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Journal of Virology, November 1999, p. 9632-9637, Vol. 73, No. 11
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Association of Murine Leukemia Virus Pol with
Virions, Independent of Gag-Pol Expression
Gary L.
Buchschacher Jr.,1
Lei
Yu,2
Fukashi
Murai,2,
Theodore
Friedmann,2,* and
Atsushi
Miyanohara2
Division of Hematology/Oncology, Department
of Medicine,1 and Department of
Pediatrics,2 Center for Molecular Genetics,
University of California
San Diego, La Jolla, California 92093-0634
Received 4 March 1999/Accepted 3 August 1999
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ABSTRACT |
During the replication cycle of murine leukemia virus (MLV), Pol is
normally synthesized as part of a Gag-Pol fusion protein. In this
study, the ability of free MLV Pol to be incorporated into virions was
examined. When MLV Gag and MLV Pol were coexpressed from separate
plasmids in cells, reverse transcriptase (RT) activity associated with
Gag core particles at a slightly lower level than did RT activity
generated from wild-type Gag-Pol expression. Particles produced in this
manner were somewhat less infectious than those produced with wild-type
Gag-Pol. A smaller amount of MLV Pol also associated with heterologous
human immunodeficiency virus type 1 Gag cores.
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TEXT |
The genomes of all retroviruses
contain gag, pol, and env genes, which
encode the major structural and enzymatic proteins necessary for
formation of virions and completion of the viral replication cycle
(reviewed in reference 8). During virus production in infected cells, the Gag precursor protein is processed to produce the matrix (MA), capsid (CA), and nucleocapsid (NC) proteins that comprise the virion core. The Pol precursor is processed to produce the
functional viral enzymes protease (PR), reverse transcriptase (RT), and
integrase (IN). Though much is known about viral replication and virion
structure, there are still relatively few details known about the
molecular interactions among viral proteins which occur during virion
assembly, maturation, and release from cells.
Pol protein is normally produced only in the form of the Gag-Pol fusion
precursor protein, either by infrequent read-through suppression of Gag
termination or by ribosomal frameshifting during translation of
viral mRNA, at a level estimated to be 5 to 10% of the amount of
free Gag produced (11, 16, 34). It is generally accepted,
though it has not been shown definitively, that retroviral pol gene products are incorporated into forming virions as
part of Gag-Pol fusion proteins via interactions of the Gag portion of
the Gag-Pol precursor with other Gag molecules constituting the virion
core (3, 10, 15, 26, 29). Subsequent proteolytic processing
of Gag and Gag-Pol precursors by the virus-encoded protease leads to
formation of mature, functional virions made up of condensed viral
cores consisting of NC, MA, and CA and active PR, RT, and IN surrounded
by the viral envelope obtained during virus budding (8, 33).
The amount of Gag-Pol that is produced relative to the level of free
Gag appears to be important to the process of virus assembly. For
murine leukemia virus (MLV), a mutation that resulted in production of
100% Gag-Pol and no free Gag prevented the proteolytic processing of
Gag-Pol and the assembly of virions (11). Similar mutations in human immunodeficiency virus (HIV) and spleen necrosis virus also
prevented virion formation (17, 18, 24, 34). It was suggested that the stoichiometry of Gag and Gag-Pol production is
important in virion formation because overproduction of Gag-Pol may
impede core formation by preventing appropriate Gag molecular interactions. Conversely, the presence of the Gag-Pol precursor is not
necessary for virion core formation, since expression of Gag alone in
cells is sufficient for the formation and release of virus-like
particles, though such particles are noninfectious since they lack a
viral genome, pol-encoded enzymatic functions, and the
envelope glycoprotein (13, 32).
The experiments described herein were designed to determine whether MLV
Pol not generated from the Gag-Pol precursor can associate with
assembling virions. The ability of MLV Gag and Pol proteins generated
from separate expression plasmids to assemble into infectious virus
particles was examined. Then, as a first step in studying the
feasibility of engineering a chimeric MLV/HIV vector system, the
studies were extended by using HIV Gag and MLV Pol to determine if MLV
Pol could associate with heterologous HIV Gag virion cores.
