Cholesterol and M2 Rendezvous in Budding and Scission of Influenza A Virus.

The cholesterol of the host cell plasma membrane and viral M2 protein plays a crucial role in multiple stages of infection and replication of the influenza A virus. Cholesterol is required for the formation of heterogeneous membrane microdomains (or rafts) in the budozone of the host cell that serves as assembly sites for the viral components. The raft microstructures act as scaffolds for several proteins. Cholesterol may further contribute to the mechanical forces necessary for membrane scission in the last stage of budding and help to maintain the stability of the virus envelope. The M2 protein has been shown to cause membrane scission in model systems by promoting the formation of curved lipid bilayer structures that, in turn, can lead to membrane vesicles budding off or scission intermediates. Membrane remodeling by M2 is intimately linked with cholesterol as it affects local lipid composition, fluidity, and stability of the membrane. Thus, both cholesterol and M2 protein contribute to the efficient and proper release of newly formed influenza viruses from the virus-infected cells.

Hypothesis: inflammatory acid-base disruption underpins Long Covid.

The mechanism of Long Covid (Post-Acute Sequelae of COVID-19; PASC) is currently unknown, with no validated diagnostics or therapeutics. SARS-CoV-2 can cause disseminated infections that result in multi-system tissue damage, dysregulated inflammation, and cellular metabolic disruptions. The tissue damage and inflammation has been shown to impair microvascular circulation, resulting in hypoxia, which coupled with virally-induced metabolic reprogramming, increases cellular anaerobic respiration. Both acute and PASC patients show systemic dysregulation of multiple markers of the acid-base balance. Based on these data, we hypothesize that the shift to anaerobic respiration causes an acid-base disruption that can affect every organ system and underpins the symptoms of PASC. This hypothesis can be tested by longitudinally evaluating acid-base markers in PASC patients and controls over the course of a month. If our hypothesis is correct, this could have significant implications for our understanding of PASC and our ability to develop effective diagnostic and therapeutic approaches.

Influence of Landscape Patterns on Exposure to Lassa Fever Virus, Guinea.

Lassa fever virus (LASV) is the causative agent of Lassa fever, a disease endemic in West Africa. Exploring the relationships between environmental factors and LASV transmission across ecologically diverse regions can provide crucial information for the design of appropriate interventions and disease monitoring. We investigated LASV exposure in 2 ecologically diverse regions of Guinea. Our results showed that exposure to LASV was heterogenous between and within sites. LASV IgG seropositivity was 11.9% (95% CI 9.7%-14.5%) in a coastal study site in Basse-Guinée, but it was 59.6% (95% CI 55.5%-63.5%) in a forested study site located in Guinée Forestière. Seropositivity increased with age in the coastal site. We also found significant associations between exposure risk for LASV and landscape fragmentation in coastal and forested regions. Our study highlights the potential link between environmental change and LASV emergence and the urgent need for research on land management practices that reduce disease risks.

Dating first cases of COVID-19.

Questions persist as to the origin of the COVID-19 pandemic. Evidence is building that its origin as a zoonotic spillover occurred prior to the officially accepted timing of early December, 2019. Here we provide novel methods to date the origin of COVID-19 cases. We show that six countries had exceptionally early cases, unlikely to represent part of their main case series. The model suggests a likely timing of the first case of COVID-19 in China as November 17 (95% CI October 4). Origination dates are discussed for the first five countries outside China and each continent. Results infer that SARS-CoV-2 emerged in China in early October to mid-November, and by January, had spread globally. This suggests an earlier and more rapid timeline of spread. Our study provides new approaches for estimating dates of the arrival of infectious diseases based on small samples that can be applied to many epidemiological situations.

Immobilising giant unilamellar vesicles with zirconium metal-organic framework anchors.

Lipid bilayer vesicles have provided a window into the function and fundamental properties of cells. However, as is the case for most living and soft matter, vesicles do not remain still. This necessitates some microscopy experiments to include a preparatory immobilisation step. Here, we describe a straightforward method to immobilise giant unilamellar vesicles (GUVs) using zirconium-based metal-organic frameworks (MOFs) and demonstrate that GUVs bound in this way will stay in position on a timescale of minutes to hours.

Cholesterol Alters the Orientation and Activity of the Influenza Virus M2 Amphipathic Helix in the Membrane.

