Comparative study on the effect of human BST-2/Tetherin on HIV-1 release in cells of various species
© Sato et al; licensee BioMed Central Ltd. 2009
Received: 03 February 2009
Accepted: 02 June 2009
Published: 02 June 2009
In this study, we first demonstrate that endogenous hBST-2 is predominantly expressed on the plasma membrane of a human T cell line, MT-4 cells, and that Vpu-deficient HIV-1 was less efficiently released than wild-type HIV-1 from MT-4 cells. In addition, surface hBST-2 was rapidly down-regulated in wild-type but not Vpu-deficient HIV-1-infected cells. This is a direct insight showing that provirus-encoded Vpu has the potential to down-regulate endogenous hBST-2 from the surface of HIV-1-infected T cells. Corresponding to previous reports, the aforementioned findings suggested that hBST-2 has the potential to suppress the release of Vpu-deficient HIV-1. However, the molecular mechanism(s) for tethering HIV-1 particles by hBST-2 remains unclear, and we speculated about the requirement for cellular co-factor(s) to trigger or assist its tethering ability. To explore this possibility, we utilize several cell lines derived from various species including human, AGM, dog, cat, rabbit, pig, mink, potoroo, and quail. We found that ectopic hBST-2 was efficiently expressed on the surface of all analyzed cells, and its expression suppressed the release of viral particles in a dose-dependent manner. These findings suggest that hBST-2 can tether HIV-1 particles without the need of additional co-factor(s) that may be expressed exclusively in primates, and thus, hBST-2 can also exert its function in many cells derived from a broad range of species. Interestingly, the suppressive effect of hBST-2 on HIV-1 release in Vero cells was much less pronounced than in the other examined cells despite the augmented surface expression of ectopic hBST-2 on Vero cells. Taken together, our findings suggest the existence of certain cell types in which hBST-2 cannot efficiently exert its inhibitory effect on virus release. The cell type-specific effect of hBST-2 may be critical to elucidate the mechanism of BST-2-dependent suppression of virus release.
To accomplish efficient release of HIV-1 particles, HIV-1 Vpu is required in certain cells (e.g., HeLa cells) but is dispensable in other cell types (e.g., HEK293 and Cos-7 cells) [1–3]. A previous report suggested that an inhibitory factor(s) for HIV-1 release is expressed in HeLa cells and the effect is attenuated by Vpu . Recently, Neil and colleagues identified the inhibitor, hBST-2 (also called CD317 or HM1.24), in HeLa cells, and referred to this protein as "Tetherin" . They also showed that the inhibitory action of hBST-2 on HIV-1 particle release was antagonized by Vpu, and they concluded that hBST-2 functions by tethering HIV-1 particles to the cell surface . In addition, Van Damme and colleagues demonstrated that Vpu down-regulates hBST-2 from the surface of HeLa cells . On the other hand, Miyagi and colleagues have recently reported that Vpu augments HIV-1 release without down-regulating surface hBST-2 in CEMx174 and H9 cells . Therefore, the relevance of surface hBST-2 down-regulation and the antagonistic action of Vpu on the tethering ability of hBST-2 remain unclear.
To analyze the sensitivity of endogenous hBST-2 on the surface of MT-4 cells to Vpu antagonism, MT-4 cells were infected with either wild-type or Vpu-deficient HIV-1, and the level of surface hBST-2 was subsequently monitored. The amount of released virions in the culture supernatant of wild-type HIV-1-infected cells was significantly higher when compared to that of Vpu-deleted HIV-1-infected cells (Figure 1E), while the percentage of p24-positive cells in wild-type HIV-1-infected culture was similar to that in Vpu-deleted HIV-1-infected culture (Figure 1F). These results suggest that the liberation of Vpu-deficient HIV-1 virions was impaired by endogenous hBST-2 in MT-4 cells. In addition, we clearly found that the surface expression of hBST-2 on wild-type but not Vpu-deleted HIV-1-infected cells (i.e., p24-positive cells) was severely down-regulated (Figures 1D and 1H). Although it has remained ambiguous in the literature whether endogenous hBST-2 on the surface of human T cells is down-regulated by HIV-1 infection [6, 7], this is the first demonstration of the significant down-regulation of endogenous hBST-2 in T cells by Vpu which resulted from HIV-1 infection and not from transfection with a Vpu-expressing plasmid [6, 8].
