Previous Article | Next Article 
Journal of Virology, December 2000, p. 11531-11537, Vol. 74, No. 24
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Translation Is Not Required To Generate Virion Precursor RNA in
Human Immunodeficiency Virus Type 1-Infected T Cells
Melinda
Butsch1,2 and
Kathleen
Boris-Lawrie1,2,3,4,5,*
Center for Retrovirus
Research,1 Departments of Veterinary
Biosciences4 and Molecular Virology,
Immunology and Medical Genetics,3 The
Ohio State Biochemistry Program,2 and
Comprehensive Cancer Center,5 The
Ohio State University, Columbus, Ohio 43210-1093
Received 18 July 2000/Accepted 25 September 2000
 |
ABSTRACT |
The retroviral primary transcription product is a multifunctional
RNA that is utilized as pre-mRNA, mRNA, and genomic RNA. The
relationship between human immunodeficiency virus type 1 (HIV-1) unspliced transcripts used as mRNA for viral protein synthesis and as
virion precursor RNA (vpRNA) for encapsidation remains an important
question. We developed a biochemical assay to evaluate the hypothesis
that prior utilization as mRNA template for protein synthesis is
necessary to generate vpRNA. HIV-1-infected T cells were treated
with translation inhibitors under conditions that maintain virus
production. Immunoprecipitation of newly synthesized HIV-1 Gag protein
revealed that de novo translation is not necessary to sustain assembly,
release, or processing of Gag structural protein. Both newly
synthesized protein and steady-state Gag are competent for assembly,
and the extracellular accumulation of Gag is proportional to the
intracellular abundance of Gag. As early as 2 h after
transcription, newly synthesized RNA is detectable in cell-free virions
and encapsidation is sustained upon inhibition of host cell
translation. Results of both [3H]uridine incorporation
assays and HIV-1-specific RNase protection assays (RPAs) indicate that
translation inhibition reduces the absolute amounts of both cytoplasmic
and virion-associated RNA. Evaluation of encapsidation efficiency by
RPA revealed that the cytoplasmic availability of vpRNA is
increased, indicating that HIV-1 unspliced mRNA can be rerouted to
function as vpRNA. Our data contrast with results from the HIV-2
and murine leukemia virus systems and indicate that HIV-1 unspliced RNA
constitutes a single functional pool that can function interchangeably
as mRNA and as vpRNA.
| |
INTRODUCTION |
The genomes of RNA viruses are
multifunctional molecules. In retroviruses, including human
immunodeficiency virus type 1 (HIV-1), the primary RNA transcript
functions as pre-mRNA for splicing, mRNA for synthesis of
viral protein, and virion precursor RNA (vpRNA) for packaging into
infectious virions. The unspliced HIV-1 mRNA and vpRNA are
physically indistinguishable and are defined experimentally by their
association with ribosomes and virions, respectively. The relationship
between mRNA and vpRNA remains poorly understood, and its
characterization may yield a new strategy to inhibit production of
infectious HIV-1 and to improve lentiviral vector systems for gene
transfer applications.
Initial investigation of the relationship between retroviral unspliced
mRNA and vpRNA focused on cells productively infected with the
genetically simple murine leukemia virus (MLV) (11, 15, 20).
Levin and colleagues (10, 11) analyzed cells treated with
the transcription inhibitor actinomycin D (actD) and showed that viral
mRNA remains available to direct viral protein synthesis, but the
particles do not contain genomic RNA. These data implied that MLV
transcripts segregate into two functionally distinct populations of
mRNA for translation or vpRNA for encapsidation (11). Stoltzfus et al. (23) applied isotopic
equilibrium assay to cells infected with avian sarcoma virus (ASV) and
observed not two but rather a single RNA population that functions as
both ASV mRNA and vpRNA. Sonstegard and Hackett (22)
came to similar conclusions in their studies of Rous sarcoma virus
(RSV) vector RNAs. Transfection studies with vectors that contain or
lack most of the RSV encapsidation signal, 
, indicate that
interaction of Gag with autogenously modulates competition between
the translational machinery and assembling viral proteins. The data
indicate that equilibrium exists between vector RNA destined for
translation or encapsidation, which is determined by the cytoplasmic
availability of Gag protein and ribosomes (22).
Investigation of the fate of vpRNA from genetically complex
retroviruses has been largely limited to genetic studies with HIV
vectors and has not been pursued for RNA expressed from HIV-1 provirus
in human T cells. Studies with HIV-1-based vectors have shown that the
RNA structure inherent in the HIV-1 encapsidation signal inhibits
efficient translation (6, 17). These results imply that
HIV-1 encapsidation and translation are competing processes. McBride et
al. (13) evaluated a subgenomic HIV-1 vector that contains a
premature gag stop codon and found that encapsidation remained efficient. These data are consistent with the successful use
of HIV-1 as a gene transfer vector (9, 18) and eliminate a
requirement for ongoing Gag protein synthesis. However, the question of
whether or not it is necessary for vpRNA to serve as mRNA
template prior to encapsidation remains open. Contrasting results were
obtained in a study of HIV-2-based vectors that contain deletions at
the 3' end of the gag open reading frame. These results indicated that Gag protein translation from vector template was necessary to generate HIV-2 vpRNA (8). HIV-1 differs
from HIV-2 in that the complete encapsidation signal exists only on
unspliced viral RNA and not on spliced RNAs as in HIV-2
(14). The requirement for prior translation of HIV-2
gag mRNA is a potential mechanism for selective
encapsidation of HIV-2 unspliced RNA into progeny virions
(8).
