Marburg Virus VP35 Can Both Fully Coat the Backbone and Cap the Ends of dsRNA for Interferon Antagonism

Filoviruses, including Marburg virus (MARV) and Ebola virus (EBOV), cause fatal hemorrhagic fever in humans and non-human primates. All filoviruses encode a unique multi-functional protein termed VP35. The C-terminal double-stranded (ds)RNA-binding domain (RBD) of VP35 has been implicated in interferon antagonism and immune evasion. Crystal structures of the VP35 RBD from two ebolaviruses have previously demonstrated that the viral protein caps the ends of dsRNA. However, it is not yet understood how the expanses of dsRNA backbone, between the ends, are masked from immune surveillance during filovirus infection. Here, we report the crystal structure of MARV VP35 RBD bound to dsRNA. In the crystal structure, molecules of dsRNA stack end-to-end to form a pseudo-continuous oligonucleotide. This oligonucleotide is continuously and completely coated along its sugar-phosphate backbone by the MARV VP35 RBD. Analysis of dsRNA binding by dot-blot and isothermal titration calorimetry reveals that multiple copies of MARV VP35 RBD can indeed bind the dsRNA sugar-phosphate backbone in a cooperative manner in solution. Further, MARV VP35 RBD can also cap the ends of the dsRNA in solution, although this arrangement was not captured in crystals. Together, these studies suggest that MARV VP35 can both coat the backbone and cap the ends, and that for MARV, coating of the dsRNA backbone may be an essential mechanism by which dsRNA is masked from backbone-sensing immune surveillance molecules.     

Exploration micromechanism of VP35 IID interaction and recognition dsRNA: A molecular dynamics simulation

Multifunctional viral protein (VP35) encoded by the highly pathogenic Ebola viruses (EBOVs) can antagonize host double‐stranded RNA (dsRNA) sensors and immune response because of the simultaneous recognition of dsRNA backbone and blunt ends. Mutation of select hydrophobic conserved basic residues within the VP35 inhibitory domain (IID) abrogates its dsRNA‐binding activity, and impairs VP35‐mediated interferon (IFN) antagonism. Herein the detailed binding mechanism between dsRNA and WT, single mutant, and double mutant were investigated by all‐atom molecular dynamics (MD) simulation and binding energy calculation. R312A/R322A double mutations results in a completely different binding site and orientation upon the structure analyses. The calculated binding free energy results reveal that R312A, R322A, and K339A single mutations decrease the binding free energies by 17.82, 13.18, and 13.68 kcal mol^?1, respectively. The binding energy decomposition indicates that the strong binding affinity of the key residues is mainly due to the contributions of electrostatic interactions in the gas phase, where come from the positively charged side chain and the negatively charged dsRNA backbone. R312A, R322A, and K339A single mutations have no significant effect on VP35 IID conformation, but the mutations influence the contributions of electrostatic interactions in the gas phase. The calculated results reveal that end‐cap residues which mainly contribute VDW interactions can recognize and capture dsRNA blunt ends, and the central basic residues (R312, R322, and K339) which mainly contribute favorable electrostatic interactions with dsRNA backbone can fix dsRNA binding site and orientation. Proteins 2017; 85:1008–1023. © 2017 Wiley Periodicals, Inc.     

Exploring the mechanism how Marburg virus VP35 recognizes and binds dsRNA by molecular dynamics simulations and free energy calculations

Filoviruses often cause terrible infectious disease which has not been successfully dealt with pharmacologically. All filoviruses encode a unique protein termed VP35 which can mask doubled‐stranded RNA to deactivate interferon. The interface of VP35–dsRNA would be a feasible target for structure‐based antiviral agent design. To explore the essence of VP35–dsRNA interaction, molecular dynamics simulation combined with MM‐GBSA calculations were performed on Marburg virus VP35–dsRNA complex and several mutational complexes. The energetic analysis indicates that nonpolar interactions provide the main driving force for the binding process. Although the intermolecular electrostatic interactions play important roles in VP35–dsRNA interaction, the whole polar interactions are unfavorable for binding which result in a low binding affinity. Compared with wild type VP35, the studied mutants F228A, R271A, and K298A have obviously reduced binding free energies with dsRNA reflecting in the reduction of polar or nonpolar interactions. The results also indicate that the loss of binding affinity for one dsRNA strand would abolish the total binding affinity. Three important residues Arg271, Arg294, and Lys298 which makes the largest contribution for binding in VP35 lose their binding affinity significantly in mutants. The uncovering of VP35–dsRNA recognition mechanism will provide some insights for development of antiviral drug. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 849–860, 2014.     

