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J Virol, February 1998, p. 1632-1639, Vol. 72, No. 2
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Mutational Analysis of the Fusion Peptide of
Moloney Murine Leukemia Virus Transmembrane Protein p15E
Nian-Ling
Zhu,
Paula M.
Cannon,*
Dagang
Chen, and
W. French
Anderson
Gene Therapy Laboratories, Norris Cancer
Center, University of Southern California School of Medicine, Los
Angeles, California 90033
Received 13 June 1997/Accepted 5 November 1997
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ABSTRACT |
Fusion peptides are hydrophobic sequences located at the N terminus
of the transmembrane (TM) envelope proteins of the orthomyxoviruses and
paramyxoviruses and several retroviruses. The Moloney murine leukemia
virus TM envelope protein, p15E, contains a hydrophobic stretch of
amino acids at its N terminus followed by a region rich in glycine and
threonine residues. A series of single amino acid substitutions were
introduced into this region, and the resulting proteins were examined
for their abilities to be properly processed and transported to the
cell surface and to induce syncytia in cells expressing the ecotropic
receptor. One substitution in the hydrophobic core and several
substitutions in the glycine/threonine-rich region that prevented both
cell-cell fusion and the transduction of NIH 3T3 cells when
incorporated into retroviral vector particles were identified. In
addition, one mutation that enhanced the fusogenicity of the resulting
envelope protein was identified. The fusion-defective mutants
trans dominantly interfered with the ability of the
wild-type envelope protein to cause syncytium formation in a cell-cell
fusion assay, although no trans-dominant inhibition of
transduction was observed. Certain substitutions in the hydrophobic
core that prevented envelope protein processing were also found. These
data indicate that the N-terminal region of p15E is important both for
viral fusion and for the correct processing and cell surface expression of the viral envelope protein.
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TEXT |
The envelope protein of the
ecotropic Moloney murine leukemia virus (MoMuLV) comprises two
polypeptides, the surface (SU) glycoprotein gp70 and the transmembrane
(TM) glycoprotein p15E (41, 42). These proteins are
processed from a common precursor, Pr85, by a host cell protease during
transport to the cell surface and remain associated following cleavage.
Infection of susceptible cells by MoMuLV is initiated by a specific
interaction between gp70 (19, 24, 37) and the ecotropic
receptor ATRC-1 (1). The interaction of retroviral SU
proteins with their cognate receptors is thought to induce a
conformational change that is transmitted to the TM subunit, thereby
triggering events that eventually lead to fusion of the viral and host
cell membranes (26). Several studies have implicated the
amino terminus of retroviral TM proteins as being important in
catalyzing the fusion of viral and cellular membranes (2-4, 7, 9,
11, 17, 21, 32, 36, 38). By analogy to the fusion proteins of the
orthomyxoviruses and paramyxoviruses, this region is referred to as the
fusion peptide.
Fusion peptides are relatively hydrophobic regions, typically rich in
alanine and glycine residues (38). It has been suggested that they may form sided helices, with bulkier, more hydrophobic residues on one side associating with the membrane and smaller residues, such as glycine and alanine, on the other (5, 7, 18, 38,
39), although this model has been contested (15). A
candidate fusion peptide at the amino terminus of the human immunodeficiency virus type 1 (HIV-1) TM protein, gp41, was identified by virtue of its sequence homology and analogous position to the fusion
peptide of the paramyxovirus ReSV F1 glycoprotein (13). It
contains a stretch of hydrophobic amino acids with the sequence LFLGFLG, and the sequence F-x-G is also present in the
fusion peptides of paramyxoviral F1 proteins. In addition, this whole region shows a periodicity of glycine residues (4, 7).
The N terminus of the MoMuLV TM protein, p15E, is also hydrophobic and
rich in alanine, glycine, and leucine residues. A stretch of
hydrophobic residues with the sequence LALLLGGL occurs 7 to 14 residues from the SU-TM cleavage site and could provide a fusion core. Previous mutational analyses of HIV-1 (2, 9, 11, 23),
simian immunodeficiency virus (4), and AKV murine leukemia virus (MuLV) (21) have demonstrated the importance of the
hydrophobic residues in this region, as well as the conserved glycine
residues (3, 7).
