Departments of Medicine, Pathology, and
Molecular Microbiology, Washington University School of Medicine,
St. Louis, Missouri 63110
Viral protein U (Vpu) is a 17-kDa phosphoprotein that enhances the
release of viral particles from human immunodeficiency virus type
1-infected cells. This study shows that the effect of Vpu on efficient
particle release depends on the rate of cell proliferation. Cells
arrested by contact inhibition, chemical arresting agents, or terminal
differentiation (i.e., macrophages) all exhibited a striking
dependence on Vpu for efficient particle release, as shown by
examination of particle production from transfections with full-length
clones, infections, and the vaccinia virus expression system. In
contrast, actively proliferating cells did not exhibit enhanced
particle release with Vpu expression. This study demonstrates the
necessity of Vpu for efficient viral particle release from quiescent cells.
 |
TEXT |
Viral protein U (Vpu) is a
unique gene product of human immunodeficiency virus (HIV) type 1 (HIV-1) with two well-described functions: CD4 degradation and
enhancement of viral particle release (32, 35). Several
studies have shown that Vpu is dispensable for HIV-1 infection in vitro
(30). However, in vivo studies have suggested that Vpu may
be a critical factor in viral pathogenesis. Although HIV strains
replicating in vitro often exhibit mutations that abrogate Vpu
expression, in vivo the Vpu open reading frame remains intact
(12). In addition, macaque models of simian-human immunodeficiency virus infections have shown that during the
nonpathogenic stages of disease, the Vpu reading frame is often
disrupted, but as the virus evolves and becomes more pathogenic, the
Vpu reading frame is often found to be intact (24-26).
Finally, HIV-1 infection of certain primary cells, such as terminally
differentiated, nondividing macrophages, generally requires Vpu
(2, 33).
Unlike many other retroviruses, HIV-1 is capable of infecting
nondividing cells. Infection of macrophages represents a
crucial step in HIV-1 pathogenesis (5, 20). For instance,
initial infection with HIV-1 generally occurs via the
macrophage-tropic R5 viruses (18, 29, 37).
Macrophages represent a longer-lived population of cells that may be a
site of viral latency, posing problems for viral eradication with
current antiretroviral therapy (19). Recently,
resting CD4 T cells have been identified as an additional site of viral
latency (40). While infection of resting T cells is
very inefficient, these cells represent the majority of cells
encountered by newly transmitted virus (40). Moreover,
HIV-1 has also been shown to induce G2 growth
arrest of infected cells via Vpr (7), leading to enhanced
viral transcription (17). Thus, infection of quiescent
cells is a major determinant of HIV-1 pathogenicity in vivo.
The role of cell growth and replication has been studied in relation to
viral entry and transport of DNA into the nuclei of nondividing cells
(9, 17, 23). This study examines viral assembly and
release in proliferating and nonproliferating cells. It shows that
efficient viral release in growth-arrested cells requires the presence
of Vpu.
Efficient viral release is dependent on Vpu in cells that are
arrested by contact inhibition.
We initially observed that viral
assembly and Vpu responses were dependent on cell growth conditions,
e.g., the CO2 concentration of the tissue culture
incubator (data not shown). This effect was not due to alterations of
the pH of the cultures but appeared to correlate with the rate of cell
proliferation (data not shown). To examine the effects of growth
arrest, we initially used contact inhibition through cell confluence as
a means of halting cells from cycling. 293T cells were grown at 10, 25, 50, 75, and 100% cell confluencies. Figure
1 shows that cells were arrested at high
confluencies, as measured by [3H]thymidine
(ICN) incorporation into DNA. Levels of incorporation at 100%
confluence were similar to those in cells arrested with nocodazole
(data not shown). At lower confluencies, these cells were actively
dividing.
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FIG. 1.