Ebolavirus VP35 suppresses IFN production from conventional but not plasmacytoid dendritic cells

Ebolaviruses naturally infect a wide variety of cells including macrophages and dendritic cells (DCs), and the resulting cytokine and interferon-α/β (IFN) responses of infected cells are thought to influence viral pathogenesis. The VP35 protein impairs RIG-I-like receptor-dependent signaling to inhibit IFN production, and this function has been suggested to promote the ineffective host immune response characteristic of ebolavirus infection. To assess the impact of VP35 on innate immunity in biologically relevant primary cells, we used a recombinant Newcastle disease virus encoding VP35 (NDV/VP35) to infect macrophages and conventional DCs, which primarily respond to RNA virus infection via RIG-I-like pathways. VP35 suppressed not only IFN but also tumor necrosis factor (TNF)-α secretion, which are normally produced from these cells upon NDV infection. Additionally, in cells susceptible to the activity of VP35, IRF7 activation is impaired. In contrast, NDV/VP35 infection of plasmacytoid DCs, which activate IRF7 and produce IFN through TLR-dependent signaling, leads to robust IFN production. When plasmacytoid DCs deficient for TLR signaling were infected, NDV/VP35 was able to inhibit IFN production. Consistent with this, VP35 was less able to inhibit TLR-dependent versus RIG-I-dependent signaling in vitro. These data demonstrate that ebolavirus VP35 suppresses both IFN and cytokine production in multiple primary human cell types. However, cells that utilize the TLR pathway can circumvent this inhibition, suggesting that the presence of multiple viral sensors enables the host to overcome viral immune evasion mechanisms.     

Characterization of the RNA silencing suppression activity of the Ebola virus VP35 protein in plants and mammalian cells

Ebola virus (EBOV) causes a lethal hemorrhagic fever for which there is no approved effective treatment or prevention strategy. EBOV VP35 is a virulence factor that blocks innate antiviral host responses, including the induction of and response to alpha/beta interferon. VP35 is also an RNA silencing suppressor (RSS). By inhibiting microRNA-directed silencing, mammalian virus RSSs have the capacity to alter the cellular environment to benefit replication. A reporter gene containing specific microRNA target sequences was used to demonstrate that prior expression of wild-type VP35 was able to block establishment of microRNA silencing in mammalian cells. In addition, wild-type VP35 C-terminal domain (CTD) protein fusions were shown to bind small interfering RNA (siRNA). Analysis of mutant proteins demonstrated that reporter activity in RSS assays did not correlate with their ability to antagonize double-stranded RNA (dsRNA)-activated protein kinase R (PKR) or bind siRNA. The results suggest that enhanced reporter activity in the presence of VP35 is a composite of nonspecific translational enhancement and silencing suppression. Moreover, most of the specific RSS activity in mammalian cells is RNA binding independent, consistent with VP35's proposed role in sequestering one or more silencing complex proteins. To examine RSS activity in a system without interferon, VP35 was tested in well-characterized plant silencing suppression assays. VP35 was shown to possess potent plant RSS activity, and the activities of mutant proteins correlated strongly, but not exclusively, with RNA binding ability. The results suggest the importance of VP35-protein interactions in blocking silencing in a system (mammalian) that cannot amplify dsRNA.     