Computer modeling predicts that the fusion core of gp41 is followed by
an internally looping structure of 8 to 17 amino acids, enriched in
serine and threonine residues (ST domain) (14). In MuLV,
there exists a similarly located region that is rich in glycine and
threonine residues. In HIV-1, this region has been implicated in the
noncovalent association between gp41 and the SU protein gp120, as both
mutations (7, 11) and insertions (23) in this
region resulted in the loss of cell-associated gp120.
Retroviral vectors based on MuLV are currently the most commonly used
system for human gene therapy. Considerable effort has been directed
towards engineering the envelope protein to enable retargeting of
virions to specific cell surface ligands (reviewed in reference
6). However, although binding of virions to specific cell surface antigens has been achieved, this does not result in
efficient transduction. We are therefore interested in understanding in
more detail the postbinding changes that occur in the envelope protein
and the role of the putative fusion peptide in this process.
The organization of the envelope protein of MoMuLV and the amino acid
sequence of the N terminus of the TM protein are shown schematically in
Fig. 1. This region contains a stretch of
hydrophobic residues that may constitute a hydrophobic core of a fusion
peptide followed by a region rich in glycine and threonine residues
(the GT region). In order to study the importance of this region for envelope function, we introduced both conservative and nonconservative substitutions into the hydrophobic core and the GT region and additionally mutated residues further downstream. Mutants were constructed in the envelope protein expression vector CEE+
(24), which expresses the MoMuLV envelope protein from a
cytomegalovirus promoter, by oligonucleotide-directed in vitro
mutagenesis (Amersham International plc).
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FIG. 1.
The N terminus of MoMuLV TM (p15E) protein. The 38 N-terminal amino acids of p15E analyzed in this study (hatched box) and
the gp70-p15E cleavage site (arrow) are indicated. Numbering is from
the start of the processed envelope protein after removal of the
33-amino-acid signal sequence (33), with the start of p15E
at residue E437. The specific mutations that were introduced are shown
below the wild-type MoMuLV sequence.
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Cell-cell fusion mediated by envelope protein mutants.
Retroviral envelope proteins can mediate both virus-cell fusion
(fusion from without) and cell-cell fusion (fusion from within) (40). The cocultivation of NIH 3T3 cells expressing
the MoMuLV envelope protein with the rat XC cell line causes cell-cell
fusion and leads to the formation of syncytia (29). The
ability of wild-type and mutant envelope proteins to direct cell-cell
fusion was tested by transfecting 1 × 105 NIH 3T3
cells with 15 µg of envelope protein expression plasmids in gridded
(2- by 2-mm) 60-mm-diameter petri dishes and overlaying the cells with
5 × 105 XC cells at 18 to 20 h posttransfection.
Following incubation for a further 36 h, the cells were fixed and
stained with 1% methylene blue in methanol and the syncytia were
counted, as described previously (21, 29).
In addition, XC cells were cocultivated with GPL cells transiently
transfected with envelope protein expression plasmids.
GPL cells
(
27) are NIH 3T3 cells expressing MoMuLV Gag-Pol and
containing the Neo
r retroviral vector LNL6. Transfection of
an envelope protein expression
plasmid into these cells results in the
production of retroviral
vector particles, so this assay is sensitive
to both fusion from
within and fusion from without.
The ability of the mutant envelope proteins to induce syncytia was
compared to that of the wild-type envelope protein (Table
1). All mutations in the hydrophobic
domain, except the L441F
substitution, drastically reduced or abolished
syncytium formation
in both GPL and NIH 3T3 transfections. In the
GT-rich domain,
the A457F, G460E, and T461P substitutions prevented
syncytium
formation, and mutant G460V exhibited decreased fusion
ability.
In contrast, several mutants with substitutions of the
threonines
at positions 463 and 464 retained nearly wild-type levels of
fusion
activity. In the more C-terminal region, the conversion of
hydrophobic
residues to charged residues for both M467E and L475R
prevented
syncytium formation. However, the A468R substitution had no
effect,
and mutant Q474E was only partially defective. These data
suggest
that mutations throughout the N-terminal region of p15E can
affect
some aspect of the fusion process.
Interestingly, mutant T464A exhibited an increased ability to induce
syncytia in XC cells cocultivated with both NIH 3T3 cells
and GPL cells
(182 and 337% of wild-type levels, respectively).
To further examine
this mutant, we carried out a time course analysis
of syncytium
formation following the addition of XC cells to transfected
GPL cells
(Fig.
2a).
