Previous Article | Next Article 
Journal of Virology, May 2001, p. 4129-4138, Vol. 75, No. 9
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.9.4129-4138.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Sequences in the Cytoplasmic Tail of the Gibbon Ape Leukemia
Virus Envelope Protein That Prevent Its Incorporation into
Lentivirus Vectors
Ilias
Christodoulopoulos and
Paula M.
Cannon*
Gene Therapy Laboratories, Norris Cancer
Center, and Department of Biochemistry and Molecular Biology,
University of Southern California Keck School of Medicine, Los Angeles,
California 90033
Received 18 August 2000/Accepted 1 February 2001
 |
ABSTRACT |
Pseudotyping retrovirus and lentivirus vectors with different viral
fusion proteins is a useful strategy to alter the host range of the
vectors. Although lentivirus vectors are efficiently pseudotyped by Env
proteins from several different subtypes of murine leukemia virus
(MuLV), the related protein from gibbon ape leukemia virus (GaLV) does
not form functional pseudotypes. We have determined that this arises
because of an inability of GaLV Env to be incorporated into lentivirus
vector particles. By exploiting the homology between the GaLV and MuLV
Env proteins, we have mapped the determinants of incompatibility in the
GaLV Env. Three modifications that allowed GaLV Env to pseudotype human immunodeficiency virus type 1 particles were identified: removal of the
R peptide (C-terminal half of the cytoplasmic domain), replacement of
the whole cytoplasmic tail with the corresponding MuLV region, and
mutation of two residues upstream of the R peptide cleavage site. In
addition, we have previously proposed that removal of the R peptide
from MuLV Env proteins enhances their fusogenicity by transmitting a
conformational change to the ectodomain of the protein (Y. Zhao et al.,
J. Virol. 72:5392-5398, 1998). Our analysis of chimeric MuLV/GaLV
Env proteins provides further evidence in support of this model and
suggests that proper Env function involves both interactions within the
cytoplasmic tail and more long-range interactions between the
cytoplasmic tail, the membrane-spanning region, and the ectodomain of
the protein.
| |
INTRODUCTION |
Retrovirus vectors derived from
murine leukemia virus (MuLV) are the most commonly used gene transfer
vectors in current human gene therapy applications (reviewed in
reference 5). Like those of their parental retroviruses,
the host ranges of such vectors are influenced in large part by
the properties of the fusion protein contained in the outer lipid
envelope of the vector particle. An attractive property of these
vectors is the relative ease with which different fusion proteins can
be incorporated into particles in place of the native envelope (Env)
protein, a process referred to as pseudotyping. This allows the vectors
to transduce ranges of cells and tissues different from
those that would be possible with just the native Env.
There are many examples of pseudotyping in the literature. Both MuLV
and retrovirus vectors derived from it can be pseudotyped by the Env
proteins from other type C mammalian retroviruses. These include
proteins from the different subtypes of MuLV, such as amphotropic
(7, 34, 46), polytropic (15, 29), and xenotropic (3, 46) subtypes and 10A1 (35), as
well as the Env proteins from gibbon ape leukemia virus (GaLV)
(36, 46, 63) and feline leukemia virus type B
(59). In addition, Env proteins from more-distantly
related retroviruses can also pseudotype MuLV particles, such as feline
endogenous virus RD114 (46, 59), Jaagsiekte sheep
retrovirus (48), human T-cell-lymphotropic virus type 1 (HTLV-1) (63), and simian immunodeficiency virus (21). Finally, MuLV-based vectors can also be pseudotyped
by nonretrovirus fusion proteins, including the
glycoproteins from vesicular stomatitis virus (VSV)
(4, 11), rabies virus (38), lymphocytic
choriomeningitis virus (LCMV) (32), and Ebola virus (64).
Despite the potentially wide host range conferred by the use of
heterologous fusion proteins, retrovirus vectors still suffer from
certain limitations. In particular, MuLV-based vectors are unable to
transduce nondividing cells (37). In contrast, lentivirus vectors derived from human immunodeficiency virus type 1 (HIV-1) are
able to transduce a variety of nondividing cells, including hematopoietic cells (39) and neurons (41,
42). HIV-1 and lentivirus vectors can also be pseudotyped by
different viral fusion proteins, including the VSV protein G
(VSV-G) (42), different MuLV subtypes (25,
30), HTLV-1 (25), and the rabies virus and Mokola
virus G proteins (40).
Most current retrovirus and lentivirus vector protocols use fusion
proteins with a broad host range, such as those from the humantropic
MuLV subtypes (amphotropic, xenotropic, and polytropic subtypes and
10A1), the GaLV Env protein, and VSV-G. Although VSV-G has an extremely
wide host range (4), its inherent cytotoxicity has made
the establishment of stable producer cell lines difficult unless
inducible systems are used (2, 65). In contrast, both the
MuLV and GaLV Env proteins are able to form stable producer cell lines
(7, 8, 29, 34, 36, 46), making them potentially more
useful for the large-scale production of pseudotyped vectors.
Although the GaLV and amphotropic MuLV receptor proteins are widely
expressed on human cells (reviewed in references 20 and
33), several side-by-side comparisons have demonstrated that
GaLV Env is better at transducing certain human cell types than the
amphotropic Env (6, 24, 57, 62). The GaLV and MuLVs are
closely related type C mammalian retroviruses, and their entry
pathways have common features (26). Their Env proteins contain two subunits, SU and TM, which are cleaved from a common precursor protein during transport to the cell surface. An additional feature of these Env proteins is that the C-terminal region of the
cytoplasmic tail, the R peptide, is cleaved by the viral protease at,
or shortly after, viral budding. R peptide cleavage is necessary to
confer full activity to the Env protein (47, 50), although not all of the Env proteins in a virion are cleaved (13,
22). The processing of the cytoplasmic tail of Env is not unique
to the mammalian type C retroviruses and has also been reported for more-distantly related retroviruses such as the Mason-Pfizer monkey virus (55) and equine infectious anemia virus
(52).
Although the combined properties of the GaLV Env and HIV-1 cores would
make lentivirus vectors pseudotyped with GaLV Env potentially useful
for human gene therapy applications, our initial attempts to produce
such vectors were unsuccessful. In agreement with a recent report
(58), we have determined that the incompatibility between
the GaLV Env and lentivirus vectors lies in the cytoplasmic tail of the
Env protein. We here describe a detailed analysis of the mechanism of
this incompatibility and describe three different strategies that allow
functional pseudotypes to form. In addition, we discuss the
implications that our findings have for Env protein function and
Env-particle interactions in the retroviruses.
| |
MATERIALS AND METHODS |
Cell lines.
293T and HeLa cells were obtained from the
American Type Culture Collection and were maintained in Dulbecco's
modified Eagle's medium plus 10 mM glutamine (Norris Cancer Center
cell culture core facility, University of Southern California) and 10%
fetal bovine serum (Hyclone, Logan, Utah).
Production of retrovirus and lentivirus vectors.
Retrovirus
vectors were produced by transient transfection of 293T cells,
essentially as described previously (15, 56). Ten
micrograms each of plasmids pCgp and pCnBg, together with 1 to 10 µg
of an appropriate fusion protein expression plasmid, was cotransfected
into 60 to 70% confluent 293T cells in 10-cm-diameter plates by
calcium phosphate precipitation. Plasmid pCgp is a packaging construct
expressing MuLV Gag-Pol (15), and pCnBg has a retrovirus vector genome carrying a nuclear 
-galactosidase marker gene
(14). Lentivirus vectors were generated in the same way
using 10 µg each of HIV-1 packaging construct pCMV
R8.2
(68) and a plasmid with the lentivirus vector genome,
pHR'-CMVLacZ (42), together with 1 to 10 µg of a fusion
protein expression plasmid. For experiments where both retrovirus and
lentivirus vectors were produced from the same cell, we cotransfected 7 µg each of plasmids pCgp and pCMVR8.2, 5 µg each of pHanPuro and
pHR'-CMVLacZ, and 7 µg of the expression plasmids for either
amphotropic MuLV or GaLV Env proteins. pHanPuro has a retrovirus vector
genome containing an internal simian virus 40 promoter driving
expression of a puromycin resistance gene. For the control experiments
where only pCgp or pCMVR8.2 was used, the total amount of DNA per
transfection was normalized to 31 µg using plasmid pBluescript
(Stratagene, La Jolla, Calif.).
