Journal
of General Virology (2001), 82, 821–830. Printed in Great Britain
...................................................................................................................................................................................................................................................................................
Analysis of the molecules involved in human T-cell leukaemia
virus type 1 entry by a vesicular stomatitis virus pseudotype
bearing its envelope glycoproteins
Kazu Okuma,1 Yoshiharu Matsuura,3 Hironobu Tatsuo,1 Yoshio Inagaki,4 Minoru Nakamura,2
Naoki Yamamoto4 and Yusuke Yanagi1
1, 2
Department of Virology1 and Department of Medicine and Biosystemic Science2, Graduate School of Medical Sciences,
Kyushu University, 812-8582, Fukuoka, Japan
3
Research Center for Emerging Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, 565-0871, Osaka, Japan
4
Department of Microbiology, Tokyo Medical and Dental University, 113-0034, Tokyo, Japan
Cellular entry of human T-cell leukaemia virus type 1 (HTLV-1) was studied by a quantitative assay
system using vesicular stomatitis virus (VSV) pseudotypes in which a recombinant VSV (VSV∆G*)
containing the gene for green fluorescent protein instead of the VSV G protein gene was
complemented with viral envelope glycoproteins in trans. Most of the cell lines tested showed
susceptibility to VSV∆G* complemented with either HTLV-1 envelope glycoproteins (VSV∆G*Env) or VSV G protein (VSV∆G*-G), but not to VSV∆G* alone, indicating that cell-free HTLV-1
could infect many cell types from several species. High concentration pronase treatment of cells
reduced their susceptibility to VSV∆G*-Env, while trypsin treatment, apparently, did not.
Treatment of the cells with sodium periodate, heparinase, heparitinase, phospholipase A2 or
phospholipase C reduced the susceptibility of cells to VSV∆G*-Env, but not to VSV∆G*
complemented with measles virus (Edmonston strain) H and F proteins (VSV∆G*-EdHF), which was
used as a control. Purified phosphatidylcholine also inhibited the infectivity of VSV∆G*-Env, but
not VSV∆G*-G. These findings indicated that, in addition to cell surface proteins, glycosaminoglycans and phospholipids play an important role in the process of cell-free HTLV-1 entry.
Introduction
Human T-cell leukaemia virus type 1 (HTLV-1) is a
retrovirus that is aetiologically associated with adult T-cell
leukaemia\lymphoma and chronic inflammatory disorders such
as HTLV-1-associated myelopathy\tropical spastic paraparesis
and uveitis (Hinuma et al., 1981 ; Osame et al., 1987 ; Mochizuki
et al., 1992). HTLV-1 infection is endemic in southern Japan,
southern USA, the Caribbean, Jamaica, South America and
equatorial Africa (Poiesz et al., 1980 ; Hinuma et al., 1981). The
main routes of HTLV-1 transmission are mother-to-child
(especially via breast feeding), sexual and parenteral (RobertGuroff et al., 1983 ; Hino et al., 1985).
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,
Author for correspondence : Kazu Okuma.
Fax j81 92 642 6140. e-mail kazu!virology.med.kyushu-u.ac.jp
0001-7487 # 2001 SGM
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 cocultivation 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
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K. Okuma and others
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-1induced 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.
Methods
Cells. HTLV-1-positive (MT-2, C91\PL) and -negative (MOLT-4,
Jurkat, Raji, Daudi, 293T, HepG2, HeLa, Vero, COS, MDCK, LMTK− and
NIH 3T3) cell lines have been maintained in our laboratory for a long
period. The MAGIC-5A cell line (a transfectant of HeLa cells expressing
CD4 and CCR5) was kindly provided by M. Tatsumi (National Institute
of Infectious Diseases, Tokyo, Japan). All the adherent cell lines were
cultured in complete Dulbecco’s Modified Eagle’s medium (DMEM,
Nissui), supplemented with 10 % foetal calf serum (FCS, Gibco), 50 µg\ml
streptomycin (Gibco) and 50 units\ml penicillin G (Gibco). Cells were
grown in 5 % CO at 37 mC. All the suspension cell lines were cultured in
#
RPMI
(Gibco) containing the same supplements and grown in 5 %
"'%!
CO at 37 mC.