The influenza virus M2 amphipathic helix (M2AH) alters membrane curvature in a cholesterol-dependent manner, mediating viral membrane scission during influenza virus budding. Here, we have investigated the biophysical effects of cholesterol on the ability of an M2AH peptide to manipulate membrane properties. We see that the ability of the M2AH to interact with membranes and form an α-helix is independent of membrane cholesterol concentration; however, cholesterol affects the angle of the M2AH peptide within the membrane. This change in membrane orientation affects the ability of the M2AH to alter lipid order. In low-cholesterol membranes, the M2AH is inserted near the level of the lipid head groups, increasing lipid order, which may contribute to generation of the membrane curvature. As the cholesterol content increases, the M2AH insertion becomes flatter and slightly deeper in the membrane below the lipid headgroups, where the polar face can continue to interact with the headgroups while the hydrophobic face binds cholesterol. This changed orientation minimizes lipid packing defects and lipid order changes, likely reducing the generation of membrane curvature. Thus, cholesterol regulates M2 membrane scission by precisely modulating M2AH positioning within the membrane. This has implications for the understanding of many of amphipathic-helix-driven cellular budding processes that occur in specific lipid environments.

Entropic forces drive clustering and spatial localization of influenza A M2 during viral budding.

The influenza A matrix 2 (M2) transmembrane protein facilitates virion release from the infected host cell. In particular, M2 plays a role in the induction of membrane curvature and/or in the scission process whereby the envelope is cut upon virion release. Here we show using coarse-grained computer simulations that various M2 assembly geometries emerge due to an entropic driving force, resulting in compact clusters or linearly extended aggregates as a direct consequence of the lateral membrane stresses. Conditions under which these protein assemblies will cause the lipid membrane to curve are explored, and we predict that a critical cluster size is required for this to happen. We go on to demonstrate that under the stress conditions taking place in the cellular membrane as it undergoes large-scale membrane remodeling, the M2 protein will, in principle, be able to both contribute to curvature induction and sense curvature to line up in manifolds where local membrane line tension is high. M2 is found to exhibit linactant behavior in liquid-disordered-liquid-ordered phase-separated lipid mixtures and to be excluded from the liquid-ordered phase, in near-quantitative agreement with experimental observations. Our findings support a role for M2 in membrane remodeling during influenza viral budding both as an inducer and a sensor of membrane curvature, and they suggest a mechanism by which localization of M2 can occur as the virion assembles and releases from the host cell, independent of how the membrane curvature is produced.

The Response to and Impact of the Ebola Epidemic: Towards an Agenda for Interdisciplinary Research.

BACKGROUND: The 2013-2016 Ebola virus disease (EVD) epidemic in West Africa was the largest in history and resulted in a huge public health burden and significant social and economic impact in those countries most affected. Its size, duration and geographical spread presents important opportunities for research than might help national and global health and social care systems to better prepare for and respond to future outbreaks. This paper examines research needs and research priorities from the perspective of those who directly experienced the EVD epidemic in Guinea. METHODS: The paper reports the findings from a research scoping exercise conducted in Guinea in 2017. This exercise explored the need for health and social care research, and identified research gaps, from the perspectives of different groups. Interviews were carried out with key stakeholders such as representatives of the Ministry of Health, non-governmental organizations (NGOs), academic and health service researchers and members of research ethics committees (N=15); health practitioners (N=12) and community representatives (N=11). Discussion groups were conducted with male and female EVD survivors (N=24) from two distinct communities. RESULTS: This research scoping exercise identified seven key questions for further research. An important research priority that emerged during this study was the need to carry out a comprehensive analysis of the wider social, economic and political impact of the epidemic on the country, communities and survivors. The social and cultural dynamics of the epidemic and the local, national and international response to it need to be better understood. Many survivors and their relatives continue to experience stigma and social isolation and have a number of complex unmet needs. It is important to understand what sort of support they need, and how that might best be provided. A better understanding of the virus and the long-term health and social implications for survivors and non-infected survivors is also needed. CONCLUSION: This study identified a need and priority for interdisciplinary research focusing on the long-term sociocultural, economic and health impact of the EVD epidemic. Experiences of survivors and other non-infected members of the community still need to be explored but in this broader context.

