Ebola Virus VP35 Research Articles

Identification of Prospective Ebola Virus VP35 and VP40 Protein Inhibitors from Myxobacterial Natural Products.

The Ebola virus (EBOV) is a lethal pathogen causing hemorrhagic fever syndrome which remains a global health challenge. In the EBOV, two multifunctional proteins, VP35 and VP40, have significant roles in replication, virion assembly, and budding from the cell and have been identified as druggable targets. In this study, we employed in silico methods comprising molecular docking, molecular dynamic simulations, and pharmacological properties to identify prospective drugs for inhibiting VP35 and VP40 proteins from the myxobacterial bioactive natural product repertoire. Cystobactamid 934-2, Cystobactamid 919-1, and Cittilin A bound firmly to VP35. Meanwhile, 2-Hydroxysorangiadenosine, Enhypyrazinone B, and Sorangiadenosine showed strong binding to the matrix protein VP40. Molecular dynamic simulations revealed that, among these compounds, Cystobactamid 919-1 and 2-Hydroxysorangiadenosine had stable interactions with their respective targets. Similarly, molecular mechanics Poisson-Boltzmann surface area (MMPBSA) calculations indicated close-fitting receptor binding with VP35 or VP40. These two compounds also exhibited good pharmacological properties. In conclusion, we identified Cystobactamid 919-1 and 2-Hydroxysorangiadenosine as potential ligands for EBOV that target VP35 and VP40 proteins. These findings signify an essential step in vitro and in vivo to validate their potential for EBOV inhibition.

Investigating the Antiviral Properties of Nyctanthes arbor-tristis Linn against the Ebola, SARS-CoV-2, Nipah, and Chikungunya Viruses: A Computational Simulation Study.

Background: The hunt for naturally occurring antiviral compounds to combat viral infection was expedited when COVID-19 and Ebola spread rapidly. Phytochemicals from Nyctanthes arbor-tristis Linn were evaluated as significant inhibitors of these viruses. Methods: Computational tools and techniques were used to assess the binding pattern of phytochemicals from Nyctanthes arbor-tristis Linn to Ebola virus VP35, SARS-CoV-2 protease, Nipah virus glycoprotein, and chikungunya virus. Results: Virtual screening and AutoDock analysis revealed that arborside-C, beta amyrin, and beta-sitosterol exhibited a substantial binding affinity for specific viral targets. The arborside-C and beta-sitosterol molecules were shown to have binding energies of -8.65 and -9.11 kcal/mol, respectively, when interacting with the major protease. Simultaneously, the medication remdesivir exhibited a control value of -6.18 kcal/mol. The measured affinity of phytochemicals for the other investigated targets was -7.52 for beta-amyrin against Ebola and -6.33 kcal/mol for nicotiflorin against Nipah virus targets. Additional molecular dynamics simulation (MDS) conducted on the molecules with significant antiviral potential, specifically the beta-amyrin-VP35 complex showing a stable RMSD pattern, yielded encouraging outcomes. Conclusions: Arborside-C, beta-sitosterol, beta-amyrin, and nicotiflorin could be established as excellent natural antiviral compounds derived from Nyctanthes arbor-tristis Linn. The virus-suppressing phytochemicals in this plant make it a compelling target for both in vitro and in vivo research in the future.

Ebola virus VP35 interacts non-covalently with ubiquitin chains to promote viral replication.

Ebolavirus (EBOV) belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans. EBOV replication requires the activity of the viral polymerase complex, which includes the cofactor and Interferon antagonist VP35. We previously showed that the covalent ubiquitination of VP35 promotes virus replication by regulating interactions with the polymerase complex. In addition, VP35 can also interact non-covalently with ubiquitin (Ub); however, the function of this interaction is unknown. Here, we report that VP35 interacts with free (unanchored) K63-linked polyUb chains. Ectopic expression of Isopeptidase T (USP5), which is known to degrade unanchored polyUb chains, reduced VP35 association with Ub and correlated with diminished polymerase activity in a minigenome assay. Using computational methods, we modeled the VP35-Ub non-covalent interacting complex, identified the VP35-Ub interacting surface, and tested mutations to validate the interface. Docking simulations identified chemical compounds that can block VP35-Ub interactions leading to reduced viral polymerase activity. Treatment with the compounds reduced replication of infectious EBOV in cells and in vivo in a mouse model. In conclusion, we identified a novel role of unanchored polyUb in regulating Ebola virus polymerase function and discovered compounds that have promising anti-Ebola virus activity.

