- Open Access
Regulation of HTLV-1 Gag budding by Vps4A, Vps4B, and AIP1/Alix
© Urata et al; licensee BioMed Central Ltd. 2007
- Received: 18 June 2007
- Accepted: 02 July 2007
- Published: 02 July 2007
HTLV-1 Gag protein is a matrix protein that contains the PTAP and PPPY sequences as L-domain motifs and which can be released from mammalian cells in the form of virus-like particles (VLPs). The cellular factors Tsg101 and Nedd4.1 interact with PTAP and PPPY, respectively, within the HTLV-1 Gag polyprotein. Tsg101 forms a complex with Vps28 and Vps37 (ESCRT-I complex) and plays an important role in the class E Vps pathway, which mediates protein sorting and invagination of vesicles into multivesicular bodies. Nedd4.1 is an E3 ubiquitin ligase that binds to the PPPY motif through its WW motif, but its function is still unknown. In the present study, to investigate the mechanism of HTLV-1 budding in detail, we analyzed HTLV-1 budding using dominant negative (DN) forms of the class E proteins.
Here, we report that DN forms of Vps4A, Vps4B, and AIP1 inhibit HTLV-1 budding.
These findings suggest that HTLV-1 budding utilizes the MVB pathway and that these class E proteins may be targets for prevention of mother-to-infant vertical transmission of the virus.
- Dominant Negative Mutant
- Equine Infectious Anemia Virus
- Dominant Negative Form
- Dominant Negative
- Lassa Virus
The Gag polyprotein of HTLV-1 is the only viral protein that is both necessary for and sufficient to drive the release of virus particles through a budding process [1–7]. During or after the process of particle release, the action of the retroviral protease cleaves Gag to produce mature matrix (MA), capsid (CA), and nucleocapsid (NC) proteins. Three functional domains that are critical for the assembly and budding processes have been identified in the Gag protein. The membrane-binding domain (M-domain) is required for myristoylation of the Gag N-terminal region and subsequent targeting of the protein to the plasma membrane. The interaction domain (I-domain) appears to be a major region involved in Gag multimerization. The late assembly domain (L-domain) plays a critical role in pinching off of virus particles from the plasma membrane of infected cells. It has also been reported that inactivation of the viral protease has no effect on the production of HTLV-1 particles, similar to our previous observations in Mason-Pfizer monkey virus (M-PMV) [7, 8].
Three L-domain consensus sequences, PPXY, PT/SAP, and YPXL, have been identified within the matrix proteins of many enveloped RNA viruses, including retro-, rhabdo-, filo-, and arenaviruses [1, 2, 4, 5, 9–21]. The majority of retroviruses possess PPXY and/or PT/SAP motifs as an L-domain, one exception being equine infectious anemia virus (EIAV), which possesses a YPXL motif. 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. Three ESCRT complexes, ESCRT-I, -II, and – III, play critical roles in the MVB sorting pathway, acting in a sequential manner. In the final step of protein sorting, AAA-type ATPase Vps4A/B interacts with ESCRT-III to catalyze disassembly of the ESCRT machinery to recycle its components.
The PTAP motif was first identified in human immunodeficiency virus (HIV) p6Gag and has been reported to interact with Tsg101, which is a ubiquitin-conjugating E2 variant and participates in vacuolar protein-sorting (Vps) machinery. The interaction between p6Gag and Tsg101 is required for HIV-1 budding, and Tsg101 appears to facilitate this budding by linking the p6 late domain to the Vps pathway [22, 23].
The PPXY motif has been shown to be the core sequence involved in binding to the WW domain, a sequence of 38 to 40 amino acids containing two widely spaced tryptophan residues, which are involved in protein-protein interaction. In fact, it has been shown that the viral PPXY sequences interact with the WW domains of the cellular Nedd4-like ubiquitin ligases, such as Nedd4 and BUL1 [4, 24–26].
It has been reported that expression of dominant negative (DN) forms and small interfering RNA (siRNA) specific for Tsg101 and AIP1/Alix inhibit L-domain-mediated VLPs or virus release [3, 12, 13, 18, 22, 30]. In addition, in many cases, DN forms of Vps4A and Vps4B lacking the ability to bind or hydrolyze ATP were shown to inhibit the budding of VLPs or virions [9, 10, 12, 22, 23, 31, 32].
