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J Virol, February 1998, p. 1146-1152, Vol. 72, No. 2
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Regulation of Human Immunodeficiency Virus
Replication by 2',5'-Oligoadenylate-Dependent RNase L
Ratan K.
Maitra1 and
Robert H.
Silverman2,*
Virus Core Facility1
and
Department of Cancer Biology,2 The
Lerner Research Institute, The Cleveland Clinic Foundation,
Cleveland, Ohio 44195
Received 3 July 1997/Accepted 4 November 1997
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ABSTRACT |
Activation of RNase L by 2',5'-linked oligoadenylates (2-5A) is one
of the antiviral pathways of interferon action. To determine the
involvement of the 2-5A system in the control of human immunodeficiency virus type 1 (HIV-1) replication, a segment of the HIV-1
nef gene was replaced with human RNase L cDNA. HIV-1
provirus containing sense orientation RNase L cDNA caused increased
expression of RNase L and 500- to 1,000-fold inhibition of virus
replication in Jurkat cells for a period of about 2 weeks.
Subsequently, a partial deletion of the RNase L cDNA which coincided
with increases in virus production occurred. The anti-HIV activity of
RNase L correlated with decreases in HIV-1 RNA and with an acceleration in cell death accompanied by DNA fragmentation. Replication of HIV-1
encoding RNase L was also transiently suppressed in peripheral blood
lymphocytes (PBL). In contrast, recombinant HIV containing reverse
orientation RNase L cDNA caused decreased levels of RNase L, increases
in HIV yields, and reductions in the anti-HIV effect of alpha
interferon in PBL and in Jurkat cells. To obtain constitutive and
continuous expression of RNase L cDNA, Jurkat cells were cotransfected with HIV-1 proviral DNA and with plasmid containing a cytomegalovirus promoter driving expression of RNase L cDNA. The RNase L plasmid suppressed HIV-1 replication by eightfold, while an antisense RNase L
construct enhanced virus production by twofold. These findings
demonstrate that RNase L can severely impair HIV replication and
suggest involvement of the 2-5A system in the anti-HIV effect of alpha
interferon.
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INTRODUCTION |
The interferons are a family of
cytokines that suppress replication of a wide range of viruses through
multiple antiviral pathways (40). Accordingly, human
immunodeficiency virus (HIV) is susceptible to inhibition by
interferons in primary and immortalized human T cells and
monocytes/macrophages (25). Several different steps in the
HIV life cycle have been reported to be affected by interferon
treatment. For example, alpha interferon treatment of chronically
infected cell lines was often due to defective assembly and release of
virus particles (8, 16, 26). In this regard, the effect of
interferons on HIV replication were similar to that previously observed
for murine retroviruses (14). However, interferons have also
been reported to inhibit early stages prior to production and
integration of proviral DNA (15, 23), perhaps at the level
of reverse transcription (33). In addition, translation of
HIV mRNAs was specifically inhibited by alpha interferon in chronically
infected, U937 promonocytic cells and in synchronously infected CEM T
cells (8).
The 2',5'-linked oligoadenylate (2-5A) system is an interferon-induced,
RNA decay pathway implicated in some of the antiviral mechanisms of
interferon action (34). Interferon treatment of cells
induces several isozymes of 2-5A synthetase and a single species of the
2-5A-dependent RNase L. The synthetases require double-stranded RNA
(dsRNA) to produce 2-5A
[pxA(2'p5'A)y, where
x = 1 to 3 and y 
2] from ATP
(18). 2-5A binds with high affinity to RNase L, converting
the inactive monomeric protein to the activated homodimer (9,
11). Many viruses produce dsRNA molecules that can activate 2-5A
synthetases, resulting in accumulation of 2-5A. Accordingly, 2-5A or
related material has been observed in interferon-treated cells
infected with encephalomyocarditis virus (EMCV), vaccinia virus,
reovirus, herpes simplex virus, and SV40 simian virus 40 (34). Furthermore, the leader region of HIV type 1 (HIV-1)
RNA is sufficiently structured to activate 2-5A synthetase activity in
vitro (32). Indeed, a chemically synthesized segment of
HIV-1 RNA corresponding to the 5'-terminal, 57-nucleotide TAR region
was capable of activating 2-5A synthetase (20). In two
studies, however, alpha interferon treatment failed to induce enhanced
degradation of either HIV RNA or rRNA (8, 33). Perhaps the
lack of RNase L activity was due to inhibition of TAR-mediated
activation of 2-5A synthetase by the HIV-1 TAT protein (30).