Ebola virus sequesters IRF3 in viral inclusion bodies to evade host antiviral immunity.

Viral inclusion bodies (IBs) commonly form during the replication of Ebola virus (EBOV) in infected cells, but their role in viral immune evasion has rarely been explored. Here, we found that interferon regulatory factor 3 (IRF3), but not TANK-binding kinase 1 (TBK1) or IκB kinase epsilon (IKKε), was recruited and sequestered in viral IBs when the cells were infected by EBOV transcription- and replication-competent virus-like particles (trVLPs). Nucleoprotein/virion protein 35 (VP35)-induced IBs formation was critical for IRF3 recruitment and sequestration, probably through interaction with STING. Consequently, the association of TBK1 and IRF3, which plays a vital role in type I interferon (IFN-I) induction, was blocked by EBOV trVLPs infection. Additionally, IRF3 phosphorylation and nuclear translocation induced by Sendai virus or poly(I:C) stimulation were suppressed by EBOV trVLPs. Furthermore, downregulation of STING significantly attenuated VP35-induced IRF3 accumulation in IBs. Coexpression of the viral proteins by which IB-like structures formed was much more potent in antagonizing IFN-I than expression of the IFN-I antagonist VP35 alone. These results suggested a novel immune evasion mechanism by which EBOV evades host innate immunity.

In-silico identification of novel natural drug leads against the Ebola virus VP40 protein: A promising approach for developing new antiviral therapeutics

Ebola, one of the deadliest known infectious diseases, was the root of epidemics in Western Africa from 2013 to 2016. Like other deadly viruses in the family Filoviridae with a high fatality rate, this virus also causes hemorrhagic fever. As a result, the Ebola virus (EBOV) represents a threat to global health. Since there are currently no effective treatments for EBOV infections, this study aims to identify potential natural drug candidates that may block the EBOV VP40 to prevent Ebola infections. The compounds were analyzed using ADMET, molecular docking, post-docking MM-GBSA, and molecular dynamics (MD) simulations. ADMET analysis identified 187 out of 452 compounds. According to molecular docking, the best three compounds were chosen from 187 compounds for further study with binding affinity −8.469, −8.175, and −7.918 kcal/mol for CID_21721878 (Kushenol L), CID_133561472 (2-[2,4-dihydroxy-5-[2-(2-hydroxypropan-2-yl)-5-methylphenyl]phenyl]-5,7-dihydroxy-2,3-dihydrochromen-4-one), and Amb_29844215 (Cathayanon I), respectively. The lead three compounds coordinated with the protein's shared amino acid residues (ILE216, PRO286, VAL287, LEU288, LEU213, PRO146, and VAL100) during molecular docking with hydrophobic bonds. Then, molecular docking results were validated using post-docking MM-GBSA of those three compounds are Kushenol L, 2-[2,4-dihydroxy-5-[2-(2-hydroxypropan-2-yl)-5-methylphenyl]phenyl]-5,7-dihydroxy-2,3-dihydrochromen-4-one and Cathayanon I had negative binding free energies of −69.53, −52.85, and −59.74 kcal/mol, respectively. All the selected compounds exhibit favorable pharmacokinetic (Pk) and toxicological properties, supporting their safety and efficacy. These three compounds were further evaluated using MD simulation, confirming the compounds' binding stability to the desired protein. After MD simulation, PCA, and DCCM analysis were performed. From all of these can suggest the best compound which is CID_21721878 (Kushenol L), which is a phytochemical derived from Cannabis sativa, another one is CID_13356472 which comes after Kushenol L, which is also a phytochemical found in several plants: Maclura tricuspidate, Euchresta japonica, Maclura pomifera. Both compounds can potentially inhibit EBOV VP40 protein activity.

