Ebola virus VP40 drives the formation of virus-like filamentous particles along with GP

Using biochemical assays, it has been demonstrated that expression of Ebola virus VP40 alone in mammalian cells induced production of particles with a density similar to that of virions. To determine the morphological properties of these particles, cells expressing VP40 and the particles released from the cells were examined by electron microscopy. VP40 induced budding from the plasma membrane of filamentous particles, which differed in length but had uniform diameters of approximately 65 nm. When the Ebola virus glycoprotein (GP) responsible for receptor binding and membrane fusion was expressed in cells, we found pleomorphic particles budding from the plasma membrane. By contrast, when GP was coexpressed with VP40, GP was found on the filamentous particles induced by VP40. These results demonstrated the central role of VP40 in formation of the filamentous structure of Ebola virions and may suggest an interaction between VP40 and GP in morphogenesis.     

VP40 octamers are essential for Ebola virus replication

Matrix protein VP40 of Ebola virus is essential for virus assembly and budding. Monomeric VP40 can oligomerize in vitro into RNA binding octamers, and the crystal structure of octameric VP40 has revealed that residues Phe125 and Arg134 are the most important residues for the coordination of a short single-stranded RNA. Here we show that full-length wild-type VP40 octamers bind RNA upon HEK 293 cell expression. While the Phe125-to-Ala mutation resulted in reduced RNA binding, the Arg134-to-Ala mutation completely abolished RNA binding and thus octamer formation. The absence of octamer formation, however, does not affect virus-like particle (VLP) formation, as the VLPs generated from the expression of wild-type VP40 and mutated VP40 in HEK 293 cells showed similar morphology and abundance and no significant difference in size. These results strongly indicate that octameric VP40 is dispensable for VLP formation. The cellular localization of mutant VP40 was different from that of wild-type VP40. While wild-type VP40 was present in small patches predominantly at the plasma membrane, the octamer-negative mutants were found in larger aggregates at the periphery of the cell and in the perinuclear region. We next introduced the Arg134-to-Ala and/or the Phe125-to-Ala mutation into the Ebola virus genome. Recombinant wild-type virus and virus expressing the VP40 Phe125-to-Ala mutation were both rescued. In contrast, no recombinant virus expressing the VP40 Arg134-to-Ala mutation could be recovered. These results suggest that RNA binding of VP40 and therefore octamer formation are essential for the Ebola virus life cycle.     

Crystal structure of the matrix protein VP40 from Ebola virus

Ebola virus maturation occurs at the plasma membrane of infected cells and involves the clustering of the viral matrix protein VP40 at the assembly site as well as its interaction with the lipid bilayer. Here we report the X-ray crystal structure of VP40 from Ebola virus at 2.0 A resolution. The crystal structure reveals that Ebola virus VP40 is topologically distinct from all other known viral matrix proteins, consisting of two domains with unique folds, connected by a flexible linker. The C-terminal domain, which is absolutely required for membrane binding, contains large hydrophobic patches that may be involved in the interaction with lipid bilayers. Likewise, a highly basic region is shared between the two domains. The crystal structure reveals how the molecule may be able to switch from a monomeric conformation to a hexameric form, as observed in vitro. Its implications for the assembly process are discussed.     

Mapping of the VP40-binding regions of the nucleoprotein of Ebola virus

Expression of Ebola virus nucleoprotein (NP) in mammalian cells leads to the formation of helical structures, which serve as a scaffold for the nucleocapsid. We recently found that NP binding with the matrix protein VP40 is important for nucleocapsid incorporation into virions (T. Noda, H. Ebihara, Y. Muramoto, K. Fujii, A. Takada, H. Sagara, J. H. Kim, H. Kida, H. Feldmann, and Y. Kawaoka, PLoS Pathog. 2:e99, 2006). To identify the region(s) on the NP molecule required for VP40 binding, we examined the interaction of a series of NP deletion mutants with VP40 biochemically and ultrastructurally. We found that both termini of NP (amino acids 2 to 150 and 601 to 739) are essential for its interaction with VP40 and for its incorporation into virus-like particles (VLPs). We also found that the C terminus of NP is important for nucleocapsid incorporation into virions. Of interest is that the formation of NP helices, which involves the N-terminal 450 amino acids of NP, is dispensable for NP incorporation into VLPs. These findings enhance our understanding of Ebola virus assembly and in so doing move us closer to the identification of targets for the development of antiviral compounds to combat Ebola virus infection.     