#
Plasmids. Mammalian expression plasmid vectors pCAGGS and
pCXN2 contain the CAG promoter, which is composed of the
cytomegalovirus immediate early enhancer and the chicken β-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. VSV∆G*-G is the recombinant VSV which was generated by
ICC
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, 0n75i10' 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
manufacturer’s protocol. Briefly, 100 µl of Opti-MEM I reduced-serum
medium (Gibco), 5 µl LipofectAMINE reagent and 1 µg pCAGHTLV-I
env, 0n7 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 0n75 ml of Opti-MEM I medium. After
a 3n5 h incubation at 37 mC, transfected cells were replenished with 1n5 ml
of DMEM, supplemented with 10 % FCS, and cultured for 24 h. Cells
were then infected with VSV∆G*-G at an m.o.i. of 2 for 1 h at 37 mC.
Virus-infected cells were washed with FCS-free DMEM seven times and
2 ml of complete medium was subsequently added. After 12–18 h of
incubation at 37 mC in 5 % CO , 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
k80 mC 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 284 000 g at 4 mC for
1 h. Purified virus pellets were dissolved in 40 µl of electrophoresis
sample buffer (0n125 M Tris–HCl pH 6n8, 20 % glycerol, 4 % SDS, 2 % βmercaptoethanol, 0n012 % bromophenol blue) and applied to SDS–PAGE
(5–20 % 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 : 10$ dilution of horseradish peroxidaseconjugated 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 5i10% of adherent cells
or 1i10& 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 mC for 1 h, 100 µl of complete
medium was added to each well. Cells were incubated at 37 mC for
12–18 h in a CO incubator. Titres of each virus in the various cell lines
#
were determined in infectious units (IU) by counting the number of GFPexpressing cells under a fluorescence microscope.
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VSV pseudotype assay analysis of HTLV-1 entry
Fig. 1. (A) Incorporation of HTLV-1 envelope glycoproteins into VSV particles. Viral proteins of purified VSV∆G* (lane 1),
VSV∆G*-G (lane 2) and VSV∆G*-Env (lane 3) particles were analysed by immunoblot analysis with LAT-27. A molecular mass
ladder (kDa) is indicated on the left. (B) Expression of GFP upon infection with VSV pseudotypes. 293T cells were infected
with VSV∆G*, VSV∆G*-G (1 : 103 dilution) or VSV∆G*-Env and GFP expression was examined by fluorescence microscopy
12–18 h after infection. Magnification, i 200. (C) Effect of LAT-27 on VSV∆G*-Env infection. VSV∆G*-Env was preincubated
with LAT-27 and then inoculated onto 293T and HepG2 cells. The effect of the antibody was examined 12–18 h after
infection. An irrelevant MAb, rat IgG, was used as the negative control. Each datum point represents the percentage infectivity
(%IU, meanpSD) calculated from three microscopic fields compared with the infectivity of each virus on untreated cells.
Inhibition of VSV∆G*-Env infection by neutralizing antibody. A 50 µl aliquot of VSV∆G*-Env was preincubated with serially
diluted LAT-27, which has HTLV-1-neutralizing activity, at 37 mC in a
CO 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 mC in 5 % CO for
#
12–18 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 mC in 5 % CO for 20 min. To
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K. Okuma and others
Fig. 2. Infectivity of VSV pseudotypes on
various cell lines derived from several
species. Suspension (A) and adherent
(B) cell lines were infected with
VSV∆G*-G, VSV∆G* or VSV∆G*-Env
from the same batch. To adjust the
infectious titre, VSV∆G*-G was diluted
10−3 with DMEM containing 10 % FCS.
After 12–18 h of infection, virus titres in
various cells were determined by
counting the number of GFP-expressing
cells under a fluorescence microscope.
The data shown are representative results
from experiments repeated with some
batches of viruses.
treat carbohydrates, 293T cells were preincubated with sodium periodate
(NaIO ) (Sigma), heparinase or heparitinase (Flavobacterium hep%
arinum) (Seikagaku) in FCS-free DMEM at 37 mC in 5 % CO 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 mC in 5 % CO 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 1n0–
2n0i10$ IU of VSV∆G*-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--serine
(PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidyl--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- β--glucopyranoside (OG)
(Sigma) at a concentration of 10 mM. Lipids were stored at 4 mC. Aliquots
of 100 µl per well of VSV∆G*-Env, VSV∆G*-EdHF or VSV∆G*-G
(1n0i10$ IU) were preincubated with 100 µM of each purified lipid at
room temperature for 1 h, inoculated onto 5i10% 293T cells and
incubated at 37 mC for 1 h. Inocula were aspirated after incubation and
cells were then incubated with 200 µl DMEM per well containing 10 %
FCS. After 12–18 h of incubation, infectivity of the viruses was evaluated
under a fluorescence microscope.