Identification of potential inhibitors targeting Ebola virus VP35 protein: a computational strategy

Ebola virus (EBOV) poses a severe threat as a highly infectious pathogen, causing devastating hemorrhagic fever in both humans and animals. The EBOV virus VP35 protein plays a crucial role in viral replication and exhibits the ability to suppress the host interferon cascade, leading to immune system depletion. As a potential drug target, VP35 protein inhibition holds promise for combating EBOV. To discover new drug candidates, we employed a computer-aided drug design approach, focusing on compounds capable of inhibiting VP35 protein replication. In this connection, a pharmacophore model was generated using molecular interactions between the VP35 protein and its inhibitor. ZINC and Cambridge database were screened using validated pharmacophore model. Further the compounds were filtered based on Lipinski’s rule of five and subjected to MD simulation and relative binding free energy calculation. Six compounds manifest a significant docking score and strong binding interaction towards VP35 protein. MD simulations further confirmed the remarkable stability of these six complexes. Relative binding free energy calculations also showed significant ΔG value in the range of −132.3 and −49.3 kcal/mol. This study paves the way for further optimization of these compounds as potential inhibitors of VP35, facilitating subsequent experimental in vitro studies. Communicated by Ramaswamy H. Sarma

Ebola virus VP35 perturbs type I interferon signaling to facilitate viral replication

As one of the deadliest viruses, Ebola virus (EBOV) causes lethal hemorrhagic fevers in humans and nonhuman primates. The suppression of innate immunity leads to robust systemic virus replication of EBOV, leading to enhanced transmission. However, the mechanism of EBOV-host interaction is not fully understood. Here, we identified multiple dysregulated genes in early stage of EBOV infection through transcriptomic analysis, which are highly clustered to Jak-STAT signaling. EBOV VP35 and VP30 were found to inhibit type I interferon (IFN) signaling. Moreover, exogenous expression of VP35 blocks the phosphorylation of endogenous STAT1, and suppresses nuclear translocation of STAT1. Using serial truncated mutations of VP35, N-terminal 1–220 amino acid residues of VP35 were identified to be essential for blocking on type I IFN signaling. Remarkably, VP35 of EBOV suppresses type I IFN signaling more efficiently than those of Bundibugyo virus (BDBV) and Marburg virus (MARV), resulting in stable replication to facilitate the pathogenesis. Altogether, this study enriches understanding on EBOV evasion of innate immune response, and provides insights into the interplay between filoviruses and host.

Ebola Virus Infection Induces HCAR2 Expression Leading to Cell Death.

Ebola virus (EBOV) induces cell death not only in infected permissive cells but also in nonpermissive, bystander cells by employing different mechanisms. Hydroxycarboxylic acid receptor 2 (HCAR2) has been reported to be involved in apoptotic cell death. We previously reported an increase in the expression of HCAR2-specific mRNA in EBOV-infected individuals with fatal outcomes. Here, we report that infection with an EBOV lacking the VP30 gene (EBOVΔVP30) results in the upregulation of HCAR2 mRNA expression in human hepatocyte Huh7.0 cells stably expressing VP30. Transient overexpression of HCAR2 reduced the viability of Huh7.0 cells and human embryonic kidney cells. Phosphatidylserine externalization and cell membrane permeabilization by HCAR2 overexpression was also observed. Interestingly, coexpression of HCAR2 with EBOV VP40 further reduced cell viability in transfected cells compared to HCAR2 coexpression with other viral proteins. Our data suggest that HCAR2 may contribute to EBOV-induced cell death.

The C-terminus of Sudan ebolavirus VP40 contains a functionally important CXnC motif, a target for redox modifications

The Ebola virus matrix protein VP40 mediates viral budding and negatively regulates viral RNA synthesis. The mechanisms by which these two functions are exerted and regulated are unknown. Using a high-resolution crystal structure of Sudan ebolavirus (SUDV) VP40, we show here that two cysteines in the flexible C-terminal arm of VP40 form a stabilizing disulfide bridge. Notably, the two cysteines are targets of posttranslational redox modifications and interact directly with the host`s thioredoxin system. Mutation of the cysteines impaired the budding function of VP40 and relaxed its inhibitory role for viral RNA synthesis. In line with these results, the growth of recombinant Ebola viruses carrying cysteine mutations was impaired and the released viral particles were elongated. Our results revealed the exact positions of the cysteines in the C-terminal arm of SUDV VP40. The cysteines and/or their redox status are critically involved in the differential regulation of viral budding and viral RNA synthesis.