MLV Pol associates with MLV Gag virion cores.
The plasmid
pCMV-(M)Gag/Pol expresses the MLV gag and pol
genes as the wild-type Gag and Gag-Pol precursors from the human cytomegalovirus (CMV) promoter. The introduction of this plasmid into
cells results in efficient production of MLV virus-like cores; when it
is coexpressed with amphotropic MLV envelope or vesicular stomatitis
virus G protein and an MLV vector, infectious virus is produced
(31). pCMV-(M)Gag/Pol was modified to generate constructs capable of expressing either the MLV gag or pol
gene separately (Fig. 1). A deletion of
approximately 2,700 bp between the two KpnI sites in the
pol gene resulted in the plasmid pCMV-(M)Gag, a construct
designed to produce MLV Gag protein; this construct also produces the
viral protease to allow proper processing of the precursor Gag protein.
A deletion of approximately 1,500 bp between the upstream
AflII and NruI sites of the gag gene
followed by linker insertion at those sites in pCMV-(M)Gag/Pol resulted in the construct pCMV-(M)Pol, which is capable of expressing MLV Pol;
this construct also contains the C terminus of the NC protein, preserved to maintain the appropriate protease cleavage site of the
amino terminus of the Pol precursor.
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FIG. 1.
Gag- and Pol-expressing constructs. pCMV-(M)Gag/Pol is
the parental construct expressing wild-type MLV Gag-Pol from the CMV
promoter. pCMV-(M)Gag, which contains the MLV gag and
pro genes, was constructed by deletion of the majority of
the pol gene (2,718 bp). The terminal 255 bp of the IN
domain remain. pCMV-(M)Pol expresses MLV Pol and was constructed by
deleting 1,476 bp from the gag gene. Twenty-three base pairs
of the amino terminus of the MA domain and 102 bp of the C terminus of
the NC domain remain. pCMV-(H)Gag(13P5) expresses HIV Gag and PR from
the CMV promoter.
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To determine if proteins expressed from these constructs could form
virions, either plasmid pCMV-(M)Gag/Pol or both pCMV-(M)Gag and
pCMV-(M)Pol, together with pCMV-ampho (a construct expressing the
amphotropic MLV envelope from the CMV promoter, previously referred to
as pHCMV-ampho-env [30]), were transfected
into 293/LZRNL cells (293 cells stably expressing LZRNL
[36], an MLV vector with the marker genes
lacZ and neo [encoding neomycin resistance])
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum, 100 U of penicillin/ml, and 0.1 mg of
streptomycin/ml. For transfections, 106 293 cells were
seeded in 60-mm-diameter plates. The next day, cells were transfected
by the calcium phosphate coprecipitation method as described elsewhere
(5), except that DNA was incubated with the cells for 8 h prior to changing of the medium and glycerol shock was not used. A
total of 15 µg of DNA was used in each transfection. Equivalent
amounts of the plasmids of interest were used in all experiments; if
necessary, the total amount of DNA used was equalized by the addition
of carrier plasmid DNA. Twenty-four hours posttransfection, the cell
medium was changed. Approximately 60 hours posttransfection, virus was
harvested for use in vector titer determinations and RT activity assays
by collecting the medium, centrifuging it briefly to remove cell
debris, and filtering it through a 0.45-µm-pore-size filter.
To determine the amount of RT activity associated with virus particles
produced from these transfections, virus was pelleted
from harvested
conditioned medium by centrifugation as previously
described
(
31). For each sample, 18 µl of virus pellet, prepared
as
noted above and resuspended in phosphate-buffered saline (PBS)
to a
volume of 30 µl, was incubated at 37°C for 60 min with 30
µl of
RT buffer: 50 mM Tris-HCl (pH 8.3), 10 mM dithiothreitol,
1 mM
MnCl
2, 60 mM NaCl, 0.02 mM dTTP, 2.5 µCi of
[
3H]dTTP, 0.25% Nonidet P-40, and 5 µg (0.04 U) of
poly(rA) · poly(dT)
10.
