Pan-ebolavirus protective therapy by two multifunctional human antibodies.

Ebolaviruses cause a severe and often fatal illness with the potential for global spread. Monoclonal antibody-based treatments that have become available recently have a narrow therapeutic spectrum and are ineffective against ebolaviruses other than Ebola virus (EBOV), including medically important Bundibugyo (BDBV) and Sudan (SUDV) viruses. Here, we report the development of a therapeutic cocktail comprising two broadly neutralizing human antibodies, rEBOV-515 and rEBOV-442, that recognize non-overlapping sites on the ebolavirus glycoprotein (GP). Antibodies in the cocktail exhibited synergistic neutralizing activity, resisted viral escape, and possessed differing requirements for their Fc-regions for optimal in vivo activities. The cocktail protected non-human primates from ebolavirus disease caused by EBOV, BDBV, or SUDV with high therapeutic effectiveness. High-resolution structures of the cocktail antibodies in complex with GP revealed the molecular determinants for neutralization breadth and potency. This study provides advanced preclinical data to support clinical development of this cocktail for pan-ebolavirus therapy.

Electron Cryo-microscopy Structure of Ebola Virus Nucleoprotein Reveals a Mechanism for Nucleocapsid-like Assembly.

Ebola virus nucleoprotein (eNP) assembles into higher-ordered structures that form the viral nucleocapsid (NC) and serve as the scaffold for viral RNA synthesis. However, molecular insights into the NC assembly process are lacking. Using a hybrid approach, we characterized the NC-like assembly of eNP, identified novel regulatory elements, and described how these elements impact function. We generated a three-dimensional structure of the eNP NC-like assembly at 5.8 Å using electron cryo-microscopy and identified a new regulatory role for eNP helices α22-α23. Biochemical, biophysical, and mutational analyses revealed that inter-eNP contacts within α22-α23 are critical for viral NC assembly and regulate viral RNA synthesis. These observations suggest that the N terminus and α22-α23 of eNP function as context-dependent regulatory modules (CDRMs). Our current study provides a framework for a structural mechanism for NC-like assembly and a new therapeutic target.

Cryo-EM structure of the Ebola virus nucleoprotein-RNA complex at 3.6 Å resolution.

Ebola virus causes haemorrhagic fever with a high fatality rate in humans and non-human primates. It belongs to the family Filoviridae in the order Mononegavirales, which are viruses that contain linear, non-segmented, negative-sense, single-stranded genomic RNA1,2. The enveloped, filamentous virion contains the nucleocapsid, consisting of the helical nucleoprotein-RNA complex, VP24, VP30, VP35 and viral polymerase1,3. The nucleoprotein-RNA complex acts as a scaffold for nucleocapsid formation and as a template for RNA replication and transcription by condensing RNA into the virion4,5. RNA binding and nucleoprotein oligomerization are synergistic and do not readily occur independently6. Although recent cryo-electron tomography studies have revealed the overall architecture of the nucleocapsid core4,5, there has been no high-resolution reconstruction of the nucleocapsid. Here we report the structure of a recombinant Ebola virus nucleoprotein-RNA complex expressed in mammalian cells without chemical fixation, at near-atomic resolution using single-particle cryo-electron microscopy. Our structure reveals how the Ebola virus nucleocapsid core encapsidates its viral genome, its sequence-independent coordination with RNA by nucleoprotein, and the dynamic transition between the RNA-free and RNA-bound states. It provides direct structural evidence for the role of the N terminus of nucleoprotein in subunit oligomerization, and for the hydrophobic and electrostatic interactions that lead to the formation of the helical assembly. The structure is validated as representative of the native biological assembly of the nucleocapsid core by consistent dimensions and symmetry with the full virion5. The atomic model provides a detailed mechanistic basis for understanding nucleocapsid assembly and highlights key structural features that may serve as targets for anti-viral drug development.

Structure and assembly of the Ebola virus nucleocapsid.

