Orthoparamyxovirinae C Proteins Have a Common Origin and a Common Structural Organization.

The protein C is a small viral protein encoded in an overlapping frame of the P gene in the subfamily Orthoparamyxovirinae. This protein, expressed by alternative translation initiation, is a virulence factor that regulates viral transcription, replication, and production of defective interfering RNA, interferes with the host-cell innate immunity systems and supports the assembly of viral particles and budding. We expressed and purified full-length and an N-terminally truncated C protein from Tupaia paramyxovirus (TupV) C protein (genus Narmovirus). We solved the crystal structure of the C-terminal part of TupV C protein at a resolution of 2.4 Å and found that it is structurally similar to Sendai virus C protein, suggesting that despite undetectable sequence conservation, these proteins are homologous. We characterized both truncated and full-length proteins by SEC-MALLS and SEC-SAXS and described their solution structures by ensemble models. We established a mini-replicon assay for the related Nipah virus (NiV) and showed that TupV C inhibited the expression of NiV minigenome in a concentration-dependent manner as efficiently as the NiV C protein. A previous study found that the Orthoparamyxovirinae C proteins form two clusters without detectable sequence similarity, raising the question of whether they were homologous or instead had originated independently. Since TupV C and SeV C are representatives of these two clusters, our discovery that they have a similar structure indicates that all Orthoparamyxovirine C proteins are homologous. Our results also imply that, strikingly, a STAT1-binding site is encoded by exactly the same RNA region of the P/C gene across Paramyxovirinae, but in different reading frames (P or C), depending on which cluster they belong to.

Structural Dynamics of the C-terminal X Domain of Nipah and Hendra Viruses Controls the Attachment to the C-terminal Tail of the Nucleocapsid Protein.

To understand the dynamic interactions between the phosphoprotein (P) and the nucleoprotein (N) within the transcription/replication complex of the Paramyxoviridae and to decipher their roles in regulating viral multiplication, we characterized the structural properties of the C-terminal X domain (PXD) of Nipah (NiV) and Hendra virus (HeV) P protein. In crystals, isolated NiV PXD adopted a two-helix dimeric conformation, which was incompetent for binding its partners, but in complex with the C-terminal intrinsically disordered tail of the N protein (NTAIL), it folded into a canonical 3H bundle conformation. In solution, SEC-MALLS, SAXS and NMR spectroscopy experiments indicated that both NiV and HeV PXD were larger in size than expected for compact proteins of the same molecular mass and were in conformational exchange between a compact three-helix (3H) bundle and partially unfolded conformations, where helix α3 is detached from the other two. Some measurements also provided strong evidence for dimerization of NiV PXD in solution but not for HeV PXD. Ensemble modeling of experimental SAXS data and statistical-dynamical modeling reconciled all these data, yielding a model where NiV and HeV PXD exchanged between different conformations, and where NiV but not HeV PXD formed dimers. Finally, recombinant NiV comprising a chimeric P carrying HeV PXD was rescued and compared with parental NiV. Experiments carried out in cellula demonstrated that the replacement of PXD did not significantly affect the replication dynamics while caused a slight virus attenuation, suggesting a possible role of the dimerization of NiV PXD in viral replication.

High Pathogenicity of Nipah Virus from Pteropus lylei Fruit Bats, Cambodia.

We conducted an in-depth characterization of the Nipah virus (NiV) isolate previously obtained from a Pteropus lylei bat in Cambodia in 2003 (CSUR381). We performed full-genome sequencing and phylogenetic analyses and confirmed CSUR381 is part of the NiV-Malaysia genotype. In vitro studies revealed similar cell permissiveness and replication of CSUR381 (compared with 2 other NiV isolates) in both bat and human cell lines. Sequence alignments indicated conservation of the ephrin-B2 and ephrin-B3 receptor binding sites, the glycosylation site on the G attachment protein, as well as the editing site in phosphoprotein, suggesting production of nonstructural proteins V and W, known to counteract the host innate immunity. In the hamster animal model, CSUR381 induced lethal infections. Altogether, these data suggest that the Cambodia bat-derived NiV isolate has high pathogenic potential and, thus, provide insight for further studies and better risk assessment for future NiV outbreaks in Southeast Asia.