Thirty microliters of each
sample was spotted onto DE81 paper
(Whatman International) in duplicate
and air dried. The filters
were washed twice in 5%
Na
2HPO
4 for 15 min, twice in H
2O
for 15
min, and once in 100% ethanol for 10 min. The filters were
dried,
and incorporated
3H was measured by scintillation
counting with a Beckman LS6500
scintillation counter. RT activity was
detected only in virus
from cells that had been transfected with
constructs that expressed
both Gag and Pol (Table
1), indicating that export of RT to the
culture supernatant is dependent on association of Pol with virion
cores. The amount of RT activity present in the virions produced
from
separate MLV Gag- and Pol-expressing plasmids was approximately
one-third that of the wild-type Gag-Pol expression level.
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TABLE 1.
Virion-associated RT activity and titer of infectious
virus upon separate MLV Gag and MLV
Pol expressiona
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For infections, virus-containing supernatant in the presence of 8 µg
of Polybrene/ml was added to 1.4 × 10
5 208F cells,
maintained in the same medium as 293 cells, plated
the day before in
60-mm-diameter dishes. Thirty-six to 48 h postinfection,
the
vector titer was determined by fixing the cells and assaying
for
-galactosidase activity. Cells infected with the LZRNL vector
were
expected to form blue-staining foci due to expression of
the
lacZ marker. Infected cells were washed in PBS and then
fixed
with 1.25% glutaraldehyde for 30 min at room temperature. The
cells were then washed four times in PBS and incubated overnight
at
37°C with PBS containing 50 mM ferricyanide, 50 mM ferrocyanide,
400 µg of 5-bromo-4-chloro-3-indolyl-
-
D-galactopyranoside
(X-Gal)
per ml, and 1 mM MgCl
2. The stain was removed, the
cells were
washed with PBS, and blue foci were scored by microscopy.
The
relative titer of infectious virus produced by separate expression
of Gag and Pol was 12% (850 of 7,400 foci were blue) compared
to the
titer of virus resulting from wild-type Gag-Pol expression
(Table
1).
When normalized for the amount of RT activity present
in the
supernatant, however, the relative infectivity of the Gag-
plus
Pol-produced virus was approximately one-third that of the
wild-type
virus. Similar titers were seen when the LZRNL vector
titer was assayed
by determination of G418 resistance, demonstrating
that the vector
produced in this system was capable of forming
a stably integrated
provirus, thereby completing the replication
cycle.
These results demonstrate that infectious virus can be
formed from the separate Gag- and Pol-expressing plasmids. To simplify
analysis of virions produced from separate Gag and Pol proteins,
transient-transfection experiments using pCMV-(M)Gag/Pol, pCMV-(M)Gag,
and pCMV-(M)Pol, but omitting a packageable viral vector and envelope
glycoprotein, were performed. This approach to studying virus
assembly
is feasible because neither packageable viral RNA nor
envelope
glycoprotein is necessary to produce virus-like particles
when
wild-type
gag and
pol genes are expressed in
cells (
31,
32). As described above, a total of 15 µg of
DNA containing
equivalent amounts of each plasmid of interest was used;
when
necessary, the total amount of DNA was held constant by addition
of carrier DNA. Sixty to 72 h posttransfection, culture
supernatant
was collected and, as before, virus was pelleted by
centrifugation.
Cell lysates of the transfected cells also were
prepared by washing
cells four times in PBS, centrifuging briefly to
pellet the cells,
resuspending the cells in 100 µl of PBS, and lysing
them by repetitive
cycles of freezing and
thawing.
