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Journal of Virology, October 2007, p. 11452-11460, Vol. 81, No. 20
0022-538X/07/$08.00+0     doi:10.1128/JVI.00853-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Role for Amino Acids ₂₁₂KLR₂₁₄ of Ebola Virus VP40 in Assembly and Budding

Sarah E. McCarthy,¹ Reed F. Johnson,¹ Yong-An Zhang,² J. Oriol Sunyer,² and Ronald N. Harty^1*

Laboratory 412,¹ Laboratory 413, Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, Pennsylvania 19104²

Received 20 April 2007/ Accepted 30 July 2007

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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 ₂₁₂KLR₂₁₄ 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) ₂₁₂KLR₂₁₄ 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 ₁₂KLR₂₁₄ residues of VP40 are important for proper assembly/oligomerization of VP40 which subsequently leads to efficient budding of VLPs.

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Ebola virus (EBOV) and Marburg virus are members of the negative-stranded RNA virus family Filoviridae (7). Filoviruses are associated with severe hemorrhagic fevers and high mortality and morbidity with fatality rates reaching 90% for EBOV Zaire strain. There are currently no approved vaccines or therapeutics for treatment of filovirus infections (8, 22).

The EBOV VP40 matrix protein is the most abundant protein in virions (6) and is able to produce virus-like particles (VLPs) in the absence of other viral proteins (13, 17, 24, 30). Ebola VP40 VLPs are virtually identical in size and morphology to infectious Ebola virions (3, 17, 18). VP40 contains overlapping late domains (L-domains) at the N terminus that interact with host proteins to mediate separation of newly made virions from the plasma membrane (13, 17, 21, 29). Previous work has shown that, in addition to the L-domain, VP40 possesses a membrane association domain(s) [M-domain(s)] and a self-interaction domain(s) [I-domain(s)] which have yet to be completely characterized. The putative M-domain and I-domain of Ebola VP40 are thought to be located in the C-terminal and N-terminal halves of the protein, respectively (5, 17, 25, 27, 29, 30). Structural studies by Dessen et al. elucidated the crystal structure of VP40 and demonstrated that the N- and C-terminal domains were structurally similar beta-sandwiches connected by a flexible linker consisting of residues 188 to 202 (5). Trypsin treatment of bacterially purified VP40 (residues 31 to 326) resulted in cleavage after Lys212 and dissociation of the N-terminal domain from the C-terminal domain (5, 27). Thus, the region of VP40 including Lys212 and surrounding residues may represent a bridging region between the N-and C-terminal domains and may be critical for overall structure and/or self-assembly of VP40.

Full-length VP40 exists as monomers in solution, while a C-terminally truncated VP40 (residues 31 to 212) spontaneously forms hexamers but does not associate with lipid membranes (27). However, in the presence of liposomes, full-length VP40 will form hexamers as well (28). This suggests that hexameric VP40 is important for assembly and budding of virus. Based upon these findings, the following working model for VP40 assembly has been proposed. Monomeric VP40 first binds to the cell membrane via the C terminus, and this membrane association leads to a conformational change in which the N-terminal domain becomes exposed and forms hexamers. Hexameric VP40 may form an organized lattice beneath the plasma membrane which leads to efficient assembly and budding of mature virions (5).

In this study, we sought to target individual residues predicted to be structurally relevant for VP40 and determine whether mutagenesis of these amino acids affected efficient budding of VP40 VLPs. We generated a series of VP40 mutants in which amino acids ₂₁₂KLR₂₁₄ were changed individually or in combination to alanine. We found that (i) VLP budding of the KLR mutants was defective compared to that of VP40-WT in a functional budding assay, (ii) intracellular localization of the KLR mutants was altered compared to VP40-WT as determined by confocal microscopy, and (iii) the oligomerization patterns of the KLR mutants were significantly different from that of VP40-WT as determined by cross-linking analysis and gel filtration. Taken together, these findings suggest that the ₂₁₂KLR₂₁₄ region of VP40 is important for proper oligomerization and assembly of VP40, leading to efficient VLP budding.