Interdomain salt‐bridges in the Ebola virus protein VP40 and their role in domain association and plasma membrane localization

The Ebola virus protein VP40 is a transformer protein that possesses an extraordinary ability to accomplish multiple functions by transforming into various oligomeric conformations. The disengagement of the C‐terminal domain (CTD) from the N‐terminal domain (NTD) is a crucial step in the conformational transformations of VP40 from the dimeric form to the hexameric form or octameric ring structure. Here, we use various molecular dynamics (MD) simulations to investigate the dynamics of the VP40 protein and the roles of interdomain interactions that are important for the domain–domain association and dissociation, and report on experimental results of the behavior of mutant variants of VP40. The MD studies find that various salt‐bridge interactions modulate the VP40 domain dynamics by providing conformational specificity through interdomain interactions. The MD simulations reveal a novel salt‐bridge between D45‐K326 when the CTD participates in a latch‐like interaction with the NTD. The D45‐K326 salt‐bridge interaction is proposed to help domain–domain association, whereas the E76‐K291 interaction is important for stabilizing the closed‐form structure. The effects of the removal of important VP40 salt‐bridges on plasma membrane (PM) localization, VP40 oligomerization, and virus like particle (VLP) budding assays were investigated experimentally by live cell imaging using an EGFP‐tagged VP40 system. It is found that the mutations K291E and D45K show enhanced PM localization but D45K significantly reduced VLP formation.     

Ebola virus matrix protein VP40 interaction with human cellular factors Tsg101 and Nedd4

The Ebola virus matrix protein VP40 is a major viral structural protein and plays a central role in virus assembly and budding at the plasma membrane of infected cells. For efficient budding, a full amino terminus of VP40 is required, which includes a PPXY and a PT/SAP motif, both of which have been proposed to interact with cellular proteins. Here, we report that Ebola VP40 can interact with cellular factors human Nedd4 and Tsg101 in vitro. We show that WW domain 3 of human Nedd4 is necessary and sufficient for binding to the PPXY motif of VP40, which requires an oligomeric conformation of VP40. Single particle electron microscopy reconstructions indicate that WW3 of Nedd4 is in close contact with the N-terminal domain of hexameric VP40. In contrast, the ubiquitin enzyme variant domain of Tsg101 was sufficient for binding to the PT/SAP motif of VP40, regardless of the oligomeric state of the matrix protein. These results suggest that hNedd4 and Tsg101 may play complimentary roles at a late stage of the assembly process, by recruiting cellular factors of two independent pathways to the site of budding at the plasma membrane.     

Ebola virus matrix protein VP40 uses the COPII transport system for its intracellular transport

The Ebola virus matrix protein VP40 plays an important role in virion formation and viral egress from cells. However, the host cell proteins and mechanisms responsible for intracellular transport of VP40 prior to its contribution to virion formation remain to be elucidated. Therefore we used coimmunoprecipitation and mass spectrometric analyses to identify host proteins interacting with VP40. We found that Sec24C, a component of the host COPII vesicular transport system, interacts specifically with VP40 via VP40 amino acids 303 to 307. Coimmunoprecipitation and dominant-negative mutant studies indicated that the COPII transport system plays a critical role in VP40 intracellular transport to the plasma membrane. Marburg virus VP40 was also shown to use the COPII transport system for intracellular transport. These findings identify a conserved intersection between a host pathway and filovirus replication, an intersection that can be targeted in the development of new antiviral drugs.     