In silico and in vitro methods to identify ebola virus VP35-dsRNA inhibitors

Ebola virus continues to be problematic as sporadic outbreaks in Africa continue to arise, and as terrorist organizations have considered the virus for bioterrorism use. Several proteins within the virus have been targeted for antiviral chemotherapy, including VP35, a dsRNA binding protein that promotes viral replication, protects dsRNA from degradation, and prevents detection of the viral genome by immune complexes. To augment the scope of our antiviral research, we have now employed molecular modeling techniques to enrich the population of compounds for further testing in vitro. In the initial docking of a static VP35 structure with an 80,000 compound library, 40 compounds were selected, of which four compounds inhibited VP35 with IC₅₀ <200 μM, with the best compounds having an IC₅₀ of 20 μM. By superimposing 26 VP35 structures, we determined four aspartic acid residues were highly flexible and the docking was repeated under flexible parameters. Of 14 compounds chosen for testing, five compounds inhibited VP35 with IC₅₀ <200 μM and one compound with an IC₅₀ of 4 μM. These studies demonstrate the value of docking in silico for enriching compounds for testing in vitro, and specifically using multiple structures as a guide for detecting flexibility and provide a foundation for further development of small molecule inhibitors directed towards VP35.     

Structural and Functional Characterization of Reston Ebola Virus VP35 Interferon Inhibitory Domain

Ebolaviruses are causative agents of lethal hemorrhagic fever in humans and nonhuman primates. Among the filoviruses characterized thus far, Reston Ebola virus (REBOV) is the only Ebola virus that is nonpathogenic to humans despite the fact that REBOV can cause lethal disease in nonhuman primates. Previous studies also suggest that REBOV is less effective at inhibiting host innate immune responses than Zaire Ebola virus (ZEBOV) or Marburg virus. Virally encoded VP35 protein is critical for immune suppression, but an understanding of the relative contributions of VP35 proteins from REBOV and other filoviruses is currently lacking. In order to address this question, we characterized the REBOV VP35 interferon inhibitory domain (IID) using structural, biochemical, and virological studies. These studies reveal differences in double-stranded RNA binding and interferon inhibition between the two species. These observed differences are likely due to increased stability and loss of flexibility in REBOV VP35 IID, as demonstrated by thermal shift stability assays. Consistent with this finding, the 1.71-Å crystal structure of REBOV VP35 IID reveals that it is highly similar to that of ZEBOV VP35 IID, with an overall backbone r.m.s.d. of 0.64 Å, but contains an additional helical element at the linker between the two subdomains of VP35 IID. Mutations near the linker, including swapping sequences between REBOV and ZEBOV, reveal that the linker sequence has limited tolerance for variability. Together with the previously solved ligand-free and double-stranded-RNA-bound forms of ZEBOV VP35 IID structures, our current studies on REBOV VP35 IID reinforce the importance of VP35 in immune suppression. Functional differences observed between REBOV and ZEBOV VP35 proteins may contribute to observed differences in pathogenicity, but these are unlikely to be the major determinant. However, the high level of similarity in structure and the low tolerance for sequence variability, coupled with the multiple critical roles played by Ebola virus VP35 proteins, highlight the viability of VP35 as a potential target for therapeutic development.     

Mutations Abrogating VP35 Interaction with Double-Stranded RNA Render Ebola Virus Avirulent in Guinea Pigs

