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Journal of Virology, January 1999, p. 290-296, Vol. 73, No. 1
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
RNase L Inhibitor Is Induced during Human Immunodeficiency
Virus Type 1 Infection and Down Regulates the 2-5A/RNase L Pathway
in Human T Cells
Camille
Martinand,
Céline
Montavon,
Tamim
Salehzada,
Michelle
Silhol,
Bernard
Lebleu, and
Catherine
Bisbal*
Institut de Génétique
Moléculaire de Montpellier (UMR 5535, CNRS-Université
de Montpellier II), 34293 Montpellier Cedex 5, France
Received 26 June 1998/Accepted 22 September 1998
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ABSTRACT |
The interferon-regulated 2-5A/RNase L pathway plays a major role in
the antiviral and antiproliferative activities of these cytokines.
Several viruses, however, have evolved strategies to escape the
antiviral activity of the 2-5A/RNase L pathway. In this context, we
have cloned a cDNA coding for the RNase L inhibitor (RLI), a protein
that specifically inhibits RNase L and whose regulated expression in
picornavirus-infected cells down regulates the activity of the
2-5A/RNase L pathway. We show here that RLI increases during the course
of human immunodeficiency virus type 1 (HIV-1) infection, which may be
related to the downregulation of RNase L activity that has been
described to occur in HIV-infected cells. In order to establish a
possible causal relationship between these observations, we have stably
transfected H9 cells with RLI sense or antisense cDNA-expressing
vectors. The overexpression of RLI causes a decrease in RNase L
activity and a twofold enhancement of HIV production. This increase in
HIV replication correlates with an increase in HIV RNA and proteins. In
contrast, reduction of RLI levels in RLI antisense cDNA-expressing
clones reverses the inhibition of RNase L activity associated with HIV
multiplication and leads to a threefold decrease in the viral load.
This anti-HIV activity correlated with a decrease in HIV RNA and
proteins. These findings demonstrate that the level of RLI, via its
modulation of RNase L activity, can severely impair HIV replication and
suggest the involvement of RLI in the inhibition of the 2-5A/RNase L
system observed during HIV infection.
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INTRODUCTION |
Interferons (IFN) control various
cellular functions and participate in host defense against viral and
microbial agents through multiple induced pathways (1). The
2-5A/RNase L pathway is one of the major pathways induced by IFN. It is
implicated in some of the antiviral mechanisms of IFN and might play a
role in the regulation of RNA turnover and stability (12).
IFN induces four different forms of human 2-5A-synthetase which, upon
activation by double-stranded RNA (dsRNA), convert ATP into an unusual
series of oligomers known as 2-5A. 2-5A then activates RNase L, a
latent endoribonuclease, which inhibits protein synthesis by
cleavage of mRNA at the 3' side of UpNp sequences (11, 13,
40). During viral infection this antiviral pathway can be
activated, since several viruses produce dsRNA structures that can
activate 2-5A-synthetase. The presence of 2-5A has been demonstrated in
cells infected with encephalomyocarditis (EMC) virus (38),
vaccinia virus (22), or reovirus (19).
Although 2-5A has long been considered to be the unique regulator of
the 2-5A/RNase L pathway, we have cloned and characterized a
polypeptide inhibitor of the 2-5A pathway (referred to as RNase L
inhibitor [RLI]). RLI cDNA codes for a 68-kDa protein whose mRNA
is not regulated by IFN. When expressed in a reticulocyte lysate, RLI
induces neither 2-5A degradation nor irreversible modification of RNase
L (3); however, it antagonizes the binding of 2-5A by the
latter and thus its nuclease activity, since 2-5A binding is a
prerequisite to RNase L dimerization and activation (10,
31).
Despite the presence of double-stranded viral RNA structures capable of
activating the 2-5A/RNase L pathway and the presence of high
concentrations of 2-5A, in several cases no RNase L activity could be
detected. Several viruses appear to have developed strategies to
counteract the antiviral activity of the 2-5A/RNase L pathway. For
example, during herpes simplex virus type 1 and 2 (HSV-1 and HSV-2)
infection, 2-5A derivatives are synthesized that behave as 2-5A
antagonists (7). Similarly, infection by vaccinia virus leads to an inhibition of 2-5A-synthetase activity and to the degradation of 2-5A (20). Recently, Rivas et al. have shown that vaccinia virus E3L protein is an inhibitor of 2-5A-synthetase (23). Finally, EMC virus downregulates RNase L activity
through the increased expression of RLI (18).