Env and fusion protein expression vectors.
All fusion
proteins were expressed from plasmids containing the human
cytomegalovirus immediate-early promoter and the origin of replication
from simian virus 40. The fusion proteins used were obtained from the
amphotropic 4070A MuLV (14, 16), the polytropic mink cell
focus-forming virus (15, 17), the xenotropic NZB MuLV
(27), 10A1 MuLV (14, 49), the SEATO strain of
GaLV (9), the Indiana strain of VSV (53),
influenza A virus/fowl plague virus/Rostock/34 (61), and
the Armstrong 53b strain of LCMV (10). Chimeric and
truncated MuLV/GaLV Env proteins were generated by splice-overlap PCR
(18), and the final constructs were fully sequenced.
Determination of vector titer.
Retrovirus and lentivirus
vector titers were measured by transduction of 293T or HeLa cells.
Serial dilutions of the vector supernatants were prepared, and 1 ml of
each dilution was added to a well of a six-well plate seeded with
105 cells the previous day in the presence of 8 µg of
Polybrene (Sigma, St. Louis, Mo.)/ml. For vectors carrying
-galactosidase markers, the titer was determined by X-Gal
(5-bromo-4-chloro-3-indolyl--D-galactopyranoside) staining at 48 h posttransduction. Cells were fixed in 0.5%
glutaraldehyde for 10 min, washed with phosphate-buffered saline for 10 min, and then incubated with staining solution (4 mM potassium
ferricyanide, 4 mM potassium ferrocyanide, 2 mM MgCl2, 80 µg of X-Gal [Sigma]/ml) at 37°C overnight. Titer was determined
by counting the blue colonies in each well under a light
microscope and multiplying this number by the appropriate
dilution factor. For experiments using vector pHanPuro, titer
was determined as the number of puromycin-resistant colonies that
grew out after 7 days of treatment with 2.5 µg of puromycin
(Sigma)/ml. Titer was expressed as CFU per milliliter of viral supernatant.
Western analysis of retrovirus and lentivirus vector
particles.
Vector particles were harvested from the supernatants
of transiently transfected 293T cells and partially purified by
centrifugation through 2 ml of 20% sucrose at 25,000 rpm at 4°C for
2 h using an SW41 rotor. The transmembrane (TM) subunit of the Env
proteins was detected using rat monoclonal antibody 42/114 against AKR MuLV TM (45) at a 1:2,000 dilution, the capsid (CA)
protein from the retrovirus vectors was detected using a goat
anti-Rauscher MuLV p30 antiserum (Quality Biotech; lot 78S221) at a
1:5,000 dilution, and the lentivirus CA protein was detected using
mouse anti-p24 monoclonal antibody 183-H12-5C (National Institutes of Health AIDS Research and Reference Reagent Program) at a 1:1,000 dilution. The secondary antibodies used were horseradish peroxidase (HRP)-conjugated rabbit anti-goat immunoglobulin G (IgG) (1:20,000), HRP-conjugated goat anti-rat IgG (1:10,000), and HRP-conjugated goat
anti-mouse IgG (1:10,000) (Pierce, Rockford, Ill.). Specific proteins
were visualized using the enhanced chemiluminescence detection
system (Amersham International plc., Arlington Heights, Ill.).
To analyze the form of the Env proteins present in cell lysates, 293T
cells were incubated in 200 µl of lysis buffer (20 mM Tris-HCl [pH
7.5], 1% Triton X-100, 0.05% sodium dodecyl sulfate [SDS], 5 mg of
sodium deoxycholate/ml, 150 mM NaCl, 1 mM phenylethanolamine fluoride)
for 10 min at 4°C, followed by centrifugation at 14,000 × g for 10 min to pellet nuclei. Fifteen microliters of the
resulting supernatants was analyzed by SDS-polyacrylamide gel
electrophoresis and Western blotting, as described above.
| |
RESULTS |
Ability of viral fusion proteins to pseudotype retrovirus or
lentivirus vectors.
We examined the efficiencies with which a
panel of different fusion proteins could pseudotype either retrovirus
(MuLV) or lentivirus (HIV-1) vectors. We included the Env proteins from the different MuLV subtypes that have tropism for human cells (amphotropic, polytropic, and xenotropic subtypes and 10A1), as well as
the related Env protein from GaLV. The heterologous viral fusion
proteins that we used were a hemagglutinin (HA) protein from an avian
pathogenic strain of influenza virus, VSV-G, and the
glycoprotein (GP) from the Armstrong 53b strain of LCMV.
Retrovirus and lentivirus vectors were generated using a three-plasmid
transient transfection system (
56) in which the Gag-Pol
and transfer vector components were kept constant but the fusion
protein was varied accordingly. The vectors so generated were
initially
titered on human 293T cells to screen for the formation
of functional
pseudotypes (Table
1) and subsequently
titered
on a range of predictive cell lines to confirm the expected
tropism
of the vectors (data not shown). Although most of the vectors
tested gave titers that were in the range of 10
4 to
10
6 CFU/ml, strikingly, the combination of the GaLV Env and
lentivirus
vector components did not result in any titer.
GaLV Env does not pseudotype lentivirus vectors.
We wished to
determine the basis for this lack of titer. As a first step, we
examined the efficiency with which the GaLV and amphotropic MuLV Env
proteins could be incorporated into retrovirus and lentivirus vector
particles. Vector particles were harvested from the supernatants of
transfected cells and analyzed by Western blotting, as were the
producer cell lysates. For Env detection, we used an antibody raised
against the MuLV TM protein that also cross-reacts with the
corresponding GaLV protein.
Both the MuLV and GaLV TM proteins were readily detected in the
pelleted retrovirus vector supernatants, indicating good Env
incorporation (Fig.
1). We did, however,
observe a difference
in the ratios of the immature (TM) and processed
(TM-R) forms
of the two proteins, with the MuLV Env being more
efficiently
processed to the TM-R form. In contrast, examination of the
lentivirus
vector supernatants revealed that only the MuLV Env protein
was
present in these vector particles. The GaLV TM was not detected,
even when the blot was overexposed (data not shown). Western analysis
of cell lysates confirmed that the GaLV TM protein was expressed
in
cells transfected with the lentivirus components, although
it was
present at a lower level than that in cells expressing
the retrovirus
vectors (Fig.
1, compare lanes 6 and 8). From these
results, it is
therefore not apparent whether the lack of pseudotyping
of
lentivirus vectors by the GaLV Env arises because of a significant
block to the incorporation of GaLV Env into HIV-1 particles or
whether
the effect is secondary to an inhibition of the steady-state
levels of
GaLV Env in cells producing lentivirus vectors.
View larger version (33K):
[in this window]
[in a new window]
|
FIG. 1.
Incorporation of GaLV and MuLV Env proteins into
retrovirus and lentivirus vectors. 293T cells were transiently
transfected with either retrovirus (R) or lentivirus (L) vector
components and the Env proteins indicated. Vector particles were
partially purified from the supernatant by centrifugation through 20%
sucrose, and both vector particles and cell lysates were subjected to
Western analysis using antibodies raised against the MuLV TM protein
that also recognize the GaLV TM. TM-R is the form of TM with the R
peptide cleaved. The CA proteins from the vectors were used as loading
controls and were detected using anti-p30 (MuLV) or anti-p24 (HIV-1)
antibodies, respectively.
|
|
Coexpression of retrovirus and lentivirus vectors reveals that GaLV
Env is available for incorporation into vector particles.
In order
to distinguish between these two possibilities, we repeated our
analyses using cells cotransfected with both retrovirus and lentivirus
vector components. We used two different marker genes on the retrovirus
and lentivirus transfer vectors (those for puromycin resistance and
-galactosidase, respectively), to enable us to distinguish between
functional pseudotypes of the two vector types. Western analysis of
vector supernatants and cell lysates confirmed that the presence of
lentivirus vector components always inhibited the steady-state levels
of GaLV Env in cell lysates, even when retrovirus vectors were also
present (Fig. 2, lanes 15 and 16).
However, as pelleted supernatants produced from cells transfected with
both retrovirus and lentivirus vectors were found to contain the GaLV
TM (lane 8), we conclude that there is still sufficient protein present
to be incorporated into vector particles.
View larger version (29K):
[in this window]
[in a new window]
|
FIG. 2.