ICE
Results
Development of an assay system with a novel VSV
pseudotype bearing HTLV-1 envelope glycoproteins
Recombinant VSV containing the GFP gene instead of the
G protein gene was either uncomplemented (VSV∆G*) or
complemented with either VSV G protein (VSV∆G*-G) or
HTLV-1 envelope glycoproteins (VSV∆G*-Env). Viruses were
purified by centrifugation and analysed by immunoblot for the
incorporation of HTLV-1 envelope glycoproteins (Fig. 1 A).
Using MAb LAT-27, HTLV-1 gp46 protein was observed
only in VSV∆G*-Env (Fig. 1 A, lane 3). To test whether HTLV1 envelope glycoproteins could rescue the infectivity of
VSV∆G* lacking the G protein, we infected 293T cells with
VSV∆G*, VSV∆G*-G or VSV∆G*-Env (Fig. 1 B). A large
number of cells expressed GFP after VSV∆G*-G or VSV∆G*Env infection, but few cells expressed GFP when infected with
VSV∆G*. The background infectivity of VSV∆G* may be
attributable to the remaining VSV∆G*-G used when the VSV
pseudotypes were prepared. Because the background infectivity was negligible (compared with the infectivity of
VSV∆G*-Env in 293T cells), VSV∆G*-Env was not treated
with anti-VSV antibody throughout this study. To ascertain
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VSV pseudotype assay analysis of HTLV-1 entry
Fig. 3. Infectivity of pseudotype viruses on cells whose surface proteins were chemically modified. 293T (A) and MAGIC-5A
(B) cells were preincubated with indicated concentrations of pronase or trypsin and infectivity of VSV∆G*-G (VSV), VSV∆G*EdHF (MV), HIV-HXB2 env (HIV-1) (only on MAGIC-5A cells) and VSV∆G*-Env (HTLV-1) was examined. The titre of each
virus was 1n5i107 IU/ml, 3n4i104 IU/ml, 1n7i104 IU/ml (on MAGIC-5A cells) and 5n3i104 IU/ml on 293T cells,
respectively. The viruses were diluted when necessary. The same viruses were also used in the experiments described in Figs
4 and 5. Each bar represents the percentage infectivity (%IU, meanpSD) calculated from three microscopic fields compared
with the infectivity of each virus on untreated cells.
the specificity of HTLV-1 envelope glycoprotein-mediated
infection, we examined the effect of MAb LAT-27, which
inhibits HTLV-1-induced syncytium formation (Fig. 1 C).
VSV∆G*-Env that had been preincubated with LAT-27 at
various concentrations was inoculated onto 293T and HepG2
cells. LAT-27, at a low concentration, inhibited the infection of
both cell lines by VSV∆G*-Env, whereas an irrelevant rat IgG,
even at high concentrations, did not inhibit infection. These
results indicated that the HTLV-1 envelope glycoproteins
incorporated into VSV particles specifically induced virus
entry by interacting with the putative cellular receptor(s) of
susceptible cells.
reported previously (Takada et al., 1997). All suspension cells,
except for HTLV-1-infected cells, exhibited susceptibility to
VSV∆G*-Env, as the infectivity titre of VSV∆G*-Env was at
least 1 log greater than that of VSV∆G* (Fig. 2 A). As HTLV1-infected MT-2 and C91\PL cells showed similar titres with
VSV∆G* and VSV∆G*-Env, virus entry dependent on HTLV-1
envelope glycoproteins could not be observed on these cells.
All adherent cell lines examined also exhibited susceptibility to
VSV∆G*-Env (Fig. 2 B) and showed titres between 1 and 4
logs greater than the titres seen with VSV∆G*. These results
suggested that cell-free HTLV-1 could infect various cell types
derived from several species.
Susceptibility of various cell lines to VSV pseudotypes
Effects of chemical modifications of cells on the
infectivity of pseudotype viruses
To analyse the presence of putative HTLV-1 receptor(s),
we examined the infectivity of VSV pseudotypes (as determined by counting the number of GFP-expressing cells) in
various cell lines derived from several species (Fig. 2). All
tested cells showed a high susceptibility to VSV∆G*-G, as
To characterize the biochemical nature of the molecule(s)
involved in HTLV-1 entry, we examined the infectivity of
VSV∆G*-Env on 293T cells that were chemically modified by
various reagents (Figs 3 and 4).