The primary goal of this project was to evaluate the hypothesis
that translation is a prerequisite to generate HIV-1 vpRNA in
chronically infected human T cells. Definition of the relationship between HIV-1 unspliced mRNA and vpRNA will shed light on
whether the transcripts constitute a single RNA pool or two
functionally independent pools of RNA that are dedicated as
either mRNA or vpRNA. If HIV-1 unspliced transcripts function
as a single pool of RNA, inhibition of protein synthesis is expected to
increase the availability of vpRNA and augment encapsidation
efficiency. However, in the case that prior utilization as mRNA is
necessary to generate vpRNA, encapsidation efficiency would be
decreased upon inhibition of protein synthesis. If HIV-1 unspliced
transcripts represent two independent pools of RNA that are committed
to either translation or encapsidation, translation inhibition would
not alter generation of vpRNA.
To examine these possibilities, HIV-1-infected human T cells were
treated with translation inhibitors under conditions that maintain
virus assembly. Comparison of ribosomal profiles of HIV-1-infected and
mock-infected T cells verifies that HIV-1 infection does not mediate
shutoff of host cell translation or disrupt the mechanistic effects of
pactamycin (pac), cycloheximide (chx), or anisomycin (aniso). Analysis
of newly synthesized HIV-1 protein and vpRNA after short-term
treatment with the inhibitors established that de novo translation is
not necessary to maintain assembly, release, and processing of Gag
precursor protein or encapsidation of vpRNA. RNase protection
assays (RPAs) demonstrate that HIV-1 encapsidation efficiency is
increased upon 80 to 90% inhibition of de novo translation. The data
indicate that prior translation of HIV-1 unspliced RNA is not a
prerequisite to generate vpRNA. HIV-1 unspliced transcripts constitute a single population of RNA that can be selected
interchangeably as vpRNA and as mRNA.
| |
MATERIALS AND METHODS |
Cells and translation inhibitors.
CEM(A) T cells infected
with HIV-1NL4-3 [CEM(A)/HIV-1 cells] were
cultured in RPMI 1640 medium supplemented with 10% fetal calf serum
and 1% antibiotic-antimycotic (Gibco-BRL). Cell viability in response
to chx, pac, or aniso was assayed by propidium iodide and flow
cytometry at 4 h after treatment (3). In subsequent experiments we used maximal concentrations having minimal cytopathic effect during a 4-h incubation: 5 × 10
8 M pac
(Pharmacia & Upjohn, Kalamazoo, Mich.), 0.5 µg of chx (Sigma, St.
Louis, Mo.) per ml, and 0.1 µg of aniso (Sigma) per ml.
Protein analysis.
CEM(A)/HIV-1 T cells were lysed in
radioimmunoprecipitation assay (RIPA) buffer (0.05 M Tris-HCl [pH 8],
0.1% sodium dodecyl sulfate [SDS], 1% Triton X-100, 2 mM
phenylmethylsulfonyl fluoride, 0.15 M NaCl) containing 1% deoxycholic
acid, and the nuclei were removed after centrifugation at
13,400 × g for 10 min. Total protein concentration was
determined by the Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, Calif.). Virion-containing medium was clarified
by centrifugation at 2,000 × g for 10 min, and virions were collected by centrifugation at 156,000 × g for
1.5 h at 4°C in a Beckman SW41 rotor. Gag enzyme-linked
immunosorbent assay (ELISA) was performed as specified by the
manufacturer (Beckman-Coulter, Brea, Calif.). 35S-labeling
experiments were performed by incubating CEM(A)/HIV-1 T cells in
cysteine/methionine-free RPMI medium with 5% dialyzed fetal bovine
serum for 30 min, followed by addition of pac, chx, or aniso coincident
with [35S]cysteine/methionine (10 µCi/ml; 1,175 Ci/mmol, 43.5 MBq/ml; ICN Biochemicals, Irvine, Calif.). The cells were
lysed in RIPA buffer containing 1% deoxycholic acid. Fifty nanograms
of cytoplasmic lysate and virion lysates equivalent to 30 ng of Gag
were precipitated by trichloroacetic acid (TCA) (20% TCA, 0.1 mg of
bovine serum albumin per ml) onto 25-mm-diameter glass fiber filters
(type A/C; Pall Corp., Ann Arbor, Mich.), washed three times in 10% TCA and in 100% ethanol, and subjected to scintillation counting. For
pulse-chase experiments, CEM(A)/HIV-1 T cells were incubated first
for 30 min in cysteine/methionine-free RPMI medium with 5% dialyzed
fetal bovine serum and then for 1 h in
[35S]cysteine/methionine-supplemented medium with or
without pac, chx, or aniso. The cells were washed, and complete RPMI
medium was added. At 1, 2, 4, 6, or 8 h postchase, cells were
harvested as above. For immunoprecipitation, the
35S-labeled lysates were incubated for 16 h with
protein A-Sepharose beads (Pharmacia) and Gag p24 antibody (gift of N. Panganiban) (12). The beads were washed once in high-salt
RIPA buffer (1 M NaCl) and once in low-salt RIPA buffer (0.15 M NaCl)
and then boiled to elute the proteins. The 35S-labeled
precipitated proteins were subjected to SDS-polyacrylamide gel
electrophoresis (PAGE), visualized, and quantified by PhosphorImager analysis (Molecular Dynamics, Inc., Sunnyvale, Calif.) with ImageQuaNT software version 4.2 (Molecular Dynamics).