Coexpression of retrovirus and lentivirus vectors. 293T
cells were transfected with the vector components and Env proteins
shown, and both vector supernatants and cell lysates were subjected to
Western analysis for the proteins indicated. R, retrovirus vectors
alone; L, lentivirus vectors alone; R/L, retrovirus and lentivirus
vectors cotransfected.
|
|
We next examined whether the GaLV TM present in Fig.
2, lane 8, was
associated with retrovirus or lentivirus vectors. We performed
titer
assays on HeLa cells and measured the numbers of both
puromycin-resistant
and
-galactosidase-expressing colonies (Table
2). This revealed
that the vector
supernatant produced from the cotransfection of
GaLV Env with both
lentivirus and retrovirus vectors was not able
to transfer
-galactosidase. In contrast, the same supernatant
produced
puromycin-resistant colonies at a titer similar to that
obtained by the
combination of the GaLV Env with retrovirus vectors
alone. Taken
together, these results suggest that the pelleted
GaLV Env was
exclusively associated with retrovirus vectors and
that the inability
of GaLV Env to pseudotype lentivirus vectors
is not primarily due to a
destabilization of the protein but arises
because of some specific
incompatibility between HIV-1 particles
and GaLV Env.
We also performed
-galactosidase titer assays with the various
vector supernatants on 293T cells (Table
2) but were unable
to
determine the corresponding retrovirus vector titers due to
the
difficulty in selecting for adherent puromycin-resistant 293T
colonies.
Surprisingly, we observed a reproducible 1-log-unit
increase in the
number of
-galactosidase-positive cells produced
by the
GaLV/retrovirus/lentivirus combination on 293T cells compared
to the
GaLV/lentivirus vectors alone, from an average of 20 to
200 CFU/ml.
Control experiments ruled out the possibility that
this increase was
caused by encapsidation of the lentivirus vector
genome by GaLV
pseudotyped retrovirus vectors (data not shown).
Although such
an enhancement was not observed when the same supernatants
were titered
on HeLa cells, we cannot rule out the possibility
that the presence of
the retrovirus vectors in some way increases
the ability of GaLV Env to
pseudotype lentivirus vectors. At present,
the mechanism of such an
effect is not
known.
Construction of chimeric MuLV/GaLV Env proteins.
To further
examine the basis for the incompatibility between the GaLV Env and
lentivirus vectors, we took advantage of the homology between the GaLV
and amphotropic MuLV Env proteins in order to construct chimeric
proteins (Fig. 3). We concentrated in
particular on the cytoplasmic domain, as previous reports of incompatibility between fusion proteins and retrovirus particles have
implicated this region. For example, both the HIV-1 (63) and human foamy virus (40) Env proteins are unable to
pseudotype MuLV cores, but modifying their cytoplasmic tails alleviates
the problem (28, 31, 54). In addition, as we have
previously shown that truncating the R peptide or the whole cytoplasmic
tail from the ecotropic MuLV Env results in proteins that still retain some function (22), we also constructed truncated versions
of the GaLV Env.
View larger version (55K):
[in this window]
[in a new window]
|
FIG. 3.
MuLV and GaLV Env proteins. (A) Comparison of the
sequences of the transmembrane (M) and cytoplasmic tail regions (T and
R) of the GaLV SEATO and amphotropic 4070A MuLV Env proteins. Numbering
for GaLV Env is from the start of the mature protein after removal of
the signal peptide (60). The C terminus of the cytoplasmic
tail (R) is cleaved from Env during virion maturation, leaving 16 amino
acids in the tail of the mature Env protein (T). (B) Schematic of the
truncated and substituted GaLV (open boxes) and MuLV (shaded boxes) Env
proteins used in this study. Construct GM(618/9) is the GaLV Env with
the substitutions K618Q and I619A, **.
|
|
Ability of MuLV/GaLV Env proteins to pseudotype retrovirus and
lentivirus vectors.
The truncated and chimeric Env proteins were
assessed for their ability to pseudotype both retrovirus and lentivirus
vectors. As before, vector particles were generated by transient
transfection and both vector supernatants and cell lysates were
analyzed by Western blotting. All of the Env proteins were found to be
efficiently incorporated into the retrovirus vector particles, except
for construct GaLVTR (Fig. 4A). Since
this protein was only present at very low levels in cells transfected
with either retrovirus or lentivirus vectors, it is likely that poor
protein stability underlies its lack of incorporation.
View larger version (46K):
[in this window]
[in a new window]
|
FIG. 4.
Incorporation of chimeric and truncated Env proteins
into vector particles. Retrovirus (A) or lentivirus (B) vectors were
generated using the Env proteins indicated, and both partially purified
vectors and cell lysates were subjected to Western analysis.
|
|
In contrast to the situation with the retrovirus vectors, the extent of
incorporation of the various Env proteins into lentivirus
vectors
varied significantly (Fig.
4B). Moreover, the nature of
the cytoplasmic
tail sequences alone was sufficient to determine
the incorporation
pattern, as the reciprocal chimeras GM(TR) and
MG(TR) displayed the
phenotypes of the Env proteins from which
the cytoplasmic tail was
derived. The whole cytoplasmic tail was
needed for this effect, as
substitution of either the T or R regions
alone did not fully alleviate
the block to incorporation in the
GaLV Env or confer total
incompatibility to the MuLV Env. We noted
that the presence of the GaLV
R peptide in particular was associated
with poor incorporation, as
GM(R) was incorporated at higher levels
than GM(T), while the converse
was true for MG(R) and MG(T). Furthermore,
the simple removal of the R
peptide from GaLV Env allowed strong
incorporation of GaLV
R into
lentivirus vectors. However, the
incorporation-competent phenotype of
mutant GM(618/9) argues against
a model whereby the GaLV R peptide
per se prevents incorporation
and, instead, suggests that some feature
of the complete cytoplasmic
tail influences the association with HIV-1
particles.
We also examined the titers directed by the various Env proteins on
293T cells (Table
3). Interestingly, for
the retrovirus
vectors, the GM series of chimeras always gave titers
that were
lower than those obtained with either the MuLV or GaLV
parental
Env proteins. The effects on titer could not simply be
attributed
to reduced Env incorporation into vector particles. For
example,
construct GM(M) gave titers that were nearly 3 orders of
magnitude
lower than those for wild-type GaLV Env, despite reasonable
levels
of incorporation into retrovirus vectors. Instead, this
suggests
that even small changes in the GaLV Env protein can
compromise
its function.
Similarly, analysis of the titers directed by the
incorporation-competent Env proteins on lentivirus vectors revealed
that
factors other than absolute incorporation levels affected titers.
The four GaLV-based proteins that were well incorporated [GaLV
R
GM(TR), GM(MTR), and GM(618/9)] gave titers only in the range
of
10
3 to 10
4 CFU/ml with lentivirus vectors,
while retrovirus vectors pseudotyped
with the same proteins
gave titers in the range of 10
5 to 10
6 CFU/ml.
This 2-order-of-magnitude difference in titers was not
simply a
property of the lentivirus vectors, as lentivirus vectors
pseudotyped with the MuLV Env gave a titer of 3.5 × 10
6, which was comparable to those achieved with the
retrovirus vectors.
Instead, it appears that these GaLV Env derivatives
have an additional
block to full function that is only manifest when
the proteins
are present on lentivirus
vectors.
R peptide cleavage patterns.
The above observations suggested
that specific interactions between the vector particle and the
pseudotyping Env protein could play a role in influencing
the overall efficiency of transduction. A clear example of such
Env-particle interactions is the fact that the viral protease is
responsible for R peptide processing. Although complete R peptide
removal is not necessary for Env function (67), the
efficiency of processing has been shown to correlate with titer in at
least one case (23). We were therefore interested to
examine whether differences in the extent of R peptide processing could
account for the differences in titer achieved with retrovirus and
lentivirus vectors.
We have repeatedly observed that cleavage of the GaLV Env by MuLV cores
is less efficient than processing of the MuLV Env
(Fig.
1,
2, and
4).
Typically two-thirds of the MuLV TM proteins
were cleaved in pelleted
vector particles, and the same degree
of processing was observed for
both the homologous MuLV particles
and the heterologous HIV-1
particles. Although the GaLV Env was
processed less efficiently, this
was clearly not a problem for
Env function as titers of 1.4 × 10
6 CFU/ml were achieved with retrovirus vectors. It is
possible
that this lower level of processing occurs naturally in GaLV
virions
and is sufficient to produce a fully functional Env. Indeed,
the
extreme fusogenicity and cytotoxicity of the R-less form of the
GaLV Env (
12) provide a rationale for such a
situation.