Along the same lines, an inhibition of RNase L activity has been
observed during the course of human immunodeficiency virus (HIV)
infection. RNase L is inactive in peripheral blood mononuclear cell
extracts from AIDS patients, despite the presence of its 2-5A activator
(5). Likewise, the 2-5A binding activity of RNase L in
lymphocytes isolated from AIDS and pre-AIDS patients was approximately
65% lower than that found in controls (39). In experimental
infection of H9 cells with HIV type 1 (HIV-1), a strong enhancement of
2-5A-synthetase activity and a small increase of RNase L activity were
observed. Both enzymes reached maximal levels at day 3 after the onset
of HIV-1 infection and declined sharply thereafter. Interestingly,
RNase L can degrade HIV-1 transcripts during the early steps of
infection, and HIV-1 transcript accumulation coincides with the
decrease of RNase L activity (29, 35). These studies suggest
that there is an accumulation of an inhibitor of the 2-5A/RNase L
pathway that interferes with 2-5A binding through the formation of an
inhibitor-RNase L complex. On the other hand, different studies have
suggested that direct expression or activation of the 2-5A/RNase L
pathway enzymes can lead to suppression of HIV replication. For
example, the stable expression of transfected 2-5A-synthetase leads to
the suppression of HIV replication (27, 28). Direct
activation of RNase L with phosphorothioate-phosphodiester 2-5A
derivatives inhibits HIV-1 reverse transcriptase and HIV-induced syncytium formation (33). Recently, Maitra and Silverman
have shown that overexpression of RNase L can suppress HIV-1
replication (17). These results show that the
2-5A/ RNase L pathway is potentially able to regulate HIV
replication but is rapidly inhibited after the early stages of HIV infection.
RLI is a logical candidate to be the inhibitor induced by HIV
infection. We have therefore studied the modulation of RNase L and RLI
expression in H9 cells following HIV-1 infection. We have established
that if RNase L activity decreased during the course of HIV-1
infection, the RNase L protein decreases only transiently and the
amount of RLI protein increases. We have stably transfected H9
cells with RLI sense or antisense cDNA-expressing vectors and followed
the course of de novo HIV-1 infection in two independent RLI sense
clones and two independent RLI antisense clones. As expected, the
overexpression of RLI decreases RNase L activity and enhances HIV-1
production while, on the contrary, the reduction of the RLI level
correlates with an increase in RNase L activity and a decrease in virus
production. Altogether, these data suggest that RLI could be a cellular
inhibitor induced by HIV-1 to inhibit the 2-5A/RNase L pathway.
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MATERIALS AND METHODS |
Cells and virus.
H9 cells (human T lymphocyte cell line)
were grown in RPMI Glutamax medium (Gibco BRL) supplemented with 10%
(vol/vol) fetal calf serum. Strain IIIB of HIV-1 was grown in H9 cells
and titrated by a reverse transcriptase (RT) assay (21).
For HIV-1 infection, H9 cells (3 × 108) were
centrifuged and the cell pellet was resuspended in 150 ml with HIV-1
strain IIIB corresponding to 4.5 × 106 cpm of RT
activity (15, 16). After 1 h of incubation at 37°C, the cells were seeded at a final concentration of 2 × 105 cells per ml in RPMI Glutamax containing 10% (vol/vol)
fetal calf serum.
Expression vectors and transfections.
The human RLI cDNA
(3) was subcloned in the sense or antisense orientation in
pcDNA3 (Invitrogen) by standard procedures (26). Then,
5 × 105 H9 cells were transfected by electroporation
(250 mV) with 10 µg of plasmid DNA. The empty pcDNA3 vector was used
as a control. Stable transfectants were selected by culturing the cells
in the presence of 1 mg of G418 (Gibco BRL) per ml. Individual clones were isolated by limit dilution and analyzed for the expression of the
transfected cDNA. Clones expressing the transfected antisense cDNA were
named VAS1 and VAS2; clones expressing the transfected sense cDNA were
named VS1 and VS2, and the clone expressing the transfected empty
vector was named VV.