Ebola virus (EBOV) protein VP35 is a double-stranded RNA (dsRNA) binding inhibitor of host interferon (IFN)-α/β responses that also functions as a viral polymerase cofactor. Recent structural studies identified key features, including a central basic patch, required for VP35 dsRNA binding activity. To address the functional significance of these VP35 structural features for EBOV replication and pathogenesis, two point mutations, K319A/R322A, that abrogate VP35 dsRNA binding activity and severely impair its suppression of IFN-α/β production were identified. Solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography reveal minimal structural perturbations in the K319A/R322A VP35 double mutant and suggest that loss of basic charge leads to altered function. Recombinant EBOVs encoding the mutant VP35 exhibit, relative to wild-type VP35 viruses, minimal growth attenuation in IFN-defective Vero cells but severe impairment in IFN-competent cells. In guinea pigs, the VP35 mutant virus revealed a complete loss of virulence. Strikingly, the VP35 mutant virus effectively immunized animals against subsequent wild-type EBOV challenge. These in vivo studies, using recombinant EBOV viruses, combined with the accompanying biochemical and structural analyses directly correlate VP35 dsRNA binding and IFN inhibition functions with viral pathogenesis. Moreover, these studies provide a framework for the development of antivirals targeting this critical EBOV virulence factor.Ebola viruses (EBOVs) are zoonotic, enveloped negative-strand RNA viruses belonging to the family Filoviridae which cause lethal viral hemorrhagic fever in humans and nonhuman primates (47). Currently, information regarding EBOV-encoded virulence determinants remains limited. This, coupled with our lack of understanding of biochemical and structural properties of virulence factors, limits efforts to develop novel prophylactic or therapeutic approaches toward these infections.It has been proposed that EBOV-encoded mechanisms to counter innate immune responses, particularly interferon (IFN) responses, are critical to EBOV pathogenesis (7). However, a role for viral immune evasion functions in the pathogenesis of lethal EBOV infection has yet to be demonstrated. Of the eight major EBOV gene products, two viral proteins have been demonstrated to counter host IFN responses. The VP35 protein is a viral polymerase cofactor and structural protein that also inhibits IFN-α/β production by preventing the activation of interferon regulatory factor (IRF)-3 and -7 (3, 4, 8, 24, 27, 34, 41). VP35 also inhibits the activation of PKR, an IFN-induced, double-stranded RNA (dsRNA)-activated kinase with antiviral activity, and inhibits RNA silencing (17, 20, 48). The VP24 protein is a minor structural protein implicated in virus assembly and regulation of viral RNA synthesis, and changes in VP24 coding sequences are also associated with adaptation of EBOVs to mice and guinea pigs (2, 13, 14, 27, 32, 37, 50, 52). Further, VP24 inhibits cellular responses to both IFN-α/β and IFN-γ by preventing the nuclear accumulation of tyrosine-phosphorylated STAT1 (44, 45). The functions of VP35 and VP24 proteins are manifested in EBOV-infected cells by the absence of IRF-3 activation, impaired production of IFN-α/β, and severely reduced expression of IFN-induced genes, even after treatment of infected cells with IFN-α (3, 19, 21, 22, 24, 25, 28).Previous studies proposed that VP35 basic residues 305, 309, and 312 are required for VP35 dsRNA binding activity (26). VP35 residues K309 and R312 were subsequently identified as critical for binding to dsRNA, and mutation of these residues impaired VP35 suppression of IFN-α/β production (8). In vivo, an EBOV engineered to carry a VP35 R312A point mutation exhibited reduced replication in mice (23). However, because the parental recombinant EBOV into which the mutation was built did not cause disease in these animals, the impact of the mutation on viral pathogenesis could not be fully evaluated. Further, the lack of available structural and biochemical data to explain how the R312A mutation affects VP35 function limited avenues for the therapeutic targeting of critical VP35 functions. Recent structural analyses of the VP35 carboxy-terminal interferon inhibitory domain (IID) suggested that additional residues from the central basic patch may contribute to VP35 dsRNA binding activity and IFN-antagonist function (30). However, a direct correlation between dsRNA and IFN inhibitory functions of VP35 with viral pathogenesis is currently lacking.In order to further define the molecular basis for VP35 dsRNA binding and IFN-antagonist function and to define the contribution of these functions to EBOV pathogenesis, an integrated molecular, structural, and virological approach was taken. The data presented below identify two VP35 carboxy-terminal basic amino acids, K319 and R322, as required for its dsRNA binding and IFN-antagonist functions. Interestingly, these residues are outside the region originally identified as being important for dsRNA binding and IFN inhibition (26). However, they lie within the central basic patch identified by prior structural studies (26, 30). Introduction of these mutations (VP35 with these mutations is designated KRA) into recombinant EBOV renders this otherwise fully lethal virus avirulent in guinea pigs. KRA-infected animals also develop EBOV-specific antibodies and become fully resistant to subsequent challenge with wild-type (WT) virus. Our data further reveal that the KRA EBOV is immunogenic and likely replicates to low levels early after infection in vivo. However, the mutant virus is subsequently cleared by host immune responses. These data demonstrate that the VP35 central basic patch is important not only for IFN-antagonist function but also for EBOV immune evasion and pathogenesis in vivo. High-resolution structural analysis, coupled with our in vitro and in vivo analyses of the recombinant Ebola viruses, provides the molecular basis for loss of function by the VP35 mutant and highlights the therapeutic potential of targeting the central basic patch with small-molecule inhibitors and for future vaccine development efforts.     