Using the panel of chimeric GaLV/MuLV proteins, we were able to examine
the factors that influenced the degree of R peptide
processing by
either retrovirus or lentivirus particles (Table
4). The viral protease recognition site
includes at least seven
residues spanning the actual cleavage site
(residues P4 to P3')
(
43), so that both the T and R
regions of the tail could influence
processing efficiency. However, for
proteins present in retrovirus
vectors, our analysis showed that only
the nature of the T region
influenced the extent of R peptide cleavage;
any protein containing
an MuLV T region was cleaved efficiently, while
any protein containing
a GaLV T region was not. This pattern held true
whatever the absolute
incorporation levels of the Env protein and is
shown most clearly
by the opposite patterns exhibited by MG(T) and
MG(R) (Fig.
4A).
However simply changing the GaLV residues in the T
region that
were part of the protease recognition site to the
corresponding
MuLV sequences, as occurs in construct GM(618/9), was not
sufficient
to produce the efficient MuLV pattern of cleavage, implying
a
contribution by more-upstream sequences in the GaLV T region.
In
contrast, for the lentivirus vectors, we observed that the
presence of
either the GaLV R or T regions reduced the levels
of processing to the
wild-type GaLV pattern and that both the
T and R regions had to be
replaced by the MuLV sequences before
the efficient MuLV pattern could
be observed.
In general, the same patterns of processing were observed for the
chimeric Env proteins regardless of whether the proteins
were
incorporated into retrovirus or lentivirus vectors. However,
a
discrepancy between the two vector types was noted for constructs
MG(R)
and GM(T). Both of these chimeras displayed the MuLV pattern
in
retrovirus vectors but the GaLV pattern in lentivirus vectors.
The
feature uniquely shared by these two proteins is the combination
of the
MuLV T region and the GaLV R region. It is possible that
the negative
effect that the GaLV R peptide has on incorporation
into lentivirus
vectors precludes efficient R peptide removal,
despite the presence of
the MuLV T region. In contrast, as incorporation
of Env proteins
into retrovirus vectors is not affected by the
presence of the GaLV R
peptide, the extent of R peptide removal
in these vectors was
determined solely by the nature of the T
region and was therefore
MuLV-like. Finally, we note that, as
construct GM(618/9) retained
the GaLV pattern of R peptide cleavage
on both vector types, we
are able to separate the two properties
conferred by the GaLV tail;
while changing residues K
618 and I
619 to those
of the MuLV sequence was sufficient to overcome the block
to
incorporation into lentivirus vectors, increasing the level
of R
peptide cleavage to the MuLV level required the substitution
of the
entire T region (retrovirus vectors) or both T and R regions
combined
(lentivirus vectors). Overall, this indicates that Env
association with
viral particles and the extent of R peptide cleavage,
while related,
are not absolutely
correlated.
Factors affecting protein stability.
Retrovirus Env proteins
are initially synthesized as a single polypeptide that is cleaved by a
host cell protease to the SU and TM subunits during transport to the
cell surface (44). Small amounts of TM in cell lysates, as
well as poor ratios of TM to uncleaved Env, can result from reduced
rates of processing and transport, as well as poor protein stability.
Furthermore, the R-less form of TM is not always apparent on Western
blots of cell lysates, reflecting the fact that this cleavage event may
occur during or after the budding of the virion from the cell.
Analysis of the lysates of cells transfected with the various truncated
and chimeric Env proteins identified certain proteins
as having
phenotypes that differed from those of either of the
parental Env
proteins. As previously mentioned, the GaLV
TR TM
signal was
barely detectable in the presence of either vector
type, suggesting an
inherently low stability of this protein.
In addition, the three
MuLV/GaLV chimeras containing the GaLV
R peptide [constructs
MG(R), MG(TR), and MG(MTR)] gave relatively
weak TM and TM-R signals
in cell lysates and had relatively stronger
bands of the uncleaved Env
(data not shown), suggesting a problem
in transport and/or processing.
Despite this defect, all three
constructs gave wild-type levels of
incorporation and titer when
expressed with retrovirus vectors (Table
2). Finally, the GaLV
Env itself was notable in that it was the only
Env protein that
was significantly inhibited or destabilized by the
coexpression
in transfected cells of lentivirus vector components.
Although
the basis for this inhibition is currently unknown, the
inhibition
could be prevented in several different ways, including the
separate
replacement of the R, T, or M region and the substitution of
residues
K
618 and I
619. Taken together, these
findings further suggest
that overall interactions between the
different regions of the
GaLV Env protein contribute to its sensitivity
to the presence
of lentivirus
vectors.
| |
DISCUSSION |
We have demonstrated that the GaLV Env protein is unable to
pseudotype lentivirus vectors because certain features of its cytoplasmic tail are incompatible with incorporation into HIV-1 particles. We have identified three different strategies that enable
such pseudotypes to form: (i) removal of the GaLV Env R peptide, (ii)
replacement of the whole (R plus T regions) GaLV Env cytoplasmic tail
with the corresponding MuLV sequences, but not either region alone, and
(iii) the replacement of residues K618 and I619
by the corresponding MuLV residues. The resulting proteins, designated
GaLVR, GM(TR), and GM(618/9), may have applications in human gene
therapy, as such pseudotyped lentivirus vectors retain the
GaLV Env host range (data not shown). In particular, GM(TR)
and GM(618/9) may prove useful for the establishment of stable cell lines, as they are not toxic when expressed in cells (data
not shown).
There are other examples in the literature of incompatibility between
retrovirus particles and heterologous fusion proteins. For example, the
Env proteins from HIV-1 (61), HIV-2 (19), Mason-Pfizer monkey virus, and simian retrovirus-1 (59) do
not pseudotype MuLV. For the HIV-1 and HIV-2 Envs, the block resides in
their long cytoplasmic tails and truncated versions of the proteins can
be incorporated (19, 31, 54). Similarly, although the
human foamy virus Env protein does not efficiently pseudotype MuLV, a
chimera containing the cytoplasmic domain of MuLV Env shows improved
incorporation (28). However, as the GaLV Env cytoplasmic
tail is relatively short (29 amino acids) and has reasonable homology
to the tail of the incorporation-compatible MuLV Env proteins, it was
surprising to observe such incompatibility between the GaLV Env and
lentivirus vectors.
Our initial attempts to understand why the GaLV Env was excluded from
lentivirus vectors were hampered by the greatly reduced levels of GaLV
TM that we observed in transfected 293T cells in the presence of
lentivirus components. However, further experiments revealed that even
the low level of protein that was present in these cells was sufficient
to pseudotype coexpressed retrovirus vectors. This in turn suggested
that the lack of GaLV Env in lentivirus vectors was not simply due to
low steady-state levels of the protein in cells or at the cell surface
but instead reflected a specific incompatibility between the Env and
vector components. Although we do not yet understand why lentivirus
vectors reduce the cellular levels of GaLV Env, we note that several
different approaches can alleviate this effect. They include the
individual replacement of either the M, T, or R region of the GaLV Env
with MuLV sequences and just the substitution of residues
K618 and I619. Finally, it is noteworthy that
even good levels of expression of a protein in transfected cells are
not sufficient to allow incorporation into lentivirus vectors, as can
be seen most clearly for construct GM(M).
Most of the variation between the cytoplasmic tails of the GaLV and
MuLV Env proteins is located in the R peptide. Although the good
incorporation of the GaLVR protein into lentivirus vectors initially
suggested that a steric block between the R peptide and HIV-1 virions
caused their incompatibility, replacing the R peptide alone with the
corresponding MuLV region was not sufficient to allow full
incorporation. Furthermore, the ability of construct GM(618/9) to be incorporated into lentivirus vectors
suggests that it was not the GaLV R peptide per se that was the problem but rather a more general structure in the whole GaLV Env tail. Overall, these findings suggest that interactions occur between the R
and T regions in the cytoplasmic tails of these Env proteins that are
important for the secondary structure of the whole of the tail and that
the R peptide cleavage site itself may be a major determinant of this property.
One of the major differences that we noted between the GaLV and MuLV
Env proteins was the different degrees of R peptide cleavage for the
two proteins when present in retrovirus vectors. Since R peptide
cleavage is performed by the viral protease, it presumably requires an
intimate association between the Env protein and the viral core. We
therefore asked whether R peptide cleavage rates would correlate well
with efficiency of incorporation into lentivirus vectors.