Cell extracts.
After infection, cells were collected at the
times indicated and were resuspended in 1 volume of hypotonic buffer
(0.5% [vol/vol] Nonidet P-40, 20 mM HEPES [pH 7.5], 10 mM
potassium acetate, 15 mM magnesium acetate, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 10 µg of aprotinin per ml, 150 µg of
leupeptin per ml), disrupted in a Dounce homogenizer, and centrifuged
at 10,000 × g as described previously (25).
The supernatant (S10) was pipetted off, and its protein
concentration was determined by spectrophotometry (37).
Western blot analysis.
Cell extracts were analyzed by
Western blot according to the method of Towbin et al. (34).
Proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred electrophoretically to a
nitrocellulose membrane. For antibody revelation, the nitrocellulose
membrane was soaked for 1 h in phosphate-buffered saline (PBS)
(140 mM NaCl-2 mM KCl-8 mM Na2HPO4-1.5 mM
KH2PO4 supplemented with 5% [wt/vol] skim
milk) and then held overnight at 4°C with the polyclonal antibodies against RNase L (1/1,000) or RLI (1/500) that we have previously described and characterized (18) or with anti-gp120 (1/100) (a generous gift of D. Misse, ORSTOM, Montpellier, France) or anti-GAPDH (1/3,000) (a generous gift of G. Cathala, IGM, Montpellier, France) in the same buffer. The filter was washed with PBS supplemented with 0.05% (vol/vol) Tween 20 and incubated for 1 h at room
temperature with donkey anti-rabbit immunoglobulin antibody conjugated
to horseradish peroxidase (1/2,000; Amersham) in PBS supplemented with
5% (wt/vol) skim milk. Chemiluminescence was generated by the NEN kit.
The gels were scanned, and the protein bands were quantified by
image analysis with the Intelligent Quantifier program (BioImage
Systems Corp.). Results are expressed as an average of three
independent experiments. Standard deviations are indicated by vertical
bars in the figures.
2-5A binding activity of RNase L during infection of cells with
HIV-1.
H9 cells, as well as the VV, VS, and VAS transfectants
(3 × 108 cells), were infected with HIV-1
(4.5 × 106 cpm of RT activity) (15, 16).
Cells were harvested at various times after the onset of
infection as indicated. Cell extracts (S10) were prepared as
described above. The radiobinding assay (14) was performed
with S10 cell extracts (600 µg of proteins) as a source of RNase
L and 2-5A4-3'-[32P]pCp (2-5ApCp)
(20,000 cpm, 3,000 Ci/mmol) as a probe. The radiobinding assay was
utilized with the modifications previously described (2, 4).
Results are expressed as an average of three independent experiments
for each cell clone (H9, VV, VS1, VS2, VAS1, and VAS2). Standard
deviations are indicated by vertical bars.
RNA analysis.
Total cellular RNA was prepared by using the
guanidine thiocyanate-lithium chloride procedure (6).
Northern blot hybridizations were performed according to standard
techniques (26). Probes (HIV long terminal repeat [LTR]
and GAPDH [glyceraldehyde-3-phosphate dehydrogenase]) were
synthesized by the multiprime procedure (random primer DNA labeling
system; Gibco-BRL). After autoradiography, mRNA was quantified by image
analysis with the Intelligent Quantifier program (BioImage Systems
Corp.). Each lane was normalized with the GAPDH probe.
RT assay.