RT assays were performed on both cell lysates and pelleted virions. The
results (Table
2) show that under these
transfection
conditions, pCMV-(M)Pol efficiently expressed MLV RT in
transfected
cells and that pelletable RT activity was detected in the
cell
supernatant only when MLV Gag and Pol were coexpressed in cells,
again suggesting an association between free Pol protein and Gag
virion
cores. In these cotransfection experiments, the amount
of pelletable RT
obtained from Gag and Pol coexpression was similar
to that from the
wild-type Gag-Pol, indicating that under these
conditions the
association of independently produced Gag and Pol
is reasonably
efficient. The difference in the pelleted RT activities
obtained in
these cotransfection experiments and the multiple-plasmid
transfection
experiments (Table
1) might reflect the higher efficiency
of DNA
couptake when only two plasmids are used (data not shown)
and the
larger amount of Gag- and Pol-expressing plasmids used
in the
cotransfection experiments.
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TABLE 2.
RT activity and p24 levels in cell lysates and virion
pellets after transfection of Gag- and
Pol-expressing plasmidsa
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To examine Gag expression and virion content, Western blot analysis of
transfected-cell lysates and of pelleted virions was
performed.
Twenty microliters of virus pellet or cell lysate resuspended
in PBS
was used for each sample. After 6 µl of loading buffer
(Tris-HCl,
glycerol, sodium dodecyl sulfate [SDS], dithiothreitol,
bromophenol
blue [
1]) was added to each sample, the samples
were
boiled for 10 min and then electrophoresed through an SDS-10%
polyacrylamide gel. Following electrophoresis, the proteins were
transferred to an Immobilon-P membrane (Millipore Corp., Bedford,
Mass.). The membrane was incubated overnight at 4°C, in blocking
reagent (5% nonfat dry milk-2% bovine serum albumin in Tris-buffered
saline [TBS]). The membrane was incubated for 2 h with primary
antibody (goat anti-MLV serum, lot no. 76S000127 and 77S000186;
Quality
Biotech, Camden, N.J.) diluted 1:1,000 in blocking reagent
which had
been diluted 1:1 with TBS. The membrane was washed three
times with
blocking reagent and incubated for 1 h with secondary
antibody
(horseradish peroxidase [HRP]-conjugated donkey anti-goat
immunoglobulin G; Santa Cruz Biotechnology, Santa Cruz, Calif.)
diluted
1:1,000 in blocking agent which had been diluted 1:1 in
TBS. The
membrane was washed once in blocking reagent, once in
0.5% Tween-TBS,
and twice in TBS (10 min per wash). Secondary
antibodies bound to viral
proteins were detected by using an enhanced
chemiluminescence kit (ECL;
Amersham) in accordance with the manufacturer's
instructions.
As can be seen in Fig.
2, pCMV-(M)Gag
(lane 2) and the wild-type Gag-Pol construct (lane 4) express and
process MLV Gag similarly.
Fully processed and partially processed
protein intermediates
are evident. The anti-MLV serum used does not
recognize the Pol
protein (lane 3). Cell lysates from three
representative trials
of cells cotransfected with pCMV-(M)Gag and
pCMV-(M)Pol are shown
in lanes 5 to 7. The Gag-protease precursor can
be seen in cells
transfected with pCMV-(M)Gag (lanes 2 and 5 to 7). The
wild-type
MLV Gag-Pol precursor (Pr200) can be seen only in lane 4 (and
is more easily visualized with a longer exposure of the membrane
to
film) at a predicted smaller amount compared to the processed
proteins.
Also, with a longer exposure, the p15 (MA) band can
be seen in lanes
expressing Gag. The amount of Gag in lanes 5
to 7 is slightly increased
compared to that of the wild-type Gag-Pol.
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FIG. 2.
Western blot analysis of cell lysates expressing MLV Gag
and Pol. Lysates of cells transfected with the indicated plasmids were
subjected to SDS-polyacrylamide gel electrophoresis and blotted, and
the blots were incubated with anti-MLV serum and probed with an
HRP-conjugated secondary antibody. Gag precursor, fully processed, and
partially processed proteins can be seen. Cells were transfected with
no DNA (lane 1), pCMV-(M)Gag (expresses MLV Gag-protease [Gag-Pro])
(lane 2), pCMV-(M)Pol (expresses MLV Pol) (lane 3), pCMV-(M)Gag/Pol
(expresses wild-type MLV Gag and Gag-Pol) (lane 4), or pCMV-(M)Gag and
pCMV-(M)Pol (lanes 5 to 7). The positions of molecular mass markers and
selected viral proteins are indicated. kD, kilodaltons.