In this study, to investigate the mechanism of HTLV-1 budding in detail, we analyzed HTLV-1 budding using DN forms of the class E proteins. Our results showed that the DN forms of Vps4A, Vps4B, and AIP1 markedly suppressed VLP production, suggesting that HTLV-1 budding utilizes the MVB pathway and that these class E proteins may be the targets for prevention of mother-to-infant vertical transmission.
HTLV-1 Gag budding utilizes Vps4A and Vps4B
DN form of AIP1/Alix suppresses the egress of the HTLV-1 VLP production
The mechanism responsible for HTLV-1 budding has not been addressed in detail. Previous studies showed that HTLV-1 Gag protein plays a key role in viral budding as in other retroviruses, and that Tsg101 and Nedd4.1 recognize PTAP and PPPY within the HTLV-1 Gag polyprotein, respectively. Although it is well known that Tsg101 and Nedd4.1 play important roles in HTLV-1 budding, further mechanisms have not been characterized. In this study, to investigate the mechanism of HTLV-1 budding in detail, we analyzed HTLV-1 budding using DN forms of the class E proteins Vps4A, Vps4B, and AIP1 (Fig. 1). The results indicated that the catalytic activities of Vps4A and Vps4B are required for budding of HTLV-1 VLPs, suggesting that HTLV-1 budding mimics the MVB pathway, similar to observations in other envelope viruses. The DN form of AIP1 expressing only the N-terminal region from residues 1–628 also suppressed the budding of HTLV-1 VLPs. The Bro1 domain of AIP1, which is present in the N-terminal region, has been reported to interact with CHMP4 [23, 33]. PRR binds to Tsg101. The V domain can bind to HIV-1 p6 and EIAV p9, and overexpression of a mutant containing only the V domain suppresses HIV-1 and EIAV particle release [28, 29]. Our results shown in Fig. 3 can be explained by binding of AIP1 (1–628) to CHMP4, thus disturbing the downstream parts of the MVB pathway. On the other hand, AIP1 (424–628) had no effect on HTLV-1 budding. AIP1 (424–628) appears to be sufficient for the function of AIP1 in HTLV-1 budding, suggesting that HIV-1 and HTLV-1 utilize AIP1 in different ways [28, 29].
Taken together, these results strongly suggest that HTLV-1 budding utilizes the MVB pathway and that these class E proteins may be useful as targets for prevention of mother-to-infant vertical transmission.
In the present study, we showed that the enzymatic activities of Vps4A and Vps4B are required for efficient budding of HTLV-1 and that endogenous Vps4A and Vps4B are sufficient for VLP production. In addition, it was shown that AIP1 (1–628) acts as a DN mutant for HTLV-1 budding.
Human 293T cells were maintained in Dulbecco's minimal essential medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum and penicillin-streptomycin at 37°C.
After PCR amplification from the K30 infectious clone, the HTLV-1 K30 gag gene was cloned into the pCAGGS-MCS vector. pVps4A, pVps4B, pVps4AEQ, and pVps4BEQ were described previously . pCAGGS-HA-AIP1, -AIP1(1–628), and -AIP1(424–628) were kind gifts from Dr. Sakaguchi .
VLP budding assay
Forty-eight hours after transfection, the cell supernatant was clarified from cell debris by centrifugation (13,000 × g, 10 min) and then VLPs were pelleted by ultracentrifugation through a 20% sucrose cushion (345,000 × g, 60 min at 4°C). Cells and VLPs were lysed with Lysis A buffer (1% TritonX-100, 25 mM Tris-HCl, pH 8.0, 50 mM NaCl, and 10% Na-deoxycholate). Cell lysates and VLPs were resolved by SDS-PAGE, and the proteins were then transferred onto nitrocellulose membranes. The mouse anti-HTLV-1 p19 monoclonal antibody TP-7 (Abcam, Cambridge, UK) was used to detect K30Gag. The mouse anti-Flag monoclonal antibody M2 (Sigma) was used for detection of Vps4A, Vps4B, Vps4AEQ, and Vps4BEQ. The mouse anti-HA monoclonal antibody 6E2 (Cell Signaling Technology, Beverley, MA) was used for detection of HA-AIP1 WT and DN series. Horseradish peroxidase-conjugated goat anti-mouse IgG antibody A-2304 (Sigma) was used as a secondary antibody. Immunoreactive bands were visualized using ECL plus (Amersham Pharmacia Biotech, Upsalla, Sweden), followed by the LAS-3000 system (Fuji Film, Tokyo, Japan). For quantification, the signal intensity on western blots was evaluated with Image Gauge version 4.1 (Fuji Film) using the LAS-3000 system.
We thank Dr. Yoko Aida for providing the HTLV-1 clone K30 plasmid DNA used for construction of pK30-Gag. This work was supported by CREST (Japan Science and Technology Agency) and JSPS (Japan Society for the Promotion of Science) Research Fellowships for Young Scientists.
- Dorweiler IJ, Ruone SJ, Wang H, Burry RW, Mansky LM: Role of the human T-cell leukemia virus type 1 PTAP motif in Gag targeting and particle release. J Virol 2006, 80: 3634-3643. 10.1128/JVI.80.7.3634-3643.2006PubMedPubMed CentralView ArticleGoogle Scholar
- Heidecker G, Lloyd PA, Fox K, Nagashima K, Derse D: Late assembly motifs of human T-cell leukemia virus type 1 and their relative roles in particle release. J Virol 2004, 78: 6636-6648. 10.1128/JVI.78.12.6636-6648.2004PubMedPubMed CentralView ArticleGoogle Scholar
- Blot V, Perugi F, Gay B, Prevost MC, Briant L, Tangy F, Abriel H, Staub O, Dokhelar MC, Pique C: Nedd4.1-mediated ubiquitination and subsequent recruitment of Tsg101 ensure HTLV-1 Gag trafficking towards the multivesicular body pathway prior to virus budding. J Cell Sci 2004, 117: 2357-2367. 10.1242/jcs.01095PubMedView ArticleGoogle Scholar
- Sakurai A, Yasuda J, Takano H, Tanaka Y, Hatakeyama M, Shida H: Regulation of human T-cell leukemia virus type 1 (HTLV-1) budding by ubiquitin ligase Nedd4. Microbes Infect 2004, 6: 150-156. 10.1016/j.micinf.2003.10.011PubMedView ArticleGoogle Scholar
- Wang H, Machesky NJ, Mansky LM: Both the PPPY and PTAP motifs are involved in human T-cell leukemia virus type 1 particle release. J Virol 2004, 78: 1503-1512. 10.1128/JVI.78.3.1503-1512.2004PubMedPubMed CentralView ArticleGoogle Scholar
- Bouamr F, Melillo JA, Wang MQ, Nagashima K, de Los Santos M, Rein A, Goff SP: 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 [corrected]. J Virol 2003, 77: 11882-11895. 10.1128/JVI.77.22.11882-11895.2003PubMedPubMed CentralView ArticleGoogle Scholar
- Le Blanc I, Prevost MC, Dokhelar MC, Rosenberg AR: The PPPY motif of human T-cell leukemia virus type 1 Gag protein is required early in the budding process. J Virol 2002, 76: 10024-10029. 10.1128/JVI.76.19.10024-10029.2002PubMedPubMed CentralView ArticleGoogle Scholar
- Yasuda J, Hunter E: A proline-rich motif (PPPY) in the Gag polyprotein of Mason-Pfizer monkey virus plays a maturation-independent role in virion release. J Virol 1998, 72: 4095-4103.PubMedPubMed CentralGoogle Scholar
- Shehu-Xhilaga M, Ablan S, Demirov DG, Chen C, Montelaro RC, Freed EO: Late domain-dependent inhibition of equine infectious anemia virus budding. J Virol 2004, 78: 724-732. 10.1128/JVI.78.2.724-732.2004PubMedPubMed CentralView ArticleGoogle Scholar
- Licata JM, Simpson-Holley M, Wright NT, Han Z, Paragas J, Harty RN: 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 2003, 77: 1812-1819. 10.1128/JVI.77.3.1812-1819.2003PubMedPubMed CentralView ArticleGoogle Scholar
- Patton GS, Morris SA, Chung W, Bieniasz PD, McClure MO: Identification of domains in gag important for prototypic foamy virus egress. J Virol 2005, 79: 6392-6399. 10.1128/JVI.79.10.6392-6399.2005PubMedPubMed CentralView ArticleGoogle Scholar
- Urata S, Noda T, Kawaoka Y, Yokosawa H, Yasuda J: Cellular factors required for Lassa virus budding. J Virol 2006, 80: 4191-4195. 10.1128/JVI.80.8.4191-4195.2006PubMedPubMed CentralView ArticleGoogle Scholar
- Urata S, Noda T, Kawaoka Y, Morikawa S, Yokosawa H, Yasuda J: 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. J Virol 2007, 81: 4895-4899. 10.1128/JVI.02829-06PubMedPubMed CentralView ArticleGoogle Scholar
- Xiang Y, Cameron CE, Wills JW, Leis J: Fine mapping and characterization of the Rous sarcoma virus Pr76gag late assembly domain. J Virol 1996, 70: 5695-5700.PubMedPubMed CentralGoogle Scholar
- Wills JW, Cameron CE, Wilson CB, Xiang Y, Bennett RP, Leis J: An assembly domain of the Rous sarcoma virus Gag protein required late in budding. J Virol 1994, 68: 6605-6618.PubMedPubMed CentralGoogle Scholar
- Noda T, Sagara H, Suzuki E, Takada A, Kida H, Kawaoka Y: Ebola virus VP40 drives the formation of virus-like filamentous particles along with GP. J Virol 2002, 76: 4855-4865. 10.1128/JVI.76.10.4855-4865.2002PubMedPubMed CentralView ArticleGoogle Scholar
- Jasenosky LD, Neumann G, Lukashevich I, Kawaoka Y: Ebola virus VP40-induced particle formation and association with the lipid bilayer. J Virol 2001, 75: 5205-5214. 10.1128/JVI.75.11.5205-5214.2001PubMedPubMed CentralView ArticleGoogle Scholar
- Perez M, Craven RC, de la Torre JC: The small RING finger protein Z drives arenavirus budding: implications for antiviral strategies. Proc Natl Acad Sci USA 2003, 100: 12978-12983. 10.1073/pnas.2133782100PubMedPubMed CentralView ArticleGoogle Scholar
- Strecker T, Eichler R, Meulen J, Weissenhorn W, Dieter Klenk H, Garten W, Lenz O: Lassa virus Z protein is a matrix protein and sufficient for the release of virus-like particles [corrected]. J Virol 2003, 77: 10700-10705. 10.1128/JVI.77.19.10700-10705.2003PubMedPubMed CentralView ArticleGoogle Scholar
- Eichler R, Strecker T, Kolesnikova L, ter Meulen J, Weissenhorn W, Becker S, Klenk HD, Garten W, Lenz O: Characterization of the Lassa virus matrix protein Z: electron microscopic study of virus-like particles and interaction with the nucleoprotein (NP). Virus Res 2004, 100: 249-255. 10.1016/j.virusres.2003.11.017PubMedView ArticleGoogle Scholar
- Yuan B, Li X, Goff SP: Mutations altering the moloney murine leukemia virus p12 Gag protein affect virion production and early events of the virus life cycle. Embo J 1999, 18: 4700-4710. 10.1093/emboj/18.17.4700PubMedPubMed CentralView ArticleGoogle Scholar
- Garrus JE, von Schwedler UK, Pornillos OW, Morham SG, Zavitz KH, Wang HE, Wettstein DA, Stray KM, Cote M, Rich RL, et al.: Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell 2001, 107: 55-65. 10.1016/S0092-8674(01)00506-2PubMedView ArticleGoogle Scholar
- von Schwedler UK, Stuchell M, Muller B, Ward DM, Chung HY, Morita E, Wang HE, Davis T, He GP, Cimbora DM, et al.: The protein network of HIV budding. Cell 2003, 114: 701-713. 10.1016/S0092-8674(03)00714-1PubMedView ArticleGoogle Scholar
- Martin-Serrano J, Eastman SW, Chung W, Bieniasz PD: HECT ubiquitin ligases link viral and cellular PPXY motifs to the vacuolar protein-sorting pathway. J Cell Biol 2005, 168: 89-101. 10.1083/jcb.200408155PubMedPubMed CentralView ArticleGoogle Scholar
- Yasuda J, Nakao M, Kawaoka Y, Shida H: Nedd4 regulates egress of Ebola virus-like particles from host cells. J Virol 2003, 77: 9987-9992. 10.1128/JVI.77.18.9987-9992.2003PubMedPubMed CentralView ArticleGoogle Scholar
- Yasuda J, Hunter E, Nakao M, Shida H: Functional involvement of a novel Nedd4-like ubiquitin ligase on retrovirus budding. EMBO Rep 2002, 3: 636-640. 10.1093/embo-reports/kvf132PubMedPubMed CentralView ArticleGoogle Scholar
- Fisher RD, Chung HY, Zhai Q, Robinson H, Sundquist WI, Hill CP: Structural and biochemical studies of ALIX/AIP1 and its role in retrovirus budding. Cell 2007, 128: 841-852. 10.1016/j.cell.2007.01.035PubMedView ArticleGoogle Scholar
- Chen C, Vincent O, Jin J, Weisz OA, Montelaro RC: Functions of early (AP-2) and late (AIP1/ALIX) endocytic proteins in equine infectious anemia virus budding. J Biol Chem 2005, 280: 40474-40480. 10.1074/jbc.M509317200PubMedView ArticleGoogle Scholar
- Munshi UM, Kim J, Nagashima K, Hurley JH, Freed EO: An Alix fragment potently inhibits HIV-1 budding: characterization of binding to retroviral YPXL late domains. J Biol Chem 2007, 282: 3847-3855. 10.1074/jbc.M607489200PubMedView ArticleGoogle Scholar
- Irie T, Shimazu Y, Yoshida T, Sakaguchi T: The YLDL sequence within Sendai virus M protein is critical for budding of virus-like particles and interacts with Alix/AIP1 independently of C protein. J Virol 2007, 81: 2263-2273. 10.1128/JVI.02218-06PubMedPubMed CentralView ArticleGoogle Scholar
- Gottwein E, Bodem J, Muller B, Schmechel A, Zentgraf H, Krausslich HG: The Mason-Pfizer monkey virus PPPY and PSAP motifs both contribute to virus release. J Virol 2003, 77: 9474-9485. 10.1128/JVI.77.17.9474-9485.2003PubMedPubMed CentralView ArticleGoogle Scholar
- Kian Chua P, Lin MH, Shih C: Potent inhibition of human Hepatitis B virus replication by a host factor Vps4. Virology 2006, 354: 1-6. 10.1016/j.virol.2006.07.018PubMedView ArticleGoogle Scholar
- Strack B, Calistri A, Craig S, Popova E, Gottlinger HG: AIP1/ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding. Cell 2003, 114: 689-699. 10.1016/S0092-8674(03)00653-6PubMedView ArticleGoogle Scholar
- Schmitt AP, Leser GP, Morita E, Sundquist WI, Lamb RA: Evidence for a new viral late-domain core sequence, FPIV, necessary for budding of a paramyxovirus. J Virol 2005, 79: 2988-2997. 10.1128/JVI.79.5.2988-2997.2005PubMedPubMed CentralView ArticleGoogle Scholar
- Irie T, Licata JM, McGettigan JP, Schnell MJ, Harty RN: Budding of PPxY-containing rhabdoviruses is not dependent on host proteins TGS101 and VPS4A. J Virol 2004, 78: 2657-2665. 10.1128/JVI.78.6.2657-2665.2004PubMedPubMed CentralView ArticleGoogle Scholar
- Goila-Gaur R, Demirov DG, Orenstein JM, Ono A, Freed EO: Defects in human immunodeficiency virus budding and endosomal sorting induced by TSG101 overexpression. J Virol 2003, 77: 6507-6519. 10.1128/JVI.77.11.6507-6519.2003PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.