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Mapping of a Region of Ebola Virus VP40 That Is Important in the Production of Virus-Like Particles

  1. Seiya Yamayoshi1,3 and
  2. Yoshihiro Kawaoka1,2,3,4
  1. 1 Division of Virology, Department of Microbiology and Immunology, University of Tokyo, Tokyo
  2. 2 International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo
  3. 3 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Saitama, Japan
  4. 4 Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison
  1. Reprints or correspondence: Dr. Yoshihiro Kawaoka, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan (kawaoka{at}ims.u-tokyo.ac.jp).

  1. Presented in part: Filoviruses: Recent Advances and Future Challenges, International Centre for Infectious Diseases Symposium, Winnipeg, Manitoba, Canada, 17–19 September 2006 (poster 35).

 
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Abstract

Ebola virus VP40 contains 2 overlapping late domains (7-PTAP-10 and 10-PPEY-13) that are essential for its interaction with Tsg101 and Nedd4 in the promotion of viral egress. Deletion of the late domains inhibits VP40-induced virus-like particles (VLPs). However, a truncated form of VP40, which lacks a late domain because of the deletion of amino acids 1–30, is released into supernatant as a VLP, indicating that the remaining portion of VP40 contains the structural elements required for VLP release. Thus, the purpose of this study was to identify the VP40 sequence essential for VLP budding, through the generation of deletion and alaninescanning mutants. We found that the amino acid sequence around the proline at position 53 plays a critical role in VLP production and intracellular transport. These data also may suggest that a novel host factor(s) is involved in virus budding.

Ebola virus (EBOV) and Marburg virus comprise the family Filoviridae in the order Mononegavirales. These viruses cause highly lethal hemorrhagic fever with extremely high mortality rates among humans and nonhuman primates [1, 2]. EBOV has a nonsegmented, negative-stranded RNA genome that encodes 7 structural proteins [1, 3]. VP40 is the major matrix protein and is essential for virus assembly and release fromhost cells [4–8]. During assembly, VP40 interacts with nucleoprotein to form, together with VP35 and VP24, nucleocapsid-like structures. These nucleocapsid-like structures are incorporated into VP40-induced virus-like particles (VLPs) [8]. VP40 also interacts with VP35 to incorporate EBOV minigenome RNA into the VLPs [7]. Expression of VP40 in mammalian cells is sufficient for the production of VLPs that resemble authentic EBOV.

Recent studies have found that EBOV VP40 contains 2 overlapping late domains, the 7-PTAP-10 and 10-PPEY-13 motifs [9–12]. A late domain was identified originally in the retroviral Gag protein as a region important for a late step in virus budding [13]. The PTAP motif is necessary for the interaction of VP40 with Tsg101, a component of endosomal sorting complex required for transport—1, and the PPxY motif is responsible for the interaction of VP40 with Nedd4, an ubiquitin ligase, via itsWWdomain. These interactions are important for VLP release. Indeed, VP40 that lacks the late domains fails to produce VLPs [9, 14]. However, Timmins et al. [6] showed that the EBOV VP40 mutant VP40(31–326), which lacks amino acids 1–30 (including the late domains), is released into supernatant as VLPs, although the efficiency of VLP release is reduced to ∼20%, compared with that for wild-type EBOV VP40. Moreover, Neumann et al. [14] reported that EBOV VP40 late domains are not essential for virus growth in cell culture, although virus with mutations in the late domains was found to be attenuated by 1 log unit. Together, these reports suggest that EBOV VP40 may have other structural elements required for VLP release. To better understand the role of these structural elements in EBOV budding, we mapped the amino acid residues involved in VP40-mediated VLP production.

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Materials and Methods

Cells. Human embryonic kidney cells (293 and 293T cells) were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fatal calf serum and a penicillin-streptomycin solution (Sigma).

Plasmids. The VP40 coding sequence of the Mayinga strain of Zaire EBOV was inserted into mammalian expression vector pCAGGS with and without a FLAG or cMyc tag at the amino terminus. The resulting constructs were designated “pCAVP40,” “pCA-FLAG-VP40,” and “pCA-cMyc-VP40.” N-terminal deletion constructs of VP40 with a FLAG tag at the amino terminus were generated by polymerase chain reaction (PCR) using the appropriate primers, and PCR products were cloned into pCAGGS. The resulting constructs were designated “pCA-FLAG-VP40_41-326,” “pCA-FLAG-VP40_51-326,” “pCA-FLAG-VP40_53-326,” “pCA-FLAG-VP40_56-326,” and “pCA-FLAG-VP40_61-326” (figure 1A). A series of double alanine-scanning mutants was generated by use of primerbased, site-directed mutagenesis, to replace the indicated amino acid with alanine or glycine (figure 2A). A series of single alanine (or glycine)-scanning mutants was similarly generated by replacement of each amino acid with alanine or glycine, resulting in constructs designated “pCA-VP40_L51A,” “pCA-VP40_ R52A,” “pCA-VP40_P53A,” “pCA-VP40_I54A,” “pCA-VP40_A55G,” and “pCA-VP40_D56A” (figure 3A). A triple-substitution mutant with a FLAG or cMyc tag at the amino terminus was generated by replacement of 52R and 53P with alanine and 55A with glycine, resulting in constructs designated “pCA-FLAG-VP40_525355” or “pCA-cMyc-VP40_525355” (figure 4A).

Figure 1
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Figure 1

Determination of the critical region in the N-terminus of Ebola virus VP40 for the production of virus-like particles (VLPs). A, Schematic diagrams of N-terminal deletion mutants of VP40. A FLAG tag was added at the N-terminus of the open reading frame. B, Results of transfection of 293T cells with a plasmid expressing either FLAG-tagged VP40 or FLAG-tagged deletion mutants of VP40. The deletion of ⩽50 aa from the N-terminus (pCA-FLAG-VP40_51-326) did not prevent VLP production. Western blot analysis of culture supernatant and transfected 293T cell lysates was done with an anti-FLAG antibody.

Figure 2
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Figure 2

Importance of amino acids 51–56 of Ebola virus VP40, in the efficient release of virus-like particles (VLPs). A, Schematic diagram of alanine and glycine scanning mutants. B, Comparison of the efficiency of VLP production among VP40 mutants. The efficiency of VLP release was compared by detection of VLPs in culture supernatant, by use of an anti-VP40 antibody, as described for figure 1. The intensity of the VP40 bands was quantified, and the amount of VP40 in lane 1 was set to 100%. Results are representative of 3 independent experiments. The bands marked by open circles represent full-length VP40. C, Intracellular localization of VP40 mutants. Transfection of 293 cells was done with a plasmid encoding wild-type VP40 or mutant VP40. After being fixed and permeabilized, transfected cells were stained with an anti-VP40 antibody (red).

Figure 3
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Figure 3

Importance of proline at amino acid 53 of Ebola virus VP40, in the release of virus-like particles (VLPs). A, Schematic diagram of single alanine-scanning mutant of VP40. B, Comparison of the efficiency of VLP production among single-alanine mutants. Experiments were performed as described for figure 1. The intensity of the VP40 bands was quantified, and the amount of VP40 in lane 1 was set to 100%. Results are representative of 3 independent experiments. C, Proline-to-alanine substitution at position 53 alters the intracellular localization of VP40. Transfection of 293 cells was done with a plasmid encoding a singlealanine mutant of VP40. After being fixed, transfected cells were stained with an anti-VP40 antibody (red). Nuclei were stained by 4′,6′-diamidino- 2-phenylindole (blue).

Figure 4
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Figure 4

Amino acids 52, 53, and 55 of Ebola virus VP40 not important for oligomerization. A, Schematic diagram of the VP40 mutant with 3 amino acid substitutions. B, Oligomerization of VP40 not negatively affected by amino acid substitutions at positions 52, 53, and 55. Cotransfection of 293T cells was done with plasmids encoding either pCA-FLAG-VP40 or pCAFLAG-VP40_525355 and either pCA-cMyc-VP40 or pCA-cMyc-VP40_525355. Then, lysis and immunoprecipitation (IP) of transfected cells was done with an anti-FLAG antibody. Western blot analysis of precipitated proteins was done with either an anti-FLAG antibody or an anti-cMyc antibody.

Immunoprecipitation assay. The 293T cells were cotransfected with plasmids expressing pCA-FLAG-VP40 or pCA-FLAG-VP40_525355 and those expressing pCA-cMyc-VP40 or pCA-cMyc-VP40_525355. At 48 h after transfection, the cells were lysed in lysis buffer (50 mmol/L Tris-HCl [pH 7.5], 150 mmol/L NaCl, 1 mmol/L EDTA, 0.5% Nonidet P-40, and protease-inhibitor cocktail Complete Mini [Roche]) and were maintained for 60 min at 4°. After clarification by low-speed centrifugation, the supernatants were incubated with anti-FLAG M2 Affinity Gel (Sigma) overnight at 4°C. Then, a fraction of supernatant was mixed with tricine sample buffer (Invitrogen) and incubated for 10 min at 95°C. The FLAG beads were washed 3 times with lysis buffer, were suspended in tricine sample buffer, and then were incubated for 10 min at 95°C. After the FLAG beads were removed by centrifugation, the samples were subjected to SDS-PAGE, followed byWestern blot analysis using a rabbit anti-FLAG antibody (Sigma) or a rabbit anti-cMyc antibody (Sigma).

VLP-release assay. At 48 h after transfection of 293T cells with plasmids encoding FLAG-tagged VP40 or VP40 mutants, cell-culture medium was harvested and cleared of cell debris, and the cleared supernatants were layered onto a 20%-sucrose cushion. Then, ultracentrifugation was done in an SW55 rotor (Beckman) at 50,000 rpm for 2 h at 4°C. The pellet containing VLPs was suspended in PBS and mixed with tricine sample buffer. The VLP sample was incubated for 10min at 95°C before being resolved on 10%–20% tricine gels (Invitrogen). The plasmid- transfected cells also were suspended in PBS, mixed with tricine sample buffer, and resolved on 10%–20% tricine gels. Resolved proteins were detected with a rabbit anti-VP40 antibody or a mouse anti-FLAG antibody, by Western blot analysis.

IFA. The 293 cells were transfected with plasmids expressing wild-type VP40 or VP40 mutants. At 24 h after transfection, the cells were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.2% Triton X-100. Antigens were detected with a rabbit anti-VP40 antibody as the primary antibody, followed by detection with Alexa Fluor 594 goat anti-rabbit IgG (Invitrogen). In some experiments, nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI; Invitrogen). Slides were viewed by an LSM510 confocal microscope (Carl Zeiss).

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Results and Discussion

VP40 region important in VLP release. To determine the region important for VLP release in the VP40 mutant VP40(31–326), we conducted a VLP-release assay using a plasmid expressing FLAG-tagged wild-type VP40 or a FLAG-tagged deletion mutant (figure 1B). The level of protein detected decreased as the extent of the deletion increased, with the expression of pCA-FLAG-VP40_61-326 being the lowest. VLP production was detected with mutants lacking the N-terminal 50 residues but not with those lacking the first 53 or more residues. These data indicate that the N-terminal 50 residues (pCA-FLAG-VP40_51-326) of VP40 are not required for VLP production and that the region of VP40 that is important starts with the amino acids at positions 51L and 52R.

Importance of amino acid residues 51–56 of VP40, in VLP release. To pinpoint the residues important for VLP release, wild-type VP40 and a double alanine-scanning mutant were tested in the VLP-release assay (figure 2B). In the cell lysates, wild-type VP40, pCA-VP40_50A, and pCA-VP40_5758A were detected as single bands (lanes 1, 2, and 6, respectively). For the other mutants, the predicted molecular-weight band (indicated by an open circle), as well as other smaller bands, was detected; the smaller bands were likely to have been degradation products of the full-length protein. The efficiency of VLP production was reduced to 10% for pCA-VP40_5152A (lane 3) and to <1% for pCA-VP40_5354A (lane 4) and wild-type VP40. We also observed a slight reduction in VLP production (to 77%) with pCA-VP40_55G56A (lane 5). However, amino acids at positions 50, 57, and 58 were not found to have a large impact on VLP production.

To assess the intracellular localization of these VP40 mutants, 293 cells were transfected with plasmids encoding wild-type or mutant VP40 and were stained with an anti-VP40 antibody (figure 3C). Wild-type VP40 clearly localized to the plasma membrane, as reported elsewhere [15, 16]. pCA-VP40_50A and pCA-VP40_5758A also exhibited intracellular localization similar to that of wild-type VP40. However, pCA-VP40_5152A and pCA-VP40_5354A failed to associate with the plasma membrane. Although most pCA-VP40_55G56A also failed to localize to the plasma membrane, some did associate with the plasma membrane. These data indicate that amino acids 51–56 of VP40 are important for efficient VLP production and for accurate intracellular localization.

Importance of proline at position 53 of VP40, in efficient VLP production. To further pinpoint the amino acids important for efficient VLP release, wild-type VP40 and a single-alanine or -glycine substitution mutant were tested in the VLP-release assay (figure 3B). The 293T cells expressing wild-type VP40, pCA-VP40_L51A, pCA-VP40_I54A, or pCA-VP40_D56A were rounded up, and some detached from the tissue-culture surface; pCA-VP40_R52A, pCA-VP40_P53A, and pCA-VP40_A55G did not induce such effects (data not shown). Western blot analysis showed that all single-alanine or -glycine mutants, with the exception of pCA-VP40_P53A, induced VLP production as efficiently as did wild-type VP40.

We next examined the intracellular localization of the pCAVP40_P53A mutant, by expressing the VP40 mutants in 293 cells and staining them with an anti-VP40 antibody (red) and DAPI (blue) (figure 3C). pCA-VP40_L51A, pCA-VP40_R52A, pCAVP40_ I54A, pCA-VP40_A55G, and pCA-VP40_D56A, which induced VLP production as efficiently as did wild-type VP40, were detected immediately beneath the plasma membrane, as was wild-type VP40 (figure 2C). pCA-VP40_P53A, however, did not accumulate under the plasma membrane and exhibited punctuated staining in the cytoplasm. This pattern of intracellular localization was similar to that for pCA-VP40_5354A (figure 2C). These data indicate that proline at position 53 is essential for VLP release and plasma-membrane localization.

Amino acids at positions 52, 53, and 55 of VP40 not important for dimerization. For VLP production, VP40 must oligomerize [16]. Several reports have demonstrated that the N-terminus of VP40 is important for oligomerization [17–20]. Phenylalanine and arginine at positions 124 and 134, respectively, are responsible for RNA binding and oligomerization [21]. To determine whether the region that we identified as important for VP40-induced VLP production (i.e., amino acids at positions 51–56) is important for VP40 oligomerization, we substituted amino acids 52R, 53P, and 55A with alanine or glycine in a VP40 construct tagged at the N-terminus with either FLAG or cMyc and expressed this construct with similarly tagged wild-type VP40. Immunoprecipitation andWestern blot assays using FLAG or cMyc were done, as described for figure 4B. FLAG-tagged VP40 precipitated with cMyc-tagged wildtype VP40 and with pCA-cMyc-VP40_525355 (and vice versa). These data indicate that the region of amino acid positions 51–56 is not important for VP40 oligomerization.

In this study, we showed that amino acids at positions 51–326 of VP40 represent a minimum structural requirement for VLP production (figure 1B) and that 51L, 52R, 53P, 55A, and 56D are important for efficient VLP production (figures 2B and 3B). The amino acids at positions 52R, 53P, and 55A are conserved among all species of EBOV (i.e., Zaire, 51-LRPIA-55; Sudan, 51-MRPVA-55; and Reston, 51-MRPVA-55). This region appears to be important for VP40 intracellular transport, unlike the late domain; mutations in the former region abolished VP40 plasma-membrane association, whereas those in the latter region did not. Interestingly, the Gag protein of human immunodeficiency virus and the Z protein of Lassa virus also have sequences (21-LRPGG-25 and 75-LRPSA-79, respectively) similar to that of EBOV VP40 (51-LRPIA-55) near the N-terminus. This domain may interact with host factors involved in the intracellular transport of these matrix proteins. As demonstrated with the late domains in the matrix proteins of many enveloped viruses, the region that we identified in this study may exist in the matrix proteins of other enveloped viruses and may play a critical role in their life cycle. The finding that VP40-mediated VLP release requires not only the late domain but also other specific amino acids provides a better understanding of the virus-budding mechanism, as well as insight into novel host factors involved in the different steps of virus budding.

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Acknowledgments

We thank Susan Watson for editing the manuscript.

Supplement sponsorship. This article was published as part of a supplement entitled “Filoviruses: Recent Advances and Future Challenges,” sponsored by the Public Health Agency of Canada, the National Institutes of Health, the Canadian Institutes of Health Research, Cangene, CUH2A, Smith Carter, Hemisphere Engineering, Crucell, and the International Centre for Infectious Diseases.

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Footnotes

  • Potential conflicts of interest: none reported.

  • Financial support: Japanese Ministry of Education, Culture, Sports Science and Technology; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency; and US Public Health Service research grant and Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research program (award 1-U54-AI-057153), National Institute of Allergy and Infectious Diseases, National Institutes of Health. Supplement sponsorship is detailed in the Acknowledgments.

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  1. J Infect Dis. (2007) 196 (Supplement 2): S291-S295. doi: 10.1086/520595
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