Ebola and Marburg viruses are filoviruses: filamentous, enveloped viruses that cause haemorrhagic fever. Filoviruses are within the order Mononegavirales, which also includes rabies virus, measles virus, and respiratory syncytial virus. Mononegaviruses have non-segmented, single-stranded negative-sense RNA genomes that are encapsidated by nucleoprotein and other viral proteins to form a helical nucleocapsid. The nucleocapsid acts as a scaffold for virus assembly and as a template for genome transcription and replication. Insights into nucleoprotein-nucleoprotein interactions have been derived from structural studies of oligomerized, RNA-encapsidating nucleoprotein, and cryo-electron microscopy of nucleocapsid or nucleocapsid-like structures. There have been no high-resolution reconstructions of complete mononegavirus nucleocapsids. Here we apply cryo-electron tomography and subtomogram averaging to determine the structure of Ebola virus nucleocapsid within intact viruses and recombinant nucleocapsid-like assemblies. These structures reveal the identity and arrangement of the nucleocapsid components, and suggest that the formation of an extended α-helix from the disordered carboxy-terminal region of nucleoprotein-core links nucleoprotein oligomerization, nucleocapsid condensation, RNA encapsidation, and accessory protein recruitment.

Three-dimensional insights into human enveloped viruses in vitro and in situ.

Viruses can be enveloped or non-enveloped, and require a host cell to replicate and package their genomes into new virions to infect new cells. To accomplish this task, viruses hijack the host-cell machinery to facilitate their replication by subverting and manipulating normal host cell function. Enveloped viruses can have severe consequences for human health, causing various diseases such as acquired immunodeficiency syndrome (AIDS), seasonal influenza, COVID-19, and Ebola virus disease. The complex arrangement and pleomorphic architecture of many enveloped viruses pose a challenge for the more widely used structural biology techniques, such as X-ray crystallography. Cryo-electron tomography (cryo-ET), however, is a particularly well-suited tool for overcoming the limitations associated with visualizing the irregular shapes and morphology enveloped viruses possess at macromolecular resolution. The purpose of this review is to explore the latest structural insights that cryo-ET has revealed about enveloped viruses, with particular attention given to their architectures, mechanisms of entry, replication, assembly, maturation and egress during infection. Cryo-ET is unique in its ability to visualize cellular landscapes at 3-5 nanometer resolution. Therefore, it is the most suited technique to study asymmetric elements and structural rearrangements of enveloped viruses during infection in their native cellular context.

Dual-axis Volta phase plate cryo-electron tomography of Ebola virus-like particles reveals actin-VP40 interactions.

Cryo-electron tomography (cryo-ET) is a pivotal imaging technique for studying the structure of pleomorphic enveloped viruses and their interactions with the host at native conditions. Owing to the limited tilting range of samples with a slab geometry, electron tomograms suffer from so-called missing wedge information in Fourier space. In dual-axis cryo-ET, two tomograms reconstructed from orthogonally oriented tilt series are combined into a tomogram with improved resolution as the missing wedge information is reduced to a pyramid. Volta phase plate (VPP) allows to perform in-focus cryo-ET with high contrast transfer at low-resolution frequencies and thus its application may improve the quality of dual-axis tomograms. Here, we compare dual-axis cryo-ET with and without VPP on Ebola virus-like particles to visualize and segment viral and host cell proteins within the membrane-enveloped filamentous particles. Dual-axis VPP cryo-ET reduces the missing wedge information and ray artifacts arising from the weighted back-projection during tomogram reconstruction, thereby minimizing ambiguity in the analysis of crowded environments and facilitating 3D segmentation. We show that dual-axis VPP tomograms provide a comprehensive description of macromolecular organizations such as nucleocapsid assembly states, the distribution of glycoproteins on the viral envelope and asymmetric arrangements of the VP40 layer in non-filamentous regions of virus-like particles. Our data reveal actin filaments within virus-like particles in close proximity to the viral VP40 scaffold, suggesting a direct interaction between VP40 and actin filaments. Dual-axis VPP cryo-ET provides more complete 3D information at high contrast and allows for better interpretation of macromolecule interactions and pleomorphic organizations.