Relationship of cell confluence to the effect of Vpu on
particle release. 293T cells were plated in 35-mm wells at 100, 75, 50, 25, and 10% confluencies. Each well was labeled with 50 µCi of
[3H]thymidine/ml overnight. The next day, equal numbers
of cells were lysed on glass filters (Fisher) and washed three times
with radioimmunoprecipitation buffer, and
[3H]thymidine incorporation was determined. The data at
the bottom show levels of 3H disintegrations per minute at
each confluence, as well as the fold above 100% confluence. A
duplicate set of cells grown at the same confluencies were used to
assess Gag protein synthesis and viral particle release as described
previously (3). For infection-transfection, cells were
first infected with a recombinant vaccinia virus expressing T7
polymerase, vTF7-3, at a multiplicity of infection of 10. At 30 min postinfection, cells were transfected with 10 µg of total
DNA by use of Lipofectamine. At 5 h posttransfection, the medium
was removed and 50 µCi of Tran35S label/ml was added.
Cell lysates and supernatants were harvested and prepared for
immunoprecipitation analysis with anti-p24 (CA) antisera. The
proteins were examined by SDS-12.5% PAGE. pTM3 is a plasmid
lacking gag and pol sequences. The top panel
shows levels in cell lysates. The bottom panel shows the amount of Gag
released into supernatants at each confluence.
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As previously described, the vaccinia virus expression system was then
used to express Gag-Pol (GPG1) alone or in the presence of Vpu
(pTM-Vpu) (11). Briefly, 293T cells were infected with a
recombinant vaccinia virus, vTF7-3, expressing the T7 polymerase. After
0.5 h, the cells were transfected with 4 µg of total DNA by use
of Lipofectamine (GIBCO-BRL). GPG1 is sufficient for virus-like particle production from a variety of cells (36). However,
Vpu is necessary for the efficient release of particles from certain cell types, such as HeLa or 293T cells (14, 33). Figure 1 demonstrates the effect of Vpu in cells infected-transfected at various
cell confluencies. The top panel of Fig. 1 shows levels of protein
expression in cell lysates. Although different levels were observed at
the various confluencies, essentially equivalent levels were seen in
cells expressing GPG1 alone or GPG1 plus Vpu at each confluence
examined. The amount of Vpu protein increased proportionally with that
of the Gag protein (data not shown). The bottom panel of Fig. 1 shows
levels of Gag protein released into supernatants as virus-like
particles (11). Densitometric analysis showed that
in cultures at 100, 75, and 50% confluencies, Vpu enhanced particle
release 5.6-, 2.1-, and 2.4-fold, respectively. In contrast, at 25 and
10% confluencies, Vpu enhanced particle release only 1.4- and
0.8-fold, respectively.
Vpu is necessary for efficient particle release from cells arrested
by cell cycle-arresting agents such as nocodazole and mimosine.
To
determine if contact inhibition enhanced the effect of Vpu by arresting
cell growth, we used the cell cycle inhibitor nocodazole to block cells
from exiting the G2 phase of the cell cycle.
Cells were grown to 25% confluence, at which Vpu does not efficiently promote particle release. At lower levels of confluence, cells exhibited toxicity in the presence of nocodazole and could not be used
in these studies (data not shown). The cells grown to 25%
confluence were left untreated or were treated with nocodazole at a concentration at which cell proliferation was halted, as determined by [3H]thymidine incorporation (data
not shown). The cells were then transfected with GPG1 or GPG1 plus Vpu
as described above. The proportion of Gag released was calculated from
densitometric measurements of the p24, p41, and p55 bands on film.
Figure 2 shows the results of this
experiment. When Vpu was present, there was some enhancement of
particle production. However, the enhancement was much more striking
when nocodazole was also present.
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FIG. 2.
Effects of nocodazole and mimosine on Vpu
activity. (A) Cells were grown to 25% confluence in the
presence or absence of the cell cycle inhibitor nocodazole (NOC) (10 µg/ml). Cells were infected, transfected, and metabolically labeled
as described in the text. Anti-p24 (CA) antiserum
immunoprecipitates were analyzed by SDS-PAGE and densitometry, and the
proportion of total Gag proteins found in the supernatants (Sup.) was
determined. Error bars represent standard deviations for the experiment
performed in triplicate. (B) Cells were grown to 25% confluence in
isoleucine-deficient medium for 48 h. This medium was then
replaced with complete medium containing 400 µM mimosine.