At each indicated time after the onset of
infection, 50 µl of cell culture supernatant was added to 10 µl of
TNE buffer (20 mM Tris [pH 7.8], 1 mM EDTA, 100 mM NaCl, 0.1%
[vol/vol] Triton X-100) and 20 µl of reagent buffer (250 mM Tris
HCl [pH 7.8], 25 mM MgCl2, 500 mM KCl, 50 mM
dithiothreitol, 0.5 mM EGTA, poly(rA)-oligo(dT10) [optical density = 0.025], 2.5 µl of
[methyl-3H]dTTP [82 Ci/mmol, 1 mCi/ml]) and
then incubated for 2 h at 37°C. For each reaction 50 µl was
spotted onto a GF/C filter (Whatman) and incubated in 10% (vol/vol)
trichloroacetic acid (TCA)-12 mM pyrophosphate. The filters were then
washed three times in 5% (vol/vol) TCA-12 mM pyrophosphate,
rinsed with 100% ethanol, dried, and counted. The results are
expressed as an average of three independent experiments for each
cell clone (H9, VV, VS1, VS2, VAS1, and VAS2). The standard
deviations are indicated by vertical bars.
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RESULTS |
RNase L activity decreases in HIV-1-infected cells.
The
nuclease activity of RNase L is strictly dependent on its activation by
2-5A (31), so we measured the RNase L activity in
HIV-1-infected H9 cells with the 2-5A radiobinding assay
(14). This assay quantifies the binding of a radioactive
2-5ApCp probe to RNase L in unfractionated cell extracts. In keeping
with the data described in the introduction, we observed a decrease in the binding of 2-5ApCp by RNase L in extracts from HIV-1-infected H9
cells. 2-5A binding decreases by as early as 3 days after the onset of
HIV-1 infection and reaches a minimum 10 days after the onset of HIV-1
infection (Fig. 1A). The virus production
was monitored by Western blot analyses of the HIV-1 gp120 polypeptides
in cell extracts. The polyclonal antibody against gp120 we have used
also recognizes its gp160 precursor. gp120-gp160 polypeptides could be
detected at day 3 postinfection and reached maximal levels at days 7 and 8 (Fig. 1B).
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FIG. 1.
RNase L activity during HIV-1 infection. H9 cells were
infected with HIV-1 and harvested at the indicated times. (A) Cell
extracts (600 µg of proteins) were tested without further
fractionation in a 2-5A radiobinding assay. Results are expressed as
the percentage of 2-5ApCp bound. 100% is the binding level in
uninfected cells at time zero. (B) H9 cells were infected with HIV-1
and harvested at the indicated times. For each time point, 200 µg of
protein was analyzed by SDS-PAGE and Western blotting with
polyclonal antibody against gp160/gp120. An autoradiograph of the gel
is presented.
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The decrease of RNase L activity monitored by the 2-5A radiobinding
assay in unfractionated cell extracts could result from
a decrease in
RNase L itself or from the induction of an inhibitor.
Polyclonal
antibodies against RNase L and RLI have been used to
quantify RNase L
and RLI during the course of HIV-1
infection.
RLI increases during HIV-1 infection.
RNase L was
quantified by a Western blot assay with the RNase L-specific
polyclonal antibody described previously (18). RNase L was
detected at all time points, decreasing transiently (by
40%) between days 4 and 6 (Fig. 2)
and then increasing. The decrease of RNase L activity observed in cell
extracts during the time course of HIV-1 infection (Fig. 1) coincides
with the increased protein level, particularly between days 6 and 9, suggesting that there is the accumulation of an inhibitor. Since
RLI inhibits the binding of 2-5ApCp to RNase L (3), it is a
logical candidate for this role and, indeed, it increases during the
time course of HIV-1 infection (Fig. 2). Variations in RNase L and RLI
during HIV-1 infection are not linked to a nonspecific variation of
protein synthesis, as shown by the behavior of the GAPDH
protein used as a control (Fig. 2A).
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FIG. 2.
Analysis of RNase L and RLI proteins during HIV-1
infection. H9 cells were infected with HIV-1 and harvested at the
indicated times. (A) For each time point, 200 µg of proteins was
analyzed by SDS-PAGE and Western blotting with polyclonal antibodies
against RNase L, RLI, or GAPDH as indicated. (B) A densitometric
analysis of the gels is presented ( , RLI;  , RNase L). Vertical
bars represent the standard deviations obtained with three independent
experiments. 100% corresponds to the amount of RLI or RNase L
protein in untreated cells at time zero.
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The stable transfection of an RLI antisense or RLI sense
construction modifies RNase L activity during HIV-1
infection.
