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The Ebola virus (EBOV) envelope glycoprotein (GP) is a membrane fusion machine required for virus entry into cells. Following endocytosis of EBOV, the GP1 domain is cleaved by cellular cathepsins in acidic endosomes, removing the glycan cap and exposing a binding site for the Niemann-Pick C1 (NPC1) receptor. NPC1 binding to cleaved GP1 is required for entry. How this interaction translates to GP2 domain-mediated fusion of viral and endosomal membranes is not known. Here, using a bulk fluorescence dequenching assay and single-molecule Förster resonance energy transfer (smFRET)-imaging, we found that acidic pH, Ca2+, and NPC1 binding synergistically induce conformational changes in GP2 and permit virus-liposome lipid mixing. Acidic pH and Ca2+ shifted the GP2 conformational equilibrium in favor of an intermediate state primed for NPC1 binding. Glycan cap cleavage on GP1 enabled GP2 to transition from a reversible intermediate to an irreversible conformation, suggestive of the postfusion 6-helix bundle; NPC1 binding further promoted transition to the irreversible conformation. Thus, the glycan cap of GP1 may allosterically protect against inactivation of EBOV by premature triggering of GP2.

Significance In 2014, Ebola virus became a household term. The ongoing outbreak in West Africa is the largest Ebola virus outbreak ever recorded, with over 20,000 cases and over 8,000 deaths to date. Very little is known about the human cellular immune response to Ebola virus infection, and this lack of knowledge has hindered development of effective therapies and vaccines. In this study, we characterize the human immune response to Ebola virus infection in four patients. We define the kinetics of T- and B-cell activation, and determine which viral proteins are targets of the Ebola virus-specific T-cell response in humans. Four Ebola patients received care at Emory University Hospital, presenting a unique opportunity to examine the cellular immune responses during acute Ebola virus infection. We found striking activation of both B and T cells in all four patients. Plasmablast frequencies were 10–50% of B cells, compared with less than 1% in healthy individuals. Many of these proliferating plasmablasts were IgG-positive, and this finding coincided with the presence of Ebola virus-specific IgG in the serum. Activated CD4 T cells ranged from 5 to 30%, compared with 1–2% in healthy controls. The most pronounced responses were seen in CD8 T cells, with over 50% of the CD8 T cells expressing markers of activation and proliferation. Taken together, these results suggest that all four patients developed robust immune responses during the acute phase of Ebola virus infection, a finding that would not have been predicted based on our current assumptions about the highly immunosuppressive nature of Ebola virus. Also, quite surprisingly, we found sustained immune activation after the virus was cleared from the plasma, observed most strikingly in the persistence of activated CD8 T cells, even 1 mo after the patients’ discharge from the hospital. These results suggest continued antigen stimulation after resolution of the disease. From these convalescent time points, we identified CD4 and CD8 T-cell responses to several Ebola virus proteins, most notably the viral nucleoprotein. Knowledge of the viral proteins targeted by T cells during natural infection should be useful in designing vaccines against Ebola virus.

The Ebola virus, a member of the Filoviridae family, causes severe hemorrhagic fever in humans. Filamentous virions contain a helical nucleocapsid responsible for genome transcription, replication, and packaging into progeny virions. The nucleocapsid consists of a helical nucleoprotein (NP)–viral genomic RNA complex forming the core structure, to which VP24 and VP35 bind externally. Two NPs, each paired with a VP24 molecule, constitute a repeating unit. However, the detailed nucleocapsid structure remains unclear. Here, we determine the nucleocapsid-like structure within virus-like particles at 4.6 Å resolution using single-particle cryo-electron microscopy. Mutational analysis identifies specific interactions between the two NPs and two VP24s and demonstrates that each of the two VP24s in different orientations distinctively regulates nucleocapsid assembly, viral RNA synthesis, intracellular transport of the nucleocapsid, and infectious virion production. Our findings highlight the sophisticated mechanisms underlying the assembly and functional regulation of the nucleocapsid and provide insights into antiviral development. Here, the authors use cryo-electron microscopy to structurally characterise the Ebola virus nucleocapsid within virus-like particles, uncovering how specific interactions regulate viral genome synthesis, assembly, and infectious particle production.