Ebolaviruses: New roles for old proteins.

In 2014, the world witnessed the largest Ebolavirus outbreak in recorded history. The subsequent humanitarian effort spurred extensive research, significantly enhancing our understanding of ebolavirus replication and pathogenicity. The main functions of each ebolavirus protein have been studied extensively since the discovery of the virus in 1976; however, the recent expansion of ebolavirus research has led to the discovery of new protein functions. These newly discovered roles are revealing new mechanisms of virus replication and pathogenicity, whilst enhancing our understanding of the broad functions of each ebolavirus viral protein (VP). Many of these new functions appear to be unrelated to the protein's primary function during virus replication. Such new functions range from bystander T-lymphocyte death caused by VP40-secreted exosomes to new roles for VP24 in viral particle formation. This review highlights the newly discovered roles of ebolavirus proteins in order to provide a more encompassing view of ebolavirus replication and pathogenicity.

Autophagy diminishes the early interferon-ß response to influenza A virus resulting in differential expression of interferon-stimulated genes.

Influenza A virus (IAV) infection perturbs metabolic pathways such as autophagy, a stress-induced catabolic pathway that crosstalks with cellular inflammatory responses. However, the impact of autophagy perturbation on IAV gene expression or host cell responses remains disputed. Discrepant results may be a reflection of in vivo studies using cell-specific autophagy-related (Atg) gene-deficient mouse strains, which do not delineate modification of developmental programmes from more proximal effects on inflammatory response. In vitro experiments can be confounded by gene expression divergence in wild-type cultivated cell lines, as compared to those experiencing long-term absence of autophagy. With the goal to investigate cellular processes within cells that are competent or incompetent for autophagy, we generated a novel experimental cell line in which autophagy can be restored by ATG5 protein stabilization in an otherwise Atg5-deficient background. We confirmed that IAV induced autophagosome formation and p62 accumulation in infected cells and demonstrated that perturbation of autophagy did not impact viral infection or replication in ATG5-stablized cells. Notably, the induction of interferon-stimulated genes (ISGs) by IAV was diminished when cells were autophagy competent. We further demonstrated that, in the absence of ATG5, IAV-induced interferon-ß (IFN-ß) expression was increased as compared to levels in autophagy-competent lines, a mechanism that was independent of IAV non-structural protein 1. In sum, we report that induction of autophagy by IAV infection reduces ISG expression in infected cells by limiting IFN-ß expression, which may benefit viral replication and spread.

Membrane remodeling by the M2 amphipathic helix drives influenza virus membrane scission.

Membrane scission is a crucial step in all budding processes, from endocytosis to viral budding. Many proteins have been associated with scission, though the underlying molecular details of how scission is accomplished often remain unknown. Here, we investigate the process of M2-mediated membrane scission during the budding of influenza viruses. Residues 50-61 of the viral M2 protein bind membrane and form an amphipathic α-helix (AH). Membrane binding requires hydrophobic interactions with the lipid tails but not charged interactions with the lipid headgroups. Upon binding, the M2AH induces membrane curvature and lipid ordering, constricting and destabilizing the membrane neck, causing scission. We further show that AHs in the cellular proteins Arf1 and Epsin1 behave in a similar manner. Together, they represent a class of membrane-induced AH domains that alter membrane curvature and fluidity, mediating the scission of constricted membrane necks in multiple biological pathways.

Changes associated with Ebola virus adaptation to novel species.

MOTIVATION: Ebola viruses are not pathogenic but can be adapted to replicate and cause disease in rodents. Here, we used a structural bioinformatics approach to analyze the mutations associated with Ebola virus adaptation to rodents to elucidate the determinants of host-specific Ebola virus pathogenicity. RESULTS: We identified 33 different mutations associated with Ebola virus adaptation to rodents in the proteins GP, NP, L, VP24 and VP35. Only VP24, GP and NP were consistently found mutated in rodent-adapted Ebola virus strains. Fewer than five mutations in these genes seem to be required for the adaptation of Ebola viruses to a new species. The role of mutations in GP and NP is not clear. However, three VP24 mutations located in the protein interface with karyopherin α5 may enable VP24 to inhibit karyopherins and subsequently the host interferon response. Three further VP24 mutations change hydrogen bonding or cause conformational changes. Hence, there is evidence that few mutations including crucial mutations in VP24 enable Ebola virus adaptation to new hosts. Since Reston virus, the only non-human pathogenic Ebolavirus species circulates in pigs in Asia, this raises concerns that few mutations may result in novel human pathogenic Ebolaviruses. CONTACT: m.n.wass@kent.ac.uk , m.michaelis@kent.ac.uk or j.s.rossman@kent.ac.uk. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Acquired resistance to oxaliplatin is not directly associated with increased resistance to DNA damage in SK-N-ASrOXALI4000, a newly established oxaliplatin-resistant sub-line of the neuroblastoma cell line SK-N-AS.