To determine the effects of regulating RLI levels
on HIV-1 infection, H9 cells were stably transfected with
RLI constructions in either the sense or the antisense
orientation. H9 cell transfected clones were characterized by a Western
blot assay with a polyclonal antibody against RLI protein (Fig.
3A) and with the 2-5ApCp binding activity
of RNase L (Fig. 3B). Clones expressing the RLI antisense construction, the RLI sense construction, and the empty vector have
been named VAS, VS, and VV, respectively. Two independent clones
expressing higher levels of RLI (VS1 and VS2) demonstrated lower 2-5A
binding activity by RNase L than cells transfected with the empty
vector. On the contrary, in two independent clones expressing lower
levels of RLI (VAS1 and VAS2), the 2-5A binding activity of RNase L is
increased (Fig. 3B).
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FIG. 3.
Expression of RLI and RNase L activity in H9 cells
stably transfected with antisense and sense RLI cDNA. (A) Extracts (200 µg of proteins) from H9 cells stably transfected with the VV
empty vector, with the sense RLI cDNA construction (VS1 and VS2), or
with the antisense RLI cDNA construction (VAS1 and VAS2) were analyzed
by SDS-PAGE and Western blotting with polyclonal antibody against RLI.
(B) Cell extracts (600 µg of protein) from the different
transfectants were tested without further fractionation in a 2-5A
radiobinding assay. Results are expressed as the percentage of 2-5ApCp
bound. 100% is the binding level in control VV cells.
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The 2-5A binding activity of RNase L during HIV-1 infection was
analyzed in these clones expressing different levels of RLI
(Fig.
4). HIV-1 infection of the VV clone gives
rise to a 68%
decrease in RNase L activity, a result in line with the
data presented
in Fig.
1 for the nontransfected H9 cells. A
significantly larger
decrease in RNase L activity is observed in the
clones expressing
the RLI sense construction: 90% for VS1 and 95% for
VS2 (Fig.
4A and B). On the other hand, the clones expressing the RLI
antisense
construction lead to a much lower inhibition of RNase L
activity:
29% for VAS1 and 22% for VAS2 (Fig.
4) upon HIV-1
infection. As
expected, therefore, overexpression of RLI increases the
HIV-1-associated
down regulation of RNase L activity, and lower
expression of RLI
antagonizes the HIV-1-associated down regulation of
RNase L activity.
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FIG. 4.
2-5A binding activity in cells stably transfected with
RLI sense and antisense constructions during HIV-1 infection. H9 cells
transfected with the empty vector ( , VV), with the antisense RLI
cDNA construction ( , VAS), or with the sense RLI cDNA construction
(, VS) were infected with HIV-1 and harvested at the indicated
times. Extracts (600 µg of protein) were tested without further
fractionation in a 2-5A radiobinding assay. Results are expressed as
the percentage of 2-5ApCp bound. 100% is the binding obtained at time
zero in uninfected cells. Vertical bars represent the standard
deviations obtained with three independent experiments. Clones VV,
VAS1, and VS1 are shown in panel A, and clones VV, VAS2, and VS2 are
shown in panel B.
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The production of HIV is proportional to the RLI level.
The
RLI protein level modulates RNase L activity. We therefore analyzed
whether the replication of HIV-1 is modified in the different
transfectants. Virus production was monitored by different techniques:
the measurement of RT activity in the cell culture supernatant,
Northern blot analysis of HIV mRNA transcripts, and Western blot
analysis of the HIV-1 gp160/gp120 polypeptides in cell extracts.
As clearly shown in Fig.
5, the
clones expressing the RLI sense constructions (VS1 and VS2) produced
significantly larger
amounts of virus than did the control cells,
as monitored by the
RT activity peak. On the contrary, the RT activity
was highly
repressed (by 75%) in the clones expressing the RLI
antisense
construction (VAS1 and VAS2) (Fig.
5).
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FIG. 5.
RT activity in clones expressing sense or antisense RLI
constructions. H9 cells transfected with the empty vector (, VV),
with the antisense RLI cDNA construction (, VAS), or with the sense
RLI cDNA construction (, VS) were infected with HIV-1. Virus
production was monitored by supernatant RT assay every day. 100% is
the RT activity observed in uninfected cells. Vertical bars refer to
the standard deviations obtained with three independent experiments.