Mannoside Glycolipid Conjugates Display Antiviral Activity Against Ebola Virus.

The West African outbreak of Ebola virus (EBOV) infection during 2013-2016 highlighted the need for development of field-applicable therapeutic drugs for this infection. Here we report that mannoside glycolipid conjugates (MGCs) consisting of a trimannose head and a lipophilic chain assembled by a linker inhibit EBOV infection not only of human monocyte-derived dendritic cells and macrophages, but also of a number of susceptible cells. Analysis of the mode of action leads us to conclude that MGCs act directly on cells, notably by preventing virus endocytosis.

Implementation of Objective PASC-Derived Taxon Demarcation Criteria for Official Classification of Filoviruses.

The mononegaviral family Filoviridae has eight members assigned to three genera and seven species. Until now, genus and species demarcation were based on arbitrarily chosen filovirus genome sequence divergence values (≈50% for genera, ≈30% for species) and arbitrarily chosen phenotypic virus or virion characteristics. Here we report filovirus genome sequence-based taxon demarcation criteria using the publicly accessible PAirwise Sequencing Comparison (PASC) tool of the US National Center for Biotechnology Information (Bethesda, MD, USA). Comparison of all available filovirus genomes in GenBank using PASC revealed optimal genus demarcation at the 55-58% sequence diversity threshold range for genera and at the 23-36% sequence diversity threshold range for species. Because these thresholds do not change the current official filovirus classification, these values are now implemented as filovirus taxon demarcation criteria that may solely be used for filovirus classification in case additional data are absent. A near-complete, coding-complete, or complete filovirus genome sequence will now be required to allow official classification of any novel "filovirus." Classification of filoviruses into existing taxa or determining the need for novel taxa is now straightforward and could even become automated using a presented algorithm/flowchart rooted in RefSeq (type) sequences.

Identification of a New Ribonucleoside Inhibitor of Ebola Virus Replication.

The current outbreak of Ebola virus (EBOV) in West Africa has claimed the lives of more than 15,000 people and highlights an urgent need for therapeutics capable of preventing virus replication. In this study we screened known nucleoside analogues for their ability to interfere with EBOV replication. Among them, the cytidine analogue ß-d-N4-hydroxycytidine (NHC) demonstrated potent inhibitory activities against EBOV replication and spread at non-cytotoxic concentrations. Thus, NHC constitutes an interesting candidate for the development of a suitable drug treatment against EBOV.

Characterization of a Novel Neutralizing Monoclonal Antibody Against Ebola Virus GP.

Ebola virus is the etiological agent of a severe hemorrhagic fever with a high mortality rate. As the only protein exposed on the surface of viral particles, the spike glycoprotein GP is the unique target for neutralizing monoclonal antibodies. In this study, we demonstrate the strong neutralization capacity of the monoclonal antibody #3327 and characterize its activity. GP residues that are required for recognition and neutralization were found to be located both in the internal fusion loop and in the receptor-binding domain. Analysis of Ebola virus entry in the presence of #3327 allows us to hypothesize that this antibody binds to the virus particle before internalization and endosomal processing of GP and likely prevents the final viral fusion step. Importantly, #3327 is able to block entry of virions bearing GP that contain the Q508 escape mutation common to a number of virus-neutralizing antibodies, and therefore provides future perspectives for treatment strategies against Ebola virus infection.

RNA Editing of the GP Gene of Ebola Virus is an Important Pathogenicity Factor.

Synthesis of the surface glycoprotein GP of Ebola virus (EBOV) is dependent on transcriptional RNA editing, whereas direct expression of the GP gene results in synthesis of nonstructural secreted glycoprotein sGP. In this study, we investigate the role of RNA editing in the pathogenicity of EBOV using a guinea pig model and recombinant guinea pig-adapted EBOV containing mutations at the editing site, allowing expression of surface GP without the need for RNA editing, and also preventing synthesis of sGP. We demonstrate that the elimination of the editing site leads to EBOV attenuation in vivo, explained by lower virus spread caused by the higher virus cytotoxicity and, most likely, by an increased ability of the host defense systems to recognize and eliminate virus-infected cells. We also demonstrate that expression of sGP does not affect pathogenicity of EBOV in guinea pigs. In conclusion, data obtained indicate that downregulation of the level of surface GP expression through a mechanism of GP gene RNA editing plays an important role in the high pathogenicity of EBOV.