After 48 h, treated cells and untreated cells, also plated
at 25% confluence, were transfected, labeled, and analyzed by
immunoprecipitation and SDS-PAGE as described in the text.
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The Vpu effect on release was also analyzed using the
G1-arresting agent mimosine. Isogenic proviral
clones that were Vpu+ and
Vpu
were transfected into arrested or untreated
cells. Transfection of proviral clones allowed for additional analysis
of viral assembly without vaccinia virus infection. The parental clone,
p125, has been previously described (20, 39). The
Vpu virus contains a T
C mutation in the
initiator codon, introduced by site-directed mutagenesis, to close the
Vpu open reading frame. 293T cells were seeded at 25% confluence in
isoleucine-deficient medium in six-well plates. At 48 h after
starvation, the medium was replaced with 400 µM mimosine-containing
complete medium. At 48 h after addition of the arresting agent,
these cells and untreated cells, also plated at 25% confluence, were
transfected with 4 µg of proviral DNA by use of Lipofectamine. At
24 h posttransfection, the cells were labeled with 50 µCi of
Tran35S label (ICN)/ml overnight. The next day,
cell lysates and supernatants were analyzed as described above by
radioimmunoprecipitation, sodium dodecyl sulfate (SDS)-polyacrylamide
gel electrophoresis (PAGE), and autoradiography. Figure 2B shows that
at a low confluence, Vpu had no effect on viral release in untreated
cells. However, in cells arrested with mimosine,
Vpu+ virus was more efficiently released than
Vpu virus (3.2-fold).
Thus, in situations where Vpu has no or little effect, i.e., low cell
confluence, the addition of a cell cycle inhibitor such as nocodazole
or mimosine can mimic the effect of cell arrest induced by contact
inhibition. In addition, blocking cells in G1
with mimosine or G2 with nocodazole
facilitates the effect of Vpu on particle assembly.
Vpu enhances particle release in differentiated macrophages
in a dose-dependent manner.
We next examined the effect of cell
proliferation on viral release in a more physiologically relevant
setting. Terminally differentiated, noncycling macrophages were
infected with equal amounts of p24 from
Vpu and Vpu+ viruses
generated in 293T cells transfected with molecular proviral clones.
Infections were initiated with 84 ng of p24 or with fivefold dilutions down to 0.7 ng of p24. At high viral inocula,
Vpu+ virus was released two- to threefold more
efficiently than Vpu virus (Fig.
3A). However, at lower viral
inocula, the Vpu effect was far more significant, with over a
1,000-fold increase in viral release from cells infected with 670 pg of
Vpu+ virus compared to cells infected with 670 pg
of Vpu virus (Fig. 3B). These results may
explain previous controversies. Some groups have reported a three- to
fourfold increase in viral release from macrophages in the
presence versus the absence of Vpu, while other investigators have
reported a more dramatic effect similar to that seen with the lower
viral inocula used above (2, 33, 39). Thus, the
differences reported earlier may reflect experimental variations in
cell proliferation and multiplicity of infection. Moreover, these
findings emphasize the important role of Vpu in quiescent cells.
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FIG. 3.
Effects on macrophages. Macrophages were
isolated by adherence from fresh peripheral blood mononuclear cells.
The macrophages were infected with equal amounts of p24 from
viruses obtained from transfection of 293T cells with isogenic proviral
clones that were Vpu+ and Vpu (ATGACG) in
the context of an HIV strain 125 backbone, which is a chimera of
the NL4-3 HXB-2 and ADA strains of HIV, as described previously
(39). Supernatants were harvested on the indicated days
and analyzed for reverse transcriptase (RT) activity as
described previously (39). Error bars show standard
deviations.