A Rapid Screen for Host-Encoded miRNAs with Inhibitory Effects against Ebola Virus Using a Transcription- and Replication-Competent Virus-Like Particle System.

MicroRNAs (miRNAs) may become efficient antiviral agents against the Ebola virus (EBOV) targeting viral genomic RNAs or transcripts. We previously conducted a genome-wide search for differentially expressed miRNAs during viral replication and transcription. In this study, we established a rapid screen for miRNAs with inhibitory effects against EBOV using a tetracistronic transcription- and replication-competent virus-like particle (trVLP) system. This system uses a minigenome comprising an EBOV leader region, luciferase reporter, VP40, GP, VP24, EBOV trailer region, and three noncoding regions from the EBOV genome and can be used to model the life cycle of EBOV under biosafety level (BSL) 2 conditions. Informatic analysis was performed to select up-regulated miRNAs targeting the coding regions of the minigenome with the highest binding energy to perform inhibitory effect screening. Among these miRNAs, miR-150-3p had the most significant inhibitory effect. Reverse transcription polymerase chain reaction (RT-PCR), Western blot, and double fluorescence reporter experiments demonstrated that miR-150-3p inhibited the reproduction of trVLPs via the regulation of GP and VP40 expression by directly targeting the coding regions of GP and VP40. This novel, rapid, and convenient screening method will efficiently facilitate the exploration of miRNAs against EBOV under BSL-2 conditions.

Discovery and Description of Ebola Zaire Virus in 1976 and Relevance to the West African Epidemic During 2013-2016.

BACKGROUND: In 1976, the first cases of Ebola virus disease in northern Democratic Republic of the Congo (then referred to as Zaire) were reported. This article addresses who was responsible for recognizing the disease; recovering, identifying, and naming the virus; and describing the epidemic. Key scientific approaches used in 1976 and their relevance to the 3-country (Guinea, Sierra Leone, and Liberia) West African epidemic during 2013-2016 are presented. METHODS: Field and laboratory investigations started soon after notification, in mid-September 1976, and included virus cell culture, electron microscopy (EM), immunofluorescence antibody (IFA) testing of sera, case tracing, containment, and epidemiological surveys. In 2013-2016, medical care and public health work were delayed for months until the Ebola virus disease epidemic was officially declared an emergency by World Health Organization, but research in pathogenesis, clinical presentation, including sequelae, treatment, and prevention, has increased more recently. RESULTS: Filoviruses were cultured and observed by EM in Antwerp, Belgium (Institute of Tropical Medicine); Porton Down, United Kingdom (Microbiological Research Establishment); and Atlanta, Georgia (Centers for Disease Control and Prevention). In Atlanta, serological testing identified a new virus. The 1976 outbreak (280 deaths among 318 cases) stopped in <11 weeks, and basic clinical and epidemiological features were defined. The recent massive epidemic during 2013-2016 (11 310 deaths among 28 616 cases) has virtually stopped after >2 years. Transmission indices (R0) are higher in all 3 countries than in 1976. CONCLUSIONS: An international commission working harmoniously in laboratories and with local communities was essential for rapid success in 1976. Control and understanding of the recent West African outbreak were delayed because of late recognition and because authorities were overwhelmed by many patients and poor community involvement. Despite obstacles, research was a priority in 1976 and recently.

Human transbodies to VP40 inhibit cellular egress of Ebola virus-like particles.