RNA analysis.
CEM(A)/HIV-1 cells were plated in T150
flasks, cultured overnight to 80% confluence, and incubated for 4 h in medium with [3H]uridine (30 µCi/ml; Amersham,
Piscataway, N.J.) and with or without pac, chx, or aniso. Cytoplasmic
extracts were prepared in 0.9 ml of cold cell lysis buffer (10 mM Tris
[pH 8.3], 150 mM NaCl, 1.5 mM MgCl2) and 0.1 ml of 5%
NP-40. Following centrifugation to pellet the nuclei, the supernatant
was mixed with TriReagent LS, and cytoplasmic RNA was isolated as
specified by the manufacturer (Molecular Research, Cincinnati, Ohio).
One microgram of cytoplasmic [3H]RNA and virions
equivalent to 30 ng of extracellular Gag were applied to glass fiber
filters, which were washed four times with 5% TCA containing 20 mM
sodium pyrophosphate and once with 100% ethanol, dried, and subjected
to scintillation counting. For ribosome profiles, clarified cytoplasmic
extract from three 80% confluent T150 flasks was layered onto a 10-ml
linear gradient of 15 to 45% sucrose (21). The gradient was
centrifuged 225,000 × g for 2.25 h at 4°C in a
Beckman SW41 rotor. Gradients were fractionated and monitored for
A254 on an ISCO (Lincoln, Neb.) model 160 gradient fractionator. To prepare virion RNA, cell medium was clarified by centrifugation at 2,000 × g for 10 min, and virions
were pelleted by centrifugation at 156,000 × g for
2.5 h at 4°C in a Beckman SW28 rotor, lysed in 1 ml Trizol
Reagent, and isolated as specified by the manufacturer (Gibco BRL,
Gaithersburg, Md.).
32P-labeled antisense RNA probes were generated by in vitro
transcription of pGEM(600-900), which contains the 5' untranslated region of HIV-1NL4-3 (12), and pGAPDH,
which contains the human glyceraldehyde dehydrogenase (gapdh) gene (2). Following digestion of
pGEM(600-900) with NotI and pGAPDH with NcoI,
antisense runoff RNA transcripts were synthesized with MAXIscript T7
RNA polymerase (Ambion, Austin, Tex.), and the probes were isolated by
gel elution. RPA was performed using RPA III (Ambion) according to the
instruction manual, with some modifications (2). Typically,
10 µg of cytoplasmic RNA or viral RNA from virions equivalent to 250 ng of Gag was precipitated by ethanol with 2 × 105
cpm of HIV-1 probe and 2 × 104 cpm of
gapdh probe. Samples were resuspended in 10 µl of
hybridization buffer, denatured at 94°C for 3 min, and
hybridized at 42°C overnight. RNase A/T1 was diluted
1:100 in Ambion RNase digestion buffer, and 150 µl was added to each
sample and incubated at 37°C for 30 min. SDS and proteinase K were
added to final concentrations of 1% and 0.5 mg/ml, respectively, and
samples were incubated at 37°C for 30 min. A 100-bp
32P-labeled DNA fragment was added to virion RNA samples,
followed by phenol-chloroform and chloroform extraction and
precipitation with ethanol in the presence of 10 µg of yeast RNA.
Following centrifugation, the pellets were dissolved in 6 µl of
loading buffer, denatured at 94°C for 3 min, and subjected to 5%
denaturing PAGE RNase protection products were visualized and
quantified by PhosphorImager analysis.
| |
RESULTS |
HIV-1 infection does not alter host cell response to pac,
chx, or aniso.
Our goal was to evaluate trafficking of HIV-1
unspliced RNA expressed from provirus in T cells. A genetic
approach using conventional transfection methods was of limited utility
for this purpose because overexpression of RNA from transfected DNA may
saturate the assembly process and obscure the natural
relationship between HIV-1 unspliced mRNA and vpRNA that is
exhibited by authentic provirus in an infected T cell. Therefore, we
developed a biochemical approach that limits de novo translation
of HIV-1 RNA under conditions that maintain virus production.
CEM(A)/HIV-1 cells were subjected to short-term incubation with
three mechanistically distinct biochemical antagonists of
translation:
pac, chx, and aniso. To determine the magnitude and
onset of inhibition
of protein synthesis, [
35S]cysteine/methionine
incorporation into whole cell protein was
evaluated after a 4-h
incubation with pac, chx, or aniso. Comparison
with mock-treated cells
indicated that incorporation of
[
35S]cysteine/methionine into whole cell protein was
inhibited 80
to 90%; the reduction in protein synthesis commenced by
30 min
posttreatment and continued up to 4 h posttreatment (Fig.
1).
Propidium iodide staining and flow
cytometry detected no overt
cytopathic effects on the cells during the
4-h incubation period.
Relative to the mock control, cell viability
remained 100% in
response to pac and 96% in response to chx but was
reduced to
86% in response to aniso. Ribosomal profile analysis
of the cells
indicated that the treatments exert the expected
mechanistic effects
on the translational machinery (Fig.
