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J Virol, March 1998, p. 1959-1966, Vol. 72, No. 3
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Identification of a Domain within the Human T-Cell
Leukemia Virus Type 2 Envelope Required for Syncytium Induction
and Replication
Betty
Poon1 and
Irvin S. Y.
Chen1,2,*
Department of Microbiology and
Immunology1 and
Division of
Hematology-Oncology,2 UCLA School of
Medicine, Los Angeles, California 90095
Received 12 May 1997/Accepted 13 November 1997
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ABSTRACT |
In vitro infection by human T-cell leukemia virus type 1 and 2 (HTLV-1 and HTLV-2) can result in syncytium formation, facilitating viral entry. Using cell lines that were susceptible to HTLV-2-mediated syncytium formation but were nonfusogenic with HTLV-1, we constructed chimeric envelopes between HTLV-1 and -2 and assayed for the ability to
induce syncytia in BJAB cells and HeLa cells. We have identified a
fusion domain composed of the first 64 amino acids at the amino terminus of the HTLV-2 transmembrane protein, p21, the retention of
which was required for syncytium induction. Construction of replication-competent HTLV genomic clones allowed us to correlate the
ability of HTLV-2 to induce syncytia with the ability to replicate in
BJAB cells. Differences in the ability to induce syncytia were not due
to differences in the levels of total or cell membrane-associated envelope or in the formation of multimers. Therefore, we have localized
a fusion domain within the amino terminus of the transmembrane protein
of HTLV-2 envelope that is necessary for syncytium induction and viral
replication.
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INTRODUCTION |
Human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2) are type C retroviruses that have been
associated with a variety of human malignancies. HTLV-1 is the
etiological agent of adult T-cell leukemia as well as a degenerative
neurological disorder, HTLV-1-associated myelopathy/tropical spastic
paraparesis (28, 40, 58, 60, 83). Recent reports have also
implicated HTLV-1 infection with arthropathy (42, 65),
polymyosis (23, 37), and uveitis (48, 49, 51).
HTLV-2 has been associated with a rare form of atypical hairy cell
leukemia (62, 63, 68) as well as some cases of neuropathy
(33, 39). It is estimated that between 10 million and 20 million individuals worldwide are infected with HTLV, with an overall
risk of 5% of disease progression in infected individuals
(14). HTLV is endemic in southern Japan, the Caribbean
Basin, and Central and South America. In the United States, recent
reports have identified a high proportion of HTLV, especially HTLV-2,
infection in intravenous-drug abusers (44, 61, 64).
Cell-to-cell contact is considered critical for the in vivo and in
vitro transmission of HTLV-1 and HTLV-2, as infection by cell-free HTLV
virus is inefficient in vitro and in vivo. By analogy with other
enveloped viruses, HTLV infection of susceptible cells is likely
mediated by the envelope glycoprotein. Antibodies against HTLV envelope
are protective against infection in vivo (71, 80), and
multiple epitopes that elicit neutralizing antibodies have been
identified throughout the protein (31, 34, 56). Initially
synthesized as a precursor protein, gp61, HTLV envelope is subsequently
modified by glycosylation and cleaved into two subunits, gp46 and p21.
The external surface glycoprotein, gp46, is anchored to the cell
surface by noncovalent association with the transmembrane envelope
glycoprotein, p21. Interaction of envelope with the as yet unidentified
cellular receptor leads to cell-to-cell fusion and can result in
syncytium formation.
We were interested in identifying the molecular determinants of HTLV
involved in syncytium formation and viral entry. Our laboratory has
several cell lines that are permissive to HTLV-2- but not
HTLV-1-mediated cell fusion. Therefore, we constructed recombinants
between the HTLV-1 and -2 envelope genes and assayed for the loss of
syncytium induction in BJAB cells and HeLa cells. Loss of a
64-amino-acid (aa) domain located at the amino terminus of the HTLV-2
transmembrane protein, p21, correlated with a loss in the ability of
the envelope chimera to induce cell fusion. When the chimeric envelopes
were expressed in the context of replication-competent genomic clones,
there was a good correlation between syncytium induction and the
ability to replicate in permissive cells. Present within the identified
fusion domain is a hydrophobic region and a heptad repeat resembling a
leucine zipper. We examined the contribution of the fusion domain to
the structural integrity of the HTLV-2 envelope by using a vaccinia
virus expression system. None of the recombinants affected the
synthesis, transport, or oligomer formation of the HTLV glycoprotein
complex.
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MATERIALS AND METHODS |
Cells.
BJAB cells were grown in RPMI 1640 medium
with 10% fetal bovine serum (FBS) (Gemini, Calabasas, Calif.). HeLa
cells were grown in Dulbecco modified Eagle medium containing 10% calf
serum. 729ph6neo and SLB1, which express infectious HTLV-2 and HTLV-1,
respectively, were grown in Iscove's medium with 20% FBS.
Oligonucleotide-directed mutagenesis and DNA manipulation.