Three-dimensional structural analysis of recombinant rotavirus-like particles with intact and amino-terminal-deleted VP2: implications for the architecture of the VP2 capsid layer.

Rotaviruses are the leading cause of severe infantile gastroenteritis worldwide. These viruses are large, complex icosahedral particles consisting of three concentric capsid layers enclosing a genome of eleven segments of double-stranded RNA (dsRNA). The amino terminus of the innermost capsid protein VP2 possesses a nonspecific single-stranded RNA and dsRNA binding activity, and the amino terminus is also essential for the incorporation of the polymerase enzyme VP1 and guanylyltransferase VP3 into the core of the virion. Biochemical and structural studies have suggested that VP2, and especially the amino terminus, appears to act as a scaffold for proper assembly of the components of the viral core. To locate the amino terminus of VP2 within the core, we have used electron cryomicroscopy and image reconstruction to determine the three-dimensional structures of recombinant virus-like particles that contain either full-length or amino-terminal-deleted forms of VP2 coexpressed with the intermediate capsid protein VP6. A comparison of these structures indicates two significant changes along the inner surface of VP2 in the structure lacking the amino terminus: a loss of mass adjacent to the fivefold axes and a redistribution of mass along the fivefold axes. Examination of the VP2 layer suggests that the proteins are arranged as dimers of 120 quasi-equivalent molecules, with each dimer extending between neighboring fivefold axes. Our results indicate that the amino termini of both quasi-equivalent VP2 molecules are located near the icosahedral vertices.     

Structural basis of double-stranded RNA recognition by the RIG-I like receptor MDA5

RIG-I, MDA5 and LGP2 are cytosolic pattern recognition receptors detecting single-stranded or double-stranded RNA in virally infected cells. The activation of RIG-I or MDA5 stimulates the secretion of type I interferons that play key roles in antiviral immune responses. The C-terminal domains (CTD) of RIG-I and LGP2 are responsible for RNA binding; however, it is not clear how MDA5 binds RNA. To understand the structural basis of dsRNA recognition by MDA5, we have determined the 1.45 Å resolution structure of the C-terminal domain of human MDA5. The structure revealed a highly conserved fold similar to the structures of RIG-I and LGP2 CTDs. NMR titration of MDA5 CTD with dsRNA demonstrated that a positively charged surface is involved in dsRNA binding. Mutagenesis and RNA binding studies showed that electrostatic interactions play primary roles in dsRNA recognition by MDA5. Like RIG-I and LGP2, MDA5 CTD preferentially binds dsRNA with blunt ends, but does not associate with dsRNA with either 5′ or 3′ overhangs. Molecular modeling of MDA5 CTD/dsRNA complex suggests that MDA5 CTD may recognize the first turn of blunt-ended dsRNA in a similar manner as LGP2.     

VP3, a structural protein of infectious pancreatic necrosis virus, interacts with RNA-dependent RNA polymerase VP1 and with double-stranded RNA

Infectious pancreatic necrosis virus (IPNV) is a bisegmented, double-stranded RNA (dsRNA) virus of the Birnaviridae family that causes widespread disease in salmonids. Its two genomic segments are encapsulated together with the viral RNA-dependent RNA polymerase, VP1, and the assumed internal protein, VP3, in a single-shell capsid composed of VP2. Major aspects of the molecular biology of IPNV, such as particle assembly and interference with host macromolecules, are as yet poorly understood. To understand the infection process, analysis of viral protein interactions is of crucial importance. In this study, we focus on the interaction properties of VP3, the suggested key organizer of particle assembly in birnaviruses. By applying the yeast two-hybrid system in combination with coimmunoprecipitation, VP3 was proven to bind to VP1 and to self-associate strongly. In addition, VP3 was shown to specifically bind to dsRNA in a sequence-independent manner by in vitro pull-down experiments. The binding between VP3 and VP1 was not dependent on the presence of dsRNA. Deletion analyses mapped the VP3 self-interaction domain within the 101 N-terminal amino acids and the VP1 interaction domain within the 62 C-terminal amino acids of VP3. The C-terminal end was also crucial but not sufficient for the dsRNA binding capacity of VP3. For VP1, the 90 C-terminal amino acids constituted the only dispensable part for maintaining VP3-binding ability. Kinetic analysis revealed the presence of VP1-VP3 complexes prior to the formation of mature virions in IPNV-infected CHSE-214 cells, which indicates a role in promoting the assembly process.     