2). Pac produced an accumulation
of 80S
monosomes, which is attributable to interference with translation
initiation (
5,
7,
24). Chx resulted in the accumulation
of
polyribosomes in response to a block in EF-2-dependent peptide
translocation (
4,
19,
24). Aniso reduced polyribosome
abundance
associated with defective peptide bond formation during
elongation
of the polypeptide (
24). Comparison with
the rRNA profiles of
mock-infected CEM(A) T cells (Fig.
2) indicated
that HIV-1 infection
did not change the rRNA profile of
CEM(A) T cells (
1), nor
did it alter the response to pac,
chx, or aniso. These results
indicate that HIV-1 infection does not
disrupt the host translation
machinery.
View larger version (13K):
[in this window]
[in a new window]
|
FIG. 1.
Incorporation of [35S]cysteine/methionine
is inhibited by incubation with pac, chx, and aniso. Inhibition of
[35S]cysteine/methionine incorporation occurs 0.5 h
posttreatment and is sustained over a 4-h period. CEM(A)/HIV-1 T
cells were incubated with cysteine/methionine-free RPMI medium for
0.5 h, followed by the addition of
[35S]cysteine/methionine with pac, chx, or aniso. Total
cell lysates were collected at 0.5, 2, or 4 h posttreatment,
and [35S]cysteine/methionine incorporation was
quantified by TCA precipitation assay. Average results of at least four
experiments are shown; error bars indicate standard deviations.
|
|
View larger version (36K):
[in this window]
[in a new window]
|
FIG. 2.
Ribosomal profile analysis in response to pac, chx, and
aniso. HIV-1 infected or uninfected CEM(A) T cells were treated for
4 h with or without pac, chx, or aniso, and cytoplasmic extracts
were placed on 10-ml linear gradients of 15 to 45% sucrose. After
ultracentrifugation, the gradients were fractionated and monitored at
A254 using an ISCO gradient fractionation
system.
|
|
De novo translation is not necessary for virion production or Gag
processing.
Pulse-chase labeling was used to define the onset and
duration of accumulation of newly synthesized Gag in virions. Cells were incubated for 0.5 h in medium lacking cysteine/methionine, followed by incubation for 1 h in [35S]
cysteine/methionine-supplemented medium, washing, and incubation with
medium without [35S]cysteine/methionine; 1 to 8 h
later, virions were isolated by ultracentrifugation and quantified by
Gag ELISA. One microgram of whole cell lysate or virions equivalent to
30 ng of Gag were subjected to precipitation assay with TCA to
determine [35S]cysteine/methionine incorporation into
newly synthesized proteins. Nonspecific accumulation of
[35S]cysteine/methionine was quantified in control
cultures that were treated with pac to inhibit protein synthesis. Level
of background incorporation of [35S]cysteine/methionine
into the pac-treated whole cell lysates and virion samples were similar
at each time point: 1,000 cpm or less for whole cell lysate and 30 cpm
or less for virion samples (Fig. 3).
Incorporation of 35S-labeled protein into virions was
maximal at 1 h postchase, indicating that newly synthesized Gag is
readily incorporated into virions (Fig. 3). Production of
35S-labeled virions continued for 6 h postchase,
indicating that newly synthesized Gag is not required for continued
virion production. [35S]cysteine/methionine incorporation
into virions diminished over time, a trend that matched the decline in
intracellular 35S-labeled protein. The results indicate a
concentration-dependent relationship between intracellular and
extracellular Gag.
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 3.
Pulse-chase analysis of Gag incorporation into virions.
CEM(A)/HIV-1 T cells were incubated with cysteine/methionine-free
medium for 0.5 h, followed by a 1-h incubation with of 10 µCi of
[35S]cysteine/methionine per ml and 5 × 108 M pac in control plates. Cells were washed and
incubated in complete medium with or without pac. Cell lysates and
virion lysates were collected at intervals between 1 and 8 h
postchase. TCA precipitation assay was performed with 50 ng of cellular
protein or virion lysate equivalent to 30 ng of Gag. Representative
results of three experiments are shown.
|
|
Gag immunoprecipitation assay was used to evaluate the effects of pac,
chx, and aniso on Gag protein synthesis and processing.
We also
evaluated the effect of actD, an RNA synthesis inhibitor
that is known
to disrupt shuttling of HIV-1 Rev between the nucleus
and the
cytoplasm (
16). The cells were incubated for 4 h in
medium supplemented with [
35S]cysteine/methionine.
Subsequent Gag ELISA of cell-free supernatant
indicated that Gag
production was reduced but not abrogated in
response to the translation
inhibitors. Compared to the mock-treated
cells, levels of Gag
production from pac-, chx-, aniso-, and actD-treated
cells were 68% ± 11%, 69% ± 16%, 71% ± 19%, and 93% ± 15%, respectively.
These
results indicate that the inhibitor treatments do not prevent
previously synthesized Gag from being released from the
cell.
Immunoprecipitation assays detected similar levels of
35S-labeled Gag in mock-treated and actD-treated cells (100 and 120%,
respectively) (Fig.
4).