Shedding of Ebola Virus Surface Glycoprotein Is a Mechanism of Self-regulation of Cellular Cytotoxicity and Has a Direct Effect on Virus Infectivity.

The surface glycoprotein (GP) is responsible for Ebola virus (EBOV) attachment and membrane fusion during virus entry. Surface expression of highly glycosylated GP causes marked cytotoxicity via masking of a wide range of cellular surface molecules, including integrins. Considerable amounts of surface GP are shed from virus-infected cells in a soluble truncated form by tumor necrosis factor α-converting enzyme. In this study, the role of GP shedding was investigated using a reverse genetics approach by comparing recombinant viruses possessing amino acid substitutions at the GP shedding site. Virus with an L635V substitution showed a substantial decrease in shedding, whereas a D637V substitution resulted in a striking increase in the release of shed GP. Variations in shedding efficacy correlated with observed differences in the amounts of shed GP in the medium, GP present in virus-infected cells, and GP present on virions. An increase in shedding appeared to be associated with a reduction in viral cytotoxicity, and, vice versa, the virus that shed less was more cytotoxic. An increase in shedding also resulted in a reduction in viral infectivity, whereas a decrease in shedding efficacy enhanced viral growth characteristics in vitro. Differences in shedding efficacy and, as a result, differences in the amount of mature GP available for incorporation into budding virions did not equate to differences in overall release of viral particles. Likewise, data suggest that the resulting differences in the amount of mature GP on the cell surface led to variations in the GP content of released particles and, as a consequence, in infectivity. In conclusion, fine-tuning of the levels of EBOV GP expressed at the surface of virus-infected cells via GP shedding plays an important role in EBOV replication by orchestrating the balance between optimal virion GP content and cytotoxicity caused by GP.

Entry of Ebola Virus is an Asynchronous Process.

Ebola virus (EBOV) is responsible for a severe fever with a high mortality rate. The diverse nature of the attachment of the virus to the cell surface, the initial step of virus entry, raises questions concerning the kinetics of the virus internalization process. We investigated EBOV entry kinetics using the activity of a particular monoclonal antibody that neutralizes virus infectivity. We demonstrate that inoculation of cells with EBOV results in an asynchronous entry process, as revealed by the ability of the virus to remain in a cell-bound state for an extended period of time before it is internalized.

Ebola Virus GP Gene Polyadenylation Versus RNA Editing.

Synthesis of Ebola virus (EBOV) surface glycoprotein (GP) is dependent on transcriptional RNA editing. Northern blot analysis of EBOV-infected cells using GP-gene-specific probes reveals that, in addition to full-length GP messenger RNAs (mRNAs), a shorter RNA is also synthesized, representing >40% of the total amount of GP mRNA. Sequence analysis demonstrates that this RNA is a truncated version of the full-length GP mRNA that is polyadenylated at the editing site and thus lacks a stop codon. An absence of detectable levels of protein synthesis in cellulo is consistent with the existence of tight regulation of the translation of such mRNA. However, nonstop GP mRNA was shown to be only slightly less stable than the same mRNA containing a stop codon, against the general belief in nonstop decay mechanisms aimed at detecting and destroying mRNAs lacking a stop codon. In conclusion, we demonstrate that the editing site indeed serves as a cryptic transcription termination/polyadenylation site, which rarely also functions to edit GP mRNA for expression of surface GP. This new data suggest that the downregulation of surface GP expression is even more dramatic than previously thought, reinforcing the importance of the GP gene editing site for EBOV replication and pathogenicity.

Shed GP of Ebola virus triggers immune activation and increased vascular permeability.