The formation of acquired drug resistance is a major reason for the failure of anti-cancer therapies after initial response. Here, we introduce a novel model of acquired oxaliplatin resistance, a sub-line of the non-MYCN-amplified neuroblastoma cell line SK-N-AS that was adapted to growth in the presence of 4000 ng/mL oxaliplatin (SK-N-ASrOXALI4000). SK-N-ASrOXALI4000 cells displayed enhanced chromosomal aberrations compared to SK-N-AS, as indicated by 24-chromosome fluorescence in situ hybridisation. Moreover, SK-N-ASrOXALI4000 cells were resistant not only to oxaliplatin but also to the two other commonly used anti-cancer platinum agents cisplatin and carboplatin. SK-N-ASrOXALI4000 cells exhibited a stable resistance phenotype that was not affected by culturing the cells for 10 weeks in the absence of oxaliplatin. Interestingly, SK-N-ASrOXALI4000 cells showed no cross resistance to gemcitabine and increased sensitivity to doxorubicin and UVC radiation, alternative treatments that like platinum drugs target DNA integrity. Notably, UVC-induced DNA damage is thought to be predominantly repaired by nucleotide excision repair and nucleotide excision repair has been described as the main oxaliplatin-induced DNA damage repair system. SK-N-ASrOXALI4000 cells were also more sensitive to lysis by influenza A virus, a candidate for oncolytic therapy, than SK-N-AS cells. In conclusion, we introduce a novel oxaliplatin resistance model. The oxaliplatin resistance mechanisms in SK-N-ASrOXALI4000 cells appear to be complex and not to directly depend on enhanced DNA repair capacity. Models of oxaliplatin resistance are of particular relevance since research on platinum drugs has so far predominantly focused on cisplatin and carboplatin.

Computational analysis of Ebolavirus data: prospects, promises and challenges.

The ongoing Ebola virus (also known as Zaire ebolavirus, a member of the Ebolavirus family) outbreak in West Africa has so far resulted in >28000 confirmed cases compared with previous Ebolavirus outbreaks that affected a maximum of a few hundred individuals. Hence, Ebolaviruses impose a much greater threat than we may have expected (or hoped). An improved understanding of the virus biology is essential to develop therapeutic and preventive measures and to be better prepared for future outbreaks by members of the Ebolavirus family. Computational investigations can complement wet laboratory research for biosafety level 4 pathogens such as Ebolaviruses for which the wet experimental capacities are limited due to a small number of appropriate containment laboratories. During the current West Africa outbreak, sequence data from many Ebola virus genomes became available providing a rich resource for computational analysis. Here, we consider the studies that have already reported on the computational analysis of these data. A range of properties have been investigated including Ebolavirus evolution and pathogenicity, prediction of micro RNAs and identification of Ebolavirus specific signatures. However, the accuracy of the results remains to be confirmed by wet laboratory experiments. Therefore, communication and exchange between computational and wet laboratory researchers is necessary to make maximum use of computational analyses and to iteratively improve these approaches.

Curvature Sensing by a Viral Scission Protein.

Membrane scission is the final step in all budding processes wherein a membrane neck is sufficiently constricted so as to allow for fission and the release of the budded particle. For influenza viruses, membrane scission is mediated by an amphipathic helix (AH) domain in the viral M2 protein. While it is known that the M2AH alters membrane curvature, it is not known how the protein is localized to the center neck of budding virions where it would be able to cause membrane scission. Here, we use molecular dynamics simulations on buckled lipid bilayers to show that the M2AH senses membrane curvature and preferentially localizes to regions of high membrane curvature, comparable to that seen at the center neck of budding influenza viruses. These results were then validated using in vitro binding assays to show that the M2AH senses membrane curvature by detecting lipid packing defects in the membrane. Our results show that the M2AH senses membrane curvature and suggest that the AH domain may localize the protein at the viral neck where it can then mediate membrane scission and the release of budding viruses.

