From the
The 2-5A/RNase L system is considered as a central pathway
of interferon (IFN) action and could possibly play a more general
physiological role as for instance in the regulation of RNA stability
in mammalian cells.
We describe here the expression cloning and
initial characterization of RLI (for RNase L inhibitor), a new type of
endoribonuclease inhibitor.
RLI cDNA codes for a 68-kDa polypeptide
whose expression is not regulated by IFN. Its expression in
reticulocyte extracts antagonizes the 2-5A binding ability and
the nuclease activity of endogenous RNase L or the cloned 2DR
polypeptide. The inhibition requires the association of RLI with the
nuclease and is dependent on the ratio between the two proteins.
Likewise RLI is co-immunoprecipitated with the RNase L complex by a
nuclease-specific antibody. RLI does not lead to 2-5A degradation
or to irreversible modification of RNase L. The overexpression of RLI
in stably transfected HeLa cells inhibits the antiviral activity of IFN
on encephalomyocarditis virus but not on vesicular stomatitis virus.
RLI therefore appears as the first described and potentially
important mediator of the 2-5A/RNase L pathway.
Interferons (IFNs)
The 2-5A system is one of the major
pathways induced by IFN; it has been implicated in some of the
antiviral activities of the IFNs and might play an essential role in
regulating RNA turnover and stability in cells
(5, 6) .
It has been described as composed of three enzymatic activities,
e.g. 2-5A-synthetases, 2-5A-phosphodiesterase, and
RNase L. 2-5A-synthetases are a family of four IFN-inducible
enzymes which, upon activation by double-stranded RNA, convert ATP into
the unusual series of oligomers known as
2-5A
(7, 8, 9) . The
2-5A-phosphodiesterase might be involved in the catabolism of
2-5A from its 2`,3` end
(10) . The steady state level of
2-5A, which is very unstable, could also be controlled by
dephosphorylation by phosphatases
(11) . The 2-5A-dependent
endoribonuclease L or RNase L is the effector enzyme of this system.
Its activation by subnanomolar levels of 2-5A leads to the
inhibition of protein synthesis by cleavage of mRNAs at the 3` side of
UpNp sequences
(12) .
Variations in intracellular 2-5A
and 2-5A-synthetase(s) levels have been observed during virus
infection, cell growth, or cell differentiation even in the absence of
exogenous IFN treatment
(13) . Published data are more
contradictory concerning RNase L; some suggest that IFN treatment or
cell growth status increase RNase L activity whereas others report no
alteration in RNase L activity in these circumstances (see Ref. 13 for
a review). These apparent contradictions might be explained by
differences in the sensitivity of the methods used to detect RNase L.
RNase L can indeed be detected by its binding to a radioactive
2-5A-3`-[
The correlations between
variations of 2-5A/RNase L and the control of cell growth and
differentiation, however, plead for a more general physiological role
of this system. It is interesting to note here that RNase L is
preferentially associated to polyribosomes
(17) .
RNase L was
first described as a 200-kDa protein
(19) . Its molecular weight
varies with the conditions of analysis, with the protein
concentrations, or with the origin of cell
extract
(20, 21) . Zhou et al.(1993) have cloned
a 80-kDa polypeptide (2DR) which binds 2-5A and cleaves
poly(uridylic acid) in a reticulocyte extract but not in a wheat-germ
extract
(22) . More recently the properties of the 2DR were
studied after expression of its cDNA in insect cells and purification
to homogeneity
(23) .
We have recently characterized a
monoclonal antibody (mAb3) which neutralizes RNase L activity; it
recognizes a RNA-binding protein of 80 kDa associated (but distinct)
with the previously known 2-5A binding 80-kDa protein and with
2DR. We have therefore proposed that the high molecular mass protein
complex (around 200 kDa) first characterized as RNase L contains at
least two distinct polypeptides e.g. a 2-5A-binding
protein (2-5ABP or 2DR) and a mAb3 recognized polypeptide which
has been called RNABP
(18) . Its rôle in the functioning
and regulation of RNase L in intact cells has not yet been elucidated.
The control of mRNA turnover rate is now recognized as a critical
element of gene expression regulation. However, the mechanisms
responsible for mRNA degradation in mammalian cells are poorly
understood
(24) . The 2-5A system appears as a well
characterized system of RNA degradation which might be involved in the
control of RNA metabolism.
We describe here the cloning of a
polypeptide inhibitor of the 2-5A pathway; it will be referred to
as RLI for RNase L inhibitor. This protein was isolated from an
expression library by binding to 2-5ApCp. In vitro translation of this cDNA gives rise to a protein of 68 kDa which
associates specifically with 2-5ABP and RNABP as shown by
immunoprecipitation with the mAb3 monoclonal antibody. This in
vitro translated 68-kDa protein can also form a complex with the
2DR protein.
Evidence is prevailed that RLI is a regulatory protein
whose co-expression inhibits the binding of 2-5A by the
endogenous RNase L or by 2DR, and as a consequence the
2-5A-dependent activation of RNase L. RLI does not promote
2-5A degradation.
Moreover the overexpression of the RLI cDNA
in HeLa cells results in the inhibition of the IFN-activated 2-5A
pathway.