The HTLV-2 envelope sequences from nucleotide (nt) 5123 to 7392 (72) from BCHTLV (30) and the HTLV-1 envelope
sequences from nt 5126 to 7480 (69) from Env1A (kindly
provided by D. Slamon, University of California, Los Angeles) were
subcloned between the SphI and MluI restriction
sites of CDM7 (kindly provided by D. Camerini, University of Virginia),
replacing a 2.6-kb fragment and creating CDM7-II and CDM7-I,
respectively. Following preparation of single-stranded DNA using M13
K07 phage, mutagenesis was performed according to the protocol for the
T7-GEN in vitro mutagenesis kit (United States Biochemical). The
nucleotide positions of the HTLV-1 sequences in clones NH, NK, KH, KM,
MH, and NKM are indicated in parentheses. Clones NH (nt 5179 to 6680),
NK (nt 5179 to 6118), KH (nt 6118 to 6680), KM (nt 6118 to 6290), MH
(nt 6290 to 6680), and NKM (nt 5179 to 6118 and 6290 to 6680) were
constructed by introducing compatible restriction sites within
HTLV-1 and -2 envelope. A novel HindIII site was
introduced at the ends of both genes. KpnI and
MstI sites, already present within the HTLV-1 envelope, were
introduced into the same locations within the HTLV-2 envelope. All
clones were confirmed by DNA sequencing. Oligonucleotides containing base pair substitutions that created the unique restriction sites (underlined) were as follows: for HTLV-2, 5' CTG CTA TTG
GTA CCG CAC GGC GGC G 3' (KpnI site 6105), 5' GCT GCA
AAG CTT GCA GGT CTA 3' (HindIII site 6650),
and 5' CGT CTA TTT TGC GCA GCA TAC TGT GC 3'
(MstI site 6292); for HTLV-1, 5' GGC TGG GGA AGC
TTG AGG CGA TGA G 3' (HindIII site 6682).
Constructs BM and KB were made by using overlapping oligonucleotides
that consisted of nt 6118 to 6200 (sense) and 6195 to 6310 (antisense)
of HTLV-2 and HTLV-1 envelope and nt 6116 to 6195 (sense) and 6184 to
6300 (antisense) of HTLV-1 and HTLV-2 envelope, respectively. The
oligonucleotides were annealed by heating in extension buffer (40 mM
Tris [pH 7.4], 20 mM MgCl2, 50 mM NaCl) at 65°C for 5 min and allowing the reaction to cool to 25°C. Extension occurred at
25°C for 10 min by the addition of 1 µl Sequenase (United States
Biochemical). The resulting 200-bp fragment, containing a unique
BamHI restriction site at nt 6200, was subsequently cloned
into CDM7-II between the newly created KpnI and
MstI restriction sites.
The genomic clones were obtained by inserting the chimeric envelopes
from CDM7 between the
SphI and
MluI restriction
sites
of H6H11. H6H11 contains the HTLV-2 genomic sequences from
H6
(
8) inserted between the
HindIII site of
pBR322.
The vaccinia virus-driven expression vectors containing the HTLV
envelopes were obtained by PCR amplification of the envelope
sequences
from CDM7 into the pTM-3 expression vector (kindly provided
by B. Moss,
National Institute of Allergy and Infectious Diseases).
The HTLV-2
envelope was amplified by using forward primer (5'
GCG GAA TTC TTT TCT
TCC TAC TTT TAT TC 3') and reverse primer
(5' GAA TCG AGT TAG GGC TGG
3'). The HTLV-1 envelope was amplified
by using forward primer (5' GGC
GAA TTC TTC TCG CCA CTT TGA TTT
3') and reverse primer (5' CGC AGA TCT
TAT CGG CGG GAG CGG GAT
CC 3'). The resulting 1.7-kb PCR fragments were
digested with
EcoRI and
PstI and inserted
downstream of the bacteriophage T7
promoter of pTM-3.
p24 assay.
Supernatants from transfected BJAB cells were
collected and clarified by centrifugation at 3,000 rpm for 5 min.
Analysis for the presence of HTLV p24 antigen was performed by
enzyme-linked immunosorbent assay (ELISA), using Coulter kit 6604252 according to the manufacturer's procedure.
Protein analysis.
Cells were lysed on ice for 10 min in OGL
lysis buffer (100 mM Tris [pH 8.0], 100 mM NaCl, 1 mM
CaCl2, 250 mM octyl-glucosidase). For detection of
denatured proteins, samples were heated at 100°C for 5 min in 1×
loading buffer (100 mM Tris [pH 6.8], 20% glycerol, 0.02%
bromophenol blue) with the addition of 4% sodium dodecyl sulfate (SDS)
and 5% 
-mercaptoethanol, and separated on SDS-10% polyacrylamide
gels. For detection of native proteins, samples were diluted in 1×
loading buffer and separated on 4 to 20% gradient acrylamide gels in
the presence of 0.01% SDS in the running buffer (79).