Mutant T464A caused the appearance of syncytia
after only 5 h of
cocultivation, while the wild-type envelope
protein and the
fusion-defective mutant Q474E started to form
syncytia only after
9 h in culture. At 12 to 15 h, T464A expression
resulted in
the appearance of large polynuclear syncytia with
over 20 nuclei, while
the wild-type protein formed smaller syncytia
with fewer than 10 nuclei. After 24 h, the number of syncytia
formed by mutant T464A
was 2-fold higher than that formed by the
wild type, while mutant Q474E
formed 3.5-fold fewer syncytia than
the wild type (Fig.
2b).
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FIG. 2.
Syncytium formation by wild-type p15E and p15E mutants.
(a) GPL cells were transfected with wild-type and mutant envelope
protein-expressing vectors and cocultured with XC cells, and the
syncytia were counted at various times.  , wild type;  , T464A;
 , Q474E. The results are the averages from two independent
experiments. (b) XC cells at the 24-h time point from one experiment
were photographed at ca. ×50 magnification. A, negative control; B,
wild type; C, T464; D, Q474E.
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Transduction properties of mutants.
We further analyzed the
ability of the mutant envelope proteins to cause virus-cell fusion by
measuring the transduction efficiencies of retroviral vectors obtained
from the transfected GPL cells. Culture supernatants were tested for
their ability to confer G418 resistance to NIH 3T3 cells as previously
described (27) by selection with 0.8 mg of G418 (Gibco/BRL)
per ml for 8 to 10 days.
The transduction efficiency of each mutant relative to that of the wild
type is shown in Table
1. In general, the results
agreed with the
fusion data. In the hydrophobic core, all of the
mutants except L441F
significantly decreased or abolished the
viral titers, and in the
GT-rich region, the nonfusogenic mutants
A457F, G460E, and T461P were
unable to transduce NIH 3T3 cells.
The hyperfusogenic mutant, T464A,
which produced two- to threefold
more syncytia than the wild type in
the cell-cell fusion assays,
showed wild-type levels of transduction.
Interestingly, two mutants
that failed to induce syncytia in
cocultivated XC cells (M467E
and L443I) were able to transduce NIH 3T3
cells at levels that
were 3 to 4% of the wild-type level, suggesting
that the process
of cell-cell fusion is more sensitive to envelope
protein mutations
than virus-cell fusion.
R-peptide cleavage does not enhance fusogenicity of defective
mutants.
In viral particles, the cytoplasmic tail of MoMuLV
envelope protein is cleaved by the viral protease to remove the
C-terminal 16 amino acids (the R peptide). It has previously been shown
that this truncation enhances the fusogenicity of the protein and
results in syncytium formation when the truncated envelope protein is expressed in NIH 3T3 cells (30, 31). We therefore examined the effect of R-peptide truncation on wild-type envelope protein and
fusion-defective and hyperfusogenic envelope protein mutants. The
results, shown in Table 2, confirmed that
expression of an R-less version of the wild-type protein resulted in
massive syncytia in NIH 3T3 cells, as previously reported (30,
31). Furthermore, in agreement with the minimal effect seen in
the XC cocultivation assays, the R-less versions of mutants L441F and
A468R also produced maximum syncytium formation in NIH 3T3 cells.
Removal of the R peptide of mutant Q474E, which displayed reduced
syncytia in the XC cocultivation assay, resulted in an envelope protein
that was still less fusogenic than the wild-type protein. Furthermore, an R-less version of the completely nonfusogenic mutant T461P did not
result in any syncytia, suggesting that the defects in fusogenicity
resulting from these mutations could not be compensated for by
R-peptide truncation. Finally, the R-less version of the hyperfusogenic
mutant, T464A, was no more fusogenic than the wild-type protein in this
system.
SU-TM protein processing and surface expression of mutant
envelopes.
The envelope proteins of retroviruses are transported
through the endoplasmic reticulum and the Golgi complex to the cell surface, where they are incorporated into budding virions. Cleavage of
the precursor protein into SU and TM subunits is required for this
transport process (12, 25). The position of the fusion peptide close to the SU-TM cleavage site made it a possibility that
some of the nonfusogenic mutants were actually defective in envelope
protein processing or cell surface expression. Accordingly, we examined
the nature of the envelope protein present in lysates of transfected
293T cells (Fig. 3). Wild-type envelope
protein was present as both the uncleaved precursor protein Pr85 and
the cleaved gp70 SU subunit. However, for certain mutants (L443A/R, G449L/W/A/S, and L475R), only Pr85 could be detected, suggesting a
block in the transport pathway and SU-TM cleavage. Not surprisingly, all of these mutants had previously been shown to be defective at
promoting cell-cell fusion and titers (Table 1). In contrast, a second
group of fusion-defective mutants (L445E, A457F, G460E, and T461P) that
were processed normally, exhibiting both Pr85 and gp70 in their cell
lysates, was identified (Fig. 3).
