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J Virol. May 2007; 81(9): 4895–4899.
Published online Feb 14, 2007. doi:  10.1128/JVI.02829-06
PMCID: PMC1900181

Interaction of Tsg101 with Marburg Virus VP40 Depends on the PPPY Motif, but Not the PT/SAP Motif as in the Case of Ebola Virus, and Tsg101 Plays a Critical Role in the Budding of Marburg Virus-Like Particles Induced by VP40, NP, and GP[down-pointing small open triangle]

Abstract

Marburg virus (MARV) VP40 is a matrix protein that can be released from mammalian cells in the form of virus-like particles (VLPs) and contains the PPPY sequence, which is an L-domain motif. Here, we demonstrate that the PPPY motif is important for VP40-induced VLP budding and that VLP production is significantly enhanced by coexpression of NP and GP. We show that Tsg101 interacts with VP40 depending on the presence of the PPPY motif, but not the PT/SAP motif as in the case of Ebola virus, and plays an important role in VLP budding. These findings provide new insights into the mechanism of MARV budding.

Marburg virus (MARV) is a member of the Filoviridae, a family of negative-sense RNA viruses which cause fatal hemorrhagic disease in both humans and nonhuman primates. At present, no approved vaccines or antiviral drugs are available to prevent and treat filoviral diseases.

The RNA genome of MARV encodes seven polypeptides, including the glycoprotein (GP), the nucleoprotein (NP), RNA-dependent RNA polymerase (L), VP35, VP30, VP40, and VP24. VP40 is the most abundant virion matrix protein and plays a key role in virus assembly and budding (15, 16, 31). GP is the only surface protein of filoviruses and is assumed to be responsible for binding to cellular receptors and for fusion of the viral envelope with the cellular membrane in the course of viral entry into the cells (2). The nucleocapsid complex, which contains NP, VP35, L, and VP30, encapsulates the viral genome.

Recent studies have indicated that viral matrix proteins play critical roles during the late stage of virus budding in many enveloped RNA viruses, including retro-, rhabdo-, filo-, arena-, and orthomyxoviruses, and when expressed alone in cells, they are released in the form of virus-like particles (VLPs). These viral proteins possess a so-called L-domain, containing PT/SAP, PPXY, and YPXL, which are motifs critical for efficient budding (3, 4, 6-13, 21, 25, 27, 30, 34-36, 39). Most of the host factors that interact with the L domain are involved in the class E vacuolar protein-sorting pathway, suggesting that budding into the lumen of multivesicular bodies (MVBs) in late endosomes and viral budding at the plasma membrane are topologically identical and share a common mechanism. MARV VP40 protein is sufficient for the release of VLPs (15, 16, 31) and contains a PPXY motif near its N terminus (Fig. (Fig.1B),1B), but the viral L domain and the cellular factors required for its budding have yet to be determined.

FIG. 1.
Analysis of the putative L domain within MARV VP40. (A) MARV VLPs produced by expression of VP40. At 24 h after transfection of 293T cells with pMV-VP40 (WT), filamentous particles bud from the membrane ruffles. Black arrows, longitudinally sectioned ...

To gain insight into the mechanism of MARV budding, we analyzed the function of the PPPY motif as an L domain as well as the cellular factors involved in VLP budding.

The PPPY motif within VP40 is important for efficient VLP production.

First, to confirm that the expression of MARV VP40 in cells can induce the budding of VLPs that are morphologically identical to virions, we constructed a VP40 expression vector for the wild type (WT), pMV-VP40, by insertion of the coding region of VP40, amplified by PCR using pTM-VP40 as a template, into the pCAAGS vector (16, 23) and analyzed the VP40-expressing cells by transmission electron microscopy (Fig. (Fig.1A).1A). MARV VP40 expression induced membrane ruffling and budding of filamentous particles, as shown previously (1, 17). These structures were not seen in cells transfected with the control plasmid (data not shown). To examine the role of the PPPY sequence within VP40 in MARV budding, we constructed expression vectors for VP40 mutants with single point mutations within the PPPY motif (Fig. (Fig.1B).1B). COS-7 cells (1 × 105) were transfected with each of these plasmids (1 μg) using TransIT-LT1 (Mirus Bio Corp., Madison, WI). At 48 h after transfection, the cell supernatants were cleared of cell debris by centrifugation (13,000 × g; 10 min), and then VLPs were pelleted through a 20% sucrose cushion by ultracentrifugation (345,000 × g; 30 min at 4°C). VLPs and cells were lysed with lysis A buffer (32, 36) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Western blotting (WB) using anti-VP40 antibody, which was prepared from rabbits immunized with the recombinant MARV VP40 protein. As shown in Fig. Fig.1C,1C, the levels of VP40-induced VLPs in the case of L-domain mutants, APPY, PAPY, and PPPA, were lower than that in the WT, although similar levels of VP40 were synthesized in cells expressing either the WT or an L-domain mutant. The efficiency of VLP production was different among the three L-domain mutants. Relative levels of production of VLPs from cells expressing APPY, PAPY, and PPPA were 72%, 52%, and 30% of the WT level, respectively (Fig. 1C and D). These results indicate that the PPPY motif functions as an L domain and that the tyrosine residue is the most critical for the budding function of this L domain. The PPXY motif has been reported to interact with cellular E3 ubiquitin ligases, such as Nedd4, LDI-1, and BUL1 (7, 14, 29, 33, 37). Although we examined the effects of overexpression of dominant-negative mutants of Nedd4 and BUL1 on MARV VP40-induced VLP budding, they had no effect on the efficiency of VLP budding (data not shown). These observations suggest the involvement of an E3 protein(s) other than Nedd4 and BUL1 in MARV budding.

Contributions of NP and GP to the VP40-induced VLP budding.

It has been reported that NP and GP enhance the release of Ebola VP40-induced VLPs (19). Therefore, we examined the contributions of NP and GP to MARV VP40-induced VLP budding. The expression plasmids for MARV NP and GP, pMV-NP and pMV-GP were constructed as described for pMV-VP40. COS-7 cells (1 × 105) were cotransfected with 0.5 μg of pMV-VP40 along with 0.5 μg of pMV-NP and/or 0.5 μg of pMV-GP. The levels of expression of cellular VP40 were approximately equivalent among all samples independent of coexpression of NP and/or GP, while the release of VLPs was markedly facilitated by coexpression of NP and GP (Fig. 2A and B).

FIG. 2.
Contributions of NP and GP to VP40-induced VLP budding. (A) COS-7 cells were transfected with pMV-VP40 (WT) in combination with pMV-NP, pMV-GP, and the control vector. Cell lysates and VLPs were collected, and WB using rabbit anti-GP, -NP, and -VP40 antibodies ...

Next, we examined whether MARV GP and NP also enhance VP40-induced VLP production in the context of an L-domain mutant, as Licata et al. showed previously that the increase in Ebola VP40-induced VLP budding by GP is independent of the viral L domain (19). COS-7 cells were cotransfected with pMV-VP40-PPPA along with pMV-NP and/or pMV-GP. The release of VLPs induced by the VP40-PPPA mutant was also enhanced by coexpression of NP and/or GP (Fig. 2C and D). However, the efficiency of VLP budding induced by the L-domain mutant in the presence of NP and GP was at most equal to that of VLP budding by sole expression of WT-VP40 and less than 10% of that induced by WT-VP40 in the presence of GP and NP (Fig. (Fig.1D1D and Fig. 2B and D). Taken together, these results show that the L domain of MARV VP40 is very important for efficient VLP budding even in the presence of GP and NP, suggesting that the L domain within VP40 also plays an important role in budding of MARV, as well as VLPs. Although we reported previously that the L domain of Ebola VP40 is not essential for viral replication (22), the results of the present study suggest that the L domain of MARV VP40 may affect the efficiency of MARV budding and also replication.

Tsg101 is involved in MARV VP40 budding.

To investigate the mechanism of MARV VP40-induced VLP budding in further detail, we examined the involvement of Tsg101—a component of the ESCRT-I complex known to participate in the cellular MVB sorting pathway and budding of some viruses—in MARV VLP budding using small interfering RNA (siRNA) (32). As shown in Fig. Fig.3A,3A, specific depletion of Tsg101 by siRNA significantly reduced the release of VP40-induced VLPs, indicating that Tsg101 is involved in VLP budding. We also examined whether exhaustion of Tsg101 has an effect on the budding of VLPs composed of VP40, GP, and NP. The VLP budding from cells coexpressing VP40, GP, and NP was also markedly decreased by specific depletion of Tsg101, although the reduction rate of VP40/GP/NP-induced VLP release by Tsg101 depletion was lower than that of VP40-induced VLPs (Fig. (Fig.3B).3B). These results clearly show that Tsg101 is involved in MARV VLP budding as a host factor.

FIG. 3.
Tsg101 is involved in MARV VP40-induced VLP budding. (A and B) 293T cells were pretreated with siRNA specific for Tsg101 (siTsg101) or control RNA (siCont) 1 day before plasmid transfection (5). The following day, these cells were transfected with pMV-VP40 ...

We further examined whether Tsg101 is incorporated into the VP40-induced VLPs. 293T cells (3 × 105) were cotransfected with pMV-VP40 (0.5 μg) or pMV-VP40, pMV-GP, and pMV-NP (0.5 μg each) along with pTsg101-Myc (1.5 μg), which expresses Myc-tagged Tsg101 (38). At 48 h after transfection, VLPs released from cells were collected. After quantification of VLPs by WB using anti-VP40 antibody, equal amounts of VLPs were assayed for Tsg101 incorporation. As shown in Fig. 3C and D, incorporation of Tsg101 into both VP40-induced and VP40/GP/NP-induced VLPs was observed. We also examined the incorporation of Tsg101 into VLPs in the context of the L-domain mutant VP40-PPPA. Interestingly, Tsg101 was observed at low levels or was completely absent in VLPs induced by VP40-PPPA (Fig. 3C and D), indicating that Tsg101 is incorporated into both VP40- and VP40/GP/NP-induced VLPs depending on the viral L-domain motif, PPPY.

Tsg101 has also been reported to be involved in the budding of human immunodeficiency virus type 1, human T-cell leukemia virus type 1, lymphocytic choriomeningitis virus (LCMV), Lassa virus, and Ebola virus. With the exception of LCMV, all these viruses possess the PT/SAP motif and interact with Tsg101 via this motif (3, 5, 20, 26, 32). Although LCMV does not have the PT/SAP motif, the STAP sequence, which is similar to PT/SAP, is present upstream of PPPY within the viral matrix Z protein. Previously, Licata et al. showed that Tsg101 was still incorporated into VLPs induced by the ATAP mutant of Ebola VP40, in which alanine is substituted for the first proline in the PTAP motif (18). In addition, it has also been reported that substitution of the first proline residue of the PTAP motif of human immunodeficiency virus p6 by alanine resulted in only moderate reduction of the binding affinity of p6 for Tsg101 (5). Therefore, in LCMV, the STAP sequence may function as an L domain instead of PT/SAP. However, there is no sequence similar to PT/SAP in MARV VP40.

We performed a glutathione S-transferase (GST) pulldown assay to examine whether Tsg101 interacts with VP40 depending on the PPPY motif. Recombinant GST-VP40 and GST-VP40ΔPPPY proteins were expressed in Escherichia coli BL21-Gold and purified using glutathione-Sepharose 4B beads (Amersham Biosciences, Uppsala, Sweden). The immobilized GST fusion proteins were incubated for 2 h at 4°C with 293T cell lysates overexpressing Tsg101-Myc, washed extensively, and eluted, followed by detection by WB. As shown in Fig. Fig.3E,3E, the interaction between VP40 and Tsg101 was confirmed, while VP40ΔPPPY abolished the ability to bind to Tsg101. Thus, we clearly showed that the interaction between Tsg101 and VP40 depends on the PPPY motif within VP40. This is the first report that Tsg101 interacts with viral matrix protein depending on PPPY, but not PT/SAP, and regulates viral budding. At present, it is not clear whether Tsg101 binds directly to the PPPY motif in VP40 or indirectly to VP40 via another cellular factor(s). Further studies are required to determine how Tsg101 interacts with MARV VP40 and participates in MARV budding.

It has been demonstrated that MARV VP40 interacts with the membranes of late endosomes in the course of viral infection (15). The transport of MARV VP40 involves its accumulation in MVBs followed by redistribution of VP40-enriched membrane clusters to the plasma membrane (16). Thus, VP40 is transported through the retrograde late-endosomal pathway, while GP is redistributed from the trans-Golgi network into the VP40-containing MVBs, suggesting that budding complexes assemble at late-endosomal surfaces and are then transported to the cell surface. MVBs would provide the platform for formation of membrane structures that bud MARV from the cell surface (17). Tsg101 is one of the components of ESCRT-I and is involved in the MVB sorting pathway. Therefore, it is plausible to suggest that Tsg101 recruits VP40 to MVB and participates in MARV budding there. Taken together, these results strongly suggest that MARV budding utilizes the cellular MVB sorting pathway.

The results of the present study provide important insights into the molecular aspects of MARV replication and will facilitate the development of anti-MARV therapy.

Acknowledgments

We thank H. D. Klenk and S. Becker (Philipps University, Marburg, Germany) for providing the plasmids used for construction of MARV VP40, GP, and NP expression vectors.

This work was supported by grants from the Japan Society for the Promotion of Science (JSPS) and the Japan Science and Technology Agency (JST).

Footnotes

[down-pointing small open triangle]Published ahead of print on 14 February 2007.

REFERENCES

1. Bamberg, S., L. Kolesnikova, P. Moller, H. D. Klenk, and S. Becker. 2005. VP24 of Marburg virus influences formation of infectious particles. J. Virol. 79:13421-13433. [PMC free article] [PubMed]
2. Becker, S., M. Spiess, and H. D. Klenk. 1995. The asialoglycoprotein receptor is a potential liver-specific receptor for Marburg virus. J. Gen. Virol. 76:393-399. [PubMed]
3. Bouamr, F., J. A. Melillo, M. Q. Wang, K. Nagashima, M. de Los Santos, A. Rein, and S. P. Goff. 2003. PPPYVEPTAP motif is the late domain of human T-cell leukemia virus type 1 Gag and mediates its functional interaction with cellular proteins Nedd4 and Tsg101. J. Virol. 77:11882-11895. [PMC free article] [PubMed]
4. Burleigh, L. M., L. J. Calder, J. J. Skehel, and D. A. Steinhauer. 2005. Influenza a viruses with mutations in the m1 helix six domain display a wide variety of morphological phenotypes. J. Virol. 79:1262-1270. [PMC free article] [PubMed]
5. Garrus, J. E., U. K. von Schwedler, O. W. Pornillos, S. G. Morham, K. H. Zavitz, H. E. Wang, D. A. Wettstein, K. M. Stray, M. Cote, R. L. Rich, D. G. Myszka, and W. I. Sundquist. 2001. Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell 107:55-65. [PubMed]
6. Gottlinger, H. G., T. Dorfman, J. G. Sodroski, and W. A. Haseltine. 1991. Effect of mutations affecting the p6 gag protein on human immunodeficiency virus particle release. Proc. Natl. Acad. Sci. USA 88:3195-3199. [PMC free article] [PubMed]
7. Harty, R. N., M. E. Brown, G. Wang, J. Huibregtse, and F. P. Hayes. 2000. A PPxY motif within the VP40 protein of Ebola virus interacts physically and functionally with a ubiquitin ligase: implications for filovirus budding. Proc. Natl. Acad. Sci. USA 97:13871-13876. [PMC free article] [PubMed]
8. Harty, R. N., J. Paragas, M. Sudol, and P. Palese. 1999. A proline-rich motif within the matrix protein of vesicular stomatitis virus and rabies virus interacts with WW domains of cellular proteins: implications for viral budding. J. Virol. 73:2921-2929. [PMC free article] [PubMed]
9. Huang, M., J. M. Orenstein, M. A. Martin, and E. O. Freed. 1995. p6Gag is required for particle production from full-length human immunodeficiency virus type 1 molecular clones expressing protease. J. Virol. 69:6810-6818. [PMC free article] [PubMed]
10. Hui, E. K., S. Barman, D. H. Tang, B. France, and D. P. Nayak. 2006. YRKL sequence of influenza virus M1 functions as the L domain motif and interacts with VPS28 and Cdc42. J. Virol. 80:2291-2308. [PMC free article] [PubMed]
11. Hui, E. K., S. Barman, T. Y. Yang, and D. P. Nayak. 2003. Basic residues of the helix six domain of influenza virus M1 involved in nuclear translocation of M1 can be replaced by PTAP and YPDL late assembly domain motifs. J. Virol. 77:7078-7092. [PMC free article] [PubMed]
12. Jasenosky, L. D., G. Neumann, I. Lukashevich, and Y. Kawaoka. 2001. Ebola virus VP40-induced particle formation and association with the lipid bilayer. J. Virol. 75:5205-5214. [PMC free article] [PubMed]
13. Jayakar, H. R., K. G. Murti, and M. A. Whitt. 2000. Mutations in the PPPY motif of vesicular stomatitis virus matrix protein reduce virus budding by inhibiting a late step in virion release. J. Virol. 74:9818-9827. [PMC free article] [PubMed]
14. Kikonyogo, A., F. Bouamr, M. L. Vana, Y. Xiang, A. Aiyar, C. Carter, and J. Leis. 2001. Proteins related to the Nedd4 family of ubiquitin protein ligases interact with the L domain of Rous sarcoma virus and are required for gag budding from cells. Proc. Natl. Acad. Sci. USA 98:11199-11204. [PMC free article] [PubMed]
15. Kolesnikova, L., H. Bugany, H. D. Klenk, and S. Becker. 2002. VP40, the matrix protein of Marburg virus, is associated with membranes of the late endosomal compartment. J. Virol. 76:1825-1838. [PMC free article] [PubMed]
16. Kolesnikova, L., S. Bamberg, B. Berghofer, and S. Becker. 2004. The matrix protein of Marburg virus is transported to the plasma membrane along cellular membranes: exploiting the retrograde late endosomal pathway. J. Virol. 78:2382-2393. [PMC free article] [PubMed]
17. Kolesnikova, L., B. Berghofer, S. Bamberg, and S. Becker. 2004. Multivesicular bodies as a platform for formation of the Marburg virus envelope. J. Virol. 78:12277-12287. [PMC free article] [PubMed]
18. Licata, J. M., M. Simpson-Holley, N. T. Wright, Z. Han, J. Paragas, and R. N. Harty. 2003. Overlapping motifs (PTAP and PPEY) within the Ebola virus VP40 protein function independently as late budding domains: involvement of host proteins TSG101 and VPS-4. J. Virol. 77:1812-1819. [PMC free article] [PubMed]
19. Licata, J. M., R. F. Johnson, Z. Han, and R. N. Harty. 2004. Contribution of Ebola virus glycoprotein, nucleoprotein, and VP24 to budding of VP40 virus-like particles. J. Virol. 78:7344-7351. [PMC free article] [PubMed]
20. Martin-Serrano, J., T. Zang, and P. D. Bieniasz. 2001. HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress. Nat. Med. 7:1313-1319. [PubMed]
21. Martin-Serrano, J., D. Perez-Caballero, and P. D. Bieniasz. 2004. Context-dependent effects of L-domains and ubiquitination on viral budding. J. Virol. 78:5554-5563. [PMC free article] [PubMed]
22. Neumann, G., H. Ebihara, A. Takada, T. Noda, D. Kobasa, L. D. Jasenosky, S. Watanabe, J. H. Kim, H. Feldmann, and Y. Kawaoka. 2005. Ebola virus VP40 late domains are not essential for viral replication in cell culture. J. Virol. 79:10300-10307. [PMC free article] [PubMed]
23. Niwa, H., K. Yamamura, and J. Miyazaki. 1991. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108:193-199. [PubMed]
24. Noda, T., H. Sagara, E. Suzuki, A. Takada, H. Kida, and Y. Kawaoka. 2002. Ebola virus VP40 drives the formation of virus-like filamentous particles along with GP. J. Virol. 76:4855-4865. [PMC free article] [PubMed]
25. Parent, L. J., R. P. Bennett, R. C. Craven, T. D. Nelle, N. K. Krishna, J. B. Bowzard, C. B. Wilson, B. A. Puffer, R. C. Montelaro, and J. W. Wills. 1995. Positionally independent and exchangeable late budding functions of the Rous sarcoma virus and human immunodeficiency virus Gag proteins. J. Virol. 69:5455-5460. [PMC free article] [PubMed]
26. Perez, M., R. C. Craven, and J. C. de la Torre. 2003. The small RING finger protein Z drives arenavirus budding: implications for antiviral strategies. Proc. Natl. Acad. Sci. USA 100:12978-12983. [PMC free article] [PubMed]
27. Puffer, B. A., L. J. Parent, J. W. Wills, and R. C. Montelaro. 1997. Equine infectious anemia virus utilizes a YXXL motif within the late assembly domain of the Gag p9 protein. J. Virol. 71:6541-6546. [PMC free article] [PubMed]
28. Saijo, M., M. Niikura, A. Maeda, T. Sata, T. Kurata, I. Kurane, and S. Morikawa. 2005. Characterization of monoclonal antibodies to Marburg virus nucleoprotein (NP) that can be used for NP-capture enzyme-linked immunosorbent assay. J. Med. Virol. 76:111-118. [PubMed]
29. Sakurai, A., J. Yasuda, H. Takano, Y. Tanaka, M. Hatakeyama, and H. Shida. 2004. Regulation of human T-cell leukemia virus type 1 (HTLV-1) budding by ubiquitin ligase Nedd4. Microbes Infect. 6:150-156. [PubMed]
30. Strecker, T., R. Eichler, J. Meulen, W. Weissenhorn, H. D. Klenk, W. Garten, and O. Lenz. 2003. Lassa virus Z protein is a matrix protein and sufficient for the release of virus-like particles. J. Virol. 77:10700-10705. [PMC free article] [PubMed]
31. Swenson, D. L., K. L. Warfield, K. Kuehl, T. Larsen, M. C. Hevey, A. Schmaljohn, S. Bavari, and M. J. Aman. 2004. Generation of Marburg virus-like particles by coexpression of glycoprotein and matrix protein. FEMS Immunol. Med. Microbiol. 40:27-31. [PubMed]
32. Urata, S., T. Noda, Y. Kawaoka, H. Yokosawa, and J. Yasuda. 2006. Cellular factors required for Lassa virus budding. J. Virol. 80:4191-4195. [PMC free article] [PubMed]
33. Vana, M. L., Y. Tang, A. Chen, G. Medina, C. Carter, and J. Leis. 2004. Role of Nedd4 and ubiquitination of Rous sarcoma virus Gag in budding of virus-like particles from cells. J. Virol. 78:13943-13953. [PMC free article] [PubMed]
34. Wills, J. W., C. E. Cameron, C. B. Wilson, Y. Xiang, R. P. Bennett, and J. Leis. 1994. An assembly domain of the Rous sarcoma virus Gag protein required late in budding. J. Virol. 68:6605-6618. [PMC free article] [PubMed]
35. Xiang, Y., C. E. Cameron, J. W. Wills, and J. Leis. 1996. Fine mapping and characterization of the Rous sarcoma virus Pr76gag late assembly domain. J. Virol. 70:5695-5700. [PMC free article] [PubMed]
36. Yasuda, J., and E. Hunter. 1998. A proline-rich motif (PPPY) in the Gag polyprotein of Mason-Pfizer monkey virus plays a maturation-independent role in virion release. J. Virol. 72:4095-4103. [PMC free article] [PubMed]
37. Yasuda, J., E. Hunter, M. Nakao, and H. Shida. 2002. Functional involvement of a novel Nedd4-like ubiquitin ligase on retrovirus budding. EMBO Rep. 3:636-640. [PMC free article] [PubMed]
38. Yasuda, J., M. Nakao, Y. Kawaoka, and H. Shida. 2003. Nedd4 regulates egress of Ebola virus-like particles from host cells. J. Virol. 77:9987-9992. [PMC free article] [PubMed]
39. Yuan, B., X. Li, and S. P. Goff. 1999. Mutations altering the Moloney murine leukemia virus p12 Gag protein affect virion production and early events of the virus life cycle. EMBO J. 18:4700-4710. [PMC free article] [PubMed]

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