Western analysis was performed with anti-HTLV-1 gp46 antibody (clone
65/6c2.2.34; Cellular Products Inc., Buffalo, N.Y.) and developed by
using the Amersham enhanced chemiluminescence assay.
Transfections and blue syncytium assay.
Transient
transfection assays were performed by resuspending 5 × 106 BJAB cells in electroporation medium (RPMI 1640 with
20% FBS) and electroporating with 15 µg of DNA at 230 V and 960 µF.
The blue syncytium assay was performed as previously described
(
45), with the following modifications. HeLa cells were
infected
with wild-type vaccinia virus (WR) or VTF7-3 (multiplicity of
infection [MOI] = 1.0) for 2 h at 37°C. Cells were
subsequently
transfected with 15 µl of Lipofectin (Gibco BRL) and 10 µg of
DNA/10
5 cells in serum-free Dulbecco modified Eagle
medium as specified
by the manufacturer. Cells infected with VTF7-3
were transfected
with vaccinia virus-driven HTLV envelope constructs.
Cells infected
with WR were transfected with pEM-C
lacZgAn
(
45). At 16 h posttransfection,
cells expressing the
HTLV envelope clones were harvested by treatment
with
phosphate-buffered saline-EDTA and washed twice with medium.
Then
10
5 cells from each of the two sources, WR infected and
VTF7-3 infected,
were mixed in the presence of actinomycin D (1 µg/ml) to prevent
vaccinia virus spread. After a 7-h incubation at
37°C, cultures
were fixed and stained in situ for 1 h at 37°C
with solution containing
4 mM potassium ferrocyanide, 2 mM
MgCl
2, and 0.5 mg of
5-bromo-4-chloro-3-indolyl-
-
D-galactopyranoside
(X-Gal)
per ml. Giant blue syncytia were counted microscopically.
Flow cytometry.
Cells were stained with 1 µg of
anti-HTLV-1 gp46 antibody clone (65/6c2.2.34; Cellular Products)
diluted in 100 µl of FACS (fluorescence-activated cell sorting)
buffer (phosphate-buffered saline with 2% FBS and 0.01% sodium azide)
at 4°C for 20 min. After one wash with FACS buffer, cells were
incubated at 4°C, with fluorescein isothiocyanate-conjugated sheep
anti-mouse antibody (diluted 1/200; Cappel) for an additional 20 min.
Cells were resuspended in FACS buffer, and data were acquired on a
FACScan (Becton Dickinson) and analyzed with the Lysis 2 software
program.
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RESULTS |
The amino terminus of the HTLV-2 transmembrane protein
contains sequences required for syncytium formation in BJAB
cells.
HTLV-1 and -2 can induce syncytium formation upon
infection of a variety of cell types, including T- and non-T-cell lines (35, 47, 80). We had previously observed that an
Epstein-Barr virus-negative human B-cell line (32), BJAB,
formed numerous multinucleated cells upon cocultivation with
729ph6neo, an HTLV-2-producing cell line. In contrast,
cocultivation with HTLV-1-producing cells, such as SLB1 cells, did not
result in visible syncytia. We took advantage of the differential
ability of HTLV-1 and -2 to form syncytia in BJAB cells to define the
regions of the HTLV-2 genome involved in syncytium formation.
Previous experiments have mapped the fusion phenotype to the HTLV
envelope protein, as expression of envelope alone was sufficient
to
induce syncytia (
18,
45,
57). HTLV-1 and -2 show 75%
amino
acid homology between the envelope proteins, allowing construction
of
recombinants which should maintain the functionality of the
protein.
The chimeric envelope genes were subsequently substituted
into H6H11,
an infectious HTLV-2 genomic clone (Fig.
1). These
full-length clones containing
HTLV-1 envelope sequences allowed
us to study not only the
syncytium-inducing phenotype but also
the contributions of the envelope
domains in viral replication.
The genomic clones were transfected into
BJAB cells and, at 3
days posttransfection, analyzed microscopically
for syncytium
formation. At this time, the ability of the clones to
cause fusion
is due to the transient expression of viral proteins from
the
transfected DNA. Syncytia were defined as giant cells greater
in
diameter than three single cells.
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FIG. 1.
The amino terminus of HTLV-2 p21 is necessary but not
sufficient for syncytium formation in BJAB cells. BJAB cells (5 × 105) were transfected with genomic constructs containing
chimeric envelopes (shown) as described in Materials and Methods. At
day 3 posttransfection, cells were analyzed for syncytia by microscopic
analysis. + and  indicate that greater than or less than 10% of
the cell population were undergoing cell fusion, respectively. HTLV-2
sequences are depicted by open boxes; HTLV-1 sequences are depicted by
dark boxes.
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Transfection with the genomic HTLV-2 clone, H6H11, produced syncytia in
10 to 30% of the cell population. In contrast, a genomic
clone in
which the entire HTLV-2 envelope had been substituted
by the HTLV-1
envelope (NH) did not cause any visible cell fusion.