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FIG. 3.
Western analysis of transfected-cell lysates. 293T cells
were transfected with envelope protein expression plasmids, lysed
48 h later in 500 µl of lysis buffer (20 mM Tris-HCl [pH 7.5],
1% Triton X-100, 0.05% sodium dodecyl sulfate, 5 mg of sodium
deoxycholate per ml, 150 mM NaCl, and 1 mM phenylmethylsulfonyl
fluoride) for 10 min at 4°C, and centrifuged at 10,000 × g for 10 min to pellet nuclei. The cell lysates were
resolved on a precast 8 to 16% gel, and the envelope protein precursor
Pr85 and the processed SU subunit gp70 were detected by using a
specific goat antiserum, as described previously (20). Lane
1, CEE+ wild-type protein; lane 2, L443A; lane 3, L443R; lane 4, L441F;
lane 5, L445E; lane 6, G449L; lane 7, G449W; lane 8, G449A; lane 9, G449S; lane 10, T461P; lane 11, T463A; lane 12, A457F; lane 13, G460V;
lane 14, G460E; lane 15, L475R; lane 16, Q474E; lane 17, A468R; lane
18, M467E; lane 19, T464K; lane 20, T464I; lane 21, T464A; lane 22, control (mock transfected).
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We also measured the levels of envelope protein on the cell surface for
some of the mutants that did not induce syncytia (Table
1). Cell
surface envelope proteins were detected by indirect
immunofluorescence
and fluorescence-activated cell sorting (FACS)
of transiently
transfected 293T cells, as described elsewhere
(
20). For the
processing-defective mutants L443R, G449L, and
L475R, levels of
envelope protein lower than for the wild type
were detected, and this
probably corresponded to uncleaved Pr85.
In contrast, mutant envelope
proteins L445E, A457F, and T461P,
which were processed normally, were
readily detectable on the
cell surface. In addition, we saw that the
hyperfusogenic mutant,
T464A, was present on the cell surface at levels
comparable to
that of the wild-type protein, suggesting that its
increased fusogenicity
did not result simply from an increased level of
cell surface
expression.
Envelope protein incorporation into virions.
Envelope protein
present at the cell surface is incorporated into viral particles during
budding. To test whether the mutations we had made at the N terminus of
p15E had any influence on this process, we analyzed the protein
composition of viral particles obtained from 293T cells transfected
with MoMuLV Gag-Pol and envelope protein expression vectors and
purified from culture supernatants by centrifugation through 20%
sucrose (20).
Most of the mutant viruses contained approximately wild-type levels of
gp70 and p15E (Fig.
4). However, no gp70
or p15E protein
could be detected for mutants L443R, G449L/A/S/W, or
L475R, although
some uncleaved precursor Pr85 appeared to be
incorporated at low
levels for mutants L443R and G449S/A/L. In
addition, the A457F
and L443A mutations reproducibly resulted in lower
levels of envelope
proteins being incorporated into virions. These
results are in
agreement with the analyses described above, as those
mutants
that exhibited defects in processing and cell surface
expression
were also not detected in virions.
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FIG. 4.
Western analysis of virions. Retrovirus particles were
generated by cotransfection of envelope protein expression plasmids and
the Gag-Pol expression plasmid pHIT60 into 293T cells, essentially as
described previously (20, 35). Virions were partially
purified from culture supernatants by pelleting through a 20% sucrose
cushion at 25,000 rpm and 4°C in an SW41 rotor for 4 h, and
samples were resolved on sodium dodecyl sulfate-8 to 16%
polyacrylamide gradient gels and transferred to membranes. The blots
were probed with anti-gp70 and anti-p30 Gag antisera, as described
previously (20). For the TM protein, both p15E and the
processed (R-peptide-cleaved) p12E proteins were detected with either
an anti-p15E antiserum (20) (lanes 1 to 15) or monoclonal
antibody 42-114 (28) (lanes 16 to 26). For the upper panel,
p15E and p12E run very closely together; the higher-migrating band (ns)
is nonspecific. The two forms of TM protein are more clearly resolved
in the lower panel. Lane 1, mock transfection; lane 2, CEE+ wild-type
envelope protein; lanes 3 to 15, mutants L441F, L443R, L445E, G449L,
A457F, G460E, T461P, T463A, T464A, M467E, A468R, Q474E, and L475R,
respectively; lanes 16 and 17, mutants L443A and L443R, respectively;
lanes 18 to 21, G449S, G449A, G449L, and G449W, respectively; lanes 22 and 23, G460E and G460V, respectively; lanes 24 to 26, T464K, T464I,
and T464A, respectively. Some Pr85 can be seen for mutants L443R and
G449S/A/L.