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Western blot analysis of pelleted virions, using the same antibodies as
employed for the cell lysate analysis, demonstrated
that the protein
contents of the various samples were similar
(Fig.
3). The Gag plus Pol samples (lanes 5 to
7) did have a slightly
higher protein content (confirmed and
quantitated by densitometric
analysis of band intensity [data not
shown]) than those with wild-type
Gag-Pol (lane 4), suggesting that
more virus particles were produced
from these transfected cells. The
approximately 60- and 50-kDa
bands evident in lanes 2 and 5 to 7 represent partially processed
Gag precursor (Pr65). They may be
somewhat more evident in these
lanes than in the lane with wild-type
Gag-Pol (lane 4) because
of the increased amount of virus protein
present (faint bands
become evident in lane 4 upon longer exposure of
the membrane
to film). However, it is possible that these bands
represent an
increase in the relative amount of partially processed Gag
produced
by pCMV-(M)Gag within virions due to a decreased efficiency of
Gag precursor processing by the viral protease compared to protease
activity in the context of wild-type Gag-Pol. A similar situation
was
reported in HIV virions resulting from
trans complementation
with Vpr-RT and Vpr-IN fusion proteins (
35). It is
conceivable
that subtle alterations in protein conformation which may
occur
when virions are not formed from wild-type Gag-Pol precursors
could affect protease activity or accessibility to precursor cleavage
sites.
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FIG. 3.
Western blot analysis of virions. SDS-polyacrylamide gel
electrophoresis analysis of protein isolated from pelleted virions and
probed with anti-MLV serum and an HRP-conjugated secondary antibody.
Fully processed and partially processed Gag proteins can be seen.
Virions were isolated from the supernatant of cells transfected with no
DNA (lane 1), pCMV-(M)Gag (expresses MLV Gag-protease) (lane 2),
pCMV-(M)Pol (expresses MLV Pol) (lane 3), pCMV-(M)Gag/Pol (expresses
wild-type MLV Gag and Gag-Pol) (lanes 4), or pCMV-(M)Gag and
pCMV-(M)Pol (lanes 5 to 7). The positions of molecular mass markers and
viral proteins are indicated. kD, kilodaltons.
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Densitometric analysis of Western blot band intensity was used to
normalize the amounts of RT activity in the virus pellets
of the
wild-type Gag-Pol and Gag plus Pol samples relative to
the amounts of
Gag present in these pellets. The ratios of pelletable
RT activity to
densitometric units of p15 and p30 band intensity
for the pelleted
virus samples were calculated and are shown in
Table
2. When normalized
for the amount of Gag present in virion
preparations (a reflection of
the number of virions formed and
released), the amount of RT in the Gag
plus Pol virus pellet is
three-fourths that of wild-type Gag-Pol,
indicating that free
Pol associates with Gag at a slightly lower level
than when Pol
is incorporated into virions as the Gag-Pol precursor. It
is important
to remember that this value represents the average for the
population
of virions, not individual particles. It does not address
the
fact that there may be a heterogeneous population of particles,
containing different numbers of RT
molecules.
These results indicate that MLV Pol produced independently from the
Gag-Pol precursor can associate with MLV Gag virion cores.
In Western
blot analyses of cell lysates, no Gag-Pol protein,
which theoretically
could result from recombination between the
Gag- and Pol-expressing
plasmids used in this system, was detected
in cotransfected cells. As
noted above, the wild-type Gag-Pol
precursor (Pr200) was identified
only in the lysate of cells transfected
with the wild-type
gag-pol construct. Longer exposure of the membrane
to film
failed to detect any Pr200 in cells transfected with separate
Gag- and
Pol-expressing constructs, though the Gag-protease precursor
protein
produced from pCMV-(M)Gag was readily apparent. In addition,
further
experiments (data not shown) involving serial dilution
of cell lysate
containing wild-type Gag-Pol with cell lysate from
control cells
demonstrated an ability to detect Pr200 even if
the amount present was
1/10 to 1/20 of that present in the wild-type
samples. Together, these
observations indicate that recombination,
leading to expression of
wild-type protein, is not occurring at
a high enough level in this
system to account for the results
seen. Also, the ability of MLV Pol to
associate with a heterologous
Gag protein (see below), in which there
is no sequence identity
that might support recombination, further
supports the observation
that Pol can associate with virion cores
independently of the
Gag-Pol
precursor.