Discovery and Mechanism of Highly Efficient Cyclic Cell-Penetrating Peptides.

Previous cell-penetrating peptides (CPPs) generally have low cytosolic delivery efficiencies, because of inefficient endosomal escape. In this study, a family of small, amphipathic cyclic peptides was found to be highly efficient CPPs, with cytosolic delivery efficiencies of up to 120% (compared to 2.0% for Tat). These cyclic CPPs bind directly to the plasma membrane phospholipids and enter mammalian cells via endocytosis, followed by efficient release from the endosome. Their total cellular uptake efficiency correlates positively with the binding affinity for the plasma membrane, whereas their endosomal escape efficiency increases with the endosomal membrane-binding affinity. The cyclic CPPs induce membrane curvature on giant unilamellar vesicles and budding of small vesicles, which subsequently collapse into amorphous lipid/peptide aggregates. These data suggest that cyclic CPPs exit the endosome by binding to the endosomal membrane and inducing CPP-enriched lipid domains to bud off as small vesicles. Together with their high proteolytic stability, low cytotoxicity, and oral bioavailability, these cyclic CPPs should provide a powerful system for intracellular delivery of therapeutic agents and chemical probes.

Conserved differences in protein sequence determine the human pathogenicity of Ebolaviruses.

Reston viruses are the only Ebolaviruses that are not pathogenic in humans. We analyzed 196 Ebolavirus genomes and identified specificity determining positions (SDPs) in all nine Ebolavirus proteins that distinguish Reston viruses from the four human pathogenic Ebolaviruses. A subset of these SDPs will explain the differences in human pathogenicity between Reston and the other four ebolavirus species. Structural analysis was performed to identify those SDPs that are likely to have a functional effect. This analysis revealed novel functional insights in particular for Ebolavirus proteins VP40 and VP24. The VP40 SDP P85T interferes with VP40 function by altering octamer formation. The VP40 SDP Q245P affects the structure and hydrophobic core of the protein and consequently protein function. Three VP24 SDPs (T131S, M136L, Q139R) are likely to impair VP24 binding to human karyopherin alpha5 (KPNA5) and therefore inhibition of interferon signaling. Since VP24 is critical for Ebolavirus adaptation to novel hosts, and only a few SDPs distinguish Reston virus VP24 from VP24 of other Ebolaviruses, human pathogenic Reston viruses may emerge. This is of concern since Reston viruses circulate in domestic pigs and can infect humans, possibly via airborne transmission.

Risks Posed by Reston, the Forgotten Ebolavirus.

Out of the five members of the Ebolavirus family, four cause life-threatening disease, whereas the fifth, Reston virus (RESTV), is nonpathogenic in humans. The reasons for this discrepancy remain unclear. In this review, we analyze the currently available information to provide a state-of-the-art summary of the factors that determine the human pathogenicity of Ebolaviruses. RESTV causes sporadic infections in cynomolgus monkeys and is found in domestic pigs throughout the Philippines and China. Phylogenetic analyses revealed that RESTV is most closely related to the Sudan virus, which causes a high mortality rate in humans. Amino acid sequence differences between RESTV and the other Ebolaviruses are found in all nine Ebolavirus proteins, though no one residue appears sufficient to confer pathogenicity. Changes in the glycoprotein contribute to differences in Ebolavirus pathogenicity but are not sufficient to confer pathogenicity on their own. Similarly, differences in VP24 and VP35 affect viral immune evasion and are associated with changes in human pathogenicity. A recent in silico analysis systematically determined the functional consequences of sequence variations between RESTV and human-pathogenic Ebolaviruses. Multiple positions in VP24 were differently conserved between RESTV and the other Ebolaviruses and may alter human pathogenicity. In conclusion, the factors that determine the pathogenicity of Ebolaviruses in humans remain insufficiently understood. An improved understanding of these pathogenicity-determining factors is of crucial importance for disease prevention and for the early detection of emergent and potentially human-pathogenic RESTVs.