These results are
in agreement with our previous observation that
HTLV-2- but not
HTLV-1-producing cells can induce syncytia in
BJAB cells. These results
also confirmed the role of the envelope
gene in syncytium formation.
The HTLV envelope is composed of the external surface glycoprotein,
gp46, and the transmembrane anchoring protein, p21. We
studied the
contributions of these domains in syncytium induction
by substituting
these individual domains from HTLV-1 into the
HTLV-2 envelope. A clone
(NK) that had the gp46 domain substituted
by HTLV-1 envelope sequences
but retained the p21 transmembrane
domain of HTLV-2 was capable of
inducing syncytia in BJAB cells.
The reciprocal clone (KH), containing
a replacement of the p21
domain with HTLV-1 sequences, lost the
fusogenic phenotype. These
results appear to map the syncytium
induction phenotype in BJAB
cells to the HTLV-2 p21 transmembrane
protein.
Additional chimeras were constructed to further define the regions
within p21 necessary for cell fusion. Replacement of the
carboxyl
portion of HTLV-2 p21 with HTLV-1 sequences (MH) did
not affect
syncytium formation. However, substitution of the amino
terminus of
HTLV-2 p21 with HTLV-1 sequences (KM) resulted in
loss of cell fusion.
Therefore, this 64-aa region within the HTLV-2
p21 transmembrane
protein appeared to be necessary for syncytium
induction.
We next substituted either the amino or carboxyl portion of these 64 aa
from HTLV-2 p21 with HTLV-1 envelope sequences. These
smaller
substitutions within the amino terminus of HTLV-2 p21
revealed that a
region from HTLV-2 envelope comprised of aa 330
to 365 was required for
fusion. Substitution of these sequences
with the corresponding HTLV-1
sequences (BM) correlated with a
loss of syncytium induction. This
conclusion is further supported
by the retention of these sequences
from HTLV-2 p21 in all clones
that were able to cause syncytia in BJAB
cells (NK, MH, and KB).
Therefore, we have defined a region essential
for HTLV-2 fusion
in BJAB cells to a 34-aa domain located at the amino
terminus
of the HTLV-2 envelope transmembrane protein.
Loss of this 34-aa domain from HTLV-2 envelope negatively affected
syncytium induction, as seen by the inability to fuse BJAB
cells. We
next tested whether the sequences from the amino terminus
of HTLV-2 p21
were sufficient for syncytium induction in the context
of a recombinant
where all other envelope sequences were derived
from HTLV-1. A clone
containing the first 64 aa of HTLV-2 p21
in the context of HTLV-1
envelope (NKM) did not induce syncytia
in BJAB cells. A substitution of
the smaller 34-aa domain from
HTLV-2 also did not result in syncytium
induction by the HTLV-1
envelope (data not shown). Therefore, the
fusion domain identified
in HTLV-2 was not sufficient on its own to
confer the syncytium
induction phenotype upon the HTLV-1 envelope.
The amino terminus of HTLV-2 p21 is required for HTLV-2 replication
in BJAB cells.
Cell fusion is believed to be the major route of
HTLV viral spread, as cell-free transmission is highly inefficient both
in vivo and in vitro (6, 46, 52, 59). Therefore, we examined the ability of the recombinants to replicate in BJAB cells and correlated it with their ability to cause syncytia. The replicative potential of the chimeras in BJAB cells was determined by ELISA for the
production and secretion of viral core antigen, p24, over several weeks
(Fig. 2). All of the clones were p24
positive on day 3, indicating that all of the chimeras were capable of
viral protein production from the transfected DNA (data not shown). As
expected, the genomic HTLV-2 clone, H6H11, replicated to high levels
over this time period, as shown by an increase in the production of p24
in the supernatant. The increased p24 production correlated with
increased cell fusion with syncytia in over 80% of the cells during
this time period. All clones that were capable of causing cell fusion
(NK, MH, and KB) were also able to replicate in BJAB cells, producing
p24 at levels similar to those of H6H11 (Fig. 2A). Similarly, clones
that did not induce syncytia (KH, KM, BM, and NKM) did not replicate,
producing an initial burst of p24 due to the transfected DNA that was
not sustained over time (Fig. 2B). These results support the premise
that HTLV-2 spread is dependent on the cell fusion activity, as only
the clones capable of syncytium induction were also able to replicate
in BJAB cells. As the replication-competent clones (NK, MH, and KB)
also retained the amino terminus of the HTLV-2 p21 domain, we conclude
that the same 34-aa sequences within the transmembrane protein p21
confer both the syncytium induction phenotype and the replicative
capability in BJAB cells.
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FIG. 2.