A direct acting anti-Ebola agent is needed. VP40, a conserved protein across Ebolavirus (EBOV) species has several pivotal roles in the virus life cycle. Inhibition of VP40 functions would lessen the virion integrity and interfere with the viral assembly, budding, and spread. In this study, cell penetrable human scFvs (HuscFvs) that bound to EBOV VP40 were produced by phage display technology. Gene sequences coding for VP40-bound-HuscFvs were subcloned from phagemids into protein expression plasmids downstream to a gene of cell penetrating peptide, i.e., nonaarginine (R9). By electron microscopy, transbodies from three clones effectively inhibited egress of the Ebola virus-like particles from human hepatic cells transduced with pseudo-typed-Lentivirus particles carrying EBOV VP40 and GP genes. Computerized simulation indicated that the effective HuscFvs bound to multiple basic residues in the cationic patch of VP40 C-terminal domain which are important in membrane-binding for viral matrix assembly and virus budding. The transbodies bound also to VP40 N-terminal domain and L domain peptide encompassed the PTAPPEY (WW binding) motif, suggesting that they might confer VP40 function inhibition through additional mechanism(s). The generated transbodies are worthwhile tested with authentic EBOV before developing to direct acting anti-Ebola agent for preclinical and clinical trials.

Spatial localization of the Ebola virus glycoprotein mucin-like domain determined by cryo-electron tomography.

The Ebola virus glycoprotein mucin-like domain (MLD) is implicated in Ebola virus cell entry and immune evasion. Using cryo-electron tomography of Ebola virus-like particles, we determined a three-dimensional structure for the full-length glycoprotein in a near-native state and compared it to that of a glycoprotein lacking the MLD. Our results, which show that the MLD is located at the apex and the sides of each glycoprotein monomer, provide a structural template for analysis of MLD function.

Structural dissection of Ebola virus and its assembly determinants using cryo-electron tomography.

Ebola virus is a highly pathogenic filovirus causing severe hemorrhagic fever with high mortality rates. It assembles heterogenous, filamentous, enveloped virus particles containing a negative-sense, single-stranded RNA genome packaged within a helical nucleocapsid (NC). We have used cryo-electron microscopy and tomography to visualize Ebola virus particles, as well as Ebola virus-like particles, in three dimensions in a near-native state. The NC within the virion forms a left-handed helix with an inner nucleoprotein layer decorated with protruding arms composed of VP24 and VP35. A comparison with the closely related Marburg virus shows that the N-terminal region of nucleoprotein defines the inner diameter of the Ebola virus NC, whereas the RNA genome defines its length. Binding of the nucleoprotein to RNA can assemble a loosely coiled NC-like structure; the loose coil can be condensed by binding of the viral matrix protein VP40 to the C terminus of the nucleoprotein, and rigidified by binding of VP24 and VP35 to alternate copies of the nucleoprotein. Four proteins (NP, VP24, VP35, and VP40) are necessary and sufficient to mediate assembly of an NC with structure, symmetry, variability, and flexibility indistinguishable from that in Ebola virus particles released from infected cells. Together these data provide a structural and architectural description of Ebola virus and define the roles of viral proteins in its structure and assembly.

Oligomerization of Ebola virus VP40 is essential for particle morphogenesis and regulation of viral transcription.

The morphogenesis and budding of virus particles represent an important stage in the life cycle of viruses. For Ebola virus, this process is driven by its major matrix protein, VP40. Like the matrix proteins of many other nonsegmented, negative-strand RNA viruses, VP40 has been demonstrated to oligomerize and to occur in at least two distinct oligomeric states: hexamers and octamers, which are composed of antiparallel dimers. While it has been shown that VP40 oligomers are essential for the viral life cycle, their function is completely unknown. Here we have identified two amino acids essential for oligomerization of VP40, the mutation of which blocked virus-like particle production. Consistent with this observation, oligomerization-deficient VP40 also showed impaired intracellular transport to budding sites and reduced binding to cellular membranes. However, other biological functions, such as the interaction of VP40 with the nucleoprotein, NP, remained undisturbed. Furthermore, both wild-type VP40 and oligomerization-deficient VP40 were found to negatively regulate viral genome replication, a novel function of VP40, which we have recently reported. Interestingly, while wild-type VP40 was also able to negatively regulate viral genome transcription, oligomerization-deficient VP40 was no longer able to fulfill this function, indicating that regulation of viral replication and transcription by VP40 are mechanistically distinct processes. These data indicate that VP40 oligomerization not only is a prerequisite for intracellular transport of VP40 and efficient membrane binding, and as a consequence virion morphogenesis, but also plays a critical role in the regulation of viral transcription by VP40.