Similar levels of
35S incorporation were also observed in
virion samples, indicating
that de novo RNA synthesis is not necessary
for synthesis and
processing of HIV-1 Gag. Treatment with pac, chx,
and aniso reduced
intracellular
35S-labeled Gag levels to
28, 40, and 28%, respectively. Extracellular
35S-labeled
Gag levels were similarly reduced to 20, 20, and 26%,
respectively,
indicating that extracellular
35S-labeled Gag levels are
proportional to the cytoplasmic abundance
of
35S-labeled
Gag. Each virion sample displayed fully processed Gag
p24, indicating
that de novo translation is not required for Gag
protein processing.
Minor differences observed in the intracellular
ratio of unprocessed
Gag p55 to Gag p24 may be attributable to
variation among the cells in
the intracellular concentration of
Gag p55. The observation that the
extracellular accumulation of
Gag is proportional to the intracellular
abundance of Gag validates
a concentration-dependent relationship
between extracellular and
intracellular Gag.
View larger version (50K):
[in this window]
[in a new window]
|
FIG. 4.
Pac, chx, and aniso significantly inhibit Gag synthesis.
CEM(A)/HIV-1 T cells were incubated with cysteine/methionine-free
RPMI medium for 0.5 h, followed by the addition of 10 µCi of
[35S]cysteine/methionine per ml with or without pac, chx,
or aniso. Cell lysates and cell-free supernatants were collected 4 h posttreatment, and virions were isolated by centrifugation.
Fifty-nanogram samples of 35S-labeled cell lysate and
virions equivalent to 100 ng Gag were subjected to
radioimmunoprecipitation assay with Gag p24 antibody followed by
SDS-PAGE and PhosphorImager analysis.
|
|
Newly synthesized RNA accumulates in virions within 2 h.
[3H]uridine labeling was performed to evaluate the time
course in which newly synthesized vpRNA becomes available for
encapsidation. CEM(A)/HIV-1 cells were incubated with
[3H]uridine over a 6-h period, and cytoplasmic and
virion-associated RNAs were isolated and subjected to the TCA
precipitation assay. One microgram of cytoplasmic RNA and virion RNA
equivalent to 30 ng of Gag were analyzed. By 2 h postlabeling,
3H-labeled RNA was present in the cytoplasm of the
mock-treated cells and was incorporated into virions (Fig.
5). These results indicate that as early
as 2 h postlabeling, changes in cytoplasmic RNA are manifested in
virions. As a negative control for nonspecific incorporation of
[3H]uridine, RNA synthesis was inhibited by treatment
with actD. The actD-treated samples exhibited low-level
[3H]uridine incorporation at each time point: 1,000 cpm
in cytoplasmic RNA and 100 cpm in virion RNA (Fig. 5).
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 5.
Incorporation of [3H]uridine into newly
synthesized virions. CEM(A)/HIV-1 cells were incubated for 2, 4, and 6 h in medium containing [3H]uridine (30 µCi/ml) with or without actD (0.5 µg/ml). [3H]uridine
levels in cytoplasmic RNA and in virion RNA were quantified by TCA
precipitation analysis. Representative results of at least three
experiments are shown.
|
|
To evaluate the possibility that de novo translation is a prerequisite
for generation of vpRNA, [
3H]uridine incorporation
into virion RNA was evaluated with or
without treatment with pac, chx,
or aniso. Again treatment with
actD was used to determine the value of
background incorporation
of [
3H]uridine into virion
preparations. [
3H]uridine incorporation into cellular RNA
of the actD-treated
cells was reduced to 15% of the level in
mock-treated cells. As
expected, background [
3H]uridine
incorporation into virions was minimal, 8% or less at
each time point
(Fig.
6). [
3H]uridine
incorporation into cytoplasmic RNA of pac-, chx-, or
aniso-treated cells was 53% ± 8%, 70% ± 21%, or 52% ± 21%, respectively,
of the level in mock-treated cells. These
reductions in [
3H]uridine incorporation are in part
attributable to turnover of
short-lived proteins that facilitate the
stability of steady-state
cellular RNA. [
3H]uridine
incorporation into virion RNA displayed coincident reduction
to 48% ± 8%, 30% ± 5%, or 31% ± 9%, respectively, of the mock-treated
control level. These results indicate that inhibition of de novo
translation decreases but does not abrogate the supply of vpRNA.
View larger version (26K):
[in this window]
[in a new window]
|
FIG. 6.
HIV-1 vpRNA remains available for encapsidation
during translation inhibition. CEM(A)/HIV-1 cells were incubated
for 4 h in medium containing 30 µCi of [3H]uridine
per ml with or without pac, chx, or aniso. [3H]uridine
levels in cytoplasmic RNA and in virion RNA were quantified by TCA
precipitation analysis. Average results of three experiments are shown;
error bars indicate standard deviations.
|
|
Encapsidation efficiency is sustained upon translation
inhibition.
To evaluate the effect of translation inhibition on
encapsidation efficiency of HIV-1 vpRNA, RPAs were
performed with an RNA probe complementary to the HIV-1 5'
untranslated region. To control for variation in cytoplasmic RNA
loading, cytoplasmic RNA samples were also hybridized to a probe
complementary to cellular gapdh RNA. To monitor for possible
variation in virion RNA processing, the virion samples were
supplemented with a 100-bp 32P-labeled DNA following probe
hybridization and digestion and before phenol extraction and ethanol
precipitation. Four independent RPAs were performed using 10 µg of
cytoplasmic RNA and virion RNA equivalent to 250 ng of Gag p24. A
representative RPA is shown in Fig. 7A,
and the data from the four RPAs are summarized in Fig. 7B.