Ebola virus infection induces a delayed type I IFN response in bystander cells and the shutdown of key liver genes in human iPSC-derived hepatocytes.

SummaryLiver damage and an exacerbated inflammatory response are hallmarks of Ebola virus (EBOV) infection. Little is known about the intrinsic response to infection in human hepatocytes and their contribution to inflammation. Here, we present an induced pluripotent stem cell (iPSC)-derived hepatocyte-like cell (HLC) platform to define the hepato-intrinsic response to EBOV infection. We used this platform to show robust EBOV infection, with characteristic ultrastructural changes and evidence for viral replication. Transcriptomics analysis revealed a delayed response with minimal early transcriptomic changes, followed by a general downregulation of hepatic function and upregulation of interferon signaling, providing a potential mechanism by which hepatocytes participate in disease severity and liver damage. Using RNA-fluorescence in situ hybridization (FISH), we showed that IFNB1 and CXCL10 were mainly expressed in non-infected bystander cells. We did not observe an inflammatory signature during infection. In conclusion, iPSC-HLCs are an immune competent platform to study responses to EBOV infection.

Ubiquitination of Ebola virus VP35 at lysine 309 regulates viral transcription and assembly.

Ebola virus (EBOV) VP35 is a polyfunctional protein involved in viral genome packaging, viral polymerase function, and host immune antagonism. The mechanisms regulating VP35's engagement in different functions are not well-understood. We previously showed that the host E3 ubiquitin ligase TRIM6 ubiquitinates VP35 at lysine 309 (K309) to facilitate virus replication. However, how K309 ubiquitination regulates the function of VP35 as the viral polymerase co-factor and the precise stage(s) of the EBOV replication cycle that require VP35 ubiquitination are not known. Here, we generated recombinant EBOVs encoding glycine (G) or arginine (R) mutations at VP35/K309 (rEBOV-VP35/K309G/-R) and show that both mutations prohibit VP35/K309 ubiquitination. The K309R mutant retains dsRNA binding and efficient type-I Interferon (IFN-I) antagonism due to the basic residue conservation. The rEBOV-VP35/K309G mutant loses the ability to efficiently antagonize the IFN-I response, while the rEBOV-VP35/K309R mutant's suppression is enhanced. The replication of both mutants was significantly attenuated in both IFN-competent and -deficient cells due to impaired interactions with the viral polymerase. The lack of ubiquitination on VP35/K309 or TRIM6 deficiency disrupts viral transcription with increasing severity along the transcriptional gradient. This disruption of the transcriptional gradient results in unbalanced viral protein production, including reduced synthesis of the viral transcription factor VP30. In addition, lack of ubiquitination on K309 results in enhanced interactions with the viral nucleoprotein and premature nucleocapsid packaging, leading to dysregulation of virus assembly. Overall, we identified a novel role of VP35 ubiquitination in coordinating viral transcription and assembly.

Development of a point-of-care immunoassay for Ebola Virus Disease through the detection of Ebola virus soluble glycoprotein

Abstract Ebola Virus Disease (EVD) is a deadly infectious disease that is caused by an infection with Ebola virus (EBOV). EVD is one of the deadliest viral diseases with a human case fatality rate of up to 90%. The largest outbreak to date was the 2013–2016 West African outbreak in which Zaire ebolavirus (ZEBOV) was responsible for over 11,000 deaths. Transmission of the virus can occur through contact with an infected animal or human. Early detection is imperative to prevent the spread of disease and reduce the risk of a potential epidemic by isolating known infected patients. The current gold standard for diagnosing EVD is polymerase chain reaction assays which requires complex laboratory equipment and highly trained personnel that is not easily accessible in resource-limited areas. There is a great need for an EBOV rapid diagnostic test that can be implemented at the point-of-care (POC). Multiple lateral flow immunoassays (LFIs) have been developed for this need and have received emergency use authorization. Many of these tests rely on the detection of Ebola virus VP40 antigen, however, soluble glycoprotein (sGP) has the potential to be a better biomarker for EVD rapid detection as it is abundant in the blood during an infection. The goal of this study was to produce an EBOV sGP LFI prototype. A library of seventeen monoclonal antibodies reactive to EBOV sGP were produced and characterized via enzyme-linked immunosorbent assay, surface plasmon resonance, and Western immunoblotting. From this mAb library, LFI prototypes were developed and assessed for the potential use as a rapid POC diagnostic for EVD. Supports by the National Institute of Allergy and Infectious Diseases under grant 1R41AI149940

Ebola virus VP35 hijacks the PKA-CREB1 pathway for replication and pathogenesis by AKIP1 association

Ebola virus (EBOV), one of the deadliest viruses, is the cause of fatal Ebola virus disease (EVD). The underlying mechanism of viral replication and EBOV-related hemorrhage is not fully understood. Here, we show that EBOV VP35, a cofactor of viral RNA-dependent RNA polymerase, binds human A kinase interacting protein (AKIP1), which consequently activates protein kinase A (PKA) and the PKA-downstream transcription factor CREB1. During EBOV infection, CREB1 is recruited into EBOV ribonucleoprotein complexes in viral inclusion bodies (VIBs) and employed for viral replication. AKIP1 depletion or PKA-CREB1 inhibition dramatically impairs EBOV replication. Meanwhile, the transcription of several coagulation-related genes, including THBD and SERPINB2, is substantially upregulated by VP35-dependent CREB1 activation, which may contribute to EBOV-related hemorrhage. The finding that EBOV VP35 hijacks the host PKA-CREB1 signal axis for viral replication and pathogenesis provides novel potential therapeutic approaches against EVD.

Identification of novel chemical compounds targeting filovirus VP40-mediated particle production

The central role of Ebola virus (EBOV) VP40 in nascent virion assembly and budding from infected host cells makes it an important therapeutic target. The mechanism of dimerization, following oligomerization of VP40 leading to the production of virus-like particles (VLP) has never been investigated for the development of therapeutic candidates against Ebola disease. Molecular dynamics-based computational screening targeted VP40 dimer with 40,000,000 compounds selected 374 compounds. A novel in vitro screening assay selected two compounds, NUSU#1 and NUSU#2. Conventional VLP assays consistently showed that both compounds inhibited EBOV VP40-mediated VLP production. Intriguingly, NUSU#1 inhibited the VP40-mediated VLP production in other ebolavirus species and the Marburg virus, but did not inhibit Lassa virus Z-mediated VLP production. These results strongly suggested that the selected compounds are potential lead drug candidates against Filovirus disease via disruption of VP40-mediated particle production.

Identification of potential inhibitor against Ebola virus VP35: insight into virtual screening, pharmacoinformatics profiling, and molecular dynamic studies

Identification of potential inhibitor against Ebola virus VP35: insight into virtual screening, pharmacoinformatics profiling, and molecular dynamic studies

Cynarin blocks Ebola virus replication by counteracting VP35 inhibition of interferon-beta production

Ebola virus (EBOV) is one of the deadliest infective agents whose lethality is linked to the ability to efficiently bypass the host's innate antiviral response. EBOV multifunctional protein VP35 plays a major role in viral replication both as polymerase cofactor and interferon (IFN) antagonist. By hiding the non-self 5′-ppp dsRNA from the cellular receptor RIG-I, VP35 prevents its activation and inhibits IFN-β production. Blocking VP35-dsRNA interaction and IFN-β suppression is a validated drug target.We screened a library of natural extracts and found that cynarin inhibits dsRNA-VP35 binding with an IC50 value of 8.5 μM. It reverts the EBOV VP35 inhibition of IFN-β production, while it does not induce IFN production by itself. Docking experiments suggest that the molecule can bind on the end-capping pocket of VP35 C-terminal Interferon Inhibitory domain (IID), and differential scanning fluorimetry confirmed that cynarin interacts with VP35-IID with a KD of 12 μM. Cynarin was further tested in an EBOV minigenome assay but did not inhibit VP35 polymerase cofactor activity. When evaluated during challenge of IFN-susceptible A549 cells with EBOV isolate derived from the 2014 West African outbreak, cynarin was able to inhibit viral replication with an EC50 value of 9.1 μM, showing no significant cytotoxicity.Our findings show that cynarin blocks EBOV replication by acting directly on VP35 and subverting its IFN antagonism function but not cofactor function, and as such identify the first EBOV inhibitor with this mode of action.