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For the mutant envelope proteins that were efficiently incorporated
into virions, no differences were seen in the relative
levels of gp70
and p15E or in their mobilities on gels, suggesting
that none of the
mutations had gross effects on the site of SU-TM
cleavage or SU-TM
protein interactions. In addition, there were
no apparent differences
in the relative levels of p15E and the
R-peptide-cleaved form, p12E.
The hyperfusogenic mutant, T464A,
also displayed wild-type levels of
virion gp70, p15E, and p12E
(Fig.
4, lane 11), so the hyperfusogenic
phenotype of this mutant
was not due to greater levels of envelope
protein in virions or
enhanced R-peptide cleavage.
Ecotropic receptor binding.
The analyses described above
identified mutants L445E, A457F, G460E, and T461P as being primarily
fusion defective. None of these mutants induced syncytia or resulted in
transduction, despite being normally processed, expressed on the
surface of cells, and incorporated into virions at wild-type levels. To
confirm that the block to fusion was not occurring because of a defect
in their ability to bind to the ecotropic receptor, we measured binding to NIH 3T3 cells by a FACS-based assay, as previously described (22, 43). As shown in Table 3,
all of these mutants could bind to cells expressing the ecotropic
receptor, indicating that these mutants are defective primarily at a
postbinding step of the viral entry process.
Fusion-defective mutants trans dominantly interfere
with cell-cell fusion.
Previous analyses of the fusion peptide
region of HIV-1 identified one mutant that was also trans
dominant when coexpressed with the wild-type protein (10).
In addition, several deletion mutations of the N terminus of gp41 have
also been shown to dominantly interfere with syncytium formation
(32). We therefore examined whether the group of
fusion-defective envelope proteins could function in trans
to inhibit fusion. Accordingly, wild-type and mutant envelope proteins
were coexpressed in GPL cells at ratios of 1:1 and 10:1, and syncytium
formation in overlaid XC cells was examined (Table
4). At a ratio of 1:1, very few syncytia were observed (2.1 to 5.4% of the levels produced by the wild-type protein alone), and even at a 10:1 excess of the wild-type protein, only 21.5 to 38.5% of the wild-type level of syncytium formation was
seen. These results are in close agreement with the percent inhibition
observed at these ratios for the trans-dominant HIV-1 fusion
peptide mutant (10). We also examined the effect of
coexpression of the fusion mutants and the wild-type protein on the
transduction ability of retroviral particles. In contrast to the
situation in the cell-cell fusion assay, there was no indication of a
trans-dominant effect on viral titer at either a 1:1 or a
10:1 ratio (data not shown).
Taken together, our data provide evidence that the amino terminus of
MoMuLV TM protein, p15E, contains a fusion peptide. This
result
confirms and extends the previous mutational analysis of
AKV MuLV by
Jones and Risser (
21). Substitution of amino acid
L445
within a hydrophobic stretch at the N terminus of this region
and
several substitutions (A457F, G460E/V, and T461P) in an adjacent
GT-rich region disrupted envelope protein-mediated cell-cell fusion
and
resulted in noninfectious retroviral particles when expressed
in a
packaging system. Furthermore, all of these proteins were
incorporated
into viral particles and could bind to the ecotropic
receptor. This
group of mutants is therefore defective primarily
in the fusion step of
the viral entry process (Table
5). Our
analyses also identified a second group of mutants that were unable
to
process Pr85 to the SU and TM subunits and were not found in
retroviral
particles. This group included several mutants with
substitutions at
residues L443 and G449 in the hydrophobic core
and the more C-terminal
L475R substitution (Table
5). In a previous
study of MoMuLV TM protein,
it was also noted that the G449R substitution
resulted in envelope
protein that was not incorporated into virions
(
3).