HTLV-2 replication in BJAB cells requires sequences at
the amino terminus of p21. BJAB cells (5 × 105) were
transfected with genomic constructs containing chimeric envelopes as
described in Materials and Methods. Every 3 days, the medium was
changed by allowing the cells to settle and replacing half of the
medium. At 3, 7, 14, and 21 days posttransfection, supernatants were
collected and cell debris was removed by centrifugation at 3,000 rpm
for 5 min as described in Materials and Methods. The amount of secreted
HTLV p24 was quantitated by ELISA as described in Materials and
Methods. (A) Clones that induced syncytia in BJAB cells (Fig. 1) which
included full-length HTLV-2 envelope (II), as well as clones NK, MH,
and KB. (B) Clones that did not induce syncytia in BJAB cells (KH, KM,
BM, and NKM). (C) Results from the full-length HTLV-2 envelope
and the full-length HTLV-1 envelope (NH). The data are representative
of three independent experiments. OD550, optical density at
550 nm.
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Interestingly, the clone containing the entire HTLV-1 envelope
sequences (NH) was able to replicate to the same degree as
the
wild-type HTLV-2 (Fig.
2C), albeit at a slightly lower rate
and without
detectable cell fusion. Cocultivation of BJAB cells
with lethally
irradiated SLB1 cells, the original source of the
HTLV-1 envelope
clone, did not result in productively infected
BJAB cells, as evidenced
by the eventual decline in p24 production
over time (data not shown).
Therefore, there may be interactions
occurring between the HTLV-1
envelope and other HTLV-2 viral components
that allow replication in
BJAB cells.
Fusion of HeLa cells by HTLV-2 envelope alone requires the amino
terminus of p21.
The ability of the construct containing the
HTLV-1 envelope to replicate in BJAB cells suggested that interactions
of envelope with other viral proteins may be occurring. Therefore, we
investigated the contributions to syncytium formation of the HTLV
envelope protein alone in the absence of other viral components. We
next constructed vaccinia virus/T7 promoter-driven vectors that
expressed only the HTLV chimeric envelopes. HTLV envelope expressed by
the vaccinia virus/T7 polymerase expression system has been reported to
be produced in a properly processed and folded form (3).
We also used a modification of a sensitive blue syncytium assay to
quantitate the amount of cell fusion caused by the envelope
chimeras
(
45). Briefly, cells were transfected with a construct
containing the
-galactosidase gene under the control of the T7
RNA
polymerase promoter. A second set of cells were transfected
with the
HTLV envelope construct, as well as infected with vaccinia
virus
expressing the T7 RNA polymerase (VTF7-3). Upon mixing of
the two
populations, cell fusion would allow the T7 RNA polymerase
expressed
from VTF7-3 to activate the
-galactosidase gene, resulting
in
a blue syncytia.
In these experiments, HeLa cells were used, as they produced the
highest level of expression of HTLV envelope (data not shown).
In
addition, syncytia were easier to score in HeLa cells due to
the
adherent nature of the cells. HeLa cells, similar to BJAB
cells, also
showed a differential ability to be fused by HTLV-1
and -2 envelope
(Fig.
3). HTLV-2 envelope gave rise to
780 blue
syncytia, compared to the 9 syncytia produced by the HTLV-1
envelope
(NH). Clones that were capable of inducing syncytia in BJAB
cells
(NK, MH, and KB) also resulted in significant amounts of blue
syncytia. The majority of the clones that were unable to cause
fusion
in BJAB cells (KH, KM, and NKM) were also unable to cause
fusion in
HeLa cells. In general, the blue syncytium assay in
HeLa cells, using
vaccinia virus-expressed envelope, appeared
to confirm the fusion
results of the genomic clones in BJAB cells,
with one exception.
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FIG. 3.
The amino terminus of HTLV-2 p21 is required for
syncytium formation in HeLa cells. HeLa cells (106) were
infected with wild-type vaccinia virus (WR) or VTF7-3 (MOI = 1.0)
for 2 h at 37°C. HeLa cells that were infected with WR were
transfected with pEM-ClacZgAn, a plasmid in which the
Escherichia coli lacZ gene has been linked to the T7
promoter. Cells that were infected with VTF7-3 were transfected as
described in Materials and Methods with vaccinia virus constructs
expressing chimeric HTLV envelopes. At 16 h posttransfection,
105 cells from each of the two sources were mixed and
scored for blue syncytia as described in Materials and Methods. These
data represent the average of two duplicate wells and are
representative of two independent experiments.  ,
HTLV-2;  , HTLV-1.
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Although loss of the first 64 aa at the amino terminus of HTLV-2 p21
(KM) resulted in loss of syncytia in both HeLa cells
and BJAB cells,
cell fusion could be induced in HeLa cells by
clones that retained
either the amino or carboxyl portion of this
region (BM or KB,
respectively). In contrast, in BJAB cells, clone
KB was fusogenic
whereas clone BM did not form visible syncytia.
Therefore, syncytium
induction in HeLa cells required two discrete
adjacent domains within
p21 that included the 34-aa fusion domain
identified in BJAB cells. It
is possible that clone BM caused
a low level of fusion in BJAB cells
that was undetectable microscopically
and caused a detectable amount of
fusion in HeLa cells due to
the greater sensitivity of the assay.
