Department of Virology1 and Department of Medicine and Biosystemic Science2, Graduate School of Medical Sciences, Kyushu University, 812-8582, Fukuoka, Japan
Research Center for Emerging Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, 565-0871, Osaka, Japan3
Department of Microbiology, Tokyo Medical and Dental University, 113-0034, Tokyo, Japan4
Author for correspondence: Kazu Okuma. Fax +81 92 642 6140. e-mail kazu{at}virology.med.kyushu-u.ac.jp
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Abstract |
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Introduction |
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HTLV-1 primarily infects CD4+ T-cells, but also infects many different types of cells, such as CD8+ T-cells, B-cells, monocyte/macrophages and endothelial cells. Experimentally, HTLV-1 infects various species, including monkeys, rabbits and rats (Poiesz et al., 1980 ; Krichbaum-Stenger et al., 1987
; Fan et al., 1992
). HTLV-1 envelope glycoproteins, which are synthesized as a precursor, gp61, consist of a cell surface (gp46) and a transmembrane (gp21) glycoprotein (Seiki et al., 1983
; Pique et al., 1992
). These glycoproteins are involved in virus entry into susceptible cells and the subsequent induction of syncytia: the formation of syncytia induced by the co-cultivation of HTLV-1-infected cells with uninfected cells is efficiently inhibited by monoclonal antibodies (MAbs) against envelope glycoproteins or several synthetic peptides of gp46 and gp21 (Baba et al., 1993
; Tanaka et al., 1994
; Sagara et al., 1996
). It is thought that gp46 mediates virus attachment and that gp21 mediates membrane fusion through its N-terminal hydrophobic domain (Ragheb et al., 1995
; Delamarre et al., 1997
).
Identification of HTLV-1 receptor(s) has been attempted mainly by using MAbs that inhibit syncytium formation. Previous studies have shown that cell adhesion molecules, such as intercellular adhesion molecule-1, lymphocyte function associated antigen-1 and vascular cell adhesion molecule-1, and members of the transmembrane 4 superfamily, such as CD82/C33/R2 and CD81/M38/TAPA-1, are required for HTLV-1-induced syncytium formation (Fukudome et al., 1992 ; Hildreth et al., 1997
; Daenke et al., 1999
). However, it was demonstrated recently that a large number of immunoglobulin (Ig) molecules (particularly MAbs against class II major histocompatibility complex molecules) bound to the surface of infected cells can non-specifically block HTLV-1-induced syncytium formation by the protein crowding or steric effects. This suggests that the results obtained from studies using MAbs against the cellular molecules described above may need to be re-evaluated in the context of the cell surface expression level of the molecules (Hildreth, 1998
). Thus, the cellular receptor(s) for HTLV-1 remain to be determined.
In the present study, we used vesicular stomatitis virus (VSV) and human immunodeficiency virus type 1 (HIV-1) pseudotypes containing the gene encoding the green fluorescent protein (GFP), which are complemented in trans with viral envelope glycoproteins in order to confer infectivity on these viruses. To analyse virus entry mediated by HTLV-1 envelope glycoproteins, we examined the infectivity of the pseudotypes both on various cell lines and on cells that were chemically modified by various reagents.
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Methods |
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Plasmids.
Mammalian expression plasmid vectors pCAGGS and pCXN2 contain the CAG promoter, which is composed of the cytomegalovirus immediate early enhancer and the chicken -actin promoter (Niwa et al., 1991
). A full-length cDNA of VSV (Indiana serotype) G protein was excised from pGL-1 (Rose & Bergmann, 1982
) and subcloned into pCAGGS, resulting in pCAG-VSVG. The HTLV-1 env gene was excised from pHAproenv (Shida et al., 1987
) and subcloned into pCAGGS, resulting in pCAGHTLV-I env (Okuma et al., 1999
). cDNA clones of measles virus (MV) (Edmonston strain) H and F genes were subcloned into pCXN2, resulting in pCXN2H and pCXN2F, respectively (Tanaka et al., 1998
). pNL-E dNefdV3 was constructed from pNL4-3, containing a provirus of HIV-1 NL4-3 strain, by digesting the BglII site of the env gene (i.e. deletion of the V3 loop domain) and replacing part of the nef gene into the EGFP gene (pEGFP-1, Clontech). A cDNA clone of HIV-1 (HXB2 strain) envelope gp160 was inserted into pSV7d, which contains the SV40 promoter, resulting in HXB2-env.
Virus.
VSVG*-G is the recombinant VSV which was generated by reverse genetics as described previously (Takada et al., 1997
) and kindly provided by M. A. Whitt (University of Tennessee, TN, USA). The virus was derived from a full-length cDNA clone of the VSV genome (Indiana serotype) in which the coding region of the G protein was replaced by the coding region of a modified version of the GFP gene (
G*) and complemented with the VSV G protein. VSV
G*-G was grown and harvested by infecting 293T cells that had been transfected previously with pCAG-VSVG.
Preparation of pseudotype viruses.
To generate VSV pseudotypes, 0·75x106 293T cells per well were grown in a 6-well flat-bottom collagen I-coated microplate (IWAKI) and transfected with the expression plasmids using LipofectAMINE reagent (Gibco), according to the manufacturers protocol. Briefly, 100 µl of Opti-MEM I reduced-serum medium (Gibco), 5 µl LipofectAMINE reagent and 1 µg pCAGHTLV-I env, 0·7 µg pCAG-VSVG or 1 µg pCAGGS were mixed and incubated at room temperature for 15 min. The mixture was then added to 293T cells that had been preincubated with 0·75 ml of Opti-MEM I medium. After a 3·5 h incubation at 37 °C, transfected cells were replenished with 1·5 ml of DMEM, supplemented with 10% FCS, and cultured for 24 h. Cells were then infected with VSVG*-G at an m.o.i. of 2 for 1 h at 37 °C. Virus-infected cells were washed with FCS-free DMEM seven times and 2 ml of complete medium was subsequently added. After 1218 h of incubation at 37 °C in 5% CO2, culture supernatants were collected and centrifuged to remove cell debris. VSV
G*-Env, VSV
G*-G and VSV
G* were recovered from 293T cells transfected with pCAGHTLV-I env, pCAG-VSVG or pCAGGS, respectively. The VSV pseudotype complemented with MV (Edmonston strain) H and F proteins (VSV
G*-EdHF) was recovered from 293T cells transfected with both pCXN2H and pCXN2F (Tatsuo et al., 2000
).
To prepare the HIV-1 pseudotype, 293T cells transfected with both pNL-E dNefdV3 and HXB2-env were incubated with DMEM containing 10% FCS and 20 mM sodium butyrate for 24 h, and then in fresh medium for 24 h. The HIV-1 pseudotype complemented with HIV-1 (HXB2 strain) envelope glycoproteins (HIV-HXB2 env) was then recovered from the transfected 293T cells. Each virus stock was stored at -80 °C until use.
Immunoblot analysis.
VSV pseudotypes were prepared, as described above. Viruses in the supernatants were filtered and partially purified through 20% sucrose by centrifugation at 284000 g at 4 °C for 1 h. Purified virus pellets were dissolved in 40 µl of electrophoresis sample buffer (0·125 M TrisHCl pH 6·8, 20% glycerol, 4% SDS, 2% -mercaptoethanol, 0·012% bromophenol blue) and applied to SDSPAGE (520% gradient). Separated proteins were electrophoretically transferred to a Sequi-Blot PVDF membrane (Bio-Rad). Transferred proteins were reacted with an anti-gp46 MAb, LAT-27 (rat IgG) (1:100 dilution), which was kindly provided by Y. Tanaka (Tanaka et al., 1994
). Bound antibodies were then reacted with a 1:103 dilution of horseradish peroxidase-conjugated anti-rat IgG (heavy chain-specific) (Cappel, ICN) and were visualized by chemiluminescence and fluorography using an ECL Western immunoblotting kit (Amersham).
Titration of VSV pseudotypes in various cell lines.
A 96-well collagen I-coated microplate was prepared with 5x104 of adherent cells or 1x105 of suspension cells per well. After aspirating off the media, 50 µl of each virus stock (serially diluted when necessary) was inoculated into each well. After incubation at 37 °C for 1 h, 100 µl of complete medium was added to each well. Cells were incubated at 37 °C for 1218 h in a CO2 incubator. Titres of each virus in the various cell lines were determined in infectious units (IU) by counting the number of GFP-expressing cells under a fluorescence microscope.
Inhibition of VSV
G*-Env infection by neutralizing antibody.
A 50 µl aliquot of VSVG*-Env was preincubated with serially diluted LAT-27, which has HTLV-1-neutralizing activity, at 37 °C in a CO2 incubator for 15 min and then the mixtures were inoculated onto 293T or HepG2 cells. After 1 h of incubation, the inocula were aspirated off and complete media were added, together with the diluted antibody, as described above. Cells expressing GFP were counted under a fluorescence microscope after incubation at 37 °C in 5% CO2 for 1218 h.
Chemical modification of cells.
To treat cell surface proteins, 293T and MAGIC-5A cells were preincubated with pronase (Sigma) or trypsin (Gibco) in FCS-free DMEM at 37 °C in 5% CO2 for 20 min. To treat carbohydrates, 293T cells were preincubated with sodium periodate (NaIO4) (Sigma), heparinase or heparitinase (Flavobacterium heparinum) (Seikagaku) in FCS-free DMEM at 37 °C in 5% CO2 for 1 h. Also, to treat cell surface phospholipids, 293T cells were preincubated with either phospholipase A2 (porcine pancreas) (Sigma) or phospholipase C (Bacillus cereus) (Boehringer Mannheim) in FCS-free DMEM at 37 °C in 5% CO2 for 30 min. After incubation, an equal volume of complete medium was added to each well to stop the reaction of each agent. Cells were subsequently infected with approximately 1·02·0x103 IU of VSVG*-G, VSV
G*-EdHF, HIV-HXB2 env (only when MAGIC-5A cells were treated) or VSV
G*-Env. Infected cells were counted under a fluorescence microscope.
Inhibition of VSV
G*-Env infection by purified lipids.
Phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidyl-L-serine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidyl-DL-glycerol (PG) and sphingomyelin (SP) were obtained from Sigma. Chloroform solutions of these purified lipids were dried and solubilized in PBS containing 50 mM n-octyl- -D-glucopyranoside (OG) (Sigma) at a concentration of 10 mM. Lipids were stored at 4 °C. Aliquots of 100 µl per well of VSV
G*-Env, VSV
G*-EdHF or VSV
G*-G (1·0x103 IU) were preincubated with 100 µM of each purified lipid at room temperature for 1 h, inoculated onto 5x104 293T cells and incubated at 37 °C for 1 h. Inocula were aspirated after incubation and cells were then incubated with 200 µl DMEM per well containing 10% FCS. After 1218 h of incubation, infectivity of the viruses was evaluated under a fluorescence microscope.
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Results |
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First, in order to determine the role of cell surface proteins in virus entry, 293T cells were preincubated with pronase or trypsin and then infected with VSVG*-G, VSV
G*-EdHF or VSV
G*-Env (Fig. 3A
). Pronase treatment of cells markedly reduced VSV
G*-EdHF infectivity and also affected VSV
G*-G infectivity. In contrast, VSV
G*-Env infectivity remained almost the same even after the treatment with 500 µg/ml pronase. Trypsin treatment of cells reduced the infectivity of all three pseudotype viruses to a similar extent. These results suggest that the cellular receptor for HTLV-1 on 293T cells is relatively resistant to both pronase and trypsin. To confirm this finding, we used another cell line, MAGIC-5A, and an additional virus, HIV-HXB2 env (the HIV-1 pseudotype complemented with envelope glycoproteins of HIV-1 HXB2 strain). MAGIC-5A cells, which express the HIV-1 receptor CD4 and co-receptor CXCR4 (Feng et al., 1996
), are susceptible to HIV-HXB2 env as well as to the three VSV pseudotypes. Treatment of MAGIC-5A cells with 100 µg/ml pronase markedly reduced the infectivity of VSV
G*-EdHF and HIV-HXB2 env, but not that of VSV
G*-Env (Fig. 3B
). When the cells were treated with pronase at 500 µg/ml, the infectivity of VSV
G*-Env was reduced to less than 20% of that on the untreated cells, although VSV
G*-G infectivity was also mildly affected. Trypsin treatment of MAGIC-5A cells reduced the infectivity of VSV
G*-EdHF and HIV-HXB2 env, but not VSV
G*-Env. Taken together, these results suggest that although the cellular receptor for HTLV-1 may contain the protein component, it is relatively resistant to treatment with both pronase and trypsin compared with CD46 and CD4/CXCR4.
Second, we treated 293T cells with NaIO4, heparinase or heparitinase before infecting with VSVG*-G, VSV
G*-EdHF or VSV
G*-Env to determine the role of carbohydrates in virus entry (Fig. 4
). The treatment of cells with these reagents reduced the infectivity of VSV
G*-Env in a dose-dependent manner, but hardly affected that of VSV
G*-G and VSV
G*-EdHF. Furthermore, we treated 293T cells with either phospholipase A2 or C to determine the role of cell surface phospholipids in virus entry (Fig. 4
). Neither reagent reduced (but sometimes augmented) the infectivity of VSV
G*-EdHF over the concentration ranges tested. In contrast, the infectivity of VSV
G*-Env was inhibited in a dose-dependent manner after the treatment with either phospholipase A2 or C. The infectivity of VSV
G*-G was affected by phospholipase C, but only slightly affected by phospholipase A2. Therefore, we examined whether purified lipids could also reduce the infectivity of VSV
G*-Env (Fig. 5
). Among the various purified lipids tested, PC significantly reduced (and PG slightly reduced) the number of GFP-expressing cells produced by VSV
G*-Env infection, but not that produced by either VSV
G*-EdHF or VSV
G*-G infection. These findings suggest that HTLV-1 envelope glycoproteins interact with some carbohydrates and phospholipids on the cell surface during virus entry.
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Discussion |
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It has been difficult to quantitatively detect cell-free HTLV-1 infection because of the inefficient infectivity of cell-free HTLV-1. The reason for its low infectivity is not clear, but it may be due to a low fusion activity of the HTLV-1 envelope glycoproteins, resulting from their relatively small size, hydrophobicity, fragility and low affinity (Delamarre et al., 1996 ; Hildreth, 1998
). To date, cell-free HTLV-1 infection has been analysed using pseudotype viruses carrying the genome of another virus and HTLV-1 envelope glycoproteins. Clapham et al. (1984)
developed a plaque assay using a VSV pseudotype bearing both HTLV-1 envelope glycoproteins and VSV G protein, obtained by infecting HTLV-1-producing cells with VSV. The infectious titres of virus in susceptible cells ranged from 103 to 104 IU/ml, depending upon the cell types. However, the plaque assay using this VSV pseudotype may be affected by factors that interfere with the plating of VSV (Hoshino et al., 1985
) and requires anti-VSV antibody with/without DEAE-dextran or -polybrene. The pseudotype viruses possessing Moloney murine leukaemia virus (Mo-MuLV) core and HTLV-1 envelope glycoproteins were also generated with a selectable marker gene (Wilson et al., 1989
; Vile et al., 1991
). Though HTLV-1 envelope glycoproteins were incorporated into Mo-MuLV particles, the titre of the viruses was so low (less than 200 c.f.u./ml) that they could not efficiently infect susceptible cells. It was thought that these results may reflect low expression levels of HTLV-1 envelope glycoproteins, toxicity of expression plasmids to transfected cells, inefficiency of virus assembly, lability of pseudotype virions and/or inefficient uptake of pseudotype virions by susceptible cells. A pseudotype system using a defective HIV-1 genome in combination with HTLV-1 envelope glycoproteins in 293T producer cells has been developed recently (Sutton & Littman, 1996
). Introduction of additional copies of the rev gene and treatment of cells with sodium butyrate resulted in cell-free virus titres of greater than 104 IU/ml in HOS cells. However, the cell surface molecules on susceptible cells that interact with HTLV-1 gp46 or gp21 have not yet been identified definitively and the molecular mechanisms underlying HTLV-1 entry/membrane fusion and HTLV-1-induced syncytium formation remain to be elucidated.
In this study, we developed a novel quantitative assay system in which a recombinant VSV possessing the GFP gene instead of the G protein gene was complemented with HTLV-1 envelope glycoproteins provided in trans to recover infectivity. Immunoblot analysis showed the incorporation of HTLV-1 envelope glycoproteins into VSV particles (Fig. 1A). Since the incorporation of foreign envelope proteins into VSV particles appears to require oligomerization of the proteins (Owens & Rose, 1993
), HTLV-1 envelope glycoproteins may form oligomers. In fact, by analysis of a chimera with maltose-binding protein, it was reported recently that HTLV-1 gp21 forms trimers (Kobe et al., 1999
).
Infection by recombinant VSV complemented with HTLV-1 envelope glycoproteins (VSVG*-Env) was markedly inhibited by the anti-gp46 neutralizing MAb LAT-27 (Fig. 1C
). Since LAT-27 recognizes aa 191196 of gp46 (Tanaka et al., 1994
), this region appears to be a critical domain for the interaction with the putative HTLV-1 receptor(s) on susceptible cells in this system.
The present VSV pseudotype assay system yielded from 104 to 105 IU/ml on 293T cells, demonstrating that this assay system might be more useful than systems already available for cell-free HTLV-1 infection. Various cell lines tested showed susceptibility to VSVG*-Env in different degrees (Fig. 2
). Since VSV
G*-Env could infect HepG2, 293T, Vero and COS cells more efficiently than the other cell lines tested, then these susceptible cell lines may express more putative HTLV-1 receptor(s) and/or virus entry-associated molecule(s). The inefficient infection of HTLV-1-infected MT-2 and C91/PL cells by VSV
G*-Env may reflect down-regulation of HTLV-1 receptor(s) on these cells.
Chemical modification of 293T and MAGIC-5A cells by various reagents influenced the infectivity of VSVG*-Env on these cells (Figs 3
and 4
). These results indicated the importance of some cell surface proteins, phospholipids and carbohydrates, such as glycosaminoglycans (GAGs), in HTLV-1 envelope glycoprotein-mediated entry. The high susceptibility of HepG2, 293T, Vero and COS cells to VSV
G*-Env (Fig. 2
) might result from high cell surface expression of these molecules on these cells. Involvement of GAGs in HTLV-1 entry is consistent with previous reports that heparin and dextran sulfate inhibit HTLV-1-induced cell fusion (Ida et al., 1994
; Okuma et al., 1999
) and that a plant lectin (wheat-germ agglutinin) inhibits adsorption of HTLV-1 (Yang et al., 1994
). Treatment of 293T cells with either phospholipase A2 or C inhibited the infectivity of VSV
G*-Env and purified lipids, such as PC, also blocked infection (Figs 4
and 5
). These data suggested that cell surface phospholipids, including unknown lipids, that are reported to bind to HTLV-1 gp21 and inhibit HTLV-1-induced syncytium formation (Sagara et al., 1997
), might be involved not only in cellcell fusion, but also in cell-free HTLV-1 entry through interaction with gp21. Although treatment of 293T cells with either phospholipase A2 or C inhibited the infectivity of VSV
G*-G, the various purified lipids tested did not (Figs 4
and 5
). PS was reported to markedly inhibit VSV plaque formation (Schlegel et al., 1983
) and it is not clear why PS did not reduce the infectivity of VSV
G*-G in our study. Another phospholipid, which was not tested in the present study, may be involved in VSV entry. Furthermore, the infectivity of VSV
G*-Env was significantly inhibited only on the MAGIC-5A cells treated with 500 µg/ml pronase, whereas the infectivity of VSV
G*-EdHF and HIV-HXB2 env could be inhibited at the lower concentrations of pronase. The difference of the effects of pronase treatment on virus infectivity might reflect the difference in pronase sensitivity among the respective virus receptors. By treating human peripheral blood mononuclear cells with a high concentration (500 µg/ml) of trypsin, Trejo & Ratner (2000)
demonstrated recently that the HTLV receptor is a protein. Together with our data, these data may also indicate that the HTLV-1 receptor is relatively resistant to treatment with proteases. On the other hand, the infectivity of VSV
G*-G was reduced to some extent by treatment with pronase and trypsin (Fig. 3
). This effect may reflect excessive cell damage, since the receptor for VSV has been reported to be a phospholipid.
Thus, this novel and quantitative assay system using VSV pseudotypes revealed that cell surface proteins, phospholipids and GAGs are involved in HTLV-1 infection. Therefore, HTLV-1 appears to utilize multiple ubiquitous molecules on the surface of susceptible cells as the cellular receptor/co-receptor(s), resulting in the wide distribution of the receptor(s) and the broad host range of HTLV-1 (Sutton & Littman, 1996 ; Okuma et al., 1999
). Because it was demonstrated that HTLV-1 Tax most efficiently enhances virus transcription in CD4+ T-cells (Newbound et al., 1996
), it is possible that post-entry events rather than virus attachment and viruscell fusion are critical in determining the tropism of HTLV-1.
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Acknowledgments |
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References |
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Received 23 October 2000;
accepted 13 December 2000.