Although not regulated by IFN treatment RLI might be a key
component of the IFN system and of the regulation of RNA stability in
mammalian cells.
Cell extracts were prepared as described
previously
(18) ; briefly, the cells were resuspended in 2
volumes of hypotonic buffer, disrupted in a Dounce homogenizer, and
centrifuged at 10,000
An internal
primer determined from the sequence of H2APB was used to isolate by
anchored PCR a full-length cDNA of 3568 bp coding for RLI (see
Fig. 1A). Briefly, poly(dT) cDNA was first tailed with
dG nucleotides with terminal transferase. This cDNA (0.5 µg) was
amplified by PCR (30 cycles) in a final volume of 50 µl using a 5`
poly(dC) primer with PstI and BamHI sites
(5`-TTTCTGCAGGATCCCCCCCCCCCC-3`) and an internal primer
(5`-CACTTAGATCATGTTCCACCACAAT-3`) as described in
Fig. 1A. This cDNA fragment was cloned in the
BglII site of H2ABP to give the full-length cDNA of 3568 bp.
In vitro translation in
rabbit reticulocyte lysates (with or without canine pancreatic
microsomes), or in wheat-germ extracts were performed according to the
manufacturer's instructions (Promega). The quantity of endogenous
RNase L was estimated by the amount of bound 2-5ApCp, e.g. 9
Reticulocyte translation incubations were used as a source of RLI,
2DR, or endogenous RNase L. Translation usually took place for 60 min
at 30 °C. Aliquots of each were mixed to generate various ratios of
these proteins in 2-5A binding or RNA cleavage assays.
For
immunoprecipitation, cell extracts or translation incubations were
incubated 3 h at room temperature with a 1/1,000 dilution of mAb3 or
control antibody. RNase L-antibody complexes were incubated
overnight at 4 °C with protein A-Sepharose (Pharmacia) and
recovered by centrifugation. The beads were washed several times with
10 mM Tris, pH 7.4, 0.5% (v/v) aprotinin, 1% (v/v) Nonidet
P-40, 2 mM EDTA, 150 mM NaCl, and resuspended in one
volume of 300 mM Tris, pH 8.9, 5% (w/v) SDS, 5% (v/v)
No
significative homology with other cDNAs in the EMBL and Genbank data
bases has been found.
In a chase
experiment using actinomycin D, the half-life of the RLI mRNAs was
estimated to be 4 h in HeLa cells (data not shown).
The expression of the full-length RLI cDNA in wheat-germ
or in rabbit reticulocyte extracts gave rise to a single polypeptide of
68 kDa as expected (Fig. 3A, lanes 2 and 4).
The translation of 2DR cDNA gave rise to a single polypeptide of 80 kDa
(Fig. 3A, lanes 1 and 3). The level of
expression was a little lower in wheat-germ extract than in
reticulocyte lysate for the two clones. No post-translational
modifications of these polypeptides were observed when they were
translated in the presence of canine pancreatic microsomal membranes
(data not shown). The 2DR and RLI cDNA translation products were not
recognized by the mAb3-specific monoclonal antibody that we have
developed against RNase L
(18) . As shown in
Fig. 3B, and as expected, only the 80 kDa polypeptide
(RNABP) was revealed in reticulocyte extract, either before or after
translation (compare lanes 1 to lanes 2 and
3). Likewise mAb3 did not recognize any proteins in the
wheat-germ extract in which the 2-5A/RNase L system, and
consequently RNABP is absent.
No binding of
2-5ApCp by in vitro expressed RLI (in wheat-germ
extracts) could be detected in the conditions described for the
radiobinding
(25) or for the radiocovalent assays
(26) (data not shown), thus confirming that the affinity of RLI
for 2-5A is poor. On the contrary the expression of RLI in
reticulocyte extracts led to a large dose-dependent decrease in the
radiobinding and radiocovalent binding of 2-5ApCp by endogenous
RNase L in reticulocyte lysate (Fig. 4, A and
C). As expected, the expression of 2DR alone in wheat-germ or
in reticulocyte extracts increased 2-5ApCp binding in both assays
(Fig. 4, A and C). The co-expression of RLI
gave rise to an inhibition of 2-5ApCp binding by 2DR whether
using the radiobinding or the radiocovalent assay (Fig. 4, A and C). As a control no inhibition of endogenous RNase L
in reticulocyte extract or of expressed 2DR was observed upon addition
of an irrelevant protein like luciferase (Fig. 4, A and
C). The same phenomenon was observed in other cell extracts.
As an example, the addition of RLI to Daudi or HeLa cell extracts
inhibited the binding of 2-5ApCp by RNase L
(Fig. 4B). It is interesting to note that the
translation of RLI in wheat-germ extracts did not inhibit the binding
of 2-5ApCp by 2DR (Fig. 4A). This difference
between the two translation systems has not been elucidated but could
reflect differences in the post-translational modifications of the
protein; another hypothesis is discussed later.
The inhibition of RNase L by RLI seems specific because we observed
no inhibition of pancreatic RNase or nuclease T2 (data not shown).
The human 2-5A-dependent RNase L inhibitor described in
this paper was cloned by the screening of an expression library with
2-5ApCp. Its 2-5A-binding characteristics differ from those
of the 2DR polypeptide recently cloned by Zhou et al.(1993)
(22) since RLI does not bind 2-5ApCp under conditions
classically used to characterize RNase L, e.g. 2-5A
binding and covalent binding. Our experimental conditions differ from
those used by Zhou et al.(1993)
(22) as following (i)
the
The expression of this clone
inhibits the activation of RNase L by 2-5A and leads to the
inhibition of its endoribonuclease activity. This clone was therefore
termed RLI for RNase L Inhibitor.
Even at high dose (10
Interestingly, the
P-loop motif of RLI and of 2DR is repeated. Whether a P-loop is
required for 2-5A binding by RLI as demonstrated for 2DR (22) has
not yet been established. The first P-loop is certainly not required
for 2-5A binding or for RNase L inhibition by RLI since these two
properties remain in the truncated (H2ABP) form of RLI.
Another
interesting motif is the CX
The radiobinding and
covalent binding assays are routinely performed with an 8-fold excess
of 2-5ApCp. A competition between RLI and RNase L for the binding
to 2-5ApCp seems therefore unlikely in these conditions.
Moreover, increasing the concentration of 2-5ApCp while keeping
constant the ratio between the two proteins does not modify inhibition
of the 2-5ApCp binding by 2DR. This confirms that the ratio
between RNase L and its RLI inhibitor is critical while the
concentration of 2-5ApCp is not a limiting factor in these
particular experimental conditions.
The inhibition of RNase L by RLI
is probably due to a direct interaction of the two proteins and not to
a stable modification of RNase L. The two proteins are indeed
immunoprecipitated in a complex with the RNase L associated RNABP which
we have already described (Fig. 6). This is not surprising since
(i) mAb3 recognizes a single RNABP which is associated with the
2-5ABP in the RNase L protein complex and inhibits the
2-5A-dependent RNA cleavage by RNase L
(18) . (ii) Hassel
et al.(1993) have described sequences allowing interactions
between 2DR and other proteins
(6) , and (iii) analogous
sequences exist in RLI cDNA (Fig. 1). The immunoprecipitation of
2DR in association with RNABP was also expected since it probably
reflects the organization of the natural RNase L complex. On the other
hand it seems logical that a regulatory protein (RLI) could be
associated with the protein it regulates (RNase L). Interestingly, the
immunoprecipitation of 2DR and RLI only happens when RNABP is present
and when these proteins are associated. This does not occur in
wheat-germ extract where RNABP is absent (Fig. 3). In addition
RLI translated in wheat-germ extract does not inhibit RNase L activity.
The inhibition of RNase L activity by RLI therefore seems to require
a direct association between the two proteins (as tentatively
illustrated in Fig. 10). This hypothesis is further supported by
the fact that the electrophoretic dissociation of the two proteins (RLI
and RNase L) restores the 2-5A binding capacity of RNase L
(Fig. 8). Some unknown factors remains. For example, we do not
know whether RLI interacts directly with 2-5ABP (or 2DR) or by
the intermediate of RNABP
(18) . The physiological role of RNABP
itself remains to be elucidated since cloned 2DR behaves as a fully
active 2-5A dependent nuclease
(22) . Antibodies directed
against each protein as well as cloned functional proteins will
hopefully allow determination of the role of each component of the
putative 2-5A-RNase L complex. Work along those lines is now in
progress in our laboratory.
The
inhibition of the anti-EMCV activity of IFN in RLI-transfected cells is
only partial which could be due to the remaining RNase L activity in
this clone. This could reflect the partial inhibition of nuclease
activity observed in vitro (Fig. 5). Likewise the
inhibition of RNase L pathway during EMCV infection needs a high level
of expression of a truncated form of 2DR behaving as a dominant
negative competitor
(6) . It was also demonstrated that a
2-5A inhibitor analog inhibited IFN protection against EMCV by
10-fold only (45). Another possibility is therefore that a second
pathway like the IFN-induced protein kinase (PKR) is also required for
a full expression of the IFN antiviral activity against picornaviruses.
It has indeed been shown that PKR is also implicated in the molecular
mechanism of EMCV inhibition
(46) .
Nevertheless, it is
apparent from this study that RLI is active in intact cells and in
vitro in keeping with a major rôle of the 2-5A/RNase L
in the antiviral action of IFN against picornaviruses but not on VSV
replication.
These two systems
(RLI and RI) can also be compared in their mode of action. The
equilibrium between RI and its RNase target plays a central role in the
regulation of mRNA turnover and protein biosynthesis, the two partners
being always present at the same time in the cell
(49) . In this
paper we demonstrate at least in vitro that the inactivation
of RNase L by RLI depends of the ratio between the two proteins. IFN
treatment, which increases RNase L, will modify the ratio between the
two proteins and shift the balance toward activation of the
2-5A/pathway.
RLI adds a new and potentially important level
of control and regulation of the 2-5A/RNase L pathway. Studies of
its regulation will hopefully be helpful in understanding the
regulation and the biological role of the 2-5A pathway.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We are very grateful to Dr. Gilles Uzé (IGMM,
Montpellier) for the gift of the
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
are produced and
secreted by mammalian cells in response to various inducers, such as
double-stranded RNAs
(1) or viral infection
(2) . IFNs
induce the transcription of a large family of genes
(3) involved
in the defense against viral infections, the control of cell
proliferation and differentiation, and the modulation of immune
responses
(4) .
P]pCp
probe
(14, 15) , by its ability to degrade
mRNAs
(16) , or by Western blotting with polyclonal (17) or
monoclonal antibodies
(18) .
Cells and Cell Extracts
Daudi cells were grown
in suspension culture and maintained in RPMI 1640 medium supplemented
with 10% (v/v) fetal calf serum, 60 IU/ml penicillin, and 50 µg/ml
streptomycin. HeLa cells were grown in the same medium with 2
mM glutamine. Cells were treated with Hu/
IFN, as
indicated; IFN was a generous gift of Dr. Ara Hovanessian (Institut
Pasteur, Paris, France).
g.
Radiobinding Assay for RNase L and Radiocovalent Affinity
Labeling of RNase L
The radiobinding assay
(25) and the
covalent labeling procedure
(26) were performed with S10 cell
extracts, rabbit-reticulocyte lysates, or wheat-germ extracts (with or
without the different cloned proteins translated) as a source of RNase
L. 2-5A-3`-[
P]pCp (3000
Ci/mmol)
(27) was used as probe, as indicated in the legends of
the figures. The radiobinding and radiocovalent labeling procedures
were utilized with the modifications previously described
(21) .
Isolation of cDNA Clones
The -Zap Daudi cell
cDNA library (10
plaques) was a generous gift from Dr. G.
Uzé (IGMM, Montpellier, France). It was screened with
2-5A
-3`-[
P]pCp (3000 Ci/mmol).
The nitrocellulose filters were soaked in 10 mM
isopropyl-1-thio-
-D-galactopyranoside, dried, and
overlaid onto phage plates for 3 h at 37 °C. The filters were
rinsed in buffer A (10 mM HEPES, pH 7.5, 80 mM KCl, 5
mM Mg(OAc)
, 1 mM EDTA, 5% (v/v) glycerol,
20 mM
-mercaptoethanol)
(15) saturated with 5%
(v/v) skimmed milk in buffer A, and rinsed again in buffer A without
milk. They were then incubated overnight at 4 °C in buffer A with
2-5A
-3`-[
P]pCp (5
10
disintegrations/min/filter). After washing in buffer A,
the filters were autoradiographed on x-ray films. This procedure has
led to the isolation of a 2861-bp clone named H2ABP.
Figure 1:
Cloning
of the full-length cDNA encoding the RLI. A, the H2ABP cDNA
(2861 bp) was isolated from a Daudi cells -Zap expression library
by screening with 2-5ApCp. The full-length cDNA clone RLI (3568
bp) was isolated using anchored PCR with poly(G) cDNA and an internal
primer determined from the H2ABP sequence. The additional fragment (924
bp) (noted PCR on the scheme) was added to H2ABP in the
BglII site. The RLI cDNA contains 707 additional base pairs on
the 5` side of H2ABP. B, nucleotide sequence and deduced amino
acid sequence of the RLI DNA. Numbers on the right of the
sequence indicate the position of nucleotides and amino acids,
respectively. The box with black squares indicates the
position of the ferredoxin-like sequence. Open boxes indicate
the two P-loop motifs. The gray box shows a potential PKC
phosphorylation site. The internal repeats of 128 amino acids are
underlined in bold. In the 3`-non-coding region, the
AATAAA polyadenylation signal sequences are underlined, and
the ATTTA putative instability sequences are double
underlined.
The 2DR cDNA was obtained by PCR with primers determined from the
published sequence
(22) .
Nucleotide Sequence Analysis
H2ABP in pSK
(pBluescript II SK, Stratagene) was sequenced by the Sanger dideoxy
sequencing method (T7 sequencing kit, Pharmacia) after nested 3`
deletions. These deletions were generated with exonuclease III-S1
nuclease digestion followed by filling in with Klenow DNA polymerase
(Pharmacia). The full-length cDNA was sequenced with the Sanger dideoxy
method and internal primers.
RNA Analysis
Total cellular RNA was prepared using
the guanidine thiocyanate-lithium chloride procedure
(28) .
Northern blot hybridizations were performed by standard
techniques
(29) . Probes were synthesized by the multiprime
procedure (Ready-to-go kit, Pharmacia). After autoradiography, mRNAs
were quantified by image analysis with the Bioimage program on a
Millipore Sun Station. Each lane was normalized with the GAPDH
(glyceraldehyde-3-phosphate dehydrogenase) probe.
In Vitro Synthesis of RNA Coding for RLI and
2DR
Transcription with T3 polymerase in the presence of
mG(5`)ppp(5`)G was performed as described by the
manufacturer (Promega). RNA was analyzed and quantified by
electrophoresis in 1.2% (w/v) agarose gels in TBE buffer (50
mM Tris, pH 8, 50 mM boric acid, 1 mM EDTA).
RNA was extracted by phenol-dichloromethan (v/v) and precipitated with
ethanol, 250 mM NaCl.
10
disintegrations/min. Since the
2-5ApCp had a specific activity of 3
10
Ci/mmol and since it is considered that RNase L binds
2-5ApCp in a 1:1 ratio
(30) , the amount of RNase L in the
reticulocyte lysate assay is 1.3
10
mol.
The translation of 2DR led to a 2-fold increase in the binding of
2-5ApCp; the amount of RNase L in the reticulocyte extract is
therefore 2.6
10
mol. The concentration of
the translated proteins was also quantified by
[
S]methionine incorporation (specific activity
10
Ci/mmol). This later technique gave the same result for
2DR and 0.8
10
mol for the RLI.
Ribosomal RNA Cleavage by RNase L
Specific
hydrolysis of rRNAs by 2-5A-activated RNase L was monitored in
reticulocyte extracts with some modifications previously
described
(18) . RLI and 2DR were produced in rabbit reticulocyte
extracts. The control extract was incubated in the same conditions as
the extracts used for cell-free expression of 2DR or RLI cDNA. The
different translation incubations were complemented or not with 100
nM 2-5A and further incubated for 30 min at
30 °C. RNAs were extracted with phenol-dichloromethan and
precipitated with 2 volumes of ethanol, 300 mM sodium acetate.
The pellet was dried and resuspended in an electrophoresis loading
buffer containing 50% (v/v) glycerol, TBE buffer, and 0.25% (w/v)
bromphenol blue. Samples were analyzed on a 1.2% (w/v) agarose gel in
TBE buffer. rRNAs and their degradation products were quantified by
image analysis with the Bioimage program on a Millipore Sun Station.
Western Blot Analysis and Immunoprecipitation
Cell
extracts or in vitro translated polypeptides were analyzed by
Western blot
(31) . Proteins were fractionated on SDS-PAGE and
transferred electrophoretically to a nitrocellulose sheet. The
nitrocellulose membrane was hybridized with 2-5ApCp
(15) or with mAb3 monoclonal antibody
(18) .
-mercaptoethanol, 20% (v/v) glycerol buffer. The protein
A-Sepharose was heated for 5 min at 95 °C, and the
immunoprecipitated proteins were analyzed by 10% (w/v) SDS-PAGE. The
gel was dried and subjected to autoradiography. Stability of 2-5ApCp-Reticulocyte extracts were complemented
or not with RLI or 2DR mRNA and incubated 1 h at 30 °C in
translation conditions. 2-5ApCp was then added, and the extracts
were incubated at 4 °C for 15 min (as for radiobinding) or at 37
°C for increasing periods of time as indicated in the legend of
Fig. 7
. The loading buffer containing 50% (v/v) formamide, 1%
(v/v) bromphenol blue, and 1% (v/;V) xylene cyanol was added. The
samples were boiled 3 min and centrifuged at 10,000
g.
The supernatants were loaded onto 20% (w/v) polyacrylamide gels
containing 7 M urea. The samples were analyzed by
electrophoresis at 1,100-1,600 V until the bromphenol blue dye
reached 15 cm from the bottom of the gel. The wet gel was exposed to a
Kodak XAR5 x-ray film
(32) .
Figure 7:
Stability of 2-5ApCp in reticulocyte
extracts with or without RLI. 2-5ApCp (lanes 0,
3, and 10) was incubated with reticulocyte extract
alone (lanes 1, 4, 6, and 8) or
with reticulocyte extract and RLI (lanes 2, 5,
7, and 9) for 15 min at 4 °C (lanes 1 and 2), or for 15 min (lanes 4 and 5),
30 min (lanes 6 and 7), 60 min (lanes 8 and
9) at 37 °C.
Expression Vectors and Transfections
The human
H2ABP cDNA which encoded a truncated active form of RLI was
directionally subcloned in pcDNAIneo (Invitrogen) by standard
methods
(29) . H2ABP/pcDNAIneo or plasmid pcDNAneoI (7 µg
each) were transfected in HeLa cells by calcium phosphate
coprecipitation
(29) . Stable transfectants were selected by
culturing the cells in the presence of 1 mg/ml G418 (Life Technologies,
Inc.). Individual clones were isolated for analysis of expression of
the transfected cDNA. The clone which expressed the cDNA at the highest
level was selected for further studies.
Antiviral Activity of Interferons
pcDNAneo or
H2ABP/pcDNAIneo cells were seeded at 10 cells/well. Cells
were treated 24 h later with different dilutions of Hu
/
interferon for 20 h. Cells were then infected with vesicular stomatitis
virus (VSV) or encephalomyocarditis virus (EMCV) at a multiplicity of
infection of 1.0 for 1 h at 37 °C in RPMI medium supplemented with
5% (v/v) fetal calf serum. Unadsorbed viruses were removed by three
washings with RPMI containing 10% (v/v) fetal calf serum. Virus titers
were determined 18 h later by an end point assay as described
previously
(33) .
Expression Cloning of Human RLI
Human Daudi
cells were chosen as a source of mRNA because they contain a high basal
level of 2-5A binding activity. The cDNA library (-Zap) was
screened with a radiolabeled 2-5ApCp probe using the technique we
have developed for the renaturation of RNase L activity on filter (15
and ``Experimental Procedures''). We identified one positive
clone among 10
plaques. This clone named H2ABP for human
2-5A binding protein contains a 2861-bp insert. It has an
initiator ATG at residue 225 and is translated as a 39-kDa protein in
rabbit reticulocyte extract or in wheat-germ extract. It is in an open
reading from the first nucleotide. The full-length cDNA was, therefore,
isolated by anchored PCR with the internal primer described in
Fig. 1A. A 924-bp fragment which overlaps the H2ABP
clone by 217 bp was obtained. This sequence was added to H2ABP by
cloning in the BglII site. The sequence of the complete
3568-bp clone (RLI) is presented in Fig. 1B.