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K. Okuma and others
Fig. 4. Infectivity of pseudotype viruses on chemically modified cells to determine the role of carbohydrates and phospholipids
in virus entry. 293T cells were preincubated with the indicated concentrations of NaIO4, heparinase, heparitinase and
phospholipase A2 or C. Virus infectivity of VSV∆G*-G (VSV), VSV∆G*-EdHF (MV) and VSV∆G*-Env (HTLV-1) was then
examined. Each bar represents the percentage infectivity (%IU, meanpSD) calculated from three microscopic fields compared
with the infectivity of each virus on untreated cells.
Cellular receptors for VSV and MV, Edmonston strain,
have been reported to be phospholipids (Schlegel et al., 1983)
and glycoprotein CD46 (Naniche et al., 1993), respectively.
VSV pseudotypes bearing their envelope proteins (VSV∆G*G and VSV∆G*-EdHF) were used as controls.
First, in order to determine the role of cell surface proteins
in virus entry, 293T cells were preincubated with pronase or
ICG
trypsin and then infected with VSV∆G*-G, VSV∆G*-EdHF or
VSV∆G*-Env (Fig. 3 A). 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.
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VSV pseudotype assay analysis of HTLV-1 entry
Fig. 5. Effect of purified lipids on
VSV∆G*-Env infection. VSV∆G*-Env
(HTLV-1), VSV∆G*-EdHF (MV) and
VSV∆G*-G (VSV) were preincubated
with each purified lipid and inoculated
onto 293T cells. After 12–18 h of
incubation, the infectivities of the viruses
were evaluated. Each bar represents the
percentage infectivity (%IU, meanpSD)
calculated from three microscopic fields
compared with the infectivity of each
virus preincubated with only OG (non).
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 HIVHXB2 env, but not that of VSV∆G*-Env (Fig. 3 B). 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 NaIO , heparinase or
%
heparitinase before infecting with VSV∆G*-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 dosedependent 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 GFPexpressing 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.
Discussion
HTLV-1 appears to infect cells more efficiently by cell-tocell transmission, which may lead to syncytium formation, than
by cell-free transmission (Fan et al., 1992 ; Delamarre et al.,
1997). However, susceptible cells do not necessarily produce
syncytia when co-cultivated with HTLV-1-infected cells
(Sommerfelt et al., 1988 ; Delamarre et al., 1997). Thus, it is
difficult to analyse the expression of HTLV-1 receptor(s) and
identify cells lacking them by using an assay based on
syncytium formation. Recently, quantitative and sensitive
assay systems for analysing envelope glycoprotein-mediated
cell fusions were developed for HIV-1, HTLV-1 and HTLV-2
(Nussbaum et al., 1994 ; Poon & Chen, 1998 ; Okuma et al.,
1999). Although analysis of HIV-1-induced cell fusion by this
assay system resulted in the identification of a HIV-1 coreceptor, fusin\CXCR4 (Feng et al., 1996), similar studies have
failed to determine the HTLV-1 receptor(s).
It has been difficult to quantitatively detect cell-free HTLV1 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 10$ to 10% IU\ml, depending upon the cell types.
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K. Okuma and others
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 10% 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 HTLV1 envelope glycoproteins provided in trans to recover
infectivity. Immunoblot analysis showed the incorporation of
HTLV-1 envelope glycoproteins into VSV particles (Fig. 1 A).
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 maltosebinding protein, it was reported recently that HTLV-1 gp21
forms trimers (Kobe et al., 1999).
Infection by recombinant VSV complemented with HTLV1 envelope glycoproteins (VSV∆G*-Env) was markedly
inhibited by the anti-gp46 neutralizing MAb LAT-27 (Fig.
1 C). Since LAT-27 recognizes aa 191–196 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
10% to 10& 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 VSV∆G*-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
ICI
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 VSV∆G*-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 cell–cell fusion, but also in cellfree 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 HIVHXB2 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\coreceptor(s), resulting in the wide distribution of the receptor(s)
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VSV pseudotype assay analysis of HTLV-1 entry
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 postentry events rather than virus attachment and virus–cell fusion
are critical in determining the tropism of HTLV-1.
We thank M. A. Whitt for allowing us to use the VSV∆G–GFP
system. We also thank Y. Tanaka, H. Shida and M. Tatsumi for providing
LAT-27, pHAproenv and MAGIC-5A cells, respectively.
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Received 23 October 2000 ; Accepted 13 December 2000
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