechanism to understand mitochondrial damage during HIV-1 replication.

BACKGROUND: HIV-1 replication results in mitochondrial damage that is enhanced during antiretroviral therapy (ART). The onset of HIV-1 replication is regulated by viral protein Tat, a 101-residue protein codified by two exons that elongates viral transcripts. Although the first exon of Tat (aa 1-72) forms itself an active protein, the presence of the second exon (aa 73-101) results in a more competent transcriptional protein with additional functions. RESULTS: Mitochondrial overall functions were analyzed in Jurkat cells stably expressing full-length Tat (Tat101) or one-exon Tat (Tat72). Representative results were confirmed in PBLs transiently expressing Tat101 and in HIV-infected Jurkat cells. The intracellular expression of Tat101 induced the deregulation of metabolism and cytoskeletal proteins which remodeled the function and distribution of mitochondria. Tat101 reduced the transcription of the mtDNA, resulting in low ATP production. The total amount of mitochondria increased likely to counteract their functional impairment. These effects were enhanced when Tat second exon was expressed. CONCLUSIONS: Intracellular Tat altered mtDNA transcription, mitochondrial content and distribution in CD4+ T cells. The importance of Tat second exon in non-transcriptional functions was confirmed. Tat101 may be responsible for mitochondrial dysfunctions found in HIV-1 infected patients.

Generation of monoclonal antibodies specific of the postfusion conformation of the Pneumovirinae fusion (F) protein.

Paramyxovirus entry into cells requires fusion of the viral and cell membranes mediated by one of the major virus glycoproteins, the fusion (F) glycoprotein which transits from a metastable pre-fusion conformation to a highly stable post-fusion structure during the membrane fusion process. F protein refolding involves large conformational changes of the protein trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) from each protomer into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of the Pneumovirinae F proteins, and as extension of previous work (Palomo et al., 2014), a general strategy was designed to obtain polyclonal and particularly monoclonal antibodies specific of the 6HB motif of the Pneumovirinae fusion protein. The antibodies reported here should assist in the characterization of the structural changes that the F protein of human metapneumovirus or respiratory syncytial virus experiences during the process of membrane fusion.

Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation.

Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV_F occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV_F, we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV_F at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule.