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.

Analysis of tissue tropism of GCRV-II infection in grass carp using a VP35 monoclonal antibody.

Grass carp, one of the major freshwater aquaculture species in China, is susceptible to grass carp reovirus (GCRV). GCRV is a non-enveloped RNA virus and has a double-layered capsid, causing hemorrhagic disease and high mortalities in infected fish. However, the tropism of GCRV infection has not been investigated. In this study, monoclonal antibodies against recombinant VP35 protein were generated in mice and characterized. The antibodies exhibited specific binding to the N terminal region (1-155 aa) of the recombinant VP35 protein expressed in the HEK293 cells, and native VP35 protein in the GCRV-II infected CIK cells. Immunofluorescent staining revealed that viruses aggregated in the cytoplasm of infected cells. In vivo challenge experiments showed that high levels of GCRV-II viruses were present in the gills, intestine, spleen and liver, indicating that they are the major sites for virus infection. Our study showed that the VP35 antibodies generated in this study exhibited high specificity, and are valuable for the development of diagnostic tools for GCRV-II infection.

Exploring plant-derived small molecules as inhibitors of Marburg virus RNA binding protein activity.

The search for antiviral medications is greatly influenced by the hunt for potent inhibitors of viral proteins. To find possible inhibitors of the RNA binding activity of the Marburg virus VP35 protein, we used a thorough in silico drug discovery approach in this investigation. A comprehensive virtual screening process, followed by a detailed MMGBSA analysis, led to the discovery of four potential inhibitory compounds viz. Kudzuisoflavone A, Miquelianin, Rutin, and Protopseudohypericin. They were identified from an extensive library of phytomolecules derived from three medicinal plants: Adiantum capillus-veneris, Hypericum perforatum, and Pueraria montana. In molecular dynamics (MD) simulations, all these compounds showed steady binding to the target protein and favourable interactions. Notably, the free binding energies of all the selected compounds were better than the myricetin, a well-known blocker of the VP35 protein of the Ebola virus. Overall, this investigation offers insightful information about the molecular interactions and binding dynamics of the identified inhibitors' binding to the VP35 protein of the Marburg virus. The findings highlight the potential of three particular medicinal plants as sources of key chemicals for the creation of brand-new Marburg virus antiviral drugs. More experimental validation and optimization of the identified inhibitors are necessary in order to transform these findings into effective medicines for treating Marburg virus infections.Communicated by Ramaswamy H. Sarma.

Structural and Dynamical Basis of VP35-RBD Inhibition by Marine Fungi Compounds to Combat Marburg Virus Infection.

The Marburg virus (MBV), a deadly pathogen, poses a serious threat to world health due to the lack of effective treatments, calling for an immediate search for targeted and efficient treatments. In this study, we focused on compounds originating from marine fungi in order to identify possible inhibitory compounds against the Marburg virus (MBV) VP35-RNA binding domain (VP35-RBD) using a computational approach. We started with a virtual screening procedure using the Lipinski filter as a guide. Based on their docking scores, 42 potential candidates were found. Four of these compounds-CMNPD17596, CMNPD22144, CMNPD25994, and CMNPD17598-as well as myricetin, the control compound, were chosen for re-docking analysis. Re-docking revealed that these particular compounds had a higher affinity for MBV VP35-RBD in comparison to the control. Analyzing the chemical interactions revealed unique binding properties for every compound, identified by a range of Pi-cation interactions and hydrogen bond types. We were able to learn more about the dynamic behaviors and stability of the protein-ligand complexes through a 200-nanosecond molecular dynamics simulation, as demonstrated by the compounds' consistent RMSD and RMSF values. The multidimensional nature of the data was clarified by the application of principal component analysis, which suggested stable conformations in the complexes with little modification. Further insight into the energy profiles and stability states of these complexes was also obtained by an examination of the free energy landscape. Our findings underscore the effectiveness of computational strategies in identifying and analyzing potential inhibitors for MBV VP35-RBD, offering promising paths for further experimental investigations and possible therapeutic development against the MBV.

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.

Novel Antiviral Molecules against Ebola Virus Infection.

Infection with Ebola virus (EBOV) is responsible for hemorrhagic fever in humans with a high mortality rate. Combined efforts of prevention and therapeutic intervention are required to tackle highly variable RNA viruses, whose infections often lead to outbreaks. Here, we have screened the 2P2I3D chemical library using a nanoluciferase-based protein complementation assay (NPCA) and isolated two compounds that disrupt the interaction of the EBOV protein fragment VP35IID with the N-terminus of the dsRNA-binding proteins PKR and PACT, involved in IFN response and/or intrinsic immunity, respectively. The two compounds inhibited EBOV infection in cell culture as well as infection by measles virus (MV) independently of IFN induction. Consequently, we propose that the compounds are antiviral by restoring intrinsic immunity driven by PACT. Given that PACT is highly conserved across mammals, our data support further testing of the compounds in other species, as well as against other negative-sense RNA viruses.

Cheminformatics Strategies Unlock Marburg Virus VP35 Inhibitors from Natural Compound Library.

The Ebola virus and its close relative, the Marburg virus, both belong to the family Filoviridae and are highly hazardous and contagious viruses. With a mortality rate ranging from 23% to 90%, depending on the specific outbreak, the development of effective antiviral interventions is crucial for reducing fatalities and mitigating the impact of Marburg virus outbreaks. In this investigation, a virtual screening approach was employed to evaluate 2042 natural compounds for their potential interactions with the VP35 protein of the Marburg virus. Average and worst binding energies were calculated for all 20 poses, and compounds that exhibited binding energies <-6 kcal/mol in both criteria were selected for further analysis. Based on binding energies, only six compounds (Estradiol benzoate, INVEGA (paliperidone), Isosilybin, Protopanaxadiol, Permethrin, and Bufalin) were selected for subsequent investigations, focusing on interaction analysis. Among these selected compounds, Estradiol benzoate, INVEGA (paliperidone), and Isosilybin showed strong hydrogen bonds, while the others did not. In this study, the compounds Myricetin, Isosilybin, and Estradiol benzoate were subjected to a molecular dynamics (MD) simulation and free binding energy calculation using MM/GBSA analysis. The reference component Myricetin served as a control. Estradiol benzoate exhibited the most stable and consistent root-mean-square deviation (RMSD) values, whereas Isosilybin showed significant fluctuations in RMSD. The compound Estradiol benzoate exhibited the lowest ΔG binding free energy (-22.89 kcal/mol), surpassing the control compound's binding energy (-9.29 kcal/mol). Overall, this investigation suggested that Estradiol benzoate possesses favorable binding free energies, indicating a potential inhibitory mechanism against the VP35 protein of the Marburg virus. The study proposes that these natural compounds could serve as a therapeutic option for preventing Marburg virus infection. However, experimental validation is required to further corroborate these findings.

Ebola Virus VP35 Interacts Non-Covalently with Ubiquitin Chains to Promote Viral Replication Creating New Therapeutic Opportunities.

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 co-factor 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 that correlated with reduced replication of infectious EBOV. 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.

Naïve antibody library derived monoclonal antibody against VP35 of Ebola virus.

Ebola virus is notorious for causing severe and even deadly haemorrhagic fever in infected humans and non-human primates. The high fatality rate of Ebola virus disease (EVD) has highlighted the need for effective diagnosis and treatment. Two monoclonal antibodies (mAbs) have been approved by USFDA for treatment of EVD. Virus surface glycoprotein is the common target for diagnostic and therapy including vaccines. Even so, VP35, a viral RNA polymerase cofactor and interferon inhibitor could be a potential target to curb EVD. The present work describes the isolation of three mAb clones from a phage-displayed human naïve scFv library against recombinant VP35. The clones showed binding against rVP35 in vitro and inhibition of VP35 in luciferase reporter gene assay. Structural modelling analysis was also carried out to identify the binding interactions involved in the antibody-antigen interaction model. This allows some insight into the "fitness" of the binding pocket between the paratope and target epitope which would be useful for the design of new mAbs through in silico means in the future. In conclusion, the information obtained from the 3 isolated mAbs could be potentially useful in the quest to improve VP35 targeting for therapeutic development in the future.

Ebola virus disease: In vivo protection provided by the PAMP restricted TLR3 agonist rintatolimod and its mechanism of action.

Ebola virus (EBOV) is a highly infectious and lethal pathogen responsible for sporadic self-limiting clusters of Ebola virus disease (EVD) in Central Africa capable of reaching epidemic status. 100% protection from lethal EBOV-Zaire in Balb/c mice was achieved by rintatolimod (Ampligen) at the well tolerated human clinical dose of 6 mg/kg. The data indicate that the mechanism of action is rintatolimod's dual ability to act as both a competitive decoy for the IID domain of VP35 blocking viral dsRNA sequestration and as a pathogen-associated molecular pattern (PAMP) restricted agonist for direct TLR3 activation but lacking RIG-1-like cytosolic helicase agonist properties. These data show promise for rintatolimod as a prophylactic therapy against human Ebola outbreaks.

Hsp90 Regulates GCRV-II Proliferation by Interacting with VP35 as Its Receptor and Chaperone.

The frequent outbreak of grass carp hemorrhagic disease caused by grass carp reovirus (GCRV), especially the mainly prevalent type II GCRV (GCRV-II), has seriously affected the grass carp culture in China. However, its pathogenic mechanism is still far from clear. In this study, the GCRV-II outer capsid protein VP35 was used as bait to capture interacting partners from Ctenopharyngon idellus kidney (CIK) cells, and heat shock protein 90 (Hsp90) was selected and confirmed interacting with VP35 through the C-terminal domain of Hsp90. Knockdown of Hsp90 or inhibition of Hsp90 activity suppressed GCRV-II proliferation, demonstrating that Hsp90 is an essential factor for GCRV-II proliferation. The confocal microscopy and flow cytometry showed that Hsp90 localized at both membrane and cytoplasm of CIK cells. The entry of GCRV-II into CIK cells was efficiently blocked by incubating the cells with Hsp90 antibody or by pretreating the virus with recombinant Hsp90 protein. Whereas overexpression of Hsp90 in CIK cells, grass carp ovary (GCO) cells, or 293T cells promoted GCRV-II entry, indicating that the membrane Hsp90 functions as a receptor of GCRV-II. Furthermore, Hsp90 interacted with clathrin and mediated GCRV-II entry into CIK cells through clathrin endocytosis pathway. In addition, we found that the cytoplasmic Hsp90 acted as a chaperone of VP35 because inhibition of Hsp90 activity enhanced VP35 polyubiquitination and degraded VP35 through the proteasome pathway. Collectively, our data suggest that Hsp90 functions both as a receptor for GCRV-II entry and a chaperone for the maturation of GCRV-II VP35, thus ensuring efficient proliferation of GCRV-II. IMPORTANCE Identification of viral receptors has always been the research hot spot in virus research field as receptor functions at the first stage of viral infection, which can be designed as efficient antiviral drug targets. GCRV-II, the causative agent of the grass carp epidemic hemorrhagic disease, has caused tremendous losses in grass carp culture in China. To date, the receptor of GCRV-II remains unknown. This study focused on identifying cellular receptor interacting with the GCRV-II outer capsid protein VP35, studying the effects of their interaction on GCRV-II proliferation, and revealing the underlying mechanisms. We demonstrated that Hsp90 acts both as a receptor of GCRV-II by interacting with VP35 and as a chaperone for the maturation of VP35, thus ensuring efficient proliferation of GCRV-II. Our data provide important insights into the role of Hsp90 in GCRV-II life cycle, which will help understand the mechanism of reovirus infection.

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-ß production. Blocking VP35-dsRNA interaction and IFN-ß 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-ß 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/ß 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.

Functional Importance of Hydrophobic Patches on the Ebola Virus VP35 IFN-Inhibitory Domain.

Viral protein 35 (VP35) of Ebola virus (EBOV) is a multifunctional protein that mainly acts as a viral polymerase cofactor and an interferon antagonist. VP35 interacts with the viral nucleoprotein (NP) and double-stranded RNA for viral RNA transcription/replication and inhibition of type I interferon (IFN) production, respectively. The C-terminal portion of VP35, which is termed the IFN-inhibitory domain (IID), is important for both functions. To further identify critical regions in this domain, we analyzed the physical properties of the surface of VP35 IID, focusing on hydrophobic patches, which are expected to be functional sites that are involved in interactions with other molecules. Based on the known structural information of VP35 IID, three hydrophobic patches were identified on its surface and their biological importance was investigated using minigenome and IFN-ß promoter-reporter assays. Site-directed mutagenesis revealed that some of the amino acid substitutions that were predicted to disrupt the hydrophobicity of the patches significantly decreased the efficiency of viral genome replication/transcription due to reduced interaction with NP, suggesting that the hydrophobic patches might be critical for the formation of a replication complex through the interaction with NP. It was also found that the hydrophobic patches were involved in the IFN-inhibitory function of VP35. These results highlight the importance of hydrophobic patches on the surface of EBOV VP35 IID and also indicate that patch analysis is useful for the identification of amino acid residues that directly contribute to protein functions.

Human Parainfluenza Virus 3 Phosphoprotein Is a Tetramer and Shares Structural and Interaction Features with Ebola Phosphoprotein VP35.

The human parainfluenza virus 3 (HPIV3) poses a risk for pneumonia development in young children and immunocompromised patients. To investigate mechanisms of HPIV3 pathogenesis, we characterized the association state and host protein interactions of HPIV3 phosphoprotein (HPIV3 P), an indispensable viral polymerase cofactor. Sequence analysis and homology modeling predict that HPIV3 P possesses a long, disordered N-terminal tail (PTAIL) a coiled-coil multimerization domain (PMD), similar to the well-characterized paramyxovirus phosphoproteins from measles and Sendai viruses. Using a recombinantly expressed and purified construct of PMD and PTAIL, we show that HPIV3 P in solution is primarily an alpha-helical tetramer that is stable up to 60 °C. Pulldown and isothermal titration calorimetry experiments revealed that HPIV3 P binds the host hub protein LC8, and turbidity experiments demonstrated a new role for LC8 in increasing the solubility of HPIV3 P in the presence of crowding agents such as RNA. For comparison, we show that the multimerization domain of the Zaire Ebola virus phosphoprotein VP35 is also a tetramer and binds LC8 but with significantly higher affinity. Comparative analysis of the domain architecture of various virus phosphoproteins in the order Mononegavirales show multiple predicted and verified LC8 binding motifs, suggesting its prevalence and importance in regulating viral phosphoprotein structures. Our work provides evidence for LC8 binding to phosphoproteins with multiple association states, either tetrameric, as in the HPIV3 and Ebola phosphoproteins shown here, or dimeric as in rabies virus phosphoprotein. Taken together the data suggest that the association states of a virus-specific phosphoprotein and the complex formed by binding of the phosphoprotein to host LC8 are important regulators of viral function.

Grass Carp Reovirus VP35 Degrades MAVS Through the Autophagy Pathway to Inhibit Fish Interferon Production.

Fish interferon (IFN) is a crucial cytokine for a host to resist external pathogens, conferring cells with antiviral capacity. Meanwhile, grass carp reovirus (GCRV) is a strong pathogen that causes high mortality in grass carp. Therefore, it is necessary to study the strategy used by GCRV to evade the cellular IFN response. In this study, we found that GCRV 35-kDa protein (VP35) inhibited the host IFN production by degrading mitochondrial antiviral signaling (MAVS) protein through the autophagy pathway. First, the overexpression of VP35 inhibited the IFN activation induced by polyinosinic-polycytidylic acid (poly I:C) and MAVS, and the expression of downstream IFN-stimulated genes (ISGs) was also decreased by using VP35 under the stimulation. Second, VP35 interacted with MAVS; the experiments of truncated mutants of MAVS demonstrated that the caspase recruitment domain (CARD) and proline-rich (PRO) domains of MAVS were not necessary for this binding. Then, MAVS was degraded by using VP35 in a dose-dependent manner, and 3-MA (the autophagy pathway inhibitor) significantly blocked the degradation, meaning that MAVS was degraded by using VP35 in the autophagy pathway. The result of MAVS degradation suggested that the antiviral capacity of MAVS was remarkably depressed when interrupted by VP35. Finally, in the host cells, VP35 reduced ifn transcription and made the cells vulnerable to virus infection. In conclusion, our results reveal that GCRV VP35 impairs the host IFN response by degrading MAVS through the autophagy pathway, supplying evidence of a fish virus immune evasion strategy.

Crystal structure of human LC8 bound to a peptide from Ebola virus VP35.

Zaire ebolavirus, commonly called Ebola virus (EBOV), is an RNA virus that causes severe hemorrhagic fever with high mortality. Viral protein 35 (VP35) is a virulence factor encoded in the EBOV genome. VP35 inhibits host innate immune responses and functions as a critical cofactor for viral RNA replication. EBOV VP35 contains a short conserved motif that interacts with dynein light chain 8 (LC8), which serves as a regulatory hub protein by associating with various LC8-binding proteins. Herein, we present the crystal structure of human LC8 bound to the peptide comprising residues 67-76 of EBOV VP35. Two VP35 peptides were found to interact with homodimeric LC8 by extending the central ß-sheets, constituting a 2:2 complex. Structural analysis demonstrated that the intermolecular binding between LC8 and VP35 is mainly sustained by a network of hydrogen bonds and supported by hydrophobic interactions in which Thr73 and Thr75 of VP35 are involved. These findings were verified by binding measurements using isothermal titration calorimetry. Biochemical analyses also verified that residues 67-76 of EBOV VP35 constitute a core region for interaction with LC8. In addition, corresponding motifs from other members of the genus Ebolavirus commonly bound to LC8 but with different binding affinities. Particularly, VP35 peptides originating from pathogenic species interacted with LC8 with higher affinity than those from noninfectious species, suggesting that the binding of VP35 to LC8 is associated with the pathogenicity of the Ebolavirus species.

Transcriptional Analysis of Lymphoid Tissues from Infected Nonhuman Primates Reveals the Basis for Attenuation and Immunogenicity of an Ebola Virus Encoding a Mutant VP35 Protein.

Infection with Zaire ebolavirus (EBOV), a member of the Filoviridae family, causes a disease characterized by high levels of viremia, aberrant inflammation, coagulopathy, and lymphopenia. EBOV initially replicates in lymphoid tissues and disseminates via dendritic cells (DCs) and monocytes to liver, spleen, adrenal gland, and other secondary organs. EBOV protein VP35 is a critical immune evasion factor that inhibits type I interferon signaling and DC maturation. Nonhuman primates (NHPs) immunized with a high dose (5 × 105 PFU) of recombinant EBOV containing a mutated VP35 (VP35m) are protected from challenge with wild-type EBOV (wtEBOV). This protection is accompanied by a transcriptional response in the peripheral blood reflecting a regulated innate immune response and a robust induction of adaptive immune genes. However, the host transcriptional response to VP35m in lymphoid tissues has not been evaluated. Therefore, we conducted a transcriptional analysis of axillary and inguinal lymph nodes and spleen tissues of NHPs infected with a low dose (2 × 104 PFU) of VP35m and then back-challenged with a lethal dose of wtEBOV. VP35m induced early transcriptional responses in lymphoid tissues that are distinct from those observed in wtEBOV challenge. Specifically, we detected robust antiviral innate and adaptive responses and fewer transcriptional changes in genes with roles in angiogenesis, apoptosis, and inflammation. Two of three macaques survived wtEBOV back-challenge, with only the nonsurvivor displaying a transcriptional response reflecting Ebola virus disease. These data suggest that VP35 is a key modulator of early host responses in lymphoid tissues, thereby regulating disease progression and severity following EBOV challenge.IMPORTANCE Zaire Ebola virus (EBOV) infection causes a severe and often fatal disease characterized by inflammation, coagulation defects, and organ failure driven by a defective host immune response. Lymphoid tissues are key sites of EBOV pathogenesis and the generation of an effective immune response to infection. A recent study demonstrated that infection with an EBOV encoding a mutant VP35, a viral protein that antagonizes host immunity, can protect nonhuman primates (NHPs) against lethal EBOV challenge. However, no studies have examined the response to this mutant EBOV in lymphoid tissues. Here, we characterize gene expression in lymphoid tissues from NHPs challenged with the mutant EBOV and subsequently with wild-type EBOV to identify signatures of a protective host response. Our findings are critical for elucidating viral pathogenesis, mechanisms of host antagonism, and the role of lymphoid organs in protective responses to EBOV to improve the development of antivirals and vaccines against EBOV.

Ebola Virus VP35 Protein: Modeling of the Tetrameric Structure and an Analysis of Its Interaction with Human PKR.

The Viral Protein 35 (VP35), a crucial protein of the Zaire Ebolavirus (EBOV), interacts with a plethora of human proteins to cripple the human immune system. Despite its importance, the entire structure of the tetrameric assembly of EBOV VP35 and the means by which it antagonizes the autophosphorylation of the kinase domain of human protein kinase R (PKRK) is still elusive. We consult existing structural information to model a tetrameric assembly of the VP35 protein where 93% of the protein is modeled using crystal structure templates. We analyze our modeled tetrameric structure to identify interchain bonding networks and use molecular dynamics simulations and normal-mode analysis to unravel the flexibility and deformability of the different regions of the VP35 protein. We establish that the C-terminal of VP35 (VP35C) directly interacts with PKRK to prevent it from autophosphorylation. Further, we identify three plausible VP35C-PKRK complexes with better affinity than the PKRK dimer formed during autophosphorylation and use protein design to establish a new stretch in VP35C that interacts with PKRK. The proposed tetrameric assembly will aid in better understanding of the VP35 protein, and the reported VP35C-PKRK complexes along with their interacting sites will help in the shortlisting of small molecule inhibitors.

Ebola virus replication is regulated by the phosphorylation of viral protein VP35.

Ebola virus (EBOV) is a zoonotic pathogen, the infection often results in severe, potentially fatal, systematic disease in human and nonhuman primates. VP35, an essential viral RNA-dependent RNA polymerase cofactor, is indispensable for Ebola viral replication and host innate immune escape. In this study, VP35 was demonstrated to be phosphorylated at Serine/Threonine by immunoblotting, and the major phosphorylation sites was S187, S205, T206, S208 and S317 as revealed by LC-MS/MS. By an EBOV minigenomic system, EBOV minigenome replication was shown to be significantly inhibited by the phosphorylation-defective mutant, VP35 S187A, but was potentiated by the phosphorylation mimic mutant VP35 S187D. Together, our findings demonstrate that EBOV VP35 is phosphorylated on multiple residues in host cells, especially on S187, which may contribute to efficient viral genomic replication and viral proliferation.