In contrast, a transient increase in 2-5A synthetase and RNase L
activity was observed in extracts of HIV-infected H9 cells
(31). In addition, several studies have suggested that direct expression or activation of 2-5A system enzymes can lead to
suppression of HIV replication. For instance, expression of 2-5A
synthetase from an HIV long-terminal repeat (LTR) inhibited HIV-1
replication in HeLa T4+ cells (29) and
phosphorothioate-phosphodiester 2-5A derivatives inhibited HIV-induced
syncytium formation (36). Also, 2-5A derivatives inhibited
HIV-1 reverse transcriptase by preventing primer complex formation
(35).
Here, we have taken a novel approach to investigating the potential of
the 2-5A system for controlling HIV infections. The availability of
cDNA encoding RNase L allowed manipulation of the enzyme which
catalyzes the biological effects of the 2-5A system (45).
Accordingly, we show that HIV-1 replication can be suppressed by the
overexpression of RNase L from either an infectious HIV-1 molecular
clone or from an expression plasmid. In contrast, decreasing endogenous
levels of RNase L with a reverse orientation cDNA enhanced HIV
replication and reduced the antiviral effect of interferon. Our
findings thus demonstrate that the 2-5A system is able to regulate HIV
replication in control and interferon-treated human cells.
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MATERIALS AND METHODS |
Construction of plasmids.
HIV provirus,
pNL4-3
nef, containing a 272-bp deletion in the
nef coding sequence flanked by two unique restriction sites, XbaI and XhoI, was a generous gift of H. Kestler
(Cleveland, Ohio) (1, 5). Human RNase L cDNA, obtained by
digesting plasmid pZC5-2 with XbaI and XhoI, was
purified and subcloned in both orientations into the
XbaI/XhoI sites of pNL4-3nef (see
Fig. 1). The RNase L cDNA was also cloned into pcDNAneo (Invitrogen) at
the HindIII site in both orientations (see Fig. 8A)
(47).
Cell culture and viruses.
Jurkat cells were cultured in RPMI
1640 medium-10% fetal bovine serum (FBS) (Gibco/BRL). Peripheral
blood mononuclear cells from healthy donors were isolated from
heparinized venous blood by density gradient centrifugation on
Ficoll-Hypaque (Organon). The cells were washed twice in
phosphate-buffered saline (PBS), resuspended to 2 × 106 cells per ml in RPMI 1640 medium-L-glutamine-antibiotics-HEPES (pH 7.2), and
plated overnight in tissue culture flasks, and the nonadherent cells
consisting of primary blood lymphocytes (PBL) depleted of monocytes
were collected and activated by treating with 10 U of interleukin 2 (Chiron) per ml and 4 µg of phytohemagglutinin-P (PHA-P) (Sigma) per
ml.
Transfections and infections of cells.
Plasmid DNAs (2.5 to
5 µg) were incubated with 10 to 15 µl of Lipofectamine (Gibco/BRL)
in 100 µl of OPTI-MEM medium at room temperature for 25 to 40 min
prior to transfection of PBL or Jurkat cells. The DNA mixtures were
incubated with cells (5 × 105 to 10 × 105) in 1.0 ml of OPTI-MEM medium for 5 h, and then 4 ml of RPMI 1640 medium-10% FBS was added.
Virus stocks were prepared by introducing 5 µg of proviral DNA into
Jurkat cells with Lipofectamine. Virus titers and 50% tissue culture
infective doses (TCID50) on about 105 cells
were measured as described elsewhere (17). Prior to
infections, PBL were incubated for 48 h, washed in PBS, and
resuspended in medium lacking PHA-P. Stimulated PBL or Jurkat cells
(5 × 105 per ml) were infected with 20 TCID50 of virus in serum-free RPMI 1640 medium for 2 h
at 37°C. The cells were washed four times with PBS to remove
unabsorbed virus particles, and then 5 ml of RPMI 1640 medium-10% FBS
was added.
Interferon treatment of cells.
Jurkat cells or stimulated
PBL (1 × 105 to 2 × 105 cells per
ml) were treated with 5,000 U of recombinant human interferon 
-2b (Intron A; Schering) per ml for 18 to 24 h followed by infection with 10 to 20 TCID50 of virus in 1 ml of serum-free medium
for 2 h. The cells were washed three times with PBS and suspended in 5 ml of RPMI medium-10% FBS. Cell-free supernatants were collected for viral assays at regular intervals, and fresh medium with or without
interferon (5,000 U per ml) was added every 2 days beginning 5 days
after infection.