Ebolavirus proteins suppress the effects of small interfering RNA by direct interaction with the mammalian RNA interference pathway

Cellular RNA interference (RNAi) provides a natural response against viral infection, but some viruses have evolved mechanisms to antagonize this form of antiviral immunity. To determine whether Ebolavirus (EBOV) counters RNAi by encoding suppressors of RNA silencing (SRSs), we screened all EBOV proteins using an RNAi assay initiated by exogenously delivered small interfering RNAs (siRNAs) against either an EBOV or a reporter gene. In addition to viral protein 35 (VP35), we found that VP30 and VP40 independently act as SRSs. Here, we present the molecular mechanisms of VP30 and VP35. VP30 interacts with Dicer independently of siRNA and with one Dicer partner, TRBP, only in the presence of siRNA. VP35 directly interacts with Dicer partners TRBP and PACT in an siRNA-independent fashion and in the absence of effects on interferon (IFN). Taken together, our findings elucidate a new mechanism of RNAi suppression that extends beyond the role of SRSs in double-stranded RNA (dsRNA) binding and IFN antagonism. The presence of three suppressors highlights the relevance of host RNAi-dependent antiviral immunity in EBOV infection and illustrates the importance of RNAi in shaping the evolution of RNA viruses.     

Structure of a VP1-VP3 Complex Suggests How Birnaviruses Package the VP1 Polymerase

Infectious pancreatic necrosis virus (IPNV), a member of the family Birnaviridae, infects young salmon, with a severe impact on the commercial sea farming industry. Of the five mature proteins encoded by the IPNV genome, the multifunctional VP3 has an essential role in morphogenesis; interacting with the capsid protein VP2, the viral double-stranded RNA (dsRNA) genome and the RNA-dependent RNA polymerase VP1. Here we investigate one of these VP3 functions and present the crystal structure of the C-terminal 12 residues of VP3 bound to the VP1 polymerase. This interaction, visualized for the first time, reveals the precise molecular determinants used by VP3 to bind the polymerase. Competition binding studies confirm that this region of VP3 is necessary and sufficient for VP1 binding, while biochemical experiments show that VP3 attachment has no effect on polymerase activity. These results indicate how VP3 recruits the polymerase into birnavirus capsids during morphogenesis.     

Rotavirus RNA polymerase requires the core shell protein to synthesize the double-stranded RNA genome.

Rotavirus cores contain the double-stranded RNA (dsRNA) genome, RNA polymerase VP1, and guanylyltransferase VP3 and are enclosed within a lattice formed by the RNA-binding protein VP2. Analysis of baculovirus-expressed core-like particles (CLPs) has shown that VP1 and VP2 assemble into the simplest core-like structures with replicase activity and that VP1, but not VP3, is essential for replicase activity. To further define the role of VP1 and VP2 in the synthesis of dsRNA from viral mRNA, recombinant baculoviruses containing gene 1 (rBVg1) and gene 2 (rBVg2) of SA11 rotavirus were generated and used to express recombinant VP1 (rVP1) and rVP2, respectively. After purification, the proteins were assayed individually and together for the ability to catalyze the synthesis of dsRNA in a cell-free replication system. The results showed that dsRNA was synthesized only in assays containing rVP1 and rVP2, thus establishing that both proteins are essential for replicase activity. Even in assays containing a primer-linked mRNA template, neither rVP1 nor rVP2 alone directed RNA synthesis. Characterization of the cis-acting replication signals in mRNA recognized by the replicase of rVP1 and rVP2 showed that they were the same as those recognized by the replicase of virion-derived cores, thus excluding a role for VP3 in recognition of the mRNA template by the replicase. Analysis of RNA-protein interactions indicated that the mRNA template binds strongly to VP2 in replicase assays but that the majority of the dsRNA product neither is packaged nor stably associates with VP2. The results of replicase assays performed with mutant VP2 containing a deletion in its RNA-binding domain suggests that the essential role for VP2 in replication is linked to the protein's ability to bind the mRNA template for minus-strand synthesis.