During Ebola virus (EBOV) infection a significant amount of surface glycoprotein GP is shed from infected cells in a soluble form due to cleavage by cellular metalloprotease TACE. Shed GP and non-structural secreted glycoprotein sGP, both expressed from the same GP gene, have been detected in the blood of human patients and experimentally infected animals. In this study we demonstrate that shed GP could play a particular role during EBOV infection. In effect it binds and activates non-infected dendritic cells and macrophages inducing the secretion of pro- and anti-inflammatory cytokines (TNFα, IL1ß, IL6, IL8, IL12p40, and IL1-RA, IL10). Activation of these cells by shed GP correlates with the increase in surface expression of co-stimulatory molecules CD40, CD80, CD83 and CD86. Contrary to shed GP, secreted sGP activates neither DC nor macrophages while it could bind DCs. In this study, we show that shed GP activity is likely mediated through cellular toll-like receptor 4 (TLR4) and is dependent on GP glycosylation. Treatment of cells with anti-TLR4 antibody completely abolishes shed GP-induced activation of cells. We also demonstrate that shed GP activity is negated upon addition of mannose-binding sera lectin MBL, a molecule known to interact with sugar arrays present on the surface of different microorganisms. Furthermore, we highlight the ability of shed GP to affect endothelial cell function both directly and indirectly, demonstrating the interplay between shed GP, systemic cytokine release and increased vascular permeability. In conclusion, shed GP released from virus-infected cells could activate non-infected DCs and macrophages causing the massive release of pro- and anti-inflammatory cytokines and effect vascular permeability. These activities could be at the heart of the excessive and dysregulated inflammatory host reactions to infection and thus contribute to high virus pathogenicity.

Marburgvirus hijacks nrf2-dependent pathway by targeting nrf2-negative regulator keap1.

Marburg virus (MARV) has a high fatality rate in humans, causing hemorrhagic fever characterized by massive viral replication and dysregulated inflammation. Here, we demonstrate that VP24 of MARV binds Kelch-like ECH-associated protein 1 (Keap1), a negative regulator of nuclear transcription factor erythroid-derived 2 (Nrf2). Binding of VP24 to Keap1 Kelch domain releases Nrf2 from Keap1-mediated inhibition promoting persistent activation of a panoply of cytoprotective genes implicated in cellular responses to oxidative stress and regulation of inflammatory responses. Increased expression of Nrf2-dependent genes was demonstrated both during MARV infection and upon ectopic expression of MARV VP24. We also show that Nrf2-deficient mice can control MARV infection when compared to lethal infection in wild-type animals, indicating that Nrf2 is critical for MARV infection. We conclude that VP24-driven activation of the Nrf2-dependent pathway is likely to contribute to dysregulation of host antiviral inflammatory responses and that it ensures survival of MARV-infected cells despite these responses.

Discussions and decisions of the 2012­2014 International Committee on Taxonomy of Viruses (ICTV) Filoviridae Study Group, January 2012­June 2013.

The International Committee on Taxonomy of Viruses (ICTV) Filoviridae Study Group prepares proposals on the classification and nomenclature of filoviruses to reflect current knowledge or to correct disagreements with the International Code of Virus Classification and Nomenclature (ICVCN). In recent years, filovirus taxonomy has been corrected and updated, but parts of it remain controversial, and several topics remain to be debated. This article summarizes the decisions and discussion of the currently acting ICTV Filoviridae Study Group since its inauguration in January 2012.

Virus nomenclature below the species level: a standardized nomenclature for filovirus strains and variants rescued from cDNA.

Specific alterations (mutations, deletions, insertions) of virus genomes are crucial for the functional characterization of their regulatory elements and their expression products, as well as a prerequisite for the creation of attenuated viruses that could serve as vaccine candidates. Virus genome tailoring can be performed either by using traditionally cloned genomes as starting materials, followed by site-directed mutagenesis, or by de novo synthesis of modified virus genomes or parts thereof. A systematic nomenclature for such recombinant viruses is necessary to set them apart from wild-type and laboratory-adapted viruses, and to improve communication and collaborations among researchers who may want to use recombinant viruses or create novel viruses based on them. A large group of filovirus experts has recently proposed nomenclatures for natural and laboratory animal-adapted filoviruses that aim to simplify the retrieval of sequence data from electronic databases. Here, this work is extended to include nomenclature for filoviruses obtained in the laboratory via reverse genetics systems. The previously developed template for natural filovirus genetic variant naming, (/)///-, is retained, but we propose to adapt the type of information added to each field for cDNA clone-derived filoviruses. For instance, the full-length designation of an Ebola virus Kikwit variant rescued from a plasmid developed at the US Centers for Disease Control and Prevention could be akin to "Ebola virus H.sapiens-rec/COD/1995/Kikwit-abc1" (with the suffix "rec" identifying the recombinant nature of the virus and "abc1" being a placeholder for any meaningful isolate designator). Such a full-length designation should be used in databases and the methods section of publications. Shortened designations (such as "EBOV H.sap/COD/95/Kik-abc1") and abbreviations (such as "EBOV/Kik-abc1") could be used in the remainder of the text, depending on how critical it is to convey information contained in the full-length name. "EBOV" would suffice if only one EBOV strain/variant/isolate is addressed.