Zaire Ebola virus entry into human dendritic cells is insensitive to cathepsin L inhibition.

Cathepsins B and L contribute to Ebola virus (EBOV) entry into Vero cells and mouse embryonic fibroblasts. However, the role of cathepsins in EBOV-infection of human dendritic cells (DCs), important targets of infection in vivo, remains undefined. Here, EBOV-like particles containing a beta-lactamase-VP40 fusion reporter and Ebola virus were used to demonstrate the cathepsin dependence of EBOV entry into human monocyte-derived DCs. However, while DC infection is blocked by cathepsin B inhibitor, it is insensitive to cathepsin L inhibitor. Furthermore, DCs pre-treated for 48 h with TNFalpha were generally less susceptible to entry and infection by EBOV. This decrease in infection was associated with a decrease in cathepsin B activity. Thus, cathepsin L plays a minimal, if any, role in EBOV infection in human DCs. The inflammatory cytokine TNFalpha modulates cathepsin B activity and affects EBOV entry into and infection of human DCs.

A filovirus-unique region of Ebola virus nucleoprotein confers aberrant migration and mediates its incorporation into virions.

The Ebola virus nucleoprotein (NP) is an essential component of the nucleocapsid, required for filovirus particle formation and replication. Together with virion protein 35 (VP35) and VP24, this gene product gives rise to the filamentous nucleocapsid within transfected cells. Ebola virus NP migrates aberrantly, with an apparent molecular mass of 115 kDa, although it is predicted to encode an approximately 85-kDa protein. In this report, we show that two domains of this protein determine this aberrant migration and that this region mediates its incorporation into virions. These regions, amino acids 439 to 492 and amino acids 589 to 739, alter the mobility of Ebola virus NP by sodium dodecyl sulfate-polyacrylamide gel electrophoresis by 5 and 15 kDa, respectively, and confer similar effects on a heterologous protein, LacZ, in a position-independent fashion. Furthermore, when coexpressed with VP40, VP35, and VP24, this region mediated incorporation of NP into released viruslike particles. When fused to chimeric paramyxovirus NPs derived from measles or respiratory syncytial virus, this domain directed these proteins into the viruslike particle. The COOH-terminal NP domain comprises a conserved highly acidic region of NP with predicted disorder, distinguishing Ebola virus NPs from paramyxovirus NPs. The acidic character of this domain is likely responsible for its aberrant biochemical properties. These findings demonstrate that this region is essential for the assembly of the filamentous nucleocapsids that give rise to filoviruses.

[Electron microscopic analysis of viral assembly and budding].

Viruses show ultrastructural changes during viral assembly and budding processes in which viral genome and proteins are systemically assembled. Electron microscopy is the only way that enables us to observe such ultrastructural changes. We have investigated the mechanisms of Ebola and influenza virion formation by electron microscopy. We have elucidated the roles of each Ebola virus protein in viral assembly and budding as well as the mechanisms of genome packaging of influenza A viruses.

Ebola Virus Uptake into Polarized Cells from the Apical Surface.

Ebola virus (EBOV) causes severe hemorrhagic fever with high mortality rates. EBOV can infect many types of cells. During severe EBOV infection, polarized epithelial and endothelial cells are damaged, which promotes vascular instability and dysregulation. However, the mechanism causing these symptoms is largely unknown. Here, we studied virus infection in polarized Vero C1008 cells grown on semipermeable Transwell by using EGFP-labeled Ebola virus-like particles (VLPs). Our results showed that Ebola VLPs preferred to enter polarized Vero cells from the apical cell surface. Furthermore, we showed that the EBOV receptors TIM-1 and Axl were distributed apically, which could be responsible for mediating efficient apical viral entry. Macropinocytosis and intracellular receptor Niemann-Pick type C1 (NPC1) had no polarized distribution, although they played roles in virus entry. This study provides a new view of EBOV uptake and cell polarization, which facilitates a further understanding of EBOV infection and pathogenesis.