MLV Pol can associate with HIV Gag virion cores.
In the
above-described system, MLV Pol was able to associate with virion cores
composed of MLV Gag. If also observed with Gag from other retroviruses,
this ability could facilitate the generation of chimeric retroviral
vector systems by allowing substitution of whole retroviral genes
between viruses. In particular, an MLV/HIV chimeric vector and
packaging system may offer unique opportunities to study virus assembly
and to characterize further the required elements that allow HIV to
infect nondividing cells (7, 14, 19). If the ability to
infect nondividing cells could be transferred to an MLV-based vector,
such a vector might offer advantages over the use of an HIV vector
system in clinical applications. For these reasons, the methods
described above were used to determine whether MLV Pol could associate
with HIV Gag virion cores.
In addition to containing the
gag,
pol, and
env genes, the HIV genome encodes a number of accessory
proteins not found in
MLV (
21). Normally, HIV
gag
and
pol expression is dependent
on the viral Rev protein,
which facilitates transport of viral
RNA from the nucleus to the
cytoplasm by overcoming the effects
of negative regulatory elements
present in
gag-pol transcripts.
Also, some of the HIV
accessory proteins are incorporated into
virions and play various roles
in the viral replication cycle,
though their roles and mechanisms of
action are not all clear.
To facilitate efficient Gag production and to
avoid confounding
variables due to the presence of accessory proteins,
an HIV
gag-pro construct containing silent mutations in
negative regulatory elements
was used to express Gag. This construct
(p55BM13P5) has been shown
to express and process Gag protein and to
form virion cores in
the absence of Rev (
28). In the
experiments described here,
the HIV
gag-pro coding region
was expressed from the CMV promoter.
Thus, this construct
[pCMV-(H)Gag(13P5)] allows Gag expression
and processing in the
absence of all HIV accessory proteins and
keeps the HIV Gag-MLV Pol
experimental system as similar as possible
to the MLV Gag-MLV Pol
system.
293 cells were transfected with pCMV-(H)Gag(13P5) and pCMV-(M)Pol in
parallel with the MLV Gag plus MLV Pol experiments. Cell
lysates and
virus pellets were prepared as described above for
RT assay and protein
analysis. HIV Gag expression and virion core
production were
quantitated by measuring p24 (CA) levels present
in cell lysates and
virus pellets, using a standard enzyme-linked
immunosorbent assay
(ELISA) performed by the University of California
San
Diego Center for
AIDS Research. Samples were prepared for analysis
by diluting them
12,500-fold in a buffer containing 0.5% Triton
X-100, pH 7.4.
The results (Table
2) show that when pCMV-(H)Gag(13P5) was introduced
into cells, p24 was expressed and incorporated into
virions that could
be pelleted by centrifugation. Coexpression
of MLV Pol in cells did not
adversely affect HIV Gag expression.
Coexpression of HIV Gag and MLV
Pol resulted in RT activity in
cells at a level equivalent to that
achieved when MLV Pol was
expressed alone (Table
2), showing that
expression of HIV Gag
does not inhibit MLV Pol expression or
activity.
MLV RT activity also was detected in pelleted virions, at about
one-fourth the level of wild-type MLV Gag-Pol, suggesting
that MLV Pol
could associate with HIV Gag cores and that the RT
retained enzymatic
activity (Table
2). A direct comparison of
the MLV RT activity in these
virions and of the HIV RT activity
in native virions is not possible
for two reasons. First, it is
not possible to express HIV RT
efficiently in the absence of at
least some of the HIV accessory
proteins (
28); the presence
of these HIV proteins would
confound analysis of experiments designed
to compare the ability of MLV
Pol to associate with MLV or HIV
Gag, since MLV does not encode
accessory proteins. More importantly,
the optimal assay conditions for
HIV RT and MLV RT differ (
8),
precluding a direct comparison
of the two enzyme
activities.