Alternatively, there may
be differences in the interaction of regions
of envelope with
cellular components from these two cell lines. There
is also the
possibility that BJAB cells and HeLa cells differ in their
cell
surface requirements for fusion, akin to the different coreceptors
in the case of human immunodeficiency virus type 1 (HIV-1) cell
fusion.
The syncytium-inducing phenotype of the chimeric envelopes is not
due to differences in the levels of intracellular envelope.
The
ability of viral envelope proteins to induce syncytia is influenced by
several factors, including intracellular expression levels of the
envelope, density of the fusion protein on the cell surface, and
correct protein configuration. We were unable to examine the envelope
expressed by transfection of nonproductive genomic clones, as the
levels of envelope expression were below detection by both
radioimmunoprecipitation and flow cytometry (data not shown). We
therefore used the high levels of HTLV envelope produced by the
vaccinia virus system to analyze expression of the recombinants.
We first examined the intracellular levels of envelope in the vaccinia
virus-infected cells, using a monoclonal antibody that
recognizes gp46
from HTLV-1 and HTLV-2 (Fig.
4). By
Western analysis
on SDS-PAGE, approximately equal amounts of precursor
gp61 envelope
were detected from all of the chimeric envelope clones.
The majority
of the envelope protein existed as uncleaved precursor,
similar
to findings for infected cells (data not shown). These results
indicate that the difference in the ability of the clones to cause
fusion in HeLa cells was not attributable to differential expression
levels of the chimeric envelopes.
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FIG. 4.
The chimeric HTLV envelopes produce equivalent amounts
of intracellular gp61. HeLa cells (105) were infected with
VTF7-3 (MOI = 1) for 2 h at 37°C and transfected with
vaccinia virus constructs expressing chimeric HTLV envelopes as
described in Materials and Methods. Cells were harvested 16 h
posttransfection. Total cell lysates (10 µg) were separated on
SDS-10% polyacrylamide gels and analyzed for the presence of HTLV
envelope by Western blot analysis as described in Materials and
Methods. Sizes (in kilodaltons) of the molecular weight standards are
indicated on the left; gp61 is indicated by the arrow on the right.
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The chimeric envelopes are expressed at similar levels on the cell
surface of infected cells.
We next compared the levels of
expression of the recombinant envelopes on the cell surface by flow
cytometric analysis (Fig. 5). Compared to
uninfected cells, both wild-type HTLV-1 and -2 envelopes were
efficiently expressed by the vaccinia virus expression system. Similar
levels of HTLV envelope were detected on the surface of cells infected
with vaccinia virus expressing the various envelope chimeras regardless
of their ability to induce syncytia. The anti-gp46 antibody was
specific for the HTLV envelope, as we detected a significant level of
HTLV envelope on the cell surface of the transfected cells over an
isotype control antibody (data not shown). The expression by all the
recombinant envelope constructs of comparable levels of gp46 on the
cell surface suggested that the loss of syncytium induction was not due
to a defect in the transport of gp46 to the membrane.
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FIG. 5.
The chimeric HTLV envelopes are expressed efficiently on
the cell surface of infected cells. HeLa cells (105) were
infected with VTF7-3 for 2 h at 37°C and transfected with
vaccinia virus constructs expressing chimeric HTLV envelopes as
described in Materials and Methods. Cells were harvested 16 h
posttransfection, and 5 × 103 cells were analyzed by
flow cytometry for expression of envelope on the cell surface as
described in Materials and Methods. The histograms indicate relative
cell number (y axis) as a function of relative amount of
gp46 on the cell surface (x axis). Mock-infected cells are
represented by the white histogram area, and cells transfected with
chimeric HTLV envelopes are represented by the dark histogram area.
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The HTLV envelope chimeras are capable of forming multimers.
Oligomerization of envelope proteins is necessary for their proper
function and viral infectivity. HTLV-1 envelope has been previously
reported to be capable of forming multimers (55). The 64-aa
domain in HTLV-2 envelope that we have identified as important in cell
fusion and viral replication in BJAB cells contains a region that bears
similarities to a leucine zipper motif (Fig. 6A) (7). The different
syncytium-inducing phenotypes may therefore be due to effects on the
multimerization of the envelope protein. We examined the
oligomerization potential of the chimeric envelopes by Western analysis
of native protein complexes separated on nondenaturing protein gels
(Fig. 6B). The wild-type HTLV-1 migrated at approximately 125 kDa,
confirming that the HTLV-1 envelope exists as a multimer. Similarly,
the HTLV-2 envelope also migrated at 125 kDa, indicating that the
HTLV-2 envelope may also function as a multimer. Analysis of the
chimeric envelopes revealed that all of the recombinant envelopes also
migrated as multimers. As an additional control, we first denatured the
HTLV protein complexes at 100°C and then separated them on the same
nondenaturing protein gels as specified above. These proteins migrated
at 61 kDa, the expected size for the monomeric form of the HTLV
envelope (Fig. 6C). There was no correlation between the ability of the
chimeric envelopes to induce syncytia and their ability to form
oligomers. Therefore, the inability of some of the chimeric envelopes
to induce syncytia was not due to a failure to multimerize.