Fusion peptides are hypothesized to insert into lipid bilayers, causing
local membrane destabilization and initiating the
fusion of host and
viral membranes (
5,
39). The hydrophobic
nature of these
peptides is important, and mutations that increase
the overall
hydrophobicity of these regions have been reported
to increase
fusogenicity, while polar or charged residues abolish
fusion (
4,
9,
11). Our data also confirm the importance
of hydrophobicity in
the fusion peptide, since the L445E substitution
produced a
fusion-defective mutant and the downstream M467E mutation
prevented
cell-cell fusion and reduced the ability of the protein
to mediate
transduction. Other substitutions at residue L445 could
not be assessed
for their effects on fusogenicity since these
changes prevented the
proper processing of the envelope protein.
Fusion peptides also contain glycine residues, which have been
suggested to be important for fusion peptide function, possibly
because
of their influence on the secondary structure of the region
(
4,
7,
39). The effects of the several substitutions we
made at position
G449 on fusogenicity could not be analyzed since
they prevented
envelope protein processing. Further downstream,
the mutation of
G460 to a charged residue (glutamic acid) prevented
fusion, while the
substitution of valine had less effect. A similar
substitution of
arginine for MoMuLV G460 (
3) also produced
a protein
that was determined to be defective in either binding
or fusion.
We also investigated the role of the multiple threonine residues at the
N terminus of MoMuLV p15E, located immediately downstream
of the fusion
peptide core in the GT-rich region. The substitution
of alanine for
T463 had little effect on cell-cell fusion, while
the substitution
T464A actually increased fusogenicity. Only the
T461P mutation
abolished fusion, and it is possible that a proline
residue at this
position had a detrimental effect on the overall
secondary structure of
this region. Similarly, the replacement
of amino acid A457 with a bulky
phenylalanine residue could have
distorted local structure, causing the
lack of fusogenicity in
this mutant.
None of the mutant proteins that were incorporated into particles
appear to have altered SU-TM protein interactions, as similar
levels of
SU and TM proteins were present in the partially purified
virions. This
included several mutations made in the GT region,
which is in the
position analogous to the HIV-1 ST region that
has been implicated in
SU-TM protein interactions (
7,
11,
23). However, the T464A
substitution in the GT region did result
in an envelope protein with a
hyperfusogenic phenotype. While
this increased fusogenicity may have
occurred by reducing the
overall polar nature of this region, it is
also possible that
this substitution destabilized the interaction
between the SU
and TM subunits, making the proposed postbinding
conformational
change easier. In addition, mutant A468R also resulted
in increased
syncytium formation when expressed in NIH 3T3 cells. Jones
and
Risser (
21) previously transfected an analogous AKV MuLV
mutant
into XC cells and found that it also produced a twofold-higher
level of syncytium formation than the wild-type protein.
Greater effects on cell-cell fusion, as measured by cocultivation of XC
cells with NIH 3T3 cells, than on virus-cell fusion,
as measured by
transduction, were seen for the mutant proteins
L443I and M467E.
Differential effects on cell-cell fusion and
virus-cell fusion have
previously been reported for MuLV (
20),
HIV-1
(
34), and influenza virus hemagglutinin (
16,
38).
This result may reflect inherent differences between these two
related
but distinct processes, in particular in the relative
number of fusion
peptides that may be required to achieve fusion
over the larger surface
area of cell-cell fusion. In addition,
while the fusion-defective
mutants caused a
trans-dominant inhibition
of cell-cell
fusion when coexpressed with the wild-type protein,
we saw very little
effect on virus-cell fusion, as measured by
titer.
In summary, we have obtained evidence to suggest that the N terminus of
MoMuLV TM protein p15E is important for envelope protein-mediated
fusion and probably constitutes a fusion peptide. Furthermore,
this
region of p15E also influences the proper processing, cell
surface
expression, and subsequent incorporation into particles
of the mature
envelope protein.
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ACKNOWLEDGMENTS |
We thank J. Ragheb for providing the LETRSN vector.
This work was supported by Genetic Therapy, Inc. (GTI)/Novartis and by
NIH grant CA59318-04.
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FOOTNOTES |
*
Corresponding author. Mailing address: Norris Cancer
Center, Rm. 633, University of Southern California School of Medicine, 1441 Eastlake Ave., Los Angeles, CA 90033. Phone: (213) 764-0673. Fax:
(213) 764-0097. E-mail: pcannon{at}hsc.usc.edu.
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J Virol, February 1998, p. 1632-1639, Vol. 72, No. 2
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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