Interestingly, this was found not to be the case, and we were able to
distinguish between the determinants that governed R peptide cleavage
and those that controlled incorporation. The most extreme example
is construct GM(618/9), where we observed good
incorporation into lentivirus vectors without high levels of R peptide
cleavage. Recently, an HIV-1 MA mutant that does not block MuLV Env
incorporation into HIV-1 particles but that does prevent R peptide
cleavage by the HIV-1 protease has been described (23).
The phenotype of this mutant also suggests that incorporation into a
lentivirus vector does not necessarily lead to a normal interaction
with the HIV-1 core.
Our study has also produced evidence for specific Env-vector
interactions that act to influence the overall rate of transduction. We
observed a 2-order-of-magnitude difference in the titers directed by
retrovirus and lentivirus vectors pseudotyped with the same incorporation-competent GaLV Env derivatives [constructs GM(TR), GM(MTR), GM(618/9), and GaLVR] that could not simply
be attributed to differences in Env levels. One possible explanation is
that the MuLV and HIV-1 virions are differentially sensitive to the entry pathway directed by the GaLV Env. A similar situation has been
reported for HIV-1 virions produced in the presence or absence of the
Nef protein, which showed a 10-fold difference in infectivity when
pseudotyped with the amphotropic MuLV Env protein but not when the
fusion protein used was VSV-G (1). However, if the GaLV
entry pathway is indeed more productive for retrovirus vectors than
lentivirus vectors, then it is an unexpected finding given that no such
differences were seen for vectors pseudotyped with either the
amphotropic or 10A1 Env proteins, which recognize similar or identical
receptors. In addition, our studies with fusion proteins that direct
entry pathways markedly different from those used by the mammalian type
C retroviruses, including the pH-dependent proteins from VSV, influenza
virus, and LCMV, did not provide evidence of any pathway-specific
differences between retrovirus and lentivirus vectors.
An alternative explanation to account for these findings is that the
MuLV or HIV-1 particles themselves in some way influence Env function
and thereby affect the efficiency of transduction. Indeed, the process
of R peptide cleavage is a clear precedent to indicate that retrovirus
particles can influence Env protein function through an interaction
with the cytoplasmic tail. Although our data show that the efficiency
of R peptide processing was not a simple predictor of vector titer, it
remains possible that an additional influence of viral particles on Env
function exists that is so subtle that our current assays cannot detect
it and that the Env-particle interactions that occur in the
retroviruses are more complex than has previously been realized.
Our studies also have implications for understanding the mechanism of
fusion enhancement of retrovirus Env proteins by R peptide cleavage. We
(66, 67) and others (51) have previously
reported that R peptide-truncated forms of MuLV Env can function in
trans within an Env protein oligomer to stimulate Env
fusogenicity. The data presented here further support this model, as
even the low levels of R peptide truncation seen for the native GaLV
Env protein were sufficient to give titers on retrovirus vectors as high as those obtained with the MuLV Env. Furthermore, even though replacing the tail of the MuLV Env with the corresponding GaLV domain
in constructs MG(TR) and MG(MTR) reduced R peptide cleavage levels to
the GaLV level, this did not reduce the titers obtained for
pseudotyped retrovirus vectors. Overall, this indicates that even low levels of R peptide cleavage can confer full function to both
the GaLV and MuLV Env proteins in the context of retrovirus vectors.
Finally, we have previously proposed that R peptide cleavage enhances
MuLV Env fusogenicity by transmitting a conformational change from the
cytoplasmic tail of Env through to the ectodomain of the protein
(67). Examination of the properties of the chimeric Env
proteins lends further support to this hypothesis by suggesting the
occurrence of long-range interactions between the different domains of
the Env protein, including the cytoplasmic tail, the membrane-spanning
region, and the ectodomain.
| |
ACKNOWLEDGMENTS |
We thank our colleagues in the Gene Therapy Laboratories for
their support, in particular Maria Barcova, Celina Ngiam, and Kathleen
Burke. We also thank French Anderson and Nori Kasahara (USC) for their
thoughtful suggestions. The following reagent was obtained from the NIH
AIDS Research and Reference Reagent Program: anti-p24 monoclonal
antibody 183-H12-5C from Bruce Chesebro and Kathy Wehrly. We thank
Maribeth Eiden (NIH) for providing the GaLV SEATO Env plasmid.
This work was supported by funding from Genetic Therapy, Inc./Novartis
and Public Health Service grant CA-59318 from the National Cancer Institute.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Norris Cancer
Center, Room 6338, University of Southern California Keck School of
Medicine, 1441 Eastlake Ave., Los Angeles, CA 90033. Phone: (323)
865-0673. Fax: (323) 865-0097. E-mail:
pcannon{at}hsc.usc.edu.
| |
REFERENCES |
| 1.
|
Aiken, C.
1997.
Pseudotyping human immunodeficiency virus type 1 (HIV-1) by the glycoprotein of vesicular stomatitis virus targets HIV-1 entry to an endocytic pathway and suppresses both the requirement for Nef and the sensitivity to cyclosporin A.
J. Virol.
71:5871-5877[Abstract].
|
| 2.
|
Arai, T.,
K. Matsumoto,
K. Saitoh,
M. Ui,
T. Ito,
M. Murakami,
Y. Kanegae,
I. Saito,
F. L. Cosset,
Y. Takeuchi, and H. Iba.
1998.
A new system for stringent, high-titer vesicular stomatitis virus G protein-pseudotyped retrovirus vector induction by introduction of Cre recombinase into stable prepackaging cell lines.
J. Virol.
72:1115-1121[Abstract/Free Full Text].
|
| 3.
|
Battini, J. L.,
O. Danos, and J. M. Heard.
1995.
Receptor-binding domain of murine leukemia virus envelope glycoproteins.
J. Virol.
69:713-719[Abstract].
|
| 4.
|
Burns, J. C.,
T. Friedmann,
W. Driever,
M. Burrascano, and J. K. Yee.
1993.
Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells.
Proc. Natl. Acad. Sci. USA
90:8033-8037[Abstract/Free Full Text].
|
| 5.
|
Cannon, P. M., and W. F. Anderson.
2000.
Retroviral vectors for human gene therapy, p. 1-16.
In
N. S. Templeton, and D. D. Lasic (ed.), Gene therapy: therapeutic mechanisms and strategies. Marcel Dekker, Inc, New York, N.Y.
|
| 6.
|
Chuah, M. K. L.,
H. Brems,
V. Vanslembrouck,
D. Collen, and T. Vandendriessche.
1998.
Bone marrow stromal cells as targets for gene therapy of hemophilia A.
Hum. Gene Ther.
9:353-365[Medline].
|
| 7.
|
Cone, R. D., and R. Mulligan.
1984.
High-efficiency gene transfer into mammalian cells: generation of helper-free recombinant retrovirus with broad mammalian host range.
Proc. Natl. Acad. Sci. USA
81:6349-6353[Abstract/Free Full Text].
|
| 8.
|
Danos, O., and R. C. Mulligan.
1988.
Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges.
Proc. Natl. Acad. Sci. USA
85:6460-6464[Abstract/Free Full Text].
|
| 9.
|
De Paoli, A.,
D. O. Johnsen, and W. W. Noll.
1973.
Granulocytic leukemia in whitehanded gibbons.
J. Am. Vet. Med. Assoc.
163:624-628[Medline].
|
| 10.
|
Dutko, F. J., and M. B. A. Oldstone.
1983.
Genomic and biological variation among commonly used lymphocytic choriomeningitis virus strains.
J. Gen. Virol.
64:1689-1698[Abstract/Free Full Text].
|
| 11.
|
Emi, N.,
T. Friedmann, and J. K. Yee.
1991.
Pseudotype formation of murine leukemia virus with the G protein of vesicular stomatitis virus.
J. Virol.
65:1202-1207[Abstract/Free Full Text].
|
| 12.
|
Fielding, A. K.,
S. Chapel-Fernandez,
M. P. Chadwick,
F. J. Bullough,
F. L. Cosset, and S. J. Russell.
2000.
A hyperfusogenic gibbon ape leukemia envelope glycoprotein: targeting of a cytotoxic gene by ligand display.
Hum. Gene Ther.