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The work presented in this study demonstrates that HIV-1 assembly is
inefficient in growth-arrested cells. The lack of efficient assembly
was seen when cells were grown at low CO2
concentrations, at high confluencies, in the presence of cell cycle
inhibitors such as nocodazole or mimosine, or in terminally
differentiated, nondividing cells such as macrophages. However,
coexpression of the Vpu protein restored efficient particle assembly
under each of these conditions.
Vpu functions described in the literature include both degradation of
newly synthesized CD4 and enhancement of the release of viral particles
from the surface of infected cells (32). While the
significance and mechanism of CD4 degradation have largely been
delineated, the significance and mechanism of enhanced particle assembly remain unclear. Several interesting observations concerning this activity have been reported in the literature: the effect of Vpu
is dependent on cell type (2, 33); variations in the effect have been reported, with some investigators showing it to be
fairly moderate (2- to 3-fold) while others have reported a more
dramatic effect (>1,000-fold) (2, 33); Vpu can enhance the release of divergent retroviral Gag proteins (16); and
HIV-2 Env may have an effect on viral assembly similar to that of HIV-1 Vpu (4).
While intriguing, these observations indicate a broad effect of Vpu on
the assembly process. Vpu enhances the release of divergent Gag
proteins from viruses that do not contain a Vpu-like protein, making a
direct interaction of HIV-1 Gag and Vpu unlikely. The finding that
another protein (HIV-2 Env) has activity similar to that of Vpu is also
indicative of a nonspecific mechanism of action. Moreover, the
variability of Vpu action in different cell types and in the magnitude
of the effect on viral release implicates factors in the cell or
cellular environment which may modulate the assembly process and the
effect of Vpu on viral assembly. The work presented here furthers the
idea of a more generalized mechanism of action of Vpu on the assembly
process, one that depends on the rate of growth of the cells.
The role of Vpu in modulating the cellular environment is intriguing.
Recently, a group of small, hydrophobic viral proteins have been
grouped together and termed viroporins. They include poliovirus 2BC and
3AB, influenza virus M2, togavirus 6K, rotavirus NSP4, hepatitis A
virus 3A, 2B, and 2BC, hepatitis C virus E1, and coxsackievirus 2B
proteins (3, 6, 22). These proteins regulate various
points of the viral life cycle by altering membrane permeability. In
particular, the coxsackievirus 2B, hepatitis virus 2BC, and influenza
virus M2 proteins alter ion and membrane permeabilities and enhance
virion assembly and the release of each of these viruses (22, 31,
38). Vpu, which has been shown to form channels and alter
permeability in bacteria, oocytes, and human cells, may act in a manner
similar to that of these other small, hydrophobic proteins (8,
13, 15, 34). It may function by altering the membrane
permeability or environment in cells that are less permissive for HIV-1 assembly.
Intriguingly, a recent report demonstrated that HIV, like several other
viruses, buds selectively through lipid raft structures at the plasma
membrane (28). Additionally, raft formation may be related
to cell activation, with dramatic reorganization of membrane raft
structures occurring during cell activation (10, 21, 27).
Moreover, raft structures may also be regulated by ionic changes
occurring in cells (1). Thus, it will be interesting to
study the effect of Vpu on raft formation. Studying the effect of Vpu
on assembly may provide greater insight into the assembly process. It
may also provide more information on membranes, their structure, and
how viruses can alter membranes to their replicative advantage.
We thank Nancy Vander Heyden for work on macrophage
infections and reverse transcriptase assays and Charlie Rice for
helpful discussions.
This work was supported by PHS grants AI34736, CA83659, and AI24745 and
training grant 5 T32 HL 07088-23 (to A.D.).
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W. Cavert,
C. J. Miller,
R. S. Veazey,
D. Notermans,
S. Little,
S. A. Danner,
D. D. Richman,
D. Havlir,
J. Wong,
H. L. Jordan,
T. W. Schacker,
P. Racz,
K. Tenner-Racz,
N. L. Letvin,
S. Wolinsky, and A. T. Haase.
1999.
Sexual transmission and propagation of SIV and HIV in resting and activated CD4(+) T cells.
Science
286:1353-1357[Abstract/Free Full Text].
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Abstract
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