Quantitation of HIV p24 levels.
Cell-free culture
supernatants (10 to 20 µl of 1:10 to 1:100 dilutions) were harvested
to determine p24 protein levels with an HIV p24 antigen capture kit
(Coulter). Briefly, cell-free culture supernatants and lyse buffer were
added to wells of microtiter strip plates coated with a monoclonal
antibody to p24 and allowed to incubate at room temperature for 1 h. The wells were washed three to four times, biotinylated anti-HIV-1
immunoglobulin G was added, and the wells were incubated at 37°C for
1 h. Following another wash, streptavidin-horseradish peroxidase
was added, and the wells were incubated for 30 min at 37°C. After
washing, tetramethylbenzidine was added at room temperature, and
development of color at 650 nm was detected as a function of time for a
15-min period. Amounts of p24 protein, which do not always correlate
with TCID50 levels, were determined in comparison to a
calibrated p24 antigen standard in a Coulter microplate reader.
PCR amplification of RNase L cDNA from HIV proviral DNA.
Total cellular DNA was used for PCR with Taq polymerase
(Perkin-Elmer Cetus). Primer pairs (5'-GCA GCC GGA TCC TTA GC-3' and 5'-GAT ATA CCA TGG ATC-3') homologous to a sequence 320 bp 5' and 1,508 bp 3' of RNase L cDNA in the recombinant HIV proviral DNA were used.
DNA (1 µg) was added to 100 µl of reaction mixture containing 0.2 mM each deoxynucleoside triphosphate (dNTP), 500 nM each
oligonucleotide primer, 4.0 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 0.01% gelatin (wt/vol), and 2.5 U of DNA
Taq polymerase (Perkin-Elmer Cetus). PCR was carried out for
30 cycles with denaturation at 94°C for 1.5 min, annealing at 50°C
for 1.5 min, and extension at 72°C for 2 min.
Detection of RNase L in Western blots.
RNase L levels were
determined with monoclonal antibody against RNase L in Western blots as
described elsewhere (11, 41). Cells were lysed with RIPA
buffer containing 10 mM Tris (pH 7.4), 1 mM EDTA, 0.15 M NaCl, 0.1%
sodium dodecyl sulfate (SDS), 1% Triton X-100, 1% sodium
deoxycholate, and 1 mM phenylmethylsulfonyl fluoride.
Northern blots for detection of HIV RNA and 18S rRNA.
Total
cellular RNAs from Jurkat cells were harvested at 48, 72, and 96 h
posttransfection with RNAzol reagent (Cinna/Biotex). RNAs were
separated in 1.2% agarose-formamide gels. RNA was transferred onto
Nylon transfer membranes (Micron Separations) with 10× SSC (1× SSC is
0.15 M NaCl, 0.015 M sodium citrate) for 18 to 20 h. The membranes
were placed in prehybridization solution (GIBCO-BRL) containing 6×
SSC, 5× Denhardt's solution, 100 µg of sheared, denatured salmon
sperm DNA per ml, 20 mM sodium phosphate buffer (pH 6.5), and 50%
formamide for 4 h at 65°C. PCR-amplified HIV LTR labeled with
[-32P]dCTP with a random priming kit (Boehringer
Mannheim) was used as a probe. About 107 cpm of probe was
added to the prehybridization buffer, and the blots were incubated for
16 h at 42°C. The membranes were washed with solutions of
SSC-SDS of increasing stringency, and the RNA levels were measured with
a PhosphorImager (Molecular Dynamics). The blots were stripped with
0.1× SSC for 10 to 15 min at 100°C and probed with a randomly
primed, 32P-labeled DNA probe of 18S rRNA (American Type
Culture Collection).
DNA fragmentation assay.
DNA fragmentation in situ was
measured with the Trevigen Apoptotic Cell System by using terminal
deoxynucleotide transferase (15 U) in 0.05 M Tris (pH 7.5)-5 mM
MgCl2-0.6 mM 
-mercaptoethanesulfonic acid-50 µg of
bovine serum albumin-0.25 mM biotinylated nucleotides (dNTPs) at
37°C for 60 min. Reactions were terminated with 0.1 M EDTA (pH 8.0).
Streptavidin-horseradish peroxidase conjugate was applied at room
temperature for 10 min. Fragmented DNA was stained blue, and labeled
cells were visualized through a microscope and counted.
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RESULTS |
Expression of RNase L cDNA from a recombinant HIV suppresses virus
replication.
To determine the effects of regulating RNase L levels
on HIV-1 replication, we subcloned human RNase L cDNA into a modified HIV-1 proviral DNA (5). RNase L cDNA was inserted in either the forward (pNL4-3/sRL) or reverse (pNL4-3/aRL) orientation in place
of a 272-bp segment of the nef gene in
pNL4-3nef DNA (Fig. 1).
Virus production after transfection of Jurkat cells or PBL with
provirus was determined by measuring levels of the viral gag or core
protein, p24, in the culture supernatants. Transfections with an HIV-1
provirus (pNL4-3, pNL4-3nef, or pNL4-3/aRL) produced similar viral growth curves in Jurkat cells (Fig.
2A). In contrast, virus replication was
very significantly repressed after transfection of Jurkat cells with
the sense RNase L construct, pNL4-3/sRL (Fig. 2A). For instance, at 16 days posttransfection of Jurkat cells with pNL4-3/sRL, the viral yield
was about 1,000-fold lower than that in pNL4-3-transfected cells.
However, by 28 days posttransfection, viral yields from pNL4-3/sRL,
pNL4-3, pNL4-3/nef, and pNL4-3/aRL were similar.
Transfections of PBL produced similar results, except that the level of
virus produced by pNL4-3/sRL was closer to 30- to 40-fold less than
that with pNL4-3 or pNL4-3/nef from 8 to 12 days and
recovery of viral growth was observed earlier, between 12 and 16 days
posttransfection (Fig. 2B). Perhaps the smaller antiviral effect of
RNase L in the PBL was because the HIV replicated more rapidly and to
much higher titers in PBL than in Jurkat cells. There was a modest but
consistent increase (as much as three- to sixfold) in viral yield
obtained after transfections with the antisense vector, pNL4-3/aRL, in
the Jurkat cells and PBL. Therefore, forward orientation RNase L cDNA
greatly but transiently suppressed virus production, while the reverse
orientation constructs modestly enhanced virus growth.
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FIG. 1.
Construction of recombinant HIV containing RNase L cDNA
in the sense and antisense orientations. The plasmids constructed or
used for this study included NL4-3 (wild-type HIV-1 proviral DNA),
NL4-3nef (272 bases deleted from nef),
NL4-3/sRL (forward orientation RNase L cDNA cloned in place of a 272-bp
segment of the nef gene), and NL4-3/aRL (reverse orientation
RNase L cDNA cloned in place of a 272-bp segment of the nef
gene).
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FIG. 2.
Expression of RNase L cDNA from a recombinant HIV
provirus suppresses HIV replication in transfected Jurkat cells (A) and
PBL (B). The cells were transfected with pNL4-3 ( ),
pNL4-3/nef ( ), pNL4-3/aRL ( ), or pNL4-3/sRL ( ).
p24 antigen in the cell-free culture supernatant was collected at
regular intervals and measured. d, days.
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RNase L expression was terminated by a deletion during long-term
cultures of transfected cells.
Viral yields were correlated with
levels of RNase L determined by probing Western blots with monoclonal
antibody against RNase L (Fig. 3A and B,
top panel). Levels of RNase L were similar in cells transfected with
pNL4-3 or pNL4-3nef. However, transfection with
pNL4-3/aRL led to a transient decrease in RNase L levels by 4 days,
followed by a gradual increase to control levels by 21 days. In
contrast, cells transfected with pNL4-3/sRL produced four- to fivefold
higher levels of RNase L by 4 to 9 days. Subsequently, there was a
gradual decline in RNase L levels in the pNL4-3/sRL-transfected cells
to basal levels by 24 days. These findings suggested that expression of
high levels of RNase L from the recombinant HIV provirus, pNL4-3/sRL,
suppressed virus production at early times posttransfection. Later,
however, a decline in expression levels which correlated with increased
virus production occurred.
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FIG. 3.
RNase L levels increase and then decline after
transfection with NL4-3/sRL due to a deletion of RNase L coding
sequence. (A) RNase L levels from Jurkat cells transfected with pNL4-3
(), pNL4-3/nef (), pNL4-3/aRL (), or pNL4-3/sRL
(). The basal level of RNase L in untreated cells was an average of
values obtained at 4, 16, and 24 days (d) of cell culture. (B) Upper
panel, Western blot analysis of RNase L from transfected Jurkat cells
probed with monoclonal antibody against human RNase L; lower panel, PCR
amplification of the integrated RNase L cDNA. RL, 3 ng of RNase L; M,
 /HindIII molecular size markers (numbers to left are
in kilobases). Plasmid names and times posttransfection in days are
indicated.
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To determine the cause of the declining levels of RNase L at late times
posttransfection with pNL4-3/sRL, the stability of
the integrated RNase
L cDNA was determined. At 4 days posttransfection,
a DNA fragment of
the expected size (4.4 kb) was obtained with
PCR primers to sequences
320 nucleotides upstream and 1,508 nucleotides
downstream of the
integrated RNase L cDNA (Fig.
3B, lower panel,
lane 2). However, at 24 days posttransfection, the amplified DNA
fragment was reduced in size
by about 2 kb (lane 3). In contrast,
the RNase L cDNA was stable in the
antisense construct, pNL4-3/aRL,
at 24 days posttransfection (lane 5).
These findings suggest that
the emergence of virus at late times
posttransfection with pNL4-3/sRL
was due to a partial deletion of the
RNase L coding sequence.
It was apparent from these experiments that
the presence of the
sense orientation RNase L cDNA in the viral genome
imposed negative
selective pressure on the recombinant virus.
Transfection with recombinant HIV provirus expressing RNase L leads
to decreased levels of HIV RNA and increased rates of cell death.
The mechanism of the anti-HIV effect of overexpressing RNase L was
investigated. Total levels of the HIV RNA were detected and measured in
Northern blots at 48, 72, and 96 h posttransfection of Jurkat
cells. During this time, amounts of HIV RNA decreased to undetectable
levels in cells transfected with pNL4-3/sRL, while viral RNA levels
increased by >10-fold in cells transfected with pNL4-3/nef and pNL4-3/aRL (Fig.
4A and B). These results were consistent
with RNase L activity against HIV RNA in the pNL4-3/sRL-transfected cells. However, we have been unable to clearly demonstrate cleavage products of either HIV RNA or rRNA in the pNL4-3/sRL-transfected cells
(Fig. 4A). Perhaps HIV RNA fragments escaped detection due to their
rapid degradation relative to the time scale of these experiments. The
reason for the lack of rRNA cleavage products was unexpected but
suggests that HIV RNA may be more susceptible to cleavage by RNase L
than rRNA.
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FIG. 4.
Reduced levels of HIV mRNAs in Jurkat cells transfected
with NL4-3/sRL. (A) Upper panel, Northern blot of HIV RNA from Jurkat
cells at 48, 72, and 96 h posttransfection with pNL4-3/sRL,
pNL4-3/nef, or pNL4-3/aRL as indicated; lower panel,
Northern blot of 18S rRNA. (B) Quantitation of total HIV mRNA with a
PhosphorImager.
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The effect of RNase L overexpression from pNL4-3/sRL on cell death was
determined by an in situ DNA fragmentation assay. Transfection
with the
control, salmon sperm DNA, did not cause cell death,
whereas
transfection of Jurkat cells with all of the HIV proviruses
led to
varying levels of cell death (Fig.
5).
However, transfection
with pNL4-3/sRL caused cell death earlier than
transfection with
pNL4-3, pNL4-3
nef, or pNL4-3/aRL. The
peak of cell death, with
35 and 40% of the cells dying, occurred by 6 days with NL4-3/sRL
and by 12 days with the other three proviruses.
Therefore, while
HIV itself causes cell death, the overexpression of
RNase L accelerates
this process.
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FIG. 5.
Overexpression of RNase L from NL4-3 recombinant
proviral DNA (NL4-3/sRL) accelerates HIV-induced cell death. Jurkat
cells were transfected with pNL4-3 (), pNL4-3/nef
(), pNL4-3/aRL (), or pNL4-3/sRL () or were mock transfected
with salmon sperm DNA ( ), and the percentage of apoptotic cells as a
function of time was determined by in situ detection of DNA
fragmentation. d, days.
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Reducing expression of RNase L increases the rate of HIV
replication and transiently reduces the anti-HIV effect of
interferon.
The effect of decreasing RNase L levels on viral
growth was further explored in infections with NL4-3/aRL virus, which
encodes RNase L in the antisense orientation. Infections rather than
transfections were performed to enable the viral genomes to enter a
higher proportion of the cells. Amounts of RNase L were measured at
different times postinfection of Jurkat cells with NL4-3 and NL4-3/aRL
(Fig. 6). RNase L concentrations
decreased to almost undetectable levels at 5 days postinfection with
NL4-3/aRL, an apparent antisense effect (Fig. 6, lane 6). Subsequently,
there was a gradual increase in RNase L levels. Interferon treatment
induced RNase L in cells infected with either NL4-3 or NL4-3/aRL.
However, at 9 days postinfection, interferon-induced levels of RNase L
were approximately fourfold higher in NL4-3 infected cells than those
in NL4-3/aRL-infected cells (Fig. 6; compare lanes 3 and 7).
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FIG. 6.
Levels of RNase L in NL4-3 and NL4-3/aRL virus-infected
Jurkat cells without or with interferon pretreatment (5,000 U per ml).
Upper panel, quantitation of RNase L; lower panel, Western blot used to
prepare graph shown in upper panel. d, days.
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NL4-3/aRL virus replicated more rapidly and to higher titers than did
either NL4-3 (Fig.
7) or
NL4-3
nef viruses (data not
shown). There was as much as
5- to 12-fold more virus from Jurkat
cells and PBL infected with
NL4-3/aRL than from those infected
with NL4-3. These results suggest
that the decrease in RNase L
levels led to an increase in viral yields.
Similar results were
seen in transfection experiments (Fig.
2). To
determine if the
2-5A system was involved in the anti-HIV effect of
interferon,
infections were performed in cells pretreated with 5,000 Units
of recombinant, human alpha interferon per ml for 18 to 24 h
with
the same concentrations of interferon added again every 2 days
postinfection beginning on day 5 (Fig.
7). In Jurkat cells, NL4-3
virus
replication was completely blocked by the interferon treatments,
whereas interferon treatment reduced production of NL4-3 virus
by
24-fold in PBL (Fig.
7A and B, respectively). The inhibitory
effects of
interferon on NL4-3 and NL4-3
nef replication were
similar
(data not shown). In contrast, NL4-3/aRL replication was
reduced but
not blocked in Jurkat cells during the first 10 days
of
interferon treatment (Fig.
7A). Subsequently, interferon prevented
further increases in NL4-3/aRL titers in the Jurkat cells. In
PBL,
there was no observable effect of interferon on NL4-3/aRL
replication
during the first 9 days of infection (Fig.
7B). However,
at later times
postinfection, NL4-3/aRL replication was suppressed
by interferon
treatment. Therefore, the antiviral effect of interferon
was
transiently reduced for NL4-3/aRL compared to the wild-type
virus,
NL4-3. The increase in the activity of interferon against
NL4-3/aRL at later times postinfection could be due to increasing
levels of RNase L (Fig.
6). These findings suggest a role for
the 2-5A
system in the antiviral mechanism of action of interferons
against HIV.
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FIG. 7.
Replication of NL4-3 virus ( and ) and pNL4-3/aRL
virus ( and ) in the presence ( and ) or absence ( and
) of 5,000 U of alpha interferon per ml in Jurkat cells (A) and PBL
(B).
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Expression of RNase L from a separate plasmid suppresses HIV
replication.
Because RNase L cDNA was unstable in recombinant HIV
provirus (Fig. 3B), the RNase L cDNA was expressed under the control of
a cytomegalovirus (CMV) promoter in a plasmid vector (Fig. 8A). In cotransfections with the
wild type, pNL4-3, the RNase L expression plasmid,
pcDNAneo/sRL, suppressed HIV-1 yields by 8.4-fold in comparison to
control transfections in which pNL4-3 was introduced together with the
empty vector or with salmon sperm DNA (Fig. 8B). In contrast,
cotransfection of pNL4-3 with plasmid containing reverse orientation
RNase L cDNA, pcDNAneo/aRL, modestly enhanced virus production by
about twofold. These findings confirm in a two-plasmid system the
ability of RNase L to control of HIV replication in infected human
cells. Presumably, therefore, stable or inducible expression of RNase L
could lead to a sustained inhibition of HIV replication.
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FIG. 8.