Following the rapid down-regulation of surface hBST-2 by infection with wild-type HIV-1, the surface expression of hBST-2 was gradually but significantly replenished along with HIV-1 expansion (Figures 1D and 1H). It is unclear how and why the surface levels of hBST-2 increased; however, our finding indicates that the level of down-regulation of surface hBST-2 on HIV-1-infected T cells would vary depending on the time after infection.
Previous studies have shown that rhTRIM5α, a well-known restriction factor for HIV-1 replication [12, 13], is able to efficiently elicit its suppressive ability for HIV-1 replication in feline CRFK cells, but not in canine D-17 cells [14, 15]. These results suggest the species-specific ability of rhTRIM5α to suppress HIV-1 replication. To investigate the species-specific tethering ability of hBST-2, we next co-transfected an hBST-2-expressing plasmid (phBST-2) with either pNL4-3 or pNL43-Udel in the above examined cell lines and harvested released virions at 24 hours post-transfection. As shown in Figure 2, exogenous hBST-2 in these cell lines clearly suppressed the release of Vpu-deficient HIV-1 in a dose-dependent manner. This result strongly indicates that hBST-2 can tether released HIV-1 particles without any other unidentified co-factors that are expressed exclusively in primates. It remains conceivable that hBST-2 could employ certain elements ubiquitously expressed in many species for the tethering of released virions. Although it has been controversial whether wild-type HIV-1 release can be suppressed by ectopic hBST-2 or not [5, 6], we observed here that the release of wild-type HIV-1 was attenuated by hBST-2 and that the efficiency of hBST-2 for the release of wild-type HIV-1 was significantly lower than that for the release of Vpu-deleted HIV-1 (Figure 2).
As previously reported , we also found that endogenous hBST-2 on HeLa cells was significantly down-regulated by transfection with pNL4-3, but not with pNL43-Udel (Figure 3B). In contrast, at 24 hours post-transfection, the down-regulation of exogenous hBST-2 on the surface of the other cell lines was hardly observed except for Vero cells (Figure 3B). However, after 48 hours post-transfection, we could detect significant down-regulation of ectopically expressed hBST-2 on the surface of cells co-transfected with either pNL4-3 or a Vpu-expressing plasmid  (data not shown). These results suggest that the level of Vpu expression at 24 hours post-transfection is sufficient to antagonize the tethering ability of hBST-2, while not down-regulating surface hBST-2. In support of our data, a recent report showed that Vpu enhances HIV-1 release in the absence of surface down-regulation of hBST-2 . Taken together, these results indicate that the down-regulation of surface hBST-2 may be dispensable for the antagonism of tethering ability of hBST-2 by Vpu.
It has recently been reported that hBST-2 has the potential to suppress the release of not only HIV-1 but also other retroviruses , Ebola virus , Lassa virus , and Marburg virus [18, 19]. Therefore, further studies on the mechanism of BST-2 function will provide beneficial information leading to novel therapeutic strategies against several virus-induced diseases including AIDS.
HEK293 cells (human kidney), Vero cells (AGM kidney), Cos-7 cells (AGM kidney), rabbit skin cells (RSC, kindly provided by Dr. B. Roizman), and TZM-bl cells (obtained from AIDS reagent program, National Institute of Health) were maintained in low-glucose DMEM (Nikken) containing 10% FCS and antibiotics. D-17 cells (canine osteosarcoma), CRFK cells (feline kidney), PK-15 cells (porcine kidney), Mv.1.Lu cells (Mustela vison, mink lung), and QT6 cells (Coturnix coturnix japonica, quail fibrosarcoma) were maintained in high-glucose DMEM (Sigma) containing 10% FCS, 2 mM GlutaMax (Invitrogen), and antibiotics. PtK2 cells (potoroo kidney) were maintained in Eagle's minimum essential medium (Sigma) supplemented with 1 mM sodium pyruvate, 2 mM GlutaMax, 10% FCS and antibiotics. MT-4 cells were maintained in RPMI1640 (Nikken) containing 10% FCS and antibiotics. Mv.1.Lu cells and QT6 cells were kindly donated by Dr. A. Koito.
To construct phBST-2, a bst-2 cDNA (GenBank: NM_004335, bases 10-552) was amplified by polymerase chain reaction from a human leukocyte cDNA library (Invitrogen), and the resulting fragment was inserted into peGFP-C1 (Clontech). Sequence of the construct was confirmed with an ABI 3130xl genetic analyzer (Applied Biosystems).
Transfection and virus preparation
Cells were seeded in 6-well plate to appropriate densities 1-day prior to transfection and were transfected by using Lipofectamine 2000 reagent (Invitrogen) according to the manufacture's protocol. Briefly, 1 μg of pNL4-3  or pNL43-Udel (kindly donated by Dr. K. Strebel)  was cotransfected with 20 or 100 ng of phBST-2. The amount of plasmid DNA for transfection was normalized to 2 μg per well. Four hour after transfection, culture medium was replaced freshly. The culture supernatant was harvested, centrifuged, and then filtrated with 0.45-μm filter (Millipore) to produce virus solutions at 24 hours post-transfection. All experiments were performed in triplicate. To prepare wild-type or Vpu-deficient HIV-1 for its infection assay, pNL4-3 or pNL43-Udel was transfected into HEK293 cells by the calcium phosphate method as previously described . The prepared viruses were titrated by using peripheral blood mononuclear cells, and the TCID50 was calculated as previously described .
Quantification of the amount of released HIV-1 virion was performed by using TZM-bl cells as previously described . Briefly, appropriate virus solution was inoculated into 1 × 105 TZM-bl cells per 12-well plate. The cells were harvested at 48 hours post-infection, and β-galactosidase assay was performed by using Galacto-Star Mammalian Reporter Gene Assay System (Applied Biosystems) according to the manufacture's procedure. Activity was measured with a 1420 ALBOSX multilabel counter (Perkin Elmer).
The amount of HIV-1 virion released from CRFK, PtK2, and MT-4 cells was quantified by using HIV-1 p24 ELISA kit (ZeptoMetrix) according to the manufacture's instructions.
Flow cytometry was performed as previously described . A mouse anti-hBST-2 monoclonal antibody (donated by Chugai Pharmaceutical Co., Japan) [6, 23] and a Cy5-conjugated donkey anti-mouse IgG antisera (Chemicon) were used. For costaining of cell surface hBST-2 and intracellular p24, the anti-hBST-2 monoclonal antibody was pre-labelled with Zenon Alexa Fluor 647 mouse IgG2a labelling kit (Invitrogen) according to the manufacture's protocol. Cell surface hBST-2 was stained with the pre-labelled anti-hBST-2 antibody, and the cells were permeabiliezed and fixed with BD Cytoperm/Cytofix solution (BD Pharmingen). Then, intracellular p24 was stained with a FITC-conjugated anti-HIV-1 p24 antibody (clone 2C2, kindly provided by Dr. Y. Tanaka) .
Western blotting was performed as previously described  with some modification. Briefly, the cells were lysed with lysis buffer (1% NP-40, 50 mM Tris-HCl [pH7.5], 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, and 1 mM PMSF). The lysates were separated by SDS-PAGE and transferred to Immobilon transfer membrane (Millipore). For detection, the mouse anti-hBST-2 monoclonal antibody, a mouse anti-Tubulin monoclonal antibody (clone DM1A; Sigma), and an HRP-conjugated horse anti-mouse IgG antibody (Cell Signalling) were used. It has been reported that hBST-2 is a highly glycosylated protein . To remove the sugar chains in hBST-2 protein and detect hBST-2 more clearly, the lysates were treated with glycopeptidase F (TaKaRa) according to the manufacture's procedure.
Student's t test was used to determine statistical significance, and P < 0.05 and P < 0.01 were considered significant. The Pearson correlation coefficient was applied to determine statistical significance for the correlation between the suppression efficiency for particle release by hBST-2 and the level of surface hBST-2 in the 9 kinds of cells lines (Figure 4), and P < 0.01 was considered significant. Repeated measure ANOVA was applied to determine statistical significance between Vero cells and the other cell lines (Figure 4), and P < 0.01 was considered significant.
bone marrow stromal cell antigen-2
human immunodeficiency virus type 1
viral protein U
African green monkey
enzyme-linked immunosorbent assay
rhesus macaque tripartite motif-containing 5 isoform α
acquired immunodeficiency syndrome
Dulbecco's modified Eagle medium
fatal calf serum
50% tissue culture infectious dose
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
multiplicity of infection
mean fluorescence intensity.
We thank Klaus Strebel (National Institute of Allergy and Infectious, Diseases, National Institutes of Health) for donating materials and helpful suggestions about this study, Atsushi Koito (Kumamoto University), Yuetsu Tanaka (University of the Ryukyus), and Bernard Roizman (The University of Chicago) for providing materials, Peter Gee, Takashi Fujita, Kazuhide Onoguchi, Takayuki Shojima (Institute for Virus Research, Kyoto University), and Shingo Iwami (Shizuoka University) for their generous help in this study. We also would like to express our appreciation for Ms. Kotubu Misawa's dedicated support. This work was supported by Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Sciences, and Technology of Japan, and a Health and Labor Science Research Grant (Research on Publicly Essential Drugs and Medical Devices) from the Ministry of Health, Labor and Welfare of Japan and Japan Human Science Foundation. KS and TY were supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. TM was supported by the Bio-oriented Technology Research Advancement Institution.
- Klimkait T, Strebel K, Hoggan MD, Martin MA, Orenstein JM: The human immunodeficiency virus type 1-specific protein vpu is required for efficient virus maturation and release. J Virol. 1990, 64: 621-629.PubMed CentralPubMedGoogle Scholar
- Nomaguchi M, Fujita M, Adachi A: Role of HIV-1 Vpu protein for virus spread and pathogenesis. Microbes Infect. 2008, 10: 960-967. 10.1016/j.micinf.2008.07.006.View ArticlePubMedGoogle Scholar
- Strebel K, Klimkait T, Martin MA: A novel gene of HIV-1, vpu, and its 16-kilodalton product. Science. 1988, 241: 1221-1223. 10.1126/science.3261888.View ArticlePubMedGoogle Scholar
- Varthakavi V, Smith RM, Bour SP, Strebel K, Spearman P: Viral protein U counteracts a human host cell restriction that inhibits HIV-1 particle production. Proc Natl Acad Sci USA. 2003, 100: 15154-15159. 10.1073/pnas.2433165100.PubMed CentralView ArticlePubMedGoogle Scholar
- Neil SJ, Zang T, Bieniasz PD: Tetherin inhibits retrovirus release and is antagonized by HIV-1 Vpu. Nature. 2008, 451: 425-430. 10.1038/nature06553.View ArticlePubMedGoogle Scholar
- Van Damme N, Goff D, Katsura C, Jorgenson RL, Mitchell R, Johnson MC, Stephens EB, Guatelli J: The interferon-induced protein BST-2 restricts HIV-1 release and is downregulated from the cell surface by the viral Vpu protein. Cell Host Microbe. 2008, 3: 245-252. 10.1016/j.chom.2008.03.001.PubMed CentralView ArticlePubMedGoogle Scholar
- Miyagi E, Andrew AJ, Kao S, Strebel K: Vpu enhances HIV-1 virus release in the absence of Bst-2 cell surface down-modulation and intracellular depletion. Proc Natl Acad Sci USA. 2009, 106: 2868-2873. 10.1073/pnas.0813223106.PubMed CentralView ArticlePubMedGoogle Scholar
- Nguyen KL, llano M, Akari H, Miyagi E, Poeschla EM, Strebel K, Bour S: Codon optimization of the HIV-1 vpu and vif genes stabilizes their mRNA and allows for highly efficient Rev-independent expression. Virology. 2004, 319: 163-175. 10.1016/j.virol.2003.11.021.View ArticlePubMedGoogle Scholar
- Koito A, Shigekane H, Matsushita S: Ability of small animal cells to support the postintegration phase of human immunodeficiency virus type-1 replication. Virology. 2003, 305: 181-191. 10.1006/viro.2002.1755.View ArticlePubMedGoogle Scholar
- Munk C, Zielonka J, Constabel H, Kloke BP, Rengstl B, Battenberg M, Bonci F, Pistello M, Lochelt M, Cichutek K: Multiple restrictions of human immunodeficiency virus type 1 in feline cells. J Virol. 2007, 81: 7048-7060. 10.1128/JVI.02714-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Neil SJ, Sandrin V, Sundquist WI, Bieniasz PD: An interferon-α-induced tethering mechanism inhibits HIV-1 and Ebola virus particle release but is counteracted by the HIV-1 Vpu protein. Cell Host Microbe. 2007, 2: 193-203. 10.1016/j.chom.2007.08.001.PubMed CentralView ArticlePubMedGoogle Scholar
- Stremlau M, Owens CM, Perron MJ, Kiessling M, Autissier P, Sodroski J: The cytoplasmic body component TRIM5α restricts HIV-1 infection in Old World monkeys. Nature. 2004, 427: 848-853. 10.1038/nature02343.View ArticlePubMedGoogle Scholar
- Sakuma R, Noser JA, Ohmine S, Ikeda Y: Rhesus monkey TRIM5α restricts HIV-1 production through rapid degradation of viral Gag polyproteins. Nat Med. 2007, 13: 631-635. 10.1038/nm1562.View ArticlePubMedGoogle Scholar
- Saenz DT, Teo W, Olsen JC, Poeschla EM: Restriction of feline immunodeficiency virus by Ref1, Lv1, and primate TRIM5α proteins. J Virol. 2005, 79: 15175-15188. 10.1128/JVI.79.24.15175-15188.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Berube J, Bouchard A, Berthoux L: Both TRIM5α and TRIMCyp have only weak antiviral activity in canine D17 cells. Retrovirology. 2007, 4: 68-10.1186/1742-4690-4-68.PubMed CentralView ArticlePubMedGoogle Scholar
- Emeny JM, Morgan MJ: Regulation of the interferon system: evidence that Vero cells have a genetic defect in interferon production. J Gen Virol. 1979, 43: 247-252. 10.1099/0022-1317-43-1-247.View ArticlePubMedGoogle Scholar
- Mosca JD, Pitha PM: Transcriptional and posttranscriptional regulation of exogenous human beta interferon gene in simian cells defective in interferon synthesis. Mol Cell Biol. 1986, 6: 2279-2283.PubMed CentralView ArticlePubMedGoogle Scholar
- Jouvenet N, Neil SJ, Zhadina M, Zang T, Kratovac Z, Lee Y, McNatt M, Hatziioannou T, Bieniasz PD: Broad-spectrum inhibition of retroviral and filoviral particle release by tetherin. J Virol. 2009, 83: 1837-1844. 10.1128/JVI.02211-08.PubMed CentralView ArticlePubMedGoogle Scholar
- Sakuma T, Noda T, Urata S, Kawaoka Y, Yasuda J: Inhibition of Lassa and Marburg virus production by tetherin. J Virol. 2009, 83: 2382-2385. 10.1128/JVI.01607-08.PubMed CentralView ArticlePubMedGoogle Scholar
- Adachi A, Gendelman HE, Koenig S, Folks T, Willey R, Rabson A, Martin MA: Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol. 1986, 59: 284-291.PubMed CentralPubMedGoogle Scholar
- Sato K, Aoki J, Misawa N, Daikoku E, Sano K, Tanaka Y, Koyanagi Y: Modulation of human immunodeficiency virus type 1 infectivity through incorporation of tetraspanin proteins. J Virol. 2008, 82: 1021-1033. 10.1128/JVI.01044-07.PubMed CentralView ArticlePubMedGoogle Scholar
- Koyanagi Y, Tanaka Y, Kira J, Ito M, Hioki K, Misawa N, Kawano Y, Yamasaki K, Tanaka R, Suzuki Y, et al: Primary human immunodeficiency virus type 1 viremia and central nervous system invasion in a novel hu-PBL-immunodeficient mouse strain. J Virol. 1997, 71: 2417-2424.PubMed CentralPubMedGoogle Scholar
- Ohtomo T, Sugamata Y, Ozaki Y, Ono K, Yoshimura Y, Kawai S, Koishihara Y, Ozaki S, Kosaka M, Hirano T, Tsuchiya M: Molecular cloning and characterization of a surface antigen preferentially overexpressed on multiple myeloma cells. Biochem Biophys Res Commun. 1999, 258: 583-591. 10.1006/bbrc.1999.0683.View ArticlePubMedGoogle Scholar
- Okuma K, Tanaka R, Ogura T, Ito M, Kumakura S, Yanaka M, Nishizawa M, Sugiura W, Yamamoto N, Tanaka Y: Interleukin-4-transgenic hu-PBL-SCID mice: a model for the screening of antiviral drugs and immunotherapeutic agents against X4 HIV-1 viruses. J Infect Dis. 2008, 197: 134-141. 10.1086/524303.View ArticlePubMedGoogle Scholar
- Rollason R, Korolchuk V, Hamilton C, Schu P, Banting G: Clathrin-mediated endocytosis of a lipid-raft-associated protein is mediated through a dual tyrosine motif. J Cell Sci. 2007, 120: 3850-3858. 10.1242/jcs.003343.View ArticlePubMedGoogle 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.