Encapsidation efficiency was calculated as the level of HIV-1
virion RNA relative to the level of cytoplasmic HIV-1 unspliced
RNA.
View larger version (44K):
[in this window]
[in a new window]
|
FIG. 7.
Encapsidation efficiency is not reduced upon inhibition
of de novo translation. (A) Representative RPA of cytoplasmic and
virion RNA that was harvested after 4 h of incubation with or
without pac, chx, or aniso. Labels indicate the sizes of the protected
RNAs and control 100-bp DNA fragment used to control for viral RNA
sample processing (virus control), cell treatment, and undigested
probes. (B) Summary of four RPAs. Average results are shown; error bars
indicate standard deviations. Encapsidation efficiency was determined
by dividing virion RNA level by the corresponding cytoplasmic RNA
level.
|
|
Consistent with the [
3H]uridine results, the absolute
abundance of cytoplasmic HIV-1 unspliced RNA was
reduced upon treatment
with pac, chx, or aniso. The absolute
amount of HIV-1 RNA in virions
was also reduced. Compared to the
mock-treated control, the levels
of vpRNA encapsidation efficiency
were 165% ± 22%, 147% ± 52%,
and 97% ± 21% in response to pac,
chx, and aniso, respectively
(Fig.
7B). Encapsidation efficiency was
also increased by treatment
with actD (200% [Fig.
7B]). These data
indicate that vpRNA remains
available for encapsidation during
inhibition of de novo translation
and that prior translation of the
HIV-1 unspliced transcripts
is not necessary for generation of
vpRNA. The actD data indicate
that de novo RNA synthesis and Rev
shuttling are not necessary
for encapsidation of
vpRNA.
| |
DISCUSSION |
We developed a biochemical assay to examine the relationship
between HIV-1 unspliced mRNA and vpRNA. The assay uses
three mechanistically distinct translation antagonists to inhibit
protein synthesis in HIV-1-infected T cells under conditions that
maintain virion production. Our ribosomal profile analysis comparing
HIV-1-infected and mock-infected T cells agrees with the ribosomal
profiles of Agy et al. (1) with the exception that we do not
observe a significant reduction in the overall abundance of rRNA in
response to HIV-1 infection. Our experiments show that uninfected
and HIV-1-infected cells exhibit the expected distinct profiles in
response to pac, chx, or aniso, which selectively inhibit either the
initiation or elongation step of translation. These data confirm that
HIV-1 infection does not disrupt the translation machinery.
Pulse-chase experiments and immunoprecipitation assays established that
de novo translation is not necessary for HIV-1 particle assembly
and release, and that a concentration-dependent relationship exists
between cell-associated Gag and virion-associated Gag. The newly
synthesized Gag can be readily assembled into virions, but
steady-state Gag is also sufficient to produce virions.
Immunoprecipitation results also indicate that inhibition of protein
synthesis does not interfere with processing of Gag precursor protein.
Examination of cytoplasmic and virion RNA by RPA and
[3H]uridine labeling demonstrated that de novo
translation is not required for encapsidation of vpRNA. The
absolute level of virion RNA is reduced upon translation inhibition.
The magnitude of this reduction was greater when measured by the
[3H]uridine labeling approach, which detects both host
and viral transcripts, than by the HIV-1-specific RPA. One possible
explanation for this difference is that the
3H-labeling technique detects changes in encapsidation of
host RNAs.
Treatment with the inhibitors increased the cytoplasmic availability of
vpRNA and yielded increased encapsidation efficiencies, indicating
that HIV-1 mRNA can also be utilized as vpRNA. This ability
to increase vpRNA availability indicates that generation of
vpRNA does not require prior utilization of the HIV-1 unspliced RNA as the mRNA template for protein synthesis. We speculate that disruption of the protein synthesis machinery reduces competition by
ribosomes, and the HIV-1 mRNA is rerouted to function as
vpRNA. Our data imply that HIV-1 vpRNA and mRNA do not
follow a separate intracellular RNA pathway. Instead HIV-1
unspliced RNA constitutes a single functional pool that can function
interchangeably as mRNA and as vpRNA. Our results are similar
to results with ASV and RSV in which Stoltzfus et al. (23)
and Sonstegard and Hackett (22) concluded that a single
metabolic pool of viral RNA exists that functions as both mRNA and
vpRNA. Contrasting results in the MLV system suggested that there
are two nonequilibrating pools of MLV RNA, each functioning as either
mRNA or vpRNA (11). In the MLV system, actD-treated
cells produced virions without genomic RNA. In our HIV-1 system,
actD-treated cells sustain production of virions with genomic RNA and
exhibit increased encapsidation efficiency.
Our biochemical results from HIV-1-infected human T cells are in
agreement with genetic analysis of HIV-1-based vectors
(13) and indicate that translation of HIV-1 vector
mRNA is not a rate-limiting step in production of vector virus. Our
data contrast with HIV-2 experiments in which continued protein
synthesis was required for encapsidation of vpRNA (8).