Molecular docking based screening of predicted potential inhibitors for VP40 from Ebola virus

Ebola virus is a member of Filoviridae and cause severe human disease with 90 percent mortality. The life cycle of Ebola contains an assembly stage which is mediated by VP40 proteins. VP40 subunits oligomerize and form ring-structures which are either octamers or hexamers. Prevention of VP40 matrix protein assembly prevents virus particle formation as well as virus budding. In the present study we simulated the biological condition for a single VP40 subunit. Then a library containing 120.000 drugs like chemicals was used as the virtual screening database. Top 10 successive hits were then analyzed regarding absorption, distribution, metabolism, and excretion properties. Moreover probable accessorial human protein targets and toxicity properties of successive hits were analyzed by in silico tools. We found 4 chemicals that could bind VP40 subunits in a manner that by making an interfering steric condense prevents matrix protein oligomerization. The pharmacokinetic and toxicity studies also validated the potential of 4 finlay successive hits to be considered as a new anti-Ebola drugs.     

Stable association of viral protein VP1 with simian virus 40 DNA.

Mild dissociation of simian virus 40 particles releases a 110S virion core nucleoprotein complex containing histones and the three viral proteins VP1, VP2, and VP3. The association of viral protein VP1 within this nucleoprotein complex is mediated at least partially through a strong interaction with the viral DNA. Treatment of the virion-derived 110S nucleoprotein complex with 0.25% Sarkosyl dissociated VP2, VP3, and histones, leaving a stable VP1-DNA complex. The VP1-DNA complex had a sedimentation value of 30S and a density of 1.460 g/cm3. The calculated molecular weight of the complex was 7.9 x 10(6), with an average of 100 VP1 molecules per DNA. Agarose gel electrophoresis of the VP1-DNA complex demonstrated that VP1 is associated not only with form I and form II simian virus 40 DNAs but also with form III simian virus 40 DNA generated by cleavage with EcoRI.     

The matrix protein VP40 from Ebola virus octamerizes into pore-like structures with specific RNA binding properties

The Ebola virus membrane-associated matrix protein VP40 is thought to be crucial for assembly and budding of virus particles. Here we present the crystal structure of a disk-shaped octameric form of VP40 formed by four antiparallel homodimers of the N-terminal domain. The octamer binds an RNA triribonucleotide containing the sequence 5'-U-G-A-3' through its inner pore surface, and its oligomerization and RNA binding properties are facilitated by two conformational changes when compared to monomeric VP40. The selective RNA interaction stabilizes the ring structure and confers in vitro SDS resistance to octameric VP40. SDS-resistant octameric VP40 is also found in Ebola virus-infected cells, which suggests that VP40 has an additional function in the life cycle of the virus besides promoting virus assembly and budding off the plasma membrane.     

Role for amino acids 212KLR214 of Ebola virus VP40 in assembly and budding

Ebola virus VP40 is able to produce virus-like particles (VLPs) in the absence of other viral proteins. At least three domains within VP40 are thought to be required for efficient VLP release: the late domain (L-domain), membrane association domain (M-domain), and self-interaction domain (I-domain). While the L-domain of Ebola VP40 has been well characterized, the exact mechanism by which VP40 mediates budding through the M- and I-domains remains unclear. To identify additional domains important for VP40 assembly/budding, amino acids (212)KLR(214) were targeted for mutagenesis based on the published crystal structure of VP40. These residues are part of a loop connecting two beta sheets in the C-terminal region and thus are potentially important for overall structure and/or oligomerization of VP40. A series of alanine substitutions were generated in the KLR region of VP40, and these mutants were examined for VLP budding, intracellular localization, and oligomerization. Our results indicated that (i) (212)KLR(214) residues of VP40 are important for efficient release of VP40 VLPs, with Leu213 being the most critical; (ii) VP40 KLR mutants displayed altered patterns of cellular localization compared to that of wild-type VP40 (VP40-WT); and (iii) self-assembly of VP40 KLR mutants into oligomers was altered compared to that of VP40-WT. These results suggest that (12)KLR(214) residues of VP40 are important for proper assembly/oligomerization of VP40 which subsequently leads to efficient budding of VLPs.     