Sequence Analysis of RLI
RLI cDNA has a
5`-non-coding region of 118 nucleotides, a predicted open reading frame
extending until nucleotide 1914, and a long 3`-untranslated region of
1654 bp. The open reading frame of 599 amino acids encodes a
polypeptide of predicted molecular weight 67,515. The ATG at the
beginning of the open reading frame has the characteristics of a
``strong'' initiator codon, e.g. a purine in
position -3 and a G in +4
(34) . RLI contains two
repeated ATP/GTP-binding sites or P-loop motifs
(35) : one from
residues 110 to 117 and another from residues 379 to 386.
Interestingly, RLI has a complete homology with the
CXCX
CX
C:4Fe4S-Ferredoxin motif
between amino acids 55 and 66 (see Ref. 36 for a review). Another
remarkable feature is an internal repeat of 128 amino acids with 53%
homology starting at amino acids 200 and 440 (Fig. 1B).
RLI has a high number
(32) of thiol groups and is very rich in
leucine residues. Its estimated isoelectric point is 8.7.
Regulation of RLI RNAs Expression by IFN
The cDNA
of RLI hybridized with two cellular mRNAs of 3.5 and 2.8 kb in HeLa
cells (Fig. 2A). The same result was obtained in Daudi
and CEM cells (data not shown). Hu/
IFN treatment did not
regulate the two mRNAs which hybridize with the RLI clones, even at
high concentration. There was no quantitative variation of these mRNAs
as a function of the duration of IFN treatment. An induction of the
15-kDa protein, which has been described to be induced by IFN treatment
(37), was observed in the same conditions thus confirming that cells
responded normally to IFN treatment.
Figure 2:
Regulation of RLI mRNA expression by human
/
-IFN. A, Northern blot analysis of total
cytoplasmic RNA extracted from non-treated HeLa cells or from cells
treated with 10
units/ml of Hu
/
IFN for 2, 4, 6,
8, 10, 18, or 24 h. RNAs (20 µg) were fractionated on a 1.2% (w/v)
agarose gel and hybridized with the RLI cDNA probe, with a human 15-kDa
probe or with a human GAPDH probe. The filter was submitted to
autoradiography for 5 h for the GAPDH probe and for 12 h for the other
probes. B, Northern blot analysis of total cytoplasmic RNA
extracted from HeLa cells. RNAs (20 µg) were fractionated on a 1.2%
(w/v) agarose gel and hybridized with the A and B RLI
cDNA probes described in Fig. 1A.
To approach the difference
between the 2.8- and 3.5-kb mRNAs, we made use of a 762-bp probe (A in Fig. 2B) specific of the 5` end of RLI and a
764-bp probe (B in Fig. 2B) representing its 3`
end; their hybridization positions are indicated in
Fig. 1A. As shown in Fig. 2B, probe A
hybridizes with the two mRNAs while probe B hybridizes uniquely to the
larger mRNA. These two mRNAs therefore differ in their 3`-untranslated
region. The similarity between the sizes of the two natural mRNAs and
those of the cDNAs we have cloned is purely coincidental.
In Vitro Expression and Properties of RLI
The
biological properties of the RLI clone were studied after expression in
two cell-free translation systems, e.g. in a wheat-germ S30
(which is deficient in 2-5A/RNase L system) and in a rabbit
reticulocyte lysate (in which the 2-5A/RNase L system is
present).
Figure 3:
In vitro translation of RLI cDNA.
A, RLI (lanes 2 and 4) or 2DR (lanes 1 and 3) cDNAs were transcribed and translated in vitro in reticulocyte (lanes 1 and 2) or in wheat-germ
(lanes 3 and 4) extracts in the presence of
[S]methionine. The products were analyzed by
SDS-PAGE and autoradiography. B, reticulocyte extracts with no
mRNA (lane 1), after translation of 2DR (lane 2), or
RLI (lane 3), and wheat-germ extracts with no mRNA (lane
4), after translation of 2DR (lane 5), or RLI (lane
6) were analyzed by SDS-PAGE and Western blot with the mAb3
monoclonal antibody specific of the RNase L complex. C,
translation reactions of 2DR or RLI in wheat-germ or reticulocyte
extracts and control extracts were spotted on nitrocellulose. No RNase
L, 2DR, or RLI in wheat germ (0); 0.8
10
mol of RLI in wheat germ or in reticulocyte extracts
(RLI); 1.3
10
mol of 2DR in wheat
germ or in reticulocyte extracts (2DR); 1.3
10
mol of endogenous RNase L in reticulocyte
extracts (0, RLI, and 2DR) (see
``Experimental Procedures''). The nitrocellulose sheet
was
hybridized with a 2-5ApCp probe and analyzed by
autoradiography.
RLI slightly bound 2-5ApCp when
the translation incubations in wheat-germ extracts were spotted on a
nitrocellulose sheet and incubated with the 2-5ApCp probe
(Fig. 3C) by the protocol used to screen the -Zap
library (``Experimental Procedures'' and data not shown).
Surprisingly, we observed a decreased binding of 2-5ApCp by the
endogenous RNase L when RLI was expressed in a rabbit reticulocyte
lysate. At variance, an increase in the binding of 2-5ApCp was
clearly observed when 2DR was expressed in the wheat-germ or in the
rabbit reticulocyte extracts (Fig. 3C).
Figure 4:
Inhibition of 2-5A-binding on RNase
L by RLI. A, mRNAs coding for luciferase, RLI, or 2DR were
translated in reticulocyte or wheat-germ extracts. Various
concentrations of the translation products were added to a reticulocyte
or to a wheat-germ extract, or to an extract in which the 2DR mRNA has
already been expressed. This gave rise to different ratios between RLI
(or luciferase), endogenous RNase L, and in vitro expressed
2DR, as indicated. The extracts were then tested in a 2-5A
binding assay. The results are expressed as percent of 2-5ApCp
bound. 100% is the binding obtained when 2DR alone was expressed.
Reticulocyte + luciferase (), reticulocyte + RLI
(
), reticulocyte + 2DR + RLI(
), wheat germ +
2DR + RLI (
). B, RLI translated in reticulocyte
extract was added to Daudi (1) or HeLa cell extracts
(2). The extracts were then tested in a 2-5ApCp binding
assay. The results are expressed in percent of 2-5ApCp bound.
100% is the 2-5ApCp binding observed in Daudi or HeLa cell
extracts supplemented with a reticulocyte lysate but not with RLI.
C, covalent 2-5ApCp binding in reticulocyte extract by
endogenous RNase L or by in vitro expressed 2DR in the
following conditions: 1, reticulocyte extract alone;
2, with translated RLI; 3, with translated 2DR;
4, with translated 2DR and luciferase; 5, with
translated 2DR and RLI.
The RLI-mediated
inhibition of 2DR activity did not result from a competition for
2-5A. Indeed, the inhibition of 2-5ApCp binding by 2DR was
not modified if the experiment was performed with increasing
concentrations of 2-5ApCp provided while the ratio between 2DR
and RLI was not modified (data not shown). It is important to notice
here that the initial truncated H2ABP clone exhibited the same
properties than the full-length one; it inhibited 2-5ApCp binding
by endogenous RNase L or by in vitro expressed 2DR (data not
shown). RLI Antagonizes the 2-5A-dependent Nuclease Activity of Endogenous
RNase L or Cloned 2DR-Since RLI inhibited 2-5ApCp binding,
it was expected to behave as a RNase L antagonist. We, therefore,
tested whether the expression of RLI also inhibited its nucleolytic
activity in a cell-free assay. The rRNAs and their specific degradation
pattern that constitute an index of 2-5A/RNase L activity (50)
were studied in rabbit reticulocyte extracts supplemented or not with
RLI, 2DR, and 2-5A (Fig. 5). Total RNAs from the extracts
were fractionated by agarose gel electrophoresis
(Fig. 5A) and their degradation products were quantified
by image analysis (Fig. 5B). RLI and 2DR have not yet be
purified and were therefore expressed by the in vitro translation of their mRNAs in reticulocyte lysates. The
2-5A/RNase L pathway is functional in these extracts even in the
absence of exogenous 2-5A as evident from the occurrence of rRNAs
degradation products (as marked by arrows in
Fig. 5A). Wheat-germ extracts in which the 2-5A
pathway is not functional could not be used as a source of recombinant
nuclease since cloned 2DR is inactive even though it is capable of
binding 2-5A
(22) . Likewise, we did not succed in
expressing RLI as a functional protein in a wheat-germ extract (see
above). Nevertheless, the addition of 2-5A in the rabbit
reticulocyte extract gave rise to a slight but reproducible increase of
rRNA degradation which no longer occurred where RLI was also present
(Fig. 5A, lanes 2 and 4, and
Fig. 5B, diagrams 1 and 2).
Figure 5:
Inhibition of RNase L activity by RLI.
A, reticulocyte extracts alone (lanes 1 and
2) and supplemented with RLI mRNA (lanes 3 and
4), 2DR mRNA (lanes 5 and 6), or 2DR and RLI
mRNAs (lanes 7 and 8) were incubated 60 min at 30
°C to generate the recombinant proteins. They were then
supplemented (lanes 2, 4, 6, and 8)
or not (lanes 1, 3, 5, and 7) with
2-5A and further incubated for 30 min at 30 °C.
rRNAs were analyzed on a 1.2% (w/v) agarose gel. Intact 28 S and 18 S
rRNAs migration is indicated on the left of A. Major rRNAs
degradation products are indicated by arrows on the right of
A. B, the rRNAs and their degradation products were
quantified by image analysis and represented on diagrams. Diagram
1, reticulocyte alone; diagram 2, reticulocyte with RLI;
diagram 3, reticulocyte with 2DR; diagram 4,
reticulocyte with 2DR and RLI.
Similar
observations have been made in extracts supplemented with cloned 2DR.