View larger version (25K):
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|
FIG. 6.
The chimeric HTLV envelopes are similar in the ability
to form multimers. HeLa cells (106) were infected with
VTF7-3 for 2 h at 37°C and transfected with vaccinia virus
constructs expressing chimeric HTLV envelopes as described in Materials
and Methods. Cells were harvested 16 h posttransfection and lysed
in 500 µl of OGL buffer. (A) Sequence comparison of the amino
terminus of HTLV-2 and HTLV-1 p21 proteins. The first 64 aa of HTLV-2
and HTLV-1 p21 are represented. The nonconservative amino acid changes
between HTLV-2 and HTLV-1 are boxed. (B) Western analysis of native
envelope proteins. Total-cell lysates (20 µl) were separated on a 5 to 20% gradient nondenaturing acrylamide gel with 0.01% SDS in the
running buffer as described in Materials and Methods. HTLV envelope was
detected by Western blot analysis as described in Materials and
Methods. Sizes (in kilodaltons) of the molecular weight standards are
indicated on the left; the multimeric forms of HTLV envelope are
indicated by the arrow on the right. (C) Western analysis of denatured
envelope proteins. Total-cell lysates (20 µl) were heated to 100°C
for 5 min and separated on a 5 to 20% gradient nondenaturing
acrylamide gel with 0.01% SDS in the running buffer as described in
Materials and Methods. HTLV envelope was detected by Western blot
analysis as described in Materials and Methods. Sizes of the molecular
weight standards are indicated on the left; gp61 is indicated by the
arrow on the right.
|
|
| |
DISCUSSION |
The amino terminus of HTLV-2 p21 encodes a fusion domain required
for HTLV-2 syncytium induction and replication.
We have analyzed
the requirements for syncytium induction by the HTLV-2 envelope
protein. Our results have identified a 64-aa domain located within the
amino terminus of the transmembrane protein as necessary for
HTLV-2-mediated cell fusion of BJAB cells and HeLa cells. This region
was not sufficient by itself to confer the syncytium-inducing
phenotype, as the presence of this domain within the HTLV-1 envelope
did not permit fusion. The ability of HTLV-2 genomic clones to
replicate in permissive cells correlated with the ability to induce
syncytia. We were unable to attribute the loss of the
syncytium-inducing phenotype to total cell levels, levels of
membrane associated, or oligomerization of the envelope protein.
In this study, we used two cell lines, BJAB and HeLa, that were
permissive for HTLV-2- but not HTLV-1-mediated cell fusion.
However,
other groups have previously reported on the ability
of HTLV-1 to
induce syncytia in HeLa cells (
16,
18). Agadjanyan
et al.
have also reported that several subclones of a BJAB cell
line were
fusogenic upon cocultivation with both HTLV-2 and HTLV-1
(
1). Our inability to observe fusion with HTLV-1 envelope in
BJAB cells or HeLa cells may be a property of the cell lines upon
propagation. Distinct properties may arise in cell lines upon
long-term
culture, as illustrated by the isolation of subclones
of BJAB cells
that have lost the ability to form syncytia with
HTLV-2 (
1).
Alternatively, the HTLV-1 envelope used in our
studies may have cell
tropisms distinct from those of other HTLV-1
envelope clones. Sequence
analysis between the Env1A envelope
from SLB1 cells and the envelope
derived from a replication-competent
HTLV-1 clone (
41)
revealed no amino acid differences within
the putative fusion domain
(data not shown). However, there are
a total of seven amino acid
substitutions elsewhere in the envelope
protein which may account for
the differences observed.
Previous work in our lab has suggested that the transmembrane protein
of HTLV-2 is required for cell fusion, as expression
of gp46 alone was
not sufficient to induce syncytia (
45). In
addition, large
substitutions within the HTLV-1 transmembrane
protein by murine
leukemia virus envelope sequences abolished
HTLV-1-mediated cell fusion
(
17,
18). These studies localized
the fusion domain within
the extracellular portion of the transmembrane
protein. We have further
defined the fusion domain in HTLV-2 to
the amino terminus of p21. It is
likely that the corresponding
region in HTLV-1 envelope serves a
similar role since linker insertion
mutations within the hydrophobic
stretch in the N-terminal part
of HTLV-1 p21 resulted in loss of
syncytium induction (
57).
Potential mechanisms of viral entry mediated by the HTLV-2 fusion
domain.