Clones VV, VAS1, and VS1 are shown in panel A, and clones VV, VAS2, and
VS2 are shown in panel B.
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The mechanism of the pro-HIV effect of overexpressing RLI was then
investigated by detection of the total levels of the HIV-1
mRNA in
Northern blots. The amounts of HIV RNA are doubled in
cells transfected
with the RLI sense vector (VS2) compared to
cells transfected with the
empty vector (VV). By contrast, the
anti-HIV effect of lower expression
of RLI was confirmed, since
viral RNA levels are halved in cells
transfected with the RLI
antisense vector (VAS2) (Fig.
6A). This difference in HIV-1 RT
activity
and mRNA was also observed at the protein level.
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FIG. 6.
HIV-1 mRNA level and expression of gp160/gp120 during
HIV-1 infection in cells transfected with RLI sense and antisense cDNA
constructions. H9 cells transfected with the antisense RLI cDNA
construction (, VAS2), with the sense RLI cDNA construction (,
VS2), or with the empty vector (, VV) were infected with HIV-1 and
harvested at the indicated times. (A) For each time point, 20 µg of
total RNA was analyzed by Northern blot. A quantification of total HIV
mRNA by the Intelligent Quantifier program is represented. (B) For each
time point, 200 µg of protein was analyzed by SDS-PAGE and
Western blotting with a polyclonal antibody against gp160/gp120.
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The amount of the HIV-1 gp160/gp120 proteins was evaluated by
Western blot assays with a polyclonal antibody. In the VV clone,
gp160/gp120 polypeptides could be detected as early as 3 days
postinfection and reached maximal levels at days 5 and 6 (Fig.
6B). The
high RT activity in the VS2 clone was correlated with
the presence of
gp160/gp120 as early as 2 days after the onset
of infection; the amount
of both proteins increased to maximum
levels at days 4, 5, and 6. On the other hand, gp160/gp120 polypeptides
were poorly expressed in
the VAS2 clone and were absent from day
7 (Fig.
6B).
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DISCUSSION |
HIV replication decreases when RLI is underexpressed in human
cells.
We show here that RLI can be a suppressor of HIV
replication when it is underexpressed in human H9 cells by transfection
with an RLI antisense cDNA construction. On the contrary, when RLI is
overexpressed in human H9 cells transfected with an RLI sense construction, HIV replication is greatly increased.
The strategy we used here bypasses the requirement for IFN treatment to
obtain inhibition of viral growth. This strategy has
been used with
success in different systems to study the role
of several IFN-inducible
proteins, and it has revealed the importance
of these proteins
in the antiviral action of IFN. Moreover, this
strategy has overcome
problems associated with viruses that have
developed powerful tools to
block specific IFN pathways. Indeed,
as outlined in the introduction,
during the course of a number
of viral infections RNase L activity is
not detectable, despite
high levels of 2-5A-synthetase, 2-5A, or even
RNase L (see reference
32 for a review). These
observations provided strong evidence
that viruses can inactivate the
2-5A/RNase L pathway. For example,
the expression of 2-5A synthetase
and/or RNase L in mouse cells
restores apoptosis inhibited by the
vaccinia virus E3L protein
(
23) and inhibits replication
of EMC virus (
8,
9,
24,
32,
36). Similarly, we have
previously demonstrated that when
RLI expression is decreased in HeLa
cells by transfected antisense
RLI cDNA constructions, RNase L activity
is increased during EMC
virus infection and EMC virus production is
lower (
18).
HIV inhibits the 2-5A/RNase L pathway during the time course of
infection (
29,
30); similarly, expression of 2-5A-synthetase
cDNA from an HIV-1 LTR reduced HIV replication in HeLa T4
+
cells (
27,
28). The overexpression of RNase L in Jurkat
cells
and in peripheral blood lymphocytes severely impairs HIV
replication
(
17). It is worth noting that the expression of
RNase L in the
different systems cited above seems to produce a much
greater
antiviral effect than did the expression of 2-5A-synthetase
(
17,
23). Levels of RNase L represent a central limiting
component
to the antiviral activity of the 2-5A/RNase L
pathway.