Attempts to determine if virus containing HIV Gag and MLV Pol is
infectious were inconclusive. In its simplest form, a viral
vector used
in such a study would have to be composed of both
HIV and MLV
cis-acting sequences. Since the virion cores are composed
of
HIV Gag, the vector must contain the HIV encapsidation sequence
(
22) recognized by the
gag-encoded NC protein. In
addition,
since the enzymatic activities of such a virus would be
supplied
by MLV Pol, other
cis-acting sequences, including
the primer binding
site and
att sites, would need to be of
MLV origin. Attempts were
made to propagate such a vector, using
the HIV Gag plus MLV Pol
system, by transfecting pCMV-(H)Gag(13P5),
pCMV-(M)Pol, pCMV-ampho,
and a vector construct into 293 cells,
infecting 208F cells with
the collected supernatant, and assaying for
vector propagation
by a focal
-galactosidase assay. However, the
chimeric vectors
studied so far have shown poor gene expression from
the viral
long terminal repeat, limiting their utility. Because the
development
of a chimeric vector containing
cis-acting
sequences from more
than one type of retrovirus is liable to be
difficult, and no
appropriate alternative vector exists, the
infectivity of the
virions produced by HIV Gag and MLV Pol cannot be
assessed at
this time. Nonetheless, the above results show that in this
system,
MLV Pol can associate with heterologous HIV cores and the viral
polymerase retains enzymatic activity, as assessed by RT
assay.
The precise steps and timing of virion assembly, processing, and
maturation are complex and incompletely understood. The main
determinants driving core formation involve interactions among
at least
the CA domains (
3,
10,
15,
26,
29) (with possible
MA and NC
contributions [
4,
9,
37]) of the Gag precursor
protein, which presumably associate to form the virion core. In
type C
retroviruses and lentiviruses, it is believed that Pol
is incorporated
into virions as part of the Gag-Pol precursor.
Activation of the viral
protease, most likely during core formation
at the cell membrane for
type C retroviruses, ensues, allowing
processing of the Gag and Pol
proteins (
33). The RT and IN proteins
may play roles in the
timing of protease activation and the efficiency
with which PR
processes the Gag and Gag-Pol precursors (
6).
In addition,
it is likely that during encapsidation of genomic
viral RNA, RT and NC
play roles in selection and annealing of
the tRNA used to prime reverse
transcription (
2,
27), further
illustrating the complex
roles Gag and Pol play during virion
assembly.
An experimental system that allows virion incorporation of HIV type 1 RT and IN that have been fused to Vpr, an HIV accessory
protein
normally incorporated into virus particles, has been described
(
12,
35). As assayed by the ability of the fusion proteins
to
trans complement RT and IN mutations in the HIV genome,
Vpr-RT
and Vpr-IN were able to mediate incorporation of RT and IN into
virus particles, demonstrating an ability to separate HIV Pol
assembly
from function. In addition, a small amount of IN could
be incorporated
into virions even when not expressed as a Vpr
fusion
protein.
The experiments described herein do not address how Pol incorporation
occurs during wild-type virus replication and do not
contradict the
as-yet-unconfirmed likelihood that Pol is normally
incorporated as the
Gag-Pol precursor. The results described herein,
however, do show that
to maintain appropriate RT activity, it
is sufficient for MLV Pol to
associate with virions but, as with
HIV RT and IN, it is not necessary
for incorporation to occur
as part of the Gag-Pol protein. One
interpretation of the results
seen upon using the described system is
that the RT activity in
virions produced from separately expressed MLV
Gag and Pol was
lower than that produced from Gag-Pol expression in
virions because
independently synthesized Pol might not be incorporated
into virus
particles as efficiently or in the same stoichiometry as
with
Gag-Pol expression. The observed difference in virus infectivity
may reflect the existence of a heterogeneous population of virions
containing various amounts of RT. Alternatively, it may be that
free
Pol, compared to Gag-Pol, results in an altered molecular
organization
within virions (with regard to the viral genome,
tRNA primer, and viral
enzymes and structural proteins) that is
subsequently reflected in
decreased particle
infectivity.