11:817-826[CrossRef][Medline].
|
| 13.
|
Gray, K. D., and M. J. Roth.
1993.
Mutational analysis of the envelpe gene of Moloney murine leukemia virus.
J. Virol.
67:3489-3496[Abstract/Free Full Text].
|
| 14.
|
Han, J. Y.,
P. M. Cannon,
K. M. Lai,
Y. Zhao,
M. V. Eiden, and W. F. Anderson.
1997.
Identification of envelope protein residues required for the expanded host range of 10A1 murine leukemia virus.
J. Virol.
71:8103-8108[Abstract].
|
| 15.
|
Han, J. Y.,
Y. Zhao,
W. F. Anderson, and P. M. Cannon.
1998.
Role of variable regions A and B in the receptor binding domain of amphotropic murine leukemia virus envelope protein.
J. Virol.
72:9101-9108[Abstract/Free Full Text].
|
| 16.
|
Hartley, J. W., and W. P. Rowe.
1976.
Naturally occurring murine leukemia viruses in wild mice: characterization of a new "amphotropic" class.
J. Virol.
19:19-25[Abstract/Free Full Text].
|
| 17.
|
Hartley, J. W.,
N. K. Wolford,
L. J. Old, and W. P. Rowe.
1977.
A new class of murine leukemia virus associated with development of spontaneous lymphomas.
Proc. Natl. Acad. Sci. USA
74:789-792[Abstract/Free Full Text].
|
| 18.
|
Ho, S. N.,
H. D. Hunt,
R. M. Horton,
J. K. Pullen, and L. R. Pease.
1989.
Site-directed mutagenesis by overlap extension using polymerase chain reaction.
Gene
77:51-59[CrossRef][Medline].
|
| 19.
|
Höhne, M.,
S. Thaler,
J. C. Dudda,
B. Groner, and B. S. Schnierle.
1999.
Truncation of the human immunodeficiency virus-type-2 envelope glycoprotein allows efficient pseudotyping of murine leukemia virus retroviral vector particles.
Virology
261:70-78[CrossRef][Medline].
|
| 20.
|
Hunter, E.
1997.
Viral entry and receptors, p. 71-119.
In
J. M. Coffin, S. H. Hughes, and H. E. Varmus (ed.), Retroviruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 21.
|
Indracollo, S.,
S. Minuzzo,
F. Feroli,
F. Mammano,
F. Calderazzo,
L. Chieco-Bianchi, and A. Amadori.
1998.
Pseudotyping of Moloney leukemia virus-based retroviral vectors with simian immunodeficiency virus leads to targeted infection of human CD4+ lymphoid cells.
Gene Ther.
5:209-217[CrossRef][Medline].
|
| 22.
|
Januszeski, M. M.,
P. M. Cannon,
D. Chen,
Y. Rozenberg, and W. F. Anderson.
1997.
Functional analysis of the cytoplasmic tail of Moloney murine leukemia virus envelope protein.
J. Virol.
71:3613-3619[Abstract].
|
| 23.
|
Kiernan, R. E., and E. O. Freed.
1998.
Cleavage of the murine leukemia virus transmembrane Env protein by human immunodeficiency virus type 1 protease: transdominant inhibition by matrix mutations.
J. Virol.
72:9621-9627[Abstract/Free Full Text].
|
| 24.
|
Lam, J. S.,
M. E. Reeves,
R. Cowherd,
S. A. Rosenberg, and P. Hwu.
1996.
Improved gene transfer into human lymphocytes using retroviruses with the gibbon ape leukemia virus envelope.
Hum. Gene Ther.
7:1415-1422[Medline].
|
| 25.
|
Landau, N. R.,
K. A. Page, and D. R. Littman.
1991.
Pseudotyping with human T-cell leukemia virus type I broadens the human immunodeficiency virus host range.
J. Virol.
65:162-169[Abstract/Free Full Text].
|
| 26.
|
Lavillette, D.,
A. Ruggieri,
S. J. Russell, and F. L. Cosset.
2000.
Activation of a cell entry pathway common to type C mammalian retroviruses by soluble envelope fragments.
J. Virol.
74:295-304[Abstract/Free Full Text].
|
| 27.
|
Levy, J. A., and T. Pincus.
1970.
Demonstration of biological activity of a murine leukemia virus of New Zealand Black mice.
Science
170:326-327[Abstract/Free Full Text].
|
| 28.
|
Lindemann, D.,
M. Bock,
M. Schweizer, and A. Rethwilm.
1997.
Efficient pseudotyping of murine leukemia virus particles with chimeric human foamy virus envelope proteins.
J. Virol.
71:4815-4820[Abstract].
|
| 29.
|
Loiler, S. A.,
N. L. Difronzo, and C. A. Holland.
1997.
Gene transfer to human cells using retrovirus vectors produced by a new polytropic packaging cell line.
J. Virol.
71:4825-4828[Abstract].
|
| 30.
|
Lusso, P.,
F. Di Marzo Veronese,
B. Ensoli,
G. Franchini,
C. Jemma,
S. E. DeRocco,
V. S. Kalyanaraman, and R. C. Gallo.
1990.
Expanded HIV-1 cellular tropism by phenotypic mixing with murine endogenous retroviruses.
Science
247:848-852[Abstract/Free Full Text].
|
| 31.
|
Mammano, F.,
F. Salvatori,
S. Indracollo,
A. De Rossi,
L. Chieco-Bianchi, and H. G. Göttlinger.
1997.
Truncation of the human immunodeficiency virus type 1 envelope glycoprotein allows efficient pseudotyping of Moloney murine leukemia virus particles and gene transfer into CD4+ cells.
J. Virol.
71:3341-3345[Abstract].
|
| 32.
|
Miletic, H.,
M. Bruns,
K. Tsiakas,
B. Vogt,
R. Rezai,
C. Baum,
K. Kuhlke,
F. L. Cosset,
W. Ostertag,
H. Lother, and D. von Laer.
1999.
Retroviral vectors pseudotyped with lymphocytic choriomeningitis virus.
J. Virol.
73:6114-6116[Abstract/Free Full Text].
|
| 33.
|
Miller, A. D.
1996.
Cell-surface receptors for retroviruses and implications for gene transfer.
Proc. Natl. Acad. Sci. USA
93:11407-11413[Abstract/Free Full Text].
|
| 34.
|
Miller, A. D., and C. Buttimore.
1986.
Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production.
Mol. Cell Biol.
8:2895-2902.
|
| 35.
|
Miller, A. D., and F. Chen.
1996.
Retrovirus packaging cells based on 10A1 murine leukemia virus for production of vectors that use multiple receptors for cell entry.
J. Virol.
70:5564-5571[Abstract/Free Full Text].
|
| 36.
|
Miller, A. D.,
J. V. Garcia,
N. von Shur,
C. M. Lynch,
C. Wilson, and M. V. Eiden.
1991.
Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus.
J. Virol.
65:2220-2224[Abstract/Free Full Text].
|
| 37.
|
Miller, D. G.,
M. A. Adam, and A. D. Miller.
1990.
Gene transfer by retrovirus vectors occurs in cells that are actively replicating at the time of infection.
Mol. Cell. Biol.
10:4239-4242[Abstract/Free Full Text].
|
| 38.
|
Mitrophanous, K. A.,
S. Yoon,
J. B. Rohll,
D. Patil,
F. J. Wilkes,
V. N. Kim,
S. M. Kingsman,
A. J. Kingsman, and N. D. Mazarakis.
1999.
Stable gene transfer to the nervous system using a non-primate lentiviral vector.
Gene Ther.
6:1808-1818[CrossRef][Medline].
|
| 39.
|
Miyoshi, H.,
K. A. Smith,
D. E. Mosier,
I. M. Verma, and B. E. Torbett.
1999.
Transduction of human CD34+ cells that mediate long-term engraftment of NOD/SCID mice by HIV vectors.
Science
283:682-686[Abstract/Free Full Text].
|
| 40.
|
Mochizuki, H.,
J. P. Schwartz,
K. Tanaka,
R. O. Brady, and J. Reiser.
1998.
High-titer human immunodeficiency virus type 1-based vector systems for gene delivery into nondividing cells.
J. Virol.
72:8873-8883[Abstract/Free Full Text].
|
| 41.
|
Naldini, L.,
U. Blömer,
F. H. Gage,
D. Trono, and I. M. Verma.
1996.
Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector.