Control of HIV replication by RNase L expression
plasmids in Jurkat cells. (A) Plasmid maps for pcDNAneo, pcDNAneo/sRL,
and pcDNAneo/aRL; (B) plasmids cotransfected with NL4-3 in Jurkat
cells. p24 antigen was measured as a function of time. NL4-3 proviral
DNA cotransfected with salmon sperm DNA (), pcDNAneo (),
pcDNAneo/aRL (), or pcDNAneo/sRL (). d, days; SV40, simian virus
40; RSV, Rous sarcoma virus.
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DISCUSSION |
RNase L, an interferon-inducible antiviral protein, suppresses HIV
replication when overexpressed in human cells.
Here, we show that
RNase L can function as a potent suppressor of HIV replication when it
is overexpressed in transfected or infected human cells. This report
thus extends several studies in which antiviral activity was achieved
by elevating levels of individual, interferon-inducible proteins from
plasmids or recombinant viruses. These strategies effectively bypass
the requirement for interferon treatment to obtain inhibition of virus
growth. For instance, expression of 2-5A synthetase cDNA under the
control of strong constitutive promoters inhibited replication of
mengovirus, EMCV, and vaccinia virus while not affecting herpes simplex
virus type 2 and VSV replication (6, 7, 10, 27). Recently, Diaz-Guerra et al. reported that expression of cDNAs encoding RNase L
and 2-5A synthetase from recombinant vaccinia virus resulted in
characteristic cleavage of rRNA and a potent suppression of vaccinia
virus protein synthesis and growth (10). These investigators found that expression of RNase L produced a much greater antiviral effect than did expression of 2-5A synthetase. Similarly, expression of
a 2-5A synthetase cDNA from an HIV-1 LTR reduced HIV replication in
HeLa T4+ cells by 10- to 20-fold as measured by HIV reverse
transcriptase activity (29). Here, we have obtained up to
1,000-fold suppression of HIV-1 replication in Jurkat cells by
overexpressing RNase L (Fig. 2).
Expression of proteins from alternative, interferon-regulated pathways
also provides differing levels of protection from viral
infections. For
example, in cotransfection experiments with wild-type
HIV provirus, a
recombinant HIV provirus encoding the dsRNA-dependent
protein kinase
PKR potently suppressed virus production (
3,
20a). The TAR
binding protein TRBP blocked the anti-HIV effect
of PKR through a
direct protein-protein interaction (
3). Similarly,
TAT/72
protein is able to directly bind with and inhibit PKR function
(
21). Overexpression of PKR also suppressed replication of
EMCV
but not of vesicular stomatitis virus (VSV) (
22). In
addition,
expression of the interferon-inducible MxA protein in cells
and/or
transgenic mice inhibited replication of several RNA viruses,
including influenza A virus, measles virus, Thogoto virus, VSV,
and
human parainfluenza virus type 3 (
24,
28,
44). Clearly,
direct expression of several interferon-regulated proteins can
have
profound antiviral effects. In addition, expression of interferon
cDNAs
has been shown to repress viral replication (
37,
39).
The anti-HIV effects of the 2-5A system are probably due to the
stimulation of 2-5A synthetase activity by highly structured
regions of
HIV RNA. In particular, the TAR region of HIV RNA is
sufficiently
double stranded to stimulate both PKR and 2-5A synthetase
activities
(
12,
20,
32). Presumably, by overexpressing RNase
L, even
low levels of 2-5A would be capable of producing enough
RNA cleavage to
result in a substantial antiviral effect. Therefore,
when the RNase L
cDNA was expressed from a recombinant HIV provirus,
a severalfold
increase in RNase L levels translated into a surprising
1,000-fold
suppression of HIV growth in Jurkat cells at 2 weeks
posttransfection
(Fig.
2 and
3A). Levels of RNase L, therefore,
represent a critical,
rate-limiting component to the 2-5A system.
Perhaps the potency of this
approach was partially due to insertion
of the RNase L cDNA in the HIV
nef gene, thus coordinating RNase
L production with virus
gene expression. The negative selective
pressure on the virus imposed
by the presence of sense orientation
RNase L cDNA led to its eventual
deletion (Fig.
3B). Following
the partial deletion of the RNase L cDNA,
virus production increased
markedly (Fig.
2). In contrast, the RNase L
cDNA was stable in
the antisense construct, pNL4-3/aRL, presumably
because of a lack
of selective pressure.