This feature of the HIV-2 system is presumed to be necessary for
sorting HIV-2 genomic RNA because the RNA encapsidation signal is
present on both the HIV-2 unspliced vpRNA and spliced mRNA
(8, 14). We speculate that for HIV-1, interaction of
HIV-1 Gag protein with the RNA encapsidation signal modulates the
competition between host translational machinery and virus assembly
complexes, similar to the mechanism originally proposed from study of
RSV (22).
| |
ACKNOWLEDGMENTS |
CEM(A), from Mark Wainberg and James McMahon, was obtained
through the AIDS Research and Reference Reagent Program, Division of
AIDS, NIAID, NIH.
This work was supported by grants from the National Institute Allergy
and Infectious Diseases (R29AI40851) and the National Cancer Institute
(P30CA16058), Bethesda, Md.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Center for
Retrovirus Research, Department of Veterinary Biosciences, The Ohio
State University, Columbus, OH 43210-1093. Phone: (614) 292-1392. Fax: (614) 292-6473. E-mail: boris-lawrie.1{at}osu.edu.
| |
REFERENCES |
| 1.
|
Agy, M. B.,
M. Wambach,
K. Foy, and M. G. Katze.
1990.
Expression of cellular genes in CD4 positive lymphoid cells infected by the human immunodeficiency virus, HIV-1: evidence for a host protein synthesis shut-off induced by cellular mRNA degradation.
Virology
177:251-258[CrossRef][Medline].
|
| 2.
|
Butsch, M.,
S. Hull,
Y. Wang,
T. M. Roberts, and K. Boris-Lawrie.
1999.
The 5' RNA terminus of spleen necrosis virus contains a novel posttranscriptional control element that facilitates human immunodeficiency virus Rev/RRE-independent Gag production.
J. Virol.
73:4847-4855[Abstract/Free Full Text].
|
| 3.
|
Coder, D. M.
1997.
Assessment of cell viability, p. 9.2.1-9.2.2.
In
J. P. Robinson, et al. (ed.), Current protocols in cytometry, vol. 1. John Wiley & Sons, Inc., New York, N.Y.
|
| 4.
|
Felicetti, L.,
B. Colombo, and C. Baglioni.
1966.
Inhibition of protein synthesis in reticulocytes by antibiotics. II. The site of action of cycloheximide, streptovitacin A and pactamycin.
Biochim. Biophys. Acta
119:120-129[Medline].
|
| 5.
|
Fresno, M.,
L. Carrasco, and D. Vazquez.
1976.
Initiation of the polypeptide chain by reticulocyte cell-free systems. Survey of different inhibitors of translation.
Eur. J. Biochem.
68:355-364[Medline].
|
| 6.
|
Geballe, A. P., and M. K. Gray.
1992.
Variable inhibition of cell-free translation by HIV-1 transcript leader sequences.
Nucleic Acids Res.
20:4291-4297[Abstract/Free Full Text].
|
| 7.
|
Goldberg, I. H.
1974.
Mechanisms of action of antitumor antibiotic inhibitors of protein synthesis.
Cancer Chemother. Rep.
58:479-489[Medline].
|
| 8.
|
Kaye, J. F., and A. M. Lever.
1999.
Human immunodeficiency virus types 1 and 2 differ in the predominant mechanism used for selection of genomic RNA for encapsidation.
J. Virol.
73:3023-3031[Abstract/Free Full Text].
|
| 9.
|
Kim, V. N.,
K. Mitrophanous,
S. M. Kingsman, and A. J. Kingsman.
1998.
Minimal requirement for a lentivirus vector based on human immunodeficiency virus type 1.
J. Virol.
72:811-816[Abstract/Free Full Text].
|
| 10.
|
Levin, J. G.,
P. M. Grimley,
J. M. Ramseur, and I. K. Berezesky.
1974.
Deficiency of 60 to 70S RNA in murine leukemia virus particles assembled in cells treated with actinomycin D.
J. Virol.
14:152-161[Abstract/Free Full Text].
|
| 11.
|
Levin, J. G., and M. J. Rosenak.
1976.
Synthesis of murine leukemia virus proteins associated with virions assembled in actinomycin D-treated cells: evidence for persistence of viral messenger RNA.
Proc. Natl. Acad. Sci. USA
73:1154-1158[Abstract/Free Full Text].
|
| 12.
|
McBride, M. S., and A. T. Panganiban.
1996.
The human immunodeficiency virus type 1 encapsidation site is a multipartite RNA element composed of functional hairpin structures.
J. Virol.
70:2963-2973[Abstract]. (Erratum, 71:858, 1997.)
|
| 13.
|
McBride, M. S.,
M. D. Schwartz, and A. T. Panganiban.
1997.
Efficient encapsidation of human immunodeficiency virus type 1 vectors and further characterization of cis elements required for encapsidation.
J. Virol.
71:4544-4554[Abstract].
|
| 14.
|
McCann, E. M., and A. M. Lever.
1997.
Location of cis-acting signals important for RNA encapsidation in the leader sequence of human immunodeficiency virus type 2.
J. Virol.
71:4133-4137[Abstract].
|
| 15.
|
Messer, L. I.,
J. G. Levin, and S. K. Chattopadhyay.
1981.
Metabolism of viral RNA in murine leukemia virus-infected cells; evidence for differential stability of viral message and virion precursor RNA.