There are two possible explanations for the observation that functional
Pol could associate with virion cores. One scenario
is that Pol
incorporation simply is a result of the experimental
system used in
this study, in which Gag and Pol were expressed
independently of CMV
promoters as opposed to the MLV long terminal
repeat. It may be that in
this artificial scenario, Pol nonspecifically
associates with virion
cores differently than it does during wild-type
replication and that
this difference is irrelevant to wild-type
virus replication.
Nevertheless, these results show that
trans complementation
is possible and demonstrate that at least some
of MLV Pol's enzymatic
functions are not dependent on the protein
being expressed and
incorporated into virions as the Gag-Pol protein.
Alteration of the
wild-type MLV genome to allow separate
gag and
pol gene expression with no Gag-Pol production would permit
further
study to determine the relevance, if any, of these findings to
the natural viral replication
cycle.
Alternatively, it is possible that in this system Pol incorporation
into particles is occurring via specific interactions
between the free
Gag and Pol molecules. Such interactions have
been postulated to exist
in spumaviruses, which incorporate free
Pol into virus particles (in
spumaviruses, no Gag-Pol precursor
is produced [
20]).
If they exist for MLV and HIV, these interactions
are likely to be at
least partially conserved among retroviruses,
as indicated by the
incorporation of MLV Pol into HIV Gag cores.
Although it is nearly
certain that under wild-type conditions
the major determinant of Pol
incorporation into virions occurs
via molecular interactions of the Gag
CA domain of Gag and Gag-Pol
(
3,
10,
15,
26,
29), it is
conceivable that there are
other, minor interactions between Gag and
Pol that are unmasked
in this system when the more native interactions
are prevented.
Such interactions might assist in Gag-Pol incorporation
into virions
or promotion and stabilization of the preferred molecular
organization
within virions. If specific interactions among precursor
molecules
during retroviral particle assembly can be identified, it is
foreseeable
that inhibitors of such interactions, capable of
interfering with
the production of infectious virus from cells, might
be developed.
Further study will be necessary to determine if specific
Gag and
Pol interactions are occurring, to define their roles in virion
formation, and to identify potential intermolecular PR or C-terminal
NC
interactions that might play roles in the association of free
Pol with
virion cores. In addition to providing information regarding
virus
structure and assembly, the development of an MLV/HIV chimeric
vector
system may also help to identify the mechanism of and minimal
requirements for infection of nondividing cells. This information
might
allow transfer of the ability to infect nondividing cells
to the
MLV-based vectors systems, which have been more thoroughly
studied, and
accepted as relatively safe for clinical use, than
the more recently
developed lentivirus vectors (
23,
25).
| |
ACKNOWLEDGMENTS |
G.L.B. and L.Y. contributed equally to the work presented.
We thank J. Corbeil and the University of CaliforniaSan Diego Center
for AIDS Research for performing the p24 quantitation and G. N. Pavlakis for supplying the plasmid p55BM13P5.
G.L.B. was supported in part by a grant from the Bank of
America-Giannini Foundation. These studies also were funded by DNAVEC Research, Inc., NIH grants HL53620 and DK49023, and the Charles H. and
Anna S. Stern Foundation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pediatrics, Center for Molecular Genetics, University of
CaliforniaSan Diego, 9500 Gilman Dr., La Jolla, CA 92093-0634. Phone:
(619) 534-4268. Fax: (619) 534-1422. E-mail:
tfriedmann{at}ucsd.edu.
Present address: Pioneer Bioscience Institute, Shinjuku-ku, Tokyo
160-0004, Japan.
| |
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Journal of Virology, November 1999, p. 9632-9637, Vol. 73, No. 11
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