The presence of RNase L cDNA in recombinant provirus-accelerated,
virus-induced cell death in transfected cells.
The depletion of
CD4+ T cells in patients with AIDS occurs as a result of
apoptosis, mostly in uninfected bystander cells (2, 13).
HIV-1 Tat and gp120 proteins have been suggested to cause apoptosis in
T cells by induction of Fas ligand expression, resulting in
sensitization to Fas-mediated apoptosis (2, 4, 19, 42).
Recently, we have shown that mice lacking RNase L have a major
apoptotic defect resulting in enlarged thymuses containing thymocytes
which are resistant to anti-Fas-mediated apoptosis (46).
Perhaps, therefore, the overexpression of RNase L sensitizes cells to
Fas-Fas ligand-induced killing. The finding that overexpression of
RNase L from NL4-3/sRL accelerates cell death accompanied by DNA
fragmentation is consistent with the involvement of RNase L in
virus-mediated apoptosis (Fig. 5).
The 2-5A system restricts HIV replication in interferon-treated and
untreated cells.
HIV replication was enhanced when RNase L levels
were depressed by expression of antisense orientation RNase L cDNA
(Fig. 7). NL4-3/aRL replicated to higher titers than the control
viruses even in the absence of interferon treatment, suggesting that
basal levels of RNase L and 2-5A synthetase were sufficient to dampen HIV replication rates. Because the 2-5A system limits the extent of HIV
replication, it could contribute to establishment of chronic infections. In cells treated with alpha interferon, NL4-3/aRL virus
also replicated to higher titers than did the control viruses NL4-3 and
NL4-3/nef. The antiviral effect of interferon was
transiently reduced for NL4-3/aRL compared to NL4-3 or
NL4-3/nef (Fig. 7 and data not shown). Long-term
treatments with interferon led to an increased inhibition of
replication of NL4-3/aRL, perhaps due to induction of RNase L by
interferon (Fig. 6). These findings suggest a role for the 2-5A system
in the anti-HIV activity of interferon. Previously, it was reported
that steady-state levels of HIV RNAs and the integrity of rRNA were
unaffected by alpha interferon treatment of chronically infected U937
and acutely infected CEM T cells, arguing against a role for the 2-5A
system in the anti-HIV effect of interferon (8, 33).
However, we used a 10-fold higher concentration of alpha interferon
(5,000 versus 500 U per ml), and treatment was extended for several
days, compared with 2 days or fewer in the other studies, which are experimental parameters that may be necessary to observe the effect of
the 2-5A system on HIV replication. In addition, different cell types
were analyzed in the different studies. Relatively high levels of
interferon (5,000 U per ml) were used here because concentrations of
less than 1,000 U per ml were ineffective (data not shown). The
relative insensitivity of HIV to interferons could be the result of
inhibition of the antiviral pathways by HIV proteins (21,
30).
Destabilizing RNA as an anti-HIV strategy.
By inducing
overexpression of RNase L, HIV replication becomes severely impaired,
presumably as the result of RNA decay. Accordingly, levels of HIV RNA
declined after 48 h of posttransfection with NL4-3/sRL (Fig. 4).
Perhaps, the anti-HIV effect could be further improved by either
controlling RNase L expression from an inducible promoter or by
selectively targeting HIV RNA for decay (38). Destabilizing
RNA with the RNases onconase (a frog RNase) and bovine seminal RNase A
also causes potent inhibition of HIV replication in H9 cells
(43). The advantage of RNase L over other RNases is that it
can exist in either a silent or an active form. Previously, we showed
that there is sufficient secondary structure in HIV TAR RNA to
stimulate 2-5A synthetase activity (20). Our results here
demonstrate that antiviral approaches involving the 2-5A system have
the potential to provide potent suppression of HIV infections in vivo.
| |
ACKNOWLEDGMENTS |
We thank Amiya Banerjee (Cleveland) for comments made
during preparation of the manuscript, Aimin Zhou (Cleveland) for
pcDNAneo/aRL and pcDNAneo/sRL, and H. Kestler (Cleveland) for
pNL4-3/nef.
This investigation was supported by U.S. Public Health Service grant CA
44059, which was awarded to R.H.S. by the Department of Health and
Human Services, National Cancer Institute.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Cancer Biology, NN1-06, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Phone: (216) 445-9650. Fax: (216) 445-6269. E-mail: silverr{at}cesmtp.ccf.org.
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Abstract
Introduction