Nonstructural Nipah virus C protein regulates both the early host proinflammatory response and viral virulence.

Nipah virus (NiV) is a highly pathogenic, negative-strand RNA paramyxovirus that has recently emerged from flying foxes to cause serious human disease. We have analyzed the role of the nonstructural NiV C protein in viral immunopathogenesis using recombinant virus lacking the expression of NiV C (NiVΔC). While wild-type NiV was highly pathogenic in the hamster animal model, NiVΔC was strongly attenuated. Replication of NiVΔC was followed by the production of NiV-specific antibodies and associated with higher recruitment of inflammatory cells and less intensive histopathological lesions in different organs than in wild-type-NiV-infected animals. To analyze the molecular basis of NiVΔC attenuation, we studied early changes in gene expression in infected primary human endothelial cells, a major cellular target of NiV infection. The transcriptomic approach revealed the striking difference between wild-type and mutant NiV in the expression of genes involved in immunity, with the particularly interesting differential patterns of proinflammatory cytokines. Compared to wild-type virus, NiVΔC induced increased expression of interleukin 1 beta (IL-1ß), IL-8, CXCL2, CXCL3, CXCL6, CCL20, and beta interferon. Furthermore, the expression of NiV C in stably transfected cells decreased the production of the same panel of cytokines, revealing a role of the C protein in the regulation of cytokine balance. Together, these results suggest that NiV C regulates expression of proinflammatory cytokines, therefore providing a signal responsible for the coordination of leukocyte recruitment and the chemokine-induced immune response and controlling the lethal outcome of the infection.

VP24 is a molecular determinant of Ebola virus virulence in guinea pigs.

In sharp contrast to human and nonhuman primates, guinea pigs and some other mammals resist Ebola virus (EBOV) replication and do not develop illness upon virus inoculation. However, serial passaging of EBOV in guinea pigs results in a selection of variants with high pathogenicity. In this report, using a reverse genetics approach, we demonstrate that this dramatic increase in EBOV pathogenicity is associated with amino acid substitutions in the structural protein VP24. We show that although replication of recombinant EBOV carrying wild-type VP24 is impaired in primary peritoneal guinea pig macrophages and in the liver of infected animals, the substitutions in VP24 allow EBOV to replicate in guinea pig macrophages and spread in the liver of infected animals. Furthermore, we demonstrate that both VP24/wild type and the guinea pig-adapted VP24/8mc are similar in their ability to block expression of interferon-induced host genes, suggesting that the increase in EBOV virulence for guinea pigs is not associated with VP24 interferon antagonist function. This study sheds light on the mechanism of resistance to EBOV infection and highlights the critical role of VP24 in EBOV pathogenesis.

Unconventional secretion of Ebola virus matrix protein VP40.

The Ebola virus matrix protein VP40 plays an essential role in virus assembly and budding. In this study we reveal that transient VP40 expression results in the release into the culture medium of substantial amounts of soluble monomeric VP40 in addition to the release of virus-like particles containing an oligomeric form of this protein as previously described. We show that VP40 secretion is endoplasmic reticulum/Golgi-independent and is not associated with cell death. Soluble VP40 was observed during Ebola virus infection of cells and was also found in the serum of virus-infected animals albeit in lower amounts. Unconventional secretion of VP40 may therefore play a role in Ebola virus pathogenicity.