Global health is threatened by emerging viral infections, which largely lack effective vaccines or therapies. Targeting host pathways that are exploited by multiple viruses could offer broad-spectrum solutions. We previously reported that AAK1 and GAK, kinase regulators of the host adaptor proteins AP1 and AP2, are essential for hepatitis C virus (HCV) infection, but the underlying mechanism and relevance to other viruses or in vivo infections remained unknown. Here, we have discovered that AP1 and AP2 cotraffic with HCV particles in live cells. Moreover, we found that multiple viruses, including dengue and Ebola, exploit AAK1 and GAK during entry and infectious virus production. In cultured cells, treatment with sunitinib and erlotinib, approved anticancer drugs that inhibit AAK1 or GAK activity, or with more selective compounds inhibited intracellular trafficking of HCV and multiple unrelated RNA viruses with a high barrier to resistance. In murine models of dengue and Ebola infection, sunitinib/erlotinib combination protected against morbidity and mortality. We validated sunitinib- and erlotinib-mediated inhibition of AAK1 and GAK activity as an important mechanism of antiviral action. Additionally, we revealed potential roles for additional kinase targets. These findings advance our understanding of virus-host interactions and establish a proof of principle for a repurposed, host-targeted approach to combat emerging viruses.

Filovirus-host interactions play important roles in all stages of the virus lifecycle. Here, we identify LATS1/2 kinases and YAP, key components of the Hippo pathway, as critical regulators of EBOV transcription and egress. Specifically, we find that when YAP is phosphorylated by LATS1/2, it localizes to the cytoplasm (Hippo “ON”) where it sequesters VP40 to prevent egress. In contrast, when the Hippo pathway is “OFF”, unphosphorylated YAP translocates to the nucleus where it transcriptionally activates host genes and promotes viral egress. Our data reveal that LATS2 indirectly modulates filoviral VP40-mediated egress through phosphorylation of AMOTp130, a positive regulator of viral egress, but more surprisingly that LATS1/2 kinases directly modulate EBOV transcription by phosphorylating VP30, an essential regulator of viral transcription. In sum, our findings highlight the potential to exploit the Hippo pathway/filovirus axis for the development of host-oriented countermeasures targeting EBOV and related filoviruses. Here, the authors identify the host proteins LATS1/2 and YAP of the Hippo pathway to regulate Ebola virus transcription and virus production/egress. They show that YAP activity is important for virus particle production and release, while LATS1/2 are critical for EBOV mRNA synthesis and viral protein expression.

The interaction of host and Ebola virus (EBOV) proteins is required for establishing infection. In this study, we use proximity-dependent biotinylation to identify cellular proteins that bind to EBOV proteins encoded by six of the seven viral genes. Hits are computationally mapped onto a human protein-protein interactome and annotated with viral proteins, confirming known EBOV-host protein interactions and revealing previously undescribed interactions and processes. This approach efficiently arranges proteins into functional complexes associated with single viral proteins. Focused characterization of interactions between EBOV VP35 and the mRNA decapping complex shows that VP35 binds the scaffold protein EDC4 through the C-terminal subdomain, with both proteins colocalizing in EBOV-infected cells. siRNA depletion of EDC4, DCP2, and EDC3 reduces virus replication by inhibiting early viral RNA synthesis. Overall, the analytical approach efficiently identifies EBOV protein interactions with cellular protein complexes, providing a deeper understanding of replication mechanisms for therapeutic intervention. BioID detection of Ebola virus–host interactions when computationally integrated with existing host protein-protein networks, revealed functional cell protein complexes supporting infection, including the RNA decapping complex.

The Ebola virus (EBOV) has emerged as a significant global health concern, notably during the 2013–2016 outbreak in West Africa. Despite the clinical approval of two EBOV antibody drugs, there is an urgent need for more diverse and effective antiviral drugs, along with comprehensive understanding of viral-host interactions. In this study, we harnessed a biologically contained EBOVΔVP30-EGFP cell culture model which could recapitulate the entire viral life cycle, to conduct a genome-wide CRISPR/Cas9 screen. Through this, we identified PIK3C3 (phosphatidylinositide 3-kinase) and SLC39A9 (zinc transporter) as crucial host factors for EBOV infection. Genetic depletion of SLC39A9 and PIK3C3 lead to reduction of EBOV entry, but not impact viral genome replication, suggesting that SLC39A9 and PIK3C3 act as entry factors, facilitating viral entry into host cells. Moreover, PIK3C3 kinase activity is indispensable for the internalization of EBOV virions, presumably through the regulation of endocytic and autophagic membrane traffic, which has been previously recognized as essential for EBOV internalization. Notably, our study demonstrated that PIK3C3 kinase inhibitor could effectively block EBOV infection, underscoring PIK3C3 as a promising drug target. Furthermore, biochemical analysis showed that recombinant SLC39A9 protein could directly bind viral GP protein, which further promotes the interaction of viral GP protein with cellular receptor NPC1. These findings suggests that SLC39A9 plays dual roles in EBOV entry. Initially, it serves as an attachment factor during the early entry phase by engaging with the viral GP protein. Subsequently, SLC39A9 functions an adaptor protein, facilitating the interaction between virions and the NPC1 receptor during the late entry phase, prior to cathepsin cleavage on the viral GP. In summary, this study offers novel insights into virus-host interactions, contributing valuable information for the development of new therapies against EBOV infection.