J. Virol.
40:683-690[Abstract/Free Full Text].
|
| 16.
|
Meyer, B. E., and M. H. Malim.
1994.
The HIV-1 Rev trans-activator shuttles between the nucleus and the cytoplasm.
Genes Dev.
8:1538-1547[Abstract/Free Full Text].
|
| 17.
|
Miele, G.,
A. Mouland,
G. P. Harrison,
E. Cohen, and A. M. Lever.
1996.
The human immunodeficiency virus type 1 5' packaging signal structure affects translation but does not function as an internal ribosome entry site structure.
J. Virol.
70:944-951[Abstract].
|
| 18.
|
Naldini, L.,
U. Blomer,
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].
|
| 19.
|
Obrig, T. G.,
W. J. Culp,
W. L. McKeehan, and B. Hardesty.
1971.
The mechanism by which cycloheximide and related glutarimide antibiotics inhibit peptide synthesis on reticulocyte ribosomes.
J. Biol. Chem.
246:174-181[Abstract/Free Full Text].
|
| 20.
|
Paskind, M. P.,
R. A. Weinberg, and D. Baltimore.
1975.
Dependence of Moloney murine leukemia virus production on cell growth.
Virology
67:242-248[CrossRef][Medline].
|
| 21.
|
Roberts, T. M., and K. Boris-Lawrie.
2000.
The 5' RNA terminus of spleen necrosis virus stimulates translation of nonviral mRNA.
J. Virol.
74:8111-8118[Abstract/Free Full Text].
|
| 22.
|
Sonstegard, T. S., and P. B. Hackett.
1996.
Autogenous regulation of RNA translation and packaging by Rous sarcoma virus Pr76gag.
J. Virol.
70:6642-6652[Abstract/Free Full Text].
|
| 23.
|
Stoltzfus, C. M.,
K. Dimock,
S. Horikami, and T. A. Ficht.
1983.
Stabilities of avian sarcoma virus RNAs: comparison of subgenomic and genomic species with cellular mRNAs.
J. Gen. Virol.
64:2191-2202[Abstract/Free Full Text].
|
| 24.
|
Vazquez, D.
1979.
Inhibitors of protein biosynthesis.
Mol. Biol. Biochem. Biophys.
30:1-14, 36-38, 136-138, 155-159.
|
Journal of Virology, December 2000, p. 11531-11537, Vol. 74, No. 24
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Aguiar, R. S., Pereira, H. S., Costa, L. J., Brindeiro, R. M., Tanuri, A.
(2006). Gag-Pol bearing a reverse transcriptase drug-resistant mutation influences viral genomic RNA incorporation into human immunodeficiency virus type 1 particles. J. Gen. Virol.
87: 2669-2677
[Abstract]
[Full Text]
-
Nikolaitchik, O., Rhodes, T. D., Ott, D., Hu, W.-S.
(2006). Effects of Mutations in the Human Immunodeficiency Virus Type 1 gag Gene on RNA Packaging and Recombination.. J. Virol.
80: 4691-4697
[Abstract]
[Full Text]
-
Flynn, J. A., An, W., King, S. R., Telesnitsky, A.
(2004). Nonrandom Dimerization of Murine Leukemia Virus Genomic RNAs. J. Virol.
78: 12129-12139
[Abstract]
[Full Text]
-
LeBlanc, J. J., Beemon, K. L.
(2004). Unspliced Rous Sarcoma Virus Genomic RNAs Are Translated and Subjected to Nonsense-Mediated mRNA Decay before Packaging. J. Virol.
78: 5139-5146
[Abstract]
[Full Text]
-
Domsic, J. K., Wang, Y., Mayeda, A., Krainer, A. R., Stoltzfus, C. M.
(2003). Human Immunodeficiency Virus Type 1 hnRNP A/B-Dependent Exonic Splicing Silencer ESSV Antagonizes Binding of U2AF65 to Viral Polypyrimidine Tracts. Mol. Cell. Biol.
23: 8762-8772
[Abstract]
[Full Text]
-
Her, S., Claycomb, R., Tai, T. C., Wong, D. L.
(2003). Regulation of the Rat Phenylethanolamine N-Methyltransferase Gene by Transcription Factors Sp1 and MAZ. Mol. Pharmacol.
64: 1180-1188
[Abstract]
[Full Text]
-
Brasey, A., Lopez-Lastra, M., Ohlmann, T., Beerens, N., Berkhout, B., Darlix, J.-L., Sonenberg, N.
(2003). The Leader of Human Immunodeficiency Virus Type 1 Genomic RNA Harbors an Internal Ribosome Entry Segment That Is Active during the G2/M Phase of the Cell Cycle. J. Virol.
77: 3939-3949
[Abstract]
[Full Text]
-
Butsch, M., Boris-Lawrie, K.
(2002). Destiny of Unspliced Retroviral RNA: Ribosome and/or Virion?. J. Virol.
76: 3089-3094
[Full Text]
-
Paillart, J.-C., Skripkin, E., Ehresmann, B., Ehresmann, C., Marquet, R.
(2002). In Vitro Evidence for a Long Range Pseudoknot in the 5'-Untranslated and Matrix Coding Regions of HIV-1 Genomic RNA. J. Biol. Chem.
277: 5995-6004
[Abstract]
[Full Text]
Top
Abstract
Introduction