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Cells. Vero and 293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Mediatech) supplemented with 10% fetal calf serum (Invitrogen) and 1x penicillin-streptomycin (Invitrogen) at 5% CO₂ at 37°C.

Plasmids and antibodies. Plasmid pCAGGS VP40-WT has been described previously (20, 21). All VP40 KLR mutants were generated in pGEM T-Easy vector (Promega) using the QuikChange site-directed mutagenesis kit (Stratagene) and then subcloned into pCAGGS using the EcoRI and XhoI restriction sites. All mutations introduced into VP40 were confirmed by automated DNA sequencing. See Table 1 for oligonucleotide primer sequences. Monoclonal antisera against Ebola virus VP40 was generously provided by Roland Grunow. Polyclonal, antipeptide antiserum against Ebola virus VP40 amino acids 38 to 60 was generated by ProSci Incorporated (Poway, CA).

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TABLE 1. Sequences of oligonucleotide primers used to generate VP40 KLR mutants

VLP budding assay. Vero cells were transfected with equivalent amounts of the indicated plasmids using Hyfect transfection reagent (Denville Scientific) in OptiMem (Invitrogen). Cells were metabolically labeled 24 h posttransfection with 185 µCi of [³⁵S]methionine-cysteine (Perkin-Elmer). Six hours later, the medium was harvested, clarified, and layered over a 20% sucrose cushion in STE buffer (0.01 M Tris-HCl [pH 7.5], 0.01 M NaCl, 0.001 M EDTA [pH 8.0]), and this mixture was then centrifuged at 36,000 rpm for 2 h at 4°C in a Beckman SW41 rotor. The pellet was suspended in STE buffer and lysed with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]). Cells were washed twice with 1x phosphate-buffered saline (PBS) and then lysed in RIPA buffer. Both cells and VLPs were immunoprecipitated with monoclonal anti-VP40 antiserum and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE). VP40 in VLPs was quantified by phosphorimager analysis (Amersham).

Confocal microscopy. Vero cells were grown on coverslips and mock transfected or transfected with VP40-WT, KLR>ALR, KLR>KLA, KLR>ALA, KLR>KAR, KLR>KAA, KLR>AAR, or KLR>AAA as described above. Twenty-four hours posttransfection, cells were washed twice with 1x PBS and fixed with 1:1 acetone-methanol. The cells were then incubated with rabbit peptide anti-VP40 and washed twice with 1x PBS. Cells were then incubated with anti-rabbit antibody conjugated to Alexa Fluor 594 (Invitrogen/Molecular Probes), washed twice with 1x PBS, stained with 4',6'-diamidino-2-phenylindole (DAPI), washed four times for 5 min each time with 1x PBS, and affixed to slides with Prolong Antifade (Invitrogen/Molecular Probes). Confocal images were obtained using a Zeiss LSM-510 Meta confocal microscope.

Chemical cross-linking. The cross-linking assay was modified from the assay of Ruigrok et al. (27). Vero cells were transfected as described above with vector alone, VP40-WT, KLR>ALA, KLR>KAR, or KLR>AAA. Twenty-four hours posttransfection, cells were washed twice with 1x PBS, lysed with PBS containing 1% Triton X-100, separated into nine tubes, and treated with ethyleneglycol bis(succinimidylsuccinate) (EGS) (Pierce) in dimethyl sulfoxide (DMSO) as follows: untreated, DMSO, or 0.05 mM, 0.1 mM, 0.25 mM, 0.5 mM, 1 mM, 2 mM, or 5 mM EGS. Samples were incubated at room temperature for 20 min and then quenched with 50 mM Tris (pH 8.8) for 5 min at room temperature. Samples were then incubated at 95°C for 10 min in SDS loading buffer, then resolved on a 4 to 15% Tris-HCl gel (Bio-Rad) in SDS-PAGE running buffer, and transferred to Hybond-ECL nitrocellulose membrane (Amersham). The blot was probed with mouse monoclonal anti-VP40 and then with anti-mouse antibody conjugated to horseradish peroxidase (Amersham). Proteins were visualized by Western blotting using ECL detection reagents (Amersham) using an EpiChemi³ darkroom (UVP Bioimaging Systems).

Gel filtration. Human 293T cells were transfected as described above with vector alone, VP40-WT, KLR>AAA, and KLR>AAA M14A. Twenty-four hours posttransfection, cells were lysed with PBS containing 1% Triton X-100 and incubated at 4°C for 10 min. Cleared lysates were filtered through a 0.22-µm filter and separated on a Superdex-200 10/30 high-resolution, fast-performance liquid chromatography column (GE Healthcare) using the ÄKTA 10 purifier system (GE Healthcare). Eluted proteins were collected in 0.5-ml fractions and analyzed by SDS-PAGE and Western blotting with anti-VP40 monoclonal antibody as described above. The chromatogram plotting absorbance (280 nm) versus elution volume was generated with Unicorn software. Molecular mass standards depicted on the chromatogram had molecular masses of 670, 158, 44, and 17 kDa (Bio-Rad).

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VP40 residues ₂₁₂KLR₂₁₄ are necessary for efficient budding of VLPs. We first sought to determine whether the VP40 KLR residues were important for efficient release of VP40 VLPs. To achieve this, we constructed several point mutants of the KLR region, in which each residue was changed to alanine individually or in various combinations (Fig. 1) The KLR mutants were then assayed for their ability to bud using a functional VLP budding assay.

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FIG. 1. Schematic representation of VP40 mutants. The gray-colored half of the protein represents the N-terminal domain (amino acids 1 to 187), while the white-colored half represents the C-terminal domain (amino acids 203 to 326) connected by a flexible linker (5, 30).

Vero cells were mock transfected or transfected with VP40-WT or with each of the KLR mutants as indicated (Fig. 2A). Cells were radiolabeled at 24 h posttransfection for 6 hours. Twenty percent sucrose-purified VLPs and cell lysates were immunoprecipitated and resolved by SDS-PAGE (Fig. 2A). As expected, VP40 was not detected in mock-transfected VLPs or cells (Fig. 2A, lanes 1), whereas VP40-WT was released efficiently as a VLP and expressed in cells (lanes 2). Overall, none of the KLR mutants were able to bud as efficiently as VP40-WT (Fig. 2A and B). KLR mutants retaining the Leu213 (KLR>ALA, KLR>KLA, and KLR>ALR) were released at 27%, 34%, and 57% that of VP40-WT, respectively (Fig. 2B). Levels of VP40 mutants KLR>AAA (Fig. 2A, lane 3), KLR>KAA (lane 4), KLR>AAR (lane 5), and KLR>KAR (lane 8) in VLPs were less than 20% of VP40-WT at 13%, 16%, and 15%, respectively (Fig. 2B). All KLR mutants were found to be expressed in cell extracts (Fig. 2A, lanes 3 to 9). These data indicate that no one residue can restore budding to wild-type levels; however, retention of Leu213 correlated with enhanced levels of KLR mutant VLP release, particularly when paired with Arg214 (KLR>ALR) (Fig. 2B).

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FIG. 2. The 212KLR214 residues of VP40 are required for efficient budding of VLPs. (A) Vero cells were mock transfected (lanes 1), transfected with VP40-WT (VP40) (lanes 2), or transfected with each VP40 KLR mutant as indicated above the top gel (lanes 3 to 9), and radiolabeled. VLPs were isolated from culture supernatant and immunoprecipitated with anti-VP40 monoclonal antibody. Cell lysates were also immunoprecipitated with anti-VP40 monoclonal antibody. The positions of molecular mass markers (in kilodaltons) are shown to the left of the gels. (B) VP40-WT was set at 100%, and each VP40 KLR mutant is represented as a percentage of VP40-WT. The bars represent the averages ± standard errors of the means (error bars) for two independent experiments quantified by a phosphorimager.

In addition to the full-length KLR mutant proteins, we also detected smaller, presumably truncated forms of the KLR mutants at 45 kDa, 32.5 kDa, and 30 kDa (Fig. 2A, lanes 3 to 9, and Fig. 3A, lane 3). Truncated forms of VP40 have been observed by us and others previously, and the use of Met14 as an internal translational start site for Ebola virus VP40 has been reported (17). To elucidate the origin of these truncated forms of VP40 and determine whether these smaller species were causing the inefficient budding phenotype, and not the KLR>AAA mutation itself, we mutated Met14 to alanine in the context of the KLR>AAA mutant (Fig. 1). Human 293T cells were mock transfected or transfected with VP40-WT, KLR>AAA, or KLR>AAA M14A as indicated (Fig. 3A). Cells were radiolabeled 24 h posttransfection for 6 hours. Purified VLPs and cell lysates were immunoprecipitated with monoclonal VP40 antiserum and resolved by SDS-PAGE (Fig. 3A). As expected, VP40 was not detected in mock-transfected VLPs or cells (Fig. 3A, lanes 1), whereas VP40-WT was released efficiently as a VLP and expressed in cells (Fig. 3A, lanes 2). The KLR>AAA and KLR>AAA M14A mutants were both released at levels below 10% that of VP40-WT at 4% and 2%, respectively (Fig. 3A, lanes 3 and 4). Full-length KLR>AAA and KLR>AAA M14A were expressed in the cell fractions; however, synthesis of the 45-kDa and 30-kDa species was abolished in the KLR>AAA M14A sample (Fig. 3A, lanes 3 and 4). These data suggest that (i) translation of the 45-kDa species is initiated at internal Met14 and is abrogated by the M14A mutation; (ii) the 32.5-kDa species likely represents a degradation product of full-length VP40, while the 30-kDa species likely represents a similar degradation product of the 45-kDa species; and (iii) the 45-kDa and 30-kDa species are not responsible for the inefficient VLP budding phenotype. The precise origin of the 32.5-kDa species remains to be determined. It should be noted that the phenotype exhibited by the KLR>AAA M14A mutant in all assays was identical to that exhibited by the KLR>AAA mutant.

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FIG. 3. Budding assay for VP40-WT and mutants KLR>AAA M14A and KLR>KIR. Human 293T cells were mock transfected (lanes 1) or transfected with VP40-WT (VP40) (lanes 2), KLR>AAA (lanes 3), and KLR>AAA M14A (lanes 4 in panel A) or (B) KLR>KIR (lanes 4 in panel B). Radiolabeled proteins were immunoprecipitated with monoclonal VP40 antiserum from cell lysates and VLPs. The positions of molecular mass markers (in kilodaltons) are shown to the left of the gels in panel A. Protein levels in VLPs were quantified by phosphorimager analysis. VP40-WT was set at 100%, and the VP40 KLR mutants are represented as a percentage of VP40-WT. The bars represent the averages ± standard errors of the means (error bars) for three independent experiments.

To determine the importance of the Leu213 in VLP budding, we constructed a mutant containing the conservative change from leucine to isoleucine at position 213 (mutant KLR>KIR) (Fig. 1). Human 293T cells were mock transfected or transfected with VP40-WT, KLR>AAA, or KLR>KIR as indicated (Fig. 3B). Cells were radiolabeled 24 h posttransfection for 6 hours. Purified VLPs and cell lysates were immunoprecipitated with monoclonal VP40 antiserum and resolved by SDS-PAGE (Fig. 3B). As expected, VP40 was not detected in mock-transfected VLPs or cells (Fig. 3B, lanes 1), whereas VP40-WT was released efficiently as a VLP and expressed in cells (Fig. 3B, lanes 2). As expected, the KLR>AAA mutant was released inefficiently at 3% that of VP40-WT (Fig. 3B), whereas the KLR>KIR mutant was released on average at 61% that of VP40-WT (Fig. 3B). Both the KLR>AAA and KLR>KIR mutants were expressed equally well in cell extracts (Fig. 3B, lanes 3 and 4). These data suggest that a conservative amino acid change at position 213 is well tolerated regarding budding efficiency, and thus, the identity of the amino acid at position 213 of VP40 is likely important for efficient budding of VLPs.

Mutation of residues ₂₁₂KLR₂₁₄ results in altered localization of VP40. Since mutations in the KLR region of VP40 were detrimental to VLP budding, we sought to determine whether intracellular localization of the KLR mutants was altered compared to that of VP40-WT. Vero cells were mock transfected or transfected with VP40-WT, KLR>ALR, KLR>KLA, KLR>ALA, KLR>KAR, KLR>KAA, KLR>AAR, and KLR>AAA. After fixation, cells were incubated with a rabbit anti-VP40 peptide antibody followed by anti-rabbit antibody conjugated to Alexa Fluor 594 and staining with DAPI. Confocal microscopy revealed the typical staining pattern and localization of VP40-WT primarily at the plasma membrane (Fig. 4) (19, 20, 25). Interestingly, mutants that retained Leu213 (KLR>ALA, KLR>KLA, and KLR>ALR) all displayed staining patterns similar to that observed for VP40-WT, albeit with somewhat more staining in the cytoplasm (Fig. 4). In contrast, the L213A VP40 mutants KLR>AAA, KLR>KAA, KLR>AAR, and KLR>KAR displayed an altered localization pattern compared to that of VP40-WT (Fig. 4). Indeed, mutants lacking Leu213 displayed some localization at the plasma membrane; however, they were clearly visible in a punctuate and diffuse pattern throughout the cytoplasm (Fig. 4). The KLR mutants showing a punctate staining pattern did not colocalize to late endosomes as determined by costaining with LAMP-3 (S. E. McCarthy and R. N. Harty, data not shown). Thus, the KLR mutants displaying altered intracellular localization were also those most severely defective in VLP budding.

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FIG. 4. Mutation of VP40 212KLR214 results in altered cellular localization. Vero cells were transfected with either VP40 WT or each of the VP40 KLR mutants as indicated. Cells were fixed, incubated with rabbit VP40 peptide antibody, secondary anti-rabbit antibody (red), and DAPI (blue), and visualized by confocal microscopy.

Oligomerization of the VP40 KLR mutants. To determine whether the KLR mutations were having an effect on the ability of VP40 to oligomerize, and therefore preventing efficient budding, we performed cross-linking experiments using EGS. Vero cells were transfected with vector alone, VP40-WT, and representative mutants KLR>ALA, KLR>KAR, and KLR>AAA. Twenty-four hours posttransfection, cells were washed and lysed in PBS containing 1% Triton X-100. Cell debris was pelleted, and the cleared lysate was separated into nine fractions that were then either untreated or treated with DMSO or with increasing concentrations of EGS in DMSO as indicated (Fig. 5).

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FIG. 5. Chemical cross-linking of VP40-WT and KLR mutants. Vero cells were transfected with VP40-WT (VP40), KLR>ALA, KLR>KAR, or KLR>AAA. Cells were lysed in PBS containing 1% Triton X-100, and cleared lysate was separated into nine fractions and either untreated (Mock) (lanes 1), treated with DMSO (lanes 2), or treated with EGS at increasing concentrations (0.05 mM [lanes 3], 0.1 mM [lanes 4], 0.25 mM [lanes 5], 0.5 mM [lanes 6], 1 mM [lanes 7], 2 mM [lanes 8], and 5 mM [lanes 9]). Cross-linking reactions were quenched with 50 mM Tris (pH 8.8), resolved on a 4 to 15% Tris-HCl gel, and detected by Western blotting with monoclonal VP40 antisera. The positions of molecular mass markers (in kilodaltons) are indicated to the left of the gels. The positions of monomers, dimers, tetramers, and oligomers/aggregates (oligomer/aggr.) are shown to the right of the gels.

The oligomerization profiles for VP40-WT and KLR>ALA in the presence of increasing concentrations of cross-linker were virtually identical (Fig. 5). For example, monomers of each protein were present in the absence of EGS (Fig. 5, lanes 1 and 2), and proteins that correlated in size to dimers were evident in the presence of low concentrations of EGS (lanes 3 and 4). However, at 0.25 mM EGS, both dimer and monomer forms began to decrease with increasing amounts of EGS (Fig. 5, lanes 5 to 9).

In contrast to our findings with VP40-WT and KLR>ALA, mutants KLR>AAA and KLR>KAR displayed dramatically different oligomerization patterns in the presence of increasing amounts of EGS cross-linker (Fig. 5). For example, monomeric forms of these mutants were present in both untreated and DMSO-treated samples (Fig. 5, lanes 1 and 2), and both monomeric and dimeric forms were present in samples containing low concentrations of EGS (0.05 mM and 0.1 mM) (Fig. 5, lanes 3 and 4). As the concentration of EGS increased, the monomeric and dimeric forms decreased, and a prominent high-molecular-weight (high-MW) species became evident (Fig. 5, lanes 5 to 9). Thus, the oligomerization pattern of mutants KLR>AAA and KLR>KAR in the presence of EGS was clearly altered compared to that exhibited by VP40-WT and the leucine-retaining mutant KLR>ALA. It should be noted that additional mutants (KLR>KAA and KLR>AAR) were employed in this assay and displayed an oligomerization pattern similar to that of KLR>AAA and KLR>KAR (McCarthy and Harty, data not shown). In contrast, the oligomerization patterns of mutants KLR>KLA and KLR>ALR were found to be similar to those of VP40-WT and KLR>ALA (McCarthy and Harty, data not shown).

To further explore oligomerization of the KLR>AAA mutant and compare it to that of VP40-WT, we performed gel filtration analysis. 293T cells were mock transfected or transfected with VP40-WT, KLR>AAA, and KLR>AAA M14A as indicated (Fig. 6). Twenty-four hours posttransfection, cells were lysed with PBS containing 1% Triton X-100, clarified, filtered through a 0.22-µm filter, and separated by using a Superdex-200 10/30 high-resolution fast-performance liquid chromatography column (GE Healthcare) and ÄKTA 10 purifier system (GE Healthcare). Eluted proteins were collected in 0.5-ml fractions and analyzed by SDS-PAGE and Western blotting with monoclonal VP40 antisera (Fig. 6). As expected, VP40 was not detected in the mock control (Fig. 6). VP40-WT eluted from the column in two peaks, one at elution volumes 8.0 to 8.5 ml and the second at 13.5 to 15.5 ml (Fig. 6). The peak at elution volumes 8.0 to 8.5 ml represents high-MW species (oligomers), whereas the peak at elution volumes 13.5 to 15.5 ml represents monomeric species (Fig. 6). Interestingly, a single prominent peak at elution volumes 8.0 to 8.5 ml was observed for both the KLR>AAA and KLR>AAA M14A mutants (Fig. 6). These data suggest that the concentration of KLR>AAA and KLR>AAA M14A monomers in cells is significantly different from that of VP40-WT. Furthermore, these data suggest that the kinetics of oligomerization of VP40-WT is likely different from that of the mutants. In sum, these findings correlate well with those obtained from the cross-linking experiments (Fig. 5) and suggest that the KLR region of VP40 is important for proper assembly (i.e., oligomerization) and subsequent release of VLPs.

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FIG. 6. Gel filtration analyses of VP40-WT and KLR mutants. Human 293T cells were either mock transfected or transfected with VP40-WT, KLR>AAA, or KLR>AAA M14A as indicated. Lysates were separated by size on a Superdex-200 10/30 high-resolution fast-performance liquid chromatography column. The chromatogram for the VP40-WT elution is shown as absorbance (Abs.) (280 nm) versus elution volume. Additionally, molecular mass standards (670, 158, 44, and 17 kDa) were plotted along the dashed line as log values versus elution volume. Western blot analysis for each sample is shown. A protein peak at 8.0 and 8.5 ml corresponds to high-MW oligomers. A peak at 13.5 to 15.5 ml roughly corresponds to monomer species.

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The importance of VP40 in assembly and budding of VLPs and infectious virus has been well studied (13, 17, 21, 24, 30). However, the exact mechanism of VP40-mediated budding is still largely unknown despite many studies examining the structure and function of VP40 both in vitro and in vivo (reviewed in references 12 and 16). In this report, we focused on residues Lys212, Leu213, and Arg214 located in the C-terminal half of VP40 as part of a loop between beta sheets 7 and 8. Lys212 and Arg214 are part of an exposed electrostatic patch on the surface of the protein (5). Lys212 is also the trypsin site that, when cleaved, causes the dissociation of the two domains (5). We asked whether ₂₁₂KLR₂₁₄ residues were important for VP40 assembly and/or oligomerization and therefore budding of VP40 VLPs.

Mutation of all of the ₂₁₂KLR₂₁₄ residues to alanine resulted in budding of VLPs to levels that were only 13% that of VP40-WT (Fig. 1 and 2B). This significant decrease in budding led us to conclude that these residues were necessary for efficient VP40 VLP budding. In addition to these single alanine substitutions, we also generated a series of combination mutants (Fig. 1). Using these mutants in a functional budding assay, we found that retention of Leu213 allowed for VLP budding of 27% (KLR>ALA), 34% (KLR>KLA), and 57% (KLR>ALR) that of VP40-WT. While retention of Leu213 seemed to be most critical for partial restoration of budding to levels approaching those of VP40-WT, the additional presence of either Lys212 or Arg214 further rescued budding of VLPs (Fig. 2B). Additionally, the conservative mutation of Leu213 to Ile resulted in VLP production up to 70% that of VP40-WT, suggesting that the identity of amino acid 213 is likely important for overall structure and/or proper oligomerization of VP40 (Fig. 3B).

At least three functional domains are likely to be required for efficient budding of VP40 VLPs, the L-domain, required for binding to cellular factors, the M-domain, and the oligomerization domain (5, 13, 17, 21, 25, 27, 29, 30). The role of the L-domain of VP40 in host interactions and virus budding has been well documented (13, 17, 21, 29). However, precise mapping of the M- and I-domains of VP40 remains to be done. Recent evidence suggests that the M-domain is contained within the C terminus, while the I-domain is in the N terminus (17, 25, 27, 29, 30). Truncated VP40 consisting of the N-terminal domain (residues 31 to 212) (VP40 31-212) and missing most of the C-terminal domain did not associate with lipid membranes (30). VP40 hexamer formation is thought to be important for VLP budding due to the formation of a hexamer conformation upon membrane binding of VP40 31-326 as well as the formation of hexamer rings in solution of VP40 31-212 (27, 28). However, the effect of abrogation of hexamer formation of VP40 on budding of VLPs has yet to be determined. Destabilization of VP40 monomer by deletion of the seven C-terminal residues which are located in the interdomain interface between the two domains (VP40 31-319) resulted in detection of a monomers, dimers, trimers, tetramers, pentamers, and hexamers, and higher-MW species by cross-linking (28). These results were also produced for VP40 31-212 as well as VP40 31-326 destabilized by urea and VP40 31-326 bound to liposomes (27, 28).

Our cross-linking data with the VP40 KLR mutants lacking Leu213 suggest that the ₂₁₂KLR₂₁₄ sequence is involved in proper oligomerization, which likely affects VLP budding efficiency (Fig. 5). In addition to the cross-linking data, the gel filtration analysis of the VP40 KLR>AAA mutant shows that the KLR>AAA mutation results in detection of primarily high-MW oligomers, whereas VP40-WT was present in fractions corresponding to monomers and high-MW oligomers (Fig. 6). These findings suggest that the KLR region is important for proper formation of VP40 oligomers, which is subsequently important for efficient budding of VP40 VLPs.

Retention of the ₂₁₂KLR₂₁₄ residues, particularly Leu213, is necessary for proper cellular localization. VP40-WT localizes primarily to the plasma membrane as expected (Fig. 4) (19, 20, 25). The KLR mutants that retain Leu213 also show similar plasma membrane localization; however, the extent of membrane association appears to be weaker than that of VP40-WT with a more diffuse cytoplasmic staining. Moreover, when Leu213 is mutated to alanine, the intracellular localization pattern is even more dramatically different from that of VP40-WT, with these mutants displaying a more punctuate staining pattern (Fig. 4). It appears that without the entire ₂₁₂KLR₂₁₄ sequence or at least the Leu213, VP40 forms small aggregates that are more loosely associated with the plasma membrane. These findings correlate well with our cross-linking and gel filtration data in that VP40 likely relies on an ordered sequence of events, including tight membrane association and self-assembly to achieve efficient membrane curvature and VLP egress. Mutations within the ₂₁₂KLR₂₁₄ sequence appear to disrupt this ordered series of events (e.g., assembly/oligomerization), leading to a decrease in VLP budding.

In sum, the data presented here and those published by others (5, 17, 23, 25, 28, 29) support our working model for VP40 assembly and budding as illustrated in Fig. 7. Our model proposes that VP40 interacts with the plasma membrane, inducing a conformational change that exposes the N-terminal domain. The N-terminal domain then forms oligomers by interacting with other VP40 N termini. An ordered lattice forms at the plasma membrane, leading to protrusion of the membrane and eventual budding of VP40-WT VLPs (Fig. 7A). Mutations in the ₂₁₂KLR₂₁₄ region of VP40 likely result in formation of a more open conformation of VP40 before it interacts with the plasma membrane. A portion of the VP40 in the cell associates with the plasma membrane; however, either (i) the amount of VP40 associated with the membrane and forming oligomers is too small to induce budding, or (ii) the VP40 lattice that does form is unordered and is not sufficient to induce budding (Fig. 7B).

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FIG. 7. Working model for the role of 212KLR214 residues in assembly/budding of VP40-WT (WT) (A) and mutant KLR>AAA (B). N and C, N- and C-terminal domains, respectively; PM, plasma membrane.

Other negative-stranded RNA virus matrix proteins have been shown to self-associate and form polymers. The matrix protein (M) of vesicular stomatitis virus, a rhabdovirus, forms polymers building upon a three- to four-molecule nucleation site which may be important for virus budding (9-11). Sendai virus and influenza virus matrix proteins have also been shown to form helical polymers and a lattice structure at the cell membrane (2, 4, 14, 15, 26). Last, the human immunodeficiency virus type 1 matrix protein has been shown recently to form hexamer rings when bound to a lipid membrane (1).

It will be of interest to further dissect the exact role of the ₂₁₂KLR₂₁₄ region of VP40 in VLP budding. Experiments are currently under way to determine the exact oligomeric forms of the KLR mutants and the effect of membrane association on oligomerization of these mutants. These findings will provide insight into the mechanism of VP40 VLP assembly and budding and may contribute to design of inhibitors of Ebola virus egress.

 
We thank members of the Harty lab for critical reviews of the manuscript. We also thank Jasmine Zhao of the Biomedical Imaging Core for assistance with confocal microscopy.

This work was supported by NIH grants to R.N.H.

 
* Corresponding author. Mailing address: Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104. Phone: (215) 573-4485. Fax: (215) 898-7887. E-mail: rharty{at}vet.upenn.edu

Published ahead of print on 15 August 2007.

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Journal of Virology, October 2007, p. 11452-11460, Vol. 81, No. 20
0022-538X/07/$08.00+0     doi:10.1128/JVI.00853-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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