Significance Ebola virus causes lethal hemorrhagic fever, and the current 2014 outbreak in western Africa is the largest on record to date. No vaccines or therapeutics are yet approved for human use. Therapeutic antibody cocktails, however, have shown efficacy against otherwise lethal Ebola virus infection and show significant promise for eventual human use. Here we provide structures of every mAb in the ZMapp cocktail, as well as additional antibodies from the MB-003 and ZMAb cocktails from which ZMapp was derived, each in complex with the Ebola glycoprotein. The set of structures illustrates sites of vulnerability of Ebola virus, and importantly, provides a roadmap to determine their mechanism of protection and for ongoing selection and improvement of immunotherapeutic cocktails against the filoviruses. Ebola virus (EBOV) and related filoviruses cause severe hemorrhagic fever, with up to 90% lethality, and no treatments are approved for human use. Multiple recent outbreaks of EBOV and the likelihood of future human exposure highlight the need for pre- and postexposure treatments. Monoclonal antibody (mAb) cocktails are particularly attractive candidates due to their proven postexposure efficacy in nonhuman primate models of EBOV infection. Two candidate cocktails, MB-003 and ZMAb, have been extensively evaluated in both in vitro and in vivo studies. Recently, these two therapeutics have been combined into a new cocktail named ZMapp, which showed increased efficacy and has been given compassionately to some human patients. Epitope information and mechanism of action are currently unknown for most of the component mAbs. Here we provide single-particle EM reconstructions of every mAb in the ZMapp cocktail, as well as additional antibodies from MB-003 and ZMAb. Our results illuminate key and recurring sites of vulnerability on the EBOV glycoprotein and provide a structural rationale for the efficacy of ZMapp. Interestingly, two of its components recognize overlapping epitopes and compete with each other for binding. Going forward, this work now provides a basis for strategic selection of next-generation antibody cocktails against Ebola and related viruses and a model for predicting the impact of ZMapp on potential escape mutations in ongoing or future Ebola outbreaks.

Most nonsegmented negative strand (NNS) RNA virus genomes have complementary 3′ and 5′ terminal nucleotides because the promoters at the 3′ ends of the genomes and antigenomes are almost identical to each other. However, according to published sequences, both ends of ebolavirus genomes show a high degree of variability, and the 3′ and 5′ terminal nucleotides are not complementary. If correct, this would distinguish the ebolaviruses from other NNS RNA viruses. Therefore, we investigated the terminal genomic and antigenomic nucleotides of three different ebolavirus species, Ebola (EBOV), Sudan, and Reston viruses. Whereas the 5′ ends of ebolavirus RNAs are highly conserved with the sequence ACAGG-5′, the 3′ termini are variable and are typically 3′-GCCUGU, ACCUGU, or CCUGU. A small fraction of analyzed RNAs had extended 3′ ends. The majority of 3′ terminal sequences are consistent with a mechanism of nucleotide addition by hairpin formation and back-priming. Using single-round replicating EBOV minigenomes, we investigated the effect of the 3′ terminal nucleotide on viral replication and found that the EBOV polymerase initiates replication opposite the 3′-CCUGU motif regardless of the identity of the 3′ terminal nucleotide(s) and of the position of this motif relative to the 3′ end. Deletion or mutation of the first residue of the 3′-CCUGU motif completely abolished replication initiation, suggesting a crucial role of this nucleotide in directing initiation. Together, our data show that ebolaviruses have evolved a unique replication strategy among NNS RNA viruses resulting in 3′ overhangs. This could be a mechanism to avoid antiviral recognition.

The West Africa Ebola outbreak was the largest outbreak ever recorded, with over 28,000 reported infections; this devastating epidemic emphasized the need to understand the mechanisms to counteract virus infection. Here, we screen a library of nearly 400 interferon-stimulated genes (ISGs) against a biologically contained Ebola virus and identify several ISGs not previously known to affect Ebola virus infection. Overexpression of the top ten ISGs attenuates virus titers by up to 1000-fold. Mechanistic studies demonstrate that three ISGs interfere with virus entry, six affect viral transcription/replication, and two inhibit virion formation and budding. A comprehensive study of one ISG (CCDC92) that shows anti-Ebola activity in our screen reveals that CCDC92 can inhibit viral transcription and the formation of complete virions via an interaction with the viral protein NP. Our findings provide insights into Ebola virus infection that could be exploited for the development of therapeutics against this virus. Here, Kuroda et al. screen a library of nearly 400 interferon-stimulated genes (ISGs) and identify several ISGs that inhibit Ebola virus entry, viral transcription/replication, or virion formation. The study provides insights into interactions between Ebola and the host cells.