Proc. Natl. Acad. Sci. USA
93:11382-11388[Abstract/Free Full Text].
|
| 42.
|
Naldini, L.,
U. Blömer,
P. Gallay,
D. Ory,
R. Mulligan,
F. H. Gage,
I. M. Verma, and D. Trono.
1996.
In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.
Science
272:263-267[Abstract].
|
| 43.
|
Pettit, S. C.,
J. Simsic,
D. D. Loeb,
L. Everitt,
C. A. Hutchison III, and R. Swanstrom.
1991.
Analysis of retroviral protease cleavage sites reveals two types of cleavage sites and the structural requirements of the P1 amino acid.
J. Biol. Chem.
266:14539-14547[Abstract/Free Full Text].
|
| 44.
|
Pinter, A., and W. J. Honnen.
1983.
Topography of murine leukemia virus envelope proteins: characterization of transmembrane components.
J. Virol.
46:1056-1060[Abstract/Free Full Text].
|
| 45.
|
Pinter, A.,
W. J. Honnen,
J. S. Tung,
P. V. O'Donnell, and U. Hammerling.
1982.
Structural domains of endogenous murine leukemia virus gp70s containing specific antigenic determinants defined by monoclonal antibodies.
Virology
116:499-516[CrossRef][Medline].
|
| 46.
|
Porter, C. D.,
M. K. Collins,
C. S. Tailor,
M. H. Parkar,
F. L. Cosset,
R. A. Weiss, and Y. Takeuchi.
1996.
Comparison of efficiency of infection of human gene therapy target cells via four different retroviral receptors.
Hum. Gene Ther.
7:913-919[Medline].
|
| 47.
|
Ragheb, J. A., and W. F. Anderson.
1994.
pH-independent Moloney murine leukemia virus ecotropic envelope-mediated cell fusion: implications for the role of the R peptide and p12E TM domain in viral entry.
J. Virol.
68:3220-3231[Abstract/Free Full Text].
|
| 48.
|
Rai, S. K.,
J. C. DeMartini, and A. D. Miller.
2000.
Retrovirus vectors bearing Jaagsiekte sheep retrovirus Env transduce human cells by using a new receptor localized to chromosome 3p21.3.
J. Virol.
74:4698-4704[Abstract/Free Full Text].
|
| 49.
|
Rasheed, S.,
B. K. Pal, and M. B. Gardner.
1982.
Characterization of a highly oncogenic murine leukemia virus from wild mice.
Intl. J. Cancer
29:345-350[Medline].
|
| 50.
|
Rein, A.,
J. Mirro,
J. G. Haynes,
S. M. Ernst, and K. Nagashima.
1994.
Function of the cytoplasmic domain of a retroviral transmembrane protein: p15E-p2E cleavage activates the membrane fusion capability of the murine leukemia virus Env protein.
J. Virol.
68:1773-1781[Abstract/Free Full Text].
|
| 51.
|
Rein, A.,
C. Yang,
J. A. Haynes,
J. Mirro, and R. Compans.
1998.
Evidence for cooperation between murine leukemia virus Env molecules in mixed oligomers.
J. Virol.
72:3432-3435[Abstract/Free Full Text].
|
| 52.
|
Rice, N. R.,
L. E. Henderson,
R. C. Soweder,
T. D. Copeland,
S. Oroszlan, and J. F. Edwards.
1990.
Synthesis and processing of the transmembrane envelope protein of equine infectious anemia virus.
J. Virol.
64:3770-3778[Abstract/Free Full Text].
|
| 53.
|
Rose, J. K., and A. Shafferman.
1981.
Conditional expression of the vesicular stomatitis virus glycoprotein gene in Escherichia coli.
Proc. Natl. Acad. Sci. USA
78:6670-6674[Abstract/Free Full Text].
|
| 54.
|
Schnierle, B. S.,
J. Stitz,
V. Bosch,
F. Nocken,
H. Merget-Millitzer,
M. Engelstädter,
R. Kurth,
B. Groner, and K. Cichutek.
1997.
Pseudotyping of murine leukemia virus with the envelope glycoproteins of HIV generates a retroviral vector with specificity of infection for CD4-expressing cells.
Proc. Natl. Acad. Sci. USA
94:8640-8645[Abstract/Free Full Text].
|
| 55.
|
Sommerfelt, M. A.,
S. R. Petteway, Jr.,
G. B. Dreyer, and E. Hunter.
1992.
Effect of retroviral proteinase inhibitors on Mason-Pfizer monkey virus maturation and transmembrane glycoprotein cleavage.
J. Virol.
66:4220-4227[Abstract/Free Full Text].
|
| 56.
|
Soneoka, Y.,
P. M. Cannon,
E. E. Ramsdale,
J. C. Griffiths,
G. Romano,
S. M. Kingsman, and A. J. Kingsman.
1995.
A transient three-plasmid expression system for the production of high titer retroviral vectors.
Nucleic Acids Res.
23:628-633[Abstract/Free Full Text].
|
| 57.
|
Song, J. J.,
J. H. Kim,
H. Lee,
E. Kim,
J. Kim,
Y. S. Park,
J. Ahn,
N. C. Yoo,
J. K. Roh, and B. S. Kim.
2000.
Enhancement of gene transfer efficiency into human cancer cells by modification of retroviral vectors and addition of chemicals.
Oncol. Rep.
7:119-124[Medline].
|
| 58.
|
Stitz, J.,
C. J. Buchholz,
M. Engelstadter,
W. Uckert,
U. Bloemer,
I. Schmitt, and K. Cichutek.
2000.
Lentiviral vectors pseudotyped with envelope glycoproteins derived from gibbon ape leukemia virus and murine leukemia virus 10A1.
Virology
273:16-20[CrossRef][Medline].
|
| 59.
|
Takeuchi, Y.,
G. Simpson,
R. G. Vile,
R. A. Weiss, and M. K. L. Collins.
1992.
Retroviral pseudotypes produced by rescue of Moloney murine leukemia virus vector by C-type, but not D-type, retroviruses.
Virology
186:792-794[CrossRef][Medline].
|
| 60.
|
Ting, Y. T.,
C. A. Wilson,
K. B. Farrell,
G. J. Chaudry, and M. V. Eiden.
1998.
Simian sarcoma-associated virus fails to infect Chinese hamster cells despite the presence of functional gibbon ape leukemia virus receptors.
J. Virol.
72:9453-9458[Abstract/Free Full Text].
|
| 61.
|
Vey, M.,
M. Orlich,
S. Alder,
H. D. Klenk,
R. Rott, and W. Garten.
1992.
Hemagglutinin activation of pathogenic avian influenza viruses of serotype H7 requires the protease recognition motif R-X-K/R-R.
Virology
188:408-413[CrossRef][Medline].
|
| 62.
|
von Kalle, C.,
H. P. Kiem,
S. Goehle,
B. Darovsky,
S. Heimfeld,
B. Dorok-Storb,
R. Storb, and F. G. Shuening.
1994.
Increased gene transfer into human hematopoietic progenitor cells by extended in vitro exposure to a pseudotyped retroviral vector.
Blood
84:2890-2897[Abstract/Free Full Text].
|
| 63.
|
Wilson, C. M.,
S. Reitz,
H. Okayma, and M. V. Eiden.
1989.
Formation of infectious hybrid virions with gibbon ape leukemia virus and human T-cell leukemia virus retroviral envelope glycoproteins and the Gag and Pol proteins of Moloney murine leukemia virus.
J. Virol.
63:2374-2378[Abstract/Free Full Text].
|
| 64.
|
Wool-Lewis, R. J., and P. Bates.
1998.
Characterization of Ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines.
J. Virol.
72:3155-3160[Abstract/Free Full Text].
|
| 65.
|
Yang, Y.,
E. F. Vanin,
M. A. Whitt,
M. Fornerod,
R. Zwart,
R. D. Schneiderman,
G. Grosveld, and A. W. Nienhuis.
1995.
Inducible, high-level production of infectious murine leukemia retroviral vector particles pseudotyped with vesicular stomatitis virus G envelope protein.
Hum. Gene Ther.
6:1203-1213[Medline].
|
| 66.
|
Zhao, Y.,
S. Lee, and W. F. Anderson.
1997.
Functional interactions between monomers of the retroviral envelope protein complex.