RNase L activity is correlated with RLI levels in HIV-1-infected
human cells.
In the present report evidence is presented that
modifying the RLI levels consequently gives rise to different levels of
RNase L activity in HIV-1-infected cells and also affects viral
production (Fig. 3 and 4).
Western blot assays with antibodies against RNase L and RLI reveal that
the amount of RNase L is only transiently decreased
upon HIV-1
infection and that RLI is simultaneously induced (Fig.
2). We provide
evidence here that the loss of RNase L activity
during HIV-1 infection
is due not only to a decrease in the level
of the RNase L protein
itself but also to the accumulation of
the RNase L inhibitor, RLI. We
have already established that the
ratio between RNase L and RLI in cell
extracts governs the overall
activity of the 2-5A/RNase L system, even
when 2-5A is present
at doses largely sufficient to activate RNase L
(
3). When RLI
is decreased by the expression of an RLI
antisense cDNA construction,
the antiviral activity of RNase L against
EMC virus is increased
(
18). These experiments demonstrate
that slight changes in the
ratio between these two proteins could
profoundly modify the 2-5A
binding activity of RNase L. The increased
RLI level observed
here is sufficient to account for the inhibition of
RNase L activity
observed during HIV-1
infection.
Moreover, the experiments described so far indicate a correlation among
HIV-1 infection, the reversible inactivation of RNase
L, and RLI
induction. Modifying the level of expression of RLI
through the
expression of antisense or sense constructions represents
an attractive
possibility to substantiate this hypothesis. The
inhibition of RNase L
activity by HIV-1 varies in function of
the amount of RLI (Fig.
4). The
overexpression of RLI giving rise
to greater inhibition of RNase L
activity correlated with a higher
viral production. On the contrary,
blocking of RLI by antisense
construction reverses the inhibition of
RNase L activity observed
during HIV-1 infection and, consequently,
inhibits viral production.
These results are clearly illustrated in
Fig.
5 and
6. We could
observe a great difference between the VAS and
VS clones in the
production of HIV-1 at both the mRNA and protein
levels.
The 2-5A/RNase L pathway is a major component of the antiviral activity
of IFN. Viruses have developed numerous strategies
to circumvent this
pathway. Many of these viral inhibitors are
now being identified,
though their presence was first described
several years ago (see
reference
32 for a review). RLI, the
RNase L
inhibitor that we have cloned and identified, is implicated
in the
inhibition of the 2-5A/RNase L pathway by EMC virus (
3,
18).
The present study describes a new strategy that HIV-1 has
evolved to
inhibit cellular antiviral defenses. The identification
of the
antagonistic pathways developed by viruses is important
in
understanding the physiopathology of viral infection and in
the
implementation of new and more efficient antiviral strategies.
In
particular, it would be of interest to understand how HIV-1
is able to
induce RLI and to inhibit the cellular antiviral
defenses.
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ACKNOWLEDGMENTS |
We are very grateful to D. Misse (ORSTOM, Montpellier, France)
for the gift of antibodies against HIV gp120, to M. Benkirane (IGH, UPR
1142, Montpellier, France) for the gift of HIV LTR plasmid, to G. Cathala (IGMM, Montpellier, France) for the gift of a polyclonal antibody against GAPDH, and to I. Robbins (IGMM, Montpellier, France)
for revising the manuscript.
This work was supported by grants from Institut National de la
Santé et de la Recherche Médicale to C.B. and from the
Agence Nationale de la Recherche contre le SIDA to B.L. C.M. holds
a predoctoral fellowship from the Fondation pour la Recherche
Médicale.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institut
de Genetique Moleculaire de Montpellier, UMR 5535, CNRS-Université de Montpellier II, 1919, route de Mende,
34293 Montpellier Cedex 5, France. Phone: 33-4-67-61-36-58. Fax:
33-4-67-04-02-45. E-mail:
bisbal{at}jones.igm.cnrs-mop.fr.
Present address: Laboratoire Retrovirus, ORSTOM, 34032 Montpellier
Cedex 1, France.
| |
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Top
Abstract
Introduction