Computational Study on Potential Novel Anti-Ebola Virus Protein VP35 Natural Compounds.

Ebola virus (EBOV) is one of the most lethal pathogens that can infect humans. The Ebola viral protein VP35 (EBOV VP35) inhibits host IFN-α/β production by interfering with host immune responses to viral invasion and is thus considered as a plausible drug target. The aim of this study was to identify potential novel lead compounds against EBOV VP35 using computational techniques in drug discovery. The 3D structure of the EBOV VP35 with PDB ID: 3FKE was used for molecular docking studies. An integrated library of 7675 African natural product was pre-filtered using ADMET risk, with a threshold of 7 and, as a result, 1470 ligands were obtained for the downstream molecular docking using AutoDock Vina, after an energy minimization of the protein via GROMACS. Five known inhibitors, namely, amodiaquine, chloroquine, gossypetin, taxifolin and EGCG were used as standard control compounds for this study. The area under the curve (AUC) value, evaluating the docking protocol obtained from the receiver operating characteristic (ROC) curve, generated was 0.72, which was considered to be acceptable. The four identified potential lead compounds of NANPDB4048, NANPDB2412, ZINC000095486250 and NANPDB2476 had binding affinities of −8.2, −8.2, −8.1 and −8.0 kcal/mol, respectively, and were predicted to possess desirable antiviral activity including the inhibition of RNA synthesis and membrane permeability, with the probable activity (Pa) being greater than the probable inactivity (Pi) values. The predicted anti-EBOV inhibition efficiency values (IC50), found using a random forest classifier, ranged from 3.35 to 11.99 μM, while the Ki values ranged from 0.97 to 1.37 μM. The compounds NANPDB4048 and NANPDB2412 had the lowest binding energy of −8.2 kcal/mol, implying a higher binding affinity to EBOV VP35 which was greater than those of the known inhibitors. The compounds were predicted to possess a low toxicity risk and to possess reasonably good pharmacological profiles. Molecular dynamics (MD) simulations of the protein–ligand complexes, lasting 50 ns, and molecular mechanisms Poisson-Boltzmann surface area (MM-PBSA) calculations corroborated the binding affinities of the identified compounds and identified novel critical interacting residues. The antiviral potential of the molecules could be confirmed experimentally, while the scaffolds could be optimized for the design of future novel anti-EBOV chemotherapeutics.

Ebola virus protein VP40 binding to Sec24c for transport to the plasma membrane.

Outbreaks of the Ebola virus (EBOV) continue to occur and while a vaccine and treatment are now available, there remains a dearth of options for those who become sick with EBOV disease. An understanding at the atomic and molecular level of the various steps in the EBOV replication cycle can provide molecular targets for disrupting the virus. An important step in the EBOV replication cycle is the transport of EBOV structural matrix VP40 protein molecules to the plasma membrane inner leaflet, which involves VP40 binding to the host cell's Sec24c protein. Though some VP40 residues involved in the binding are known, the molecular details of VP40-Sec24c binding are not known. We use various molecular computational techniques to investigate the molecular details of how EBOV VP40 binds with the Sec24c complex of the ESCRT-I pathway. We employed different docking programs to identify the VP40-binding site on Sec24c and then performed molecular dynamics simulations to determine the atomic details and binding interactions of the complex. We also investigated how the inter-protein interactions of the complex are affected upon mutations of VP40 amino acids in the Sec24c-binding region. Our results provide a molecular basis for understanding previous coimmunoprecipitation experimental studies. In addition, we found that VP40 can bind to a site on Sec24c that can also bind Sec23 and suggests that VP40 may use the COPII transport mechanism in a manner that may not need the Sec23 protein in order for VP40 to be transported to the plasma membrane.

Marburg and Ebola Virus mRNA 3' Untranslated Regions Contain Negative Regulators of Translation That Are Modulated by ADAR1 Editing.

ABSTRACTThe filovirus family includes deadly pathogens such as Ebola virus (EBOV) and Marburg virus (MARV). A substantial portion of filovirus genomes encode 5′ and 3′ untranslated regions (UTRs) of viral mRNAs. Select viral genomic RNA sequences corresponding to 3′ UTRs are prone to editing by adenosine deaminase acting on RNA 1 (ADAR1). A reporter mRNA approach, in which different 5′ or 3′ UTRs were inserted into luciferase-encoding mRNAs, demonstrates that MARV 3′ UTRs yield different levels of reporter gene expression, suggesting modulation of translation. The modulation occurs in cells unable to produce microRNAs (miRNAs) and can be recapitulated in a MARV minigenome assay. Deletion mutants identified negative regulatory regions at the ends of the MARV nucleoprotein (NP) and large protein (L) 3′ UTRs. Apparent ADAR1 editing mutants were previously identified within the MARV NP 3′ UTR. Introduction of these changes into the MARV nucleoprotein (NP) 3′ UTR or deletion of the region targeted for editing enhances translation, as indicated by reporter assays and polysome analysis. In addition, the parental NP 3′ UTR, but not the edited or deletion mutant NP 3′ UTRs, induces a type I interferon (IFN) response upon transfection into cells. Because some EBOV isolates from the West Africa outbreak exhibited ADAR1 editing of the viral protein of 40 kDa (VP40) 3′ UTR, VP40 3′ UTRs with parental and edited sequences were similarly assayed. The EBOV VP40 3′ UTR edits also enhanced translation, but neither the wild-type nor the edited 3′ UTRs induced IFN. These findings implicate filoviral mRNA 3′ UTRs as negative regulators of translation that can be inactivated by innate immune responses that induce ADAR1.IMPORTANCE UTRs comprise a large percentage of filovirus genomes and are apparent targets of editing by ADAR1, an enzyme with pro- and antiviral activities. However, the functional significance of the UTRs and ADAR1 editing has been uncertain. This study demonstrates that MARV and EBOV 3′ UTRs can modulate translation, in some cases negatively. ADAR1 editing or deletion of select regions within the translation suppressing 3′ UTRs relieves the negative effects of the UTRs. These data indicate that filovirus 3′ UTRs contain translation regulatory elements that are modulated by activation of ADAR1, suggesting a complex interplay between filovirus gene expression and innate immunity.

Non-canonical proline-tyrosine interactions with multiple host proteins regulate Ebola virus infection.

The Ebola virus VP30 protein interacts with the viral nucleoprotein and with host protein RBBP6 via PPxPxY motifs that adopt non-canonical orientations, as compared to other proline-rich motifs. An affinity tag-purification mass spectrometry approach identified additional PPxPxY-containing host proteins hnRNP L, hnRNPUL1, and PEG10, as VP30 interactors. hnRNP L and PEG10, like RBBP6, inhibit viral RNA synthesis and EBOV infection, whereas hnRNPUL1 enhances. RBBP6 and hnRNP L modulate VP30 phosphorylation, increase viral transcription, andexert additive effects on viral RNA synthesis. PEG10 has more modest inhibitory effects on EBOV replication. hnRNPUL1 positively affects viral RNA synthesis but in a VP30-independent manner. Binding studies demonstrate variable capacity of the PPxPxY motifs from these proteins to bind VP30, define PxPPPPxY as an optimal binding motif, and identify the fifth proline and the tyrosine as most critical for interaction. Competition binding and hydrogen-deuterium exchange mass spectrometry studies demonstrate that each protein binds a similar interface on VP30. VP30 therefore presents a novel proline recognition domain that is targeted by multiple host proteins to modulate viral transcription.