Fusion domains have been localized to the transmembrane
protein, and specifically to the amino terminus, of other retroviral envelopes, including HIV-1 and simian immunodeficiency virus (25, 81). These domains are highly hydrophobic with a predicted
-helical structure. It has been hypothesized that these domains form
sided helixes with most of the bulky hydrophobic residues on one side of the helix (15). Located adjacent to these hydrophobic
sequences are heptad repeat sequences with nonpolar residues at the
first and fourth positions, similar to a leucine zipper motif
(7). This region has been postulated to play a role in
stabilizing the oligomeric form of these molecules. However, mutations
in the HIV-1 leucine zipper, while abolishing syncytium formation with
CD4+ cells and impairing infectivity, did not affect the
ability of the envelope protein to form oligomers (10, 22).
Mutagenesis of the HIV-1 envelope point to a critical role of the
leucine zipper motif in HIV-1 membrane fusion and virus entry, likely at a post-CD4 binding step (82).
The HTLV-2 fusion domain that we have identified contains a hydrophobic
domain adjacent to a region with homology to a leucine
zipper motif. In
BJAB cells, the syncytium-inducing phenotype
appeared to map to the
leucine zipper region. However, substitution
of the HTLV-2 leucine
zipper domain did not ablate fusion in HeLa
cells, indicating that in
some cell types this domain alone may
not be necessary for fusion. The
mechanism by which the HTLV-2
fusion domain contributes to syncytium
induction remains to be
clarified. There was no apparent correlation
between the ability
of the HTLV envelope chimeras to induce syncytia
and the intracellular
or cell surface expression, nor was there an
apparent effect of
the recombinants on the formation of multimers,
similar to the
HIV-1 results. One possible hypothesis is that the
fusion domain
in HTLV-2 p21 is required for receptor-mediated
conformational
changes. It is known that conformational changes occur
upon HIV-1
envelope binding to CD4. Post-CD4 binding events included
enhanced
antibody binding and cleavage by an exogenous proteinase of
the
V3 loop, a major neutralizing determinant in gp120 (
13,
66),
as well as exposure of previously cryptic epitopes (
67,
77).
One indicator of overall changes in the HIV-1 envelope
protein
is the observed enhanced shedding of gp120 upon CD4 binding
(
4,
76,
78). Introduction of prolines into highly conserved
leucine
or isoleucine residues of the HIV-1 leucine zipper affected
secretion
of gp120 (
9), suggesting the involvement of this
region in
modifying the envelope tertiary structure. Other viral fusion
proteins similarly undergo conformational changes in order to
acquire
their fusion potential (
29,
81). Therefore, the loss
of
syncytium induction and infectivity by our chimeric envelope
constructs
may be due to an effect on the ability of the envelope
to undergo the
appropriate conformational changes required for
HTLV viral entry.
A second hypothesis for the role of the fusion domain in HTLV-2 in
syncytium induction may be at the level of receptor utilization.
Precedence for this possibility exists for HIV-1, where syncytium
formation can be mediated by sequences which include the V3 loop
in
gp120 (
13,
26,
54). The V3 loop has also been implicated
in
the cell tropism observed for various strains of HIV-1. Changes
in the
V3 loop can influence the efficiency of entry of different
HIV-1
strains into different cell types, such as T-cell lines,
macrophages,
and microglial cells (
11,
36,
38,
50,
70,
73,
74). Although
the primary receptor for HIV-1 binding to
cells is CD4, recent reports
have identified secondary receptors,
CCR-5 and CXCR-4, that modulate
viral entry. CCR-5 and CXCR-4
are both members of the chemokine
receptor family and mediate
infection at an early stage by
macrophage-tropic and T-cell-tropic
viruses, respectively (
2,
5,
19-21,
24,
43). The ability
of HIV-1 to utilize CCR-5 or CXCR-4
has been mapped to the V3
loop (
12,
53), previously
implicated in syncytium formation
and viral entry. Using a highly
sensitive syncytium assay, we
have described cell lines that display
differential abilities
to be fused by HTLV-1 and -2. Yet, based on
receptor interference
studies, HTLV-1 and -2 have been postulated to
share a common
receptor on the tk-1 region of human chromosome 17 (
27,
75).
Therefore, it is conceivable that, analogous to
HIV-1, the fusion
capability of HTLV-1 and -2 in BJAB and HeLa cells is
mediated
by interaction of the fusion domain with accessory molecules
that
determine cellular tropism.
| |
ACKNOWLEDGMENTS |
We thank Qi-Xiang Li, Jia-Qi Zhao, and Marilee Greenwald for
assistance in preparation of vaccinia virus stocks, and we thank Yi-ming Xie for assistance with the blue syncytium assay.
This work was supported by NIH grant CA38597 and the Leukemia Society
of America. B.P. was supported by Public Health Service training grant
GM07185, and I.S.Y.C. was a Scholar of the Leukemia Society of America.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: 11-934 Factor,
Division of Hematology-Oncology, Department of Medicine, UCLA School of
Medicine, Los Angeles, CA 90095-1678. Phone: (310) 825-4793. Fax: (310)
794-7682. E-mail: rtaweesu{at}MED1.MEDSCH.ucla.edu.
| |
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J Virol, March 1998, p. 1959-1966, Vol. 72, No. 3
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