Ebola virus VP40 late domains are not essential for viral replication in cell culture

Ebola virus particle formation and budding are mediated by the VP40 protein, which possesses overlapping PTAP and PPXY late domain motifs (7-PTAPPXY-13). These late domain motifs have also been found in the Gag proteins of retroviruses and the matrix proteins of rhabdo- and arenaviruses. While in vitro studies suggest a critical role for late domain motifs in the budding of these viruses, including Ebola virus, it remains unclear as to whether the VP40 late domains play a role in Ebola virus replication. Alteration of both late domain motifs drastically reduced VP40 particle formation in vitro. However, using reverse genetics, we were able to generate recombinant Ebola virus containing mutations in either or both of the late domains. Viruses containing mutations in one or both of their late domain motifs were attenuated by one log unit. Transmission and scanning electron microscopy did not reveal appreciable differences between the mutant and wild-type viruses released from infected cells. These findings indicate that the Ebola VP40 late domain motifs enhance virus replication but are not absolutely required for virus replication in cell culture.     

Kinetics of amphiphile association with two-phase lipid bilayer vesicles

We examined the consequences of membrane heterogeneity for the association of a simple amphiphilic molecule with phospholipid vesicles with solid-liquid and liquid-liquid phase coexistence. To address this problem we studied the association of a single-chain, fluorescent amphiphile with dimyristoylphosphatidylcholine (DMPC) vesicles containing varying amounts of cholesterol. DMPC bilayers containing 15 mol% cholesterol show a region of solid-liquid-ordered (s-l(o)) coexistence below the T(m) of pure DMPC (23.9 degrees C) and a region of liquid-disordered-liquid-ordered coexistence (l(d)-l(o)) above the T(m). We first examined equilibrium binding and kinetics of amphiphile insertion into single-phase vesicles (s, l(d), and l(o) phase). The data obtained were then used to predict the behavior of the equivalent process in a two-phase system, taking into account the fractions of phases present. Next, the predicted kinetics were compared to experimental kinetics obtained from a two-phase system. We found that association of the amphiphile with lipid vesicles is not influenced by the existence of l(d)-l(o) phase boundaries but occurs much more slowly in the s-l(o) phase coexistence region than expected on the basis of phase composition.     

Serological reactivity of baculovirus-expressed Ebola virus VP35 and nucleoproteins

Ebola virus (EBOV) is a member of the family Filoviridae and is classified as a biosafety level 4 virus. This classification makes the preparation of antigen and performance of diagnostic assays time-consuming and complicated. The objective of this study was to evaluate the value of EBOV immunoassays based on recombinant nucleoprotein (r-NP) and recombinant VP35 (r-VP35) using large serum panels of African origin and from primates. Furthermore, we investigated whether the results obtained with EBOV r-VP35 enzyme-linked immunosorbent assay (ELISA) could improve on the findings obtained with the EBOV r-NP ELISA. The full-length EBOV NP and VP35 of the EBOV subtype Zaire were expressed as histidine-tagged recombinant proteins in the baculovirus expression system. The antigenic reactivity and specificity of these recombinant proteins were determined by Western blotting and ELISA using EBOV specific monoclonal antibodies. The results obtained with the r-NP and r-VP35 ELISAs were compared with the results obtained in an indirect immunofluorescence assay based on native EBOV subtype Zaire. EBOV specific monoclonal antibodies reacted specifically with the respective proteins in both Western blot and ELISA. Five hundred and twenty six samples from humans and primates were tested with r-NP and r-VP35 ELISAs. Monkey serum samples positive for EBOV subtype Reston and Zaire were both positive in the EBOV r-NP ELISA, whereas only the EBOV Zaire infected monkeys were positive in the r-VP35 ELISA. The sensitivity and specificity values of the EBOV recombinants' ELISAs compared to those of the immunofluorescence assay were 92% and 99% for r-NP and 44% and 100% for r-VP35. r-NP ELISA proved to be a sensitive and specific assay for EBOV diagnosis and for epidemiological studies for both EBOV subtypes Reston and Zaire. The use of r-VP35 in an ELISA format has no additional value for EBOV serodiagnosis.