J. Virol.
71:6967-6972[Abstract].
|
| 67.
|
Zhao, Y.,
L. Zhu,
C. A. Benedict,
D. Chen,
W. F. Anderson, and P. M. Cannon.
1998.
Functional domains in the retroviral transmembrane protein.
J. Virol.
72:5392-5398[Abstract/Free Full Text].
|
| 68.
|
Zufferey, R.,
D. Nagy,
R. J. Mandel,
L. Naldini, and D. Trono.
1997.
Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo.
Nat. Biotechnol.
15:871-875[CrossRef][Medline].
|
Journal of Virology, May 2001, p. 4129-4138, Vol. 75, No. 9
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.9.4129-4138.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Verhoeyen, E., Cosset, F.-L.
(2009). Engineering the Surface Glycoproteins of Lentiviral Vectors for Targeted Gene Transfer. CSH Protocols
2009: pdb.top59-pdb.top59
[Abstract]
[Full Text]
-
Jorgenson, R. L., Vogt, V. M., Johnson, M. C.
(2009). Foreign Glycoproteins Can Be Actively Recruited to Virus Assembly Sites during Pseudotyping. J. Virol.
83: 4060-4067
[Abstract]
[Full Text]
-
Frecha, C., Costa, C., Negre, D., Gauthier, E., Russell, S. J., Cosset, F.-L., Verhoeyen, E.
(2008). Stable transduction of quiescent T cells without induction of cycle progression by a novel lentiviral vector pseudotyped with measles virus glycoproteins. Blood
112: 4843-4852
[Abstract]
[Full Text]
-
Cote, M., Zheng, Y.-M., Albritton, L. M., Liu, S.-L.
(2008). Fusogenicity of Jaagsiekte Sheep Retrovirus Envelope Protein Is Dependent on Low pH and Is Enhanced by Cytoplasmic Tail Truncations. J. Virol.
82: 2543-2554
[Abstract]
[Full Text]
-
Khurana, S., Krementsov, D. N., de Parseval, A., Elder, J. H., Foti, M., Thali, M.
(2007). Human Immunodeficiency Virus Type 1 and Influenza Virus Exit via Different Membrane Microdomains. J. Virol.
81: 12630-12640
[Abstract]
[Full Text]
-
Long, G., Pan, X., Westenberg, M., Vlak, J. M.
(2006). Functional Role of the Cytoplasmic Tail Domain of the Major Envelope Fusion Protein of Group II Baculoviruses.. J. Virol.
80: 11226-11234
[Abstract]
[Full Text]
-
Claus, C., Hofmann, J., Uberla, K., Liebert, U. G.
(2006). Rubella virus pseudotypes and a cell-cell fusion assay as tools for functional analysis of the rubella virus E2 and E1 envelope glycoproteins.. J. Gen. Virol.
87: 3029-3037
[Abstract]
[Full Text]
-
Stitz, J., Wolfrum, N., Buchholz, C. J., Cichutek, K.
(2006). Envelope proteins of spleen necrosis virus form infectious human immunodeficiency virus type 1 pseudotype vector particles, but fail to incorporate upon substitution of the cytoplasmic domain with that of Gibbon ape leukemia virus. J. Gen. Virol.
87: 1577-1581
[Abstract]
[Full Text]
-
Merten, C. A., Stitz, J., Braun, G., Medvedovska, J., Cichutek, K., Buchholz, C. J.
(2006). Fusoselect: cell-cell fusion activity engineered by directed evolution of a retroviral glycoprotein. Nucleic Acids Res
34: e41-e41
[Abstract]
[Full Text]
-
Sandrin, V., Cosset, F.-L.
(2006). Intracellular Versus Cell Surface Assembly of Retroviral Pseudotypes Is Determined by the Cellular Localization of the Viral Glycoprotein, Its Capacity to Interact with Gag, and the Expression of the Nef Protein. J. Biol. Chem.
281: 528-542
[Abstract]
[Full Text]
-
Wyss, S., Dimitrov, A. S., Baribaud, F., Edwards, T. G., Blumenthal, R., Hoxie, J. A.
(2005). Regulation of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Fusion by a Membrane-Interactive Domain in the gp41 Cytoplasmic Tail. J. Virol.
79: 12231-12241
[Abstract]
[Full Text]
-
Song, C., Micoli, K., Bauerova, H., Pichova, I., Hunter, E.
(2005). Amino Acid Residues in the Cytoplasmic Domain of the Mason-Pfizer Monkey Virus Glycoprotein Critical for Its Incorporation into Virions. J. Virol.
79: 11559-11568
[Abstract]
[Full Text]
-
Song, C., Micoli, K., Hunter, E.
(2005). Activity of the Mason-Pfizer Monkey Virus Fusion Protein Is Modulated by Single Amino Acids in the Cytoplasmic Tail. J. Virol.
79: 11569-11579
[Abstract]
[Full Text]
-
Abada, P., Noble, B., Cannon, P. M.
(2005). Functional Domains within the Human Immunodeficiency Virus Type 2 Envelope Protein Required To Enhance Virus Production. J. Virol.
79: 3627-3638
[Abstract]
[Full Text]
-
Merten, C. A., Stitz, J., Braun, G., Poeschla, E. M., Cichutek, K., Buchholz, C. J.
(2005). Directed Evolution of Retrovirus Envelope Protein Cytoplasmic Tails Guided by Functional Incorporation into Lentivirus Particles. J. Virol.
79: 834-840
[Abstract]
[Full Text]
-
Niebert, M., Tonjes, R. R.
(2005). Evolutionary Spread and Recombination of Porcine Endogenous Retroviruses in the Suiformes. J. Virol.
79: 649-654
[Abstract]
[Full Text]
-
Sandrin, V., Muriaux, D., Darlix, J.-L., Cosset, F.-L.
(2004). Intracellular Trafficking of Gag and Env Proteins and Their Interactions Modulate Pseudotyping of Retroviruses. J. Virol.
78: 7153-7164
[Abstract]
[Full Text]
-
Liu, S.-L., Halbert, C. L., Miller, A. D.
(2004). Jaagsiekte Sheep Retrovirus Envelope Efficiently Pseudotypes Human Immunodeficiency Virus Type 1-Based Lentiviral Vectors. J. Virol.
78: 2642-2647
[Abstract]
[Full Text]
-
Zhang, X.-Y., La Russa, V. F., Reiser, J.
(2004). Transduction of Bone-Marrow-Derived Mesenchymal Stem Cells by Using Lentivirus Vectors Pseudotyped with Modified RD114 Envelope Glycoproteins. J. Virol.
78: 1219-1229
[Abstract]
[Full Text]
-
Sinn, P. L., Hickey, M. A., Staber, P. D., Dylla, D. E., Jeffers, S. A., Davidson, B. L., Sanders, D. A., McCray, P. B. Jr.
(2003). Lentivirus Vectors Pseudotyped with Filoviral Envelope Glycoproteins Transduce Airway Epithelia from the Apical Surface Independently of Folate Receptor Alpha. J. Virol.
77: 5902-5910
[Abstract]
[Full Text]
-
Aguilar, H. C., Anderson, W. F., Cannon, P. M.
(2002). Cytoplasmic Tail of Moloney Murine Leukemia Virus Envelope Protein Influences the Conformation of the Extracellular Domain: Implications for Mechanism of Action of the R Peptide. J. Virol.
77: 1281-1291
[Abstract]
[Full Text]
-
Bobkova, M., Stitz, J., Engelstadter, M., Cichutek, K., Buchholz, C. J.
(2002). Identification of R-peptides in envelope proteins of C-type retroviruses. J. Gen. Virol.
83: 2241-2246
[Abstract]
[Full Text]
-
Sandrin, V., Boson, B., Salmon, P., Gay, W., Negre, D., Le Grand, R., Trono, D., Cosset, F.-L.
(2002). Lentiviral vectors pseudotyped with a modified RD114 envelope glycoprotein show increased stability in sera and augmented transduction of primary lymphocytes and CD34+ cells derived from human and nonhuman primates. Blood
100: 823-832
[Abstract]
[Full Text]
-
Lavillette, D., Marin, M., Ruggieri, A., Mallet, F., Cosset, F.-L., Kabat, D.
(2002). The Envelope Glycoprotein of Human Endogenous Retrovirus Type W Uses a Divergent Family of Amino Acid Transporters/Cell Surface Receptors. J. Virol.
76: 6442-6452
[Abstract]
[Full Text]
Top
Abstract
Introduction