Here again rRNA degradation had already taken place in the control
extract and exogenous 2-5A largely increased degradation
(Fig. 5A, lanes 5 and 6, and
Fig. 5B, diagram 3). The latter was antagonized
by the co-expression of RLI (Fig. 5A, lanes 7 and 8 and Fig. 5B, diagram 4).
The degradation of rRNAs observed in extracts which were not
supplemented with 2-5A was only partially inhibited by RLI.
Whatever the explanation it is clear that RLI expression antagonizes
the 2-5A-dependent nucleolytic activity of RNase L whether
endogenous to the reticulocyte extract or provided from cloned 2DR.
Association of RLI with RNase L
We have previously
established that the protein which binds 2-5A (2-5ABP) was
co-immunoprecipitated with a RNABP by mAb3 in a high molecular weight
complex. mAb3 is a specific monoclonal antibody which recognizes only
RNABP and neutralizes RNase L activity
(18) . In vitro translated 2DR was immunoprecipitated with RNABP as expected from
our previous results
(18) (Fig. 6, lane 4).
Likewise, in vitro translated RLI was co-immunoprecipitated
with RNABP from reticulocyte extracts (Fig. 6, lane 2).
When present together RLI and 2DR were also immunoprecipitated by mAb3
(Fig. 6, lane 5). On the contrary, when the two proteins
were translated in wheat-germ extract (in which RNABP is absent, see
Fig. 3B) they were not immunoprecipitated by mAb3
(Fig. 6, lanes 1 and 3). RLI Does Not Degrade 2-5A-The RLI-induced inhibition of
2-5ApCp binding by endogenous RNase L or by in vitro expressed 2DR could result from 2-5ApCp degradation. In
order to eliminate this possibility, 2-5ApCp was incubated with a
reticulocyte extract alone or with a reticulocyte extract supplemented
with translated 2DR or RLI, at 4 °C (in the radiobinding assay
condition) or at 37 °C for increasing periods of time. Undegraded
2-5ApCp was quantified by acrylamide-urea gel
electrophoresis
(32) . A dephosphorylation of 2-5ApCp was
observed (at 4 or 37 °C) in the reticulocyte extract, whether
incubated alone or supplemented with RLI (Fig. 7). The
degradation of 2-5ApCp was not increased when RLI was added
(compare, for example, lanes 1 and 2, or 4 and 5 in Fig. 7) in experimental conditions in
which an inhibition of RNase L was observed. In a separate experiment
2-5A was incubated without terminal pCp and analyzed
by high performance liquid chromatography on a C18 µBondapack
column
(11) . Once again, no increased degradation was observed
upon RLI addition (data not shown).
Figure 6:
Immunoprecipitation of RLI and 2DR by
mAb3. RLI or 2DR were translated in reticulocyte or wheat-germ extracts
in the presence of [S]methionine. mAb3 (1/1000)
was then added in the translation incubation. The mAb3
protein
complex was precipitated with protein A-Sepharose and analyzed on
SDS-PAGE. 1, RLI translated in wheat-germ extract. 2,
RLI translated in reticulocyte extract. 3, 2DR translated in
wheat-germ extract. 4, 2DR translated in reticulocyte extract.
5, co-immunoprecipitation of 2DR and RLI by mAb3 in
reticulocyte extract.
Separation of RLI and RNase L Reactivates RNase
L
To determine whether RLI stably modifies the 2-5A
binding capacity of RNase L, the two proteins were dissociated by gel
electrophoresis and the capacity of RNase L to bind 2-5ApCp was
determined by the Western blot assay
(15) . As shown in
Fig. 8
, no more difference in the binding of 2-5ApCp was
observed whether the reticulocyte extract had been incubated alone
(Fig. 8, lane 1) or had been supplemented with RLI mRNA
(Fig. 8, lane 2). This should be compared with the data
reported in Fig. 4where a 80% inhibition of 2-5ApCp
binding was observed when RLI and RNase L were not separated. Inhibition of the 2-5A/RNase L Pathway in Intact Cells-RLI
inhibits 2-5ApCp binding and behaves as a RNase L inhibitor in
cell-free extracts. We therefore tested whether its overexpression
antagonized the IFN-regulated 2-5A/RNase L pathway in intact
cells. HeLa cells were stably transfected with the H2ABP cDNA clone
which codes for a truncated but fully functional protein as already
mentioned.
Figure 8:
2-5A binding activity of RNase L on
filter. 1, reticulocyte-extract alone, or 2, with
translated RLI was analyzed by SDS-PAGE, transferred to nitrocellulose
sheet, and incubated with 2-5ApCp as indicated under
``Experimental Procedures.'' After washing, the membrane was
submitted to autoradiography.
The 2-5ApCp binding by RNase L in the
H2ABP/pcDNAIneo selected clone is reduced by 30% as compared to non
transfected HeLa cells or to cells transfected with the vector alone
(data not shown). This clone (H2ABP/pcDNAIneo) and a clone transfected
with the empty vector (pcDNAIneo) were compared for the antiviral
effect of IFN against VSV and EMCV (Fig. 9).
Figure 9:
Expression of the truncated active RLI
supresses a part of the anti-EMCV activity of IFN. Control pcDNAIneo
cells () or H2ABP/pcDNAIneo cells (
) were incubated for
20 h in the absence or presence of Hu
/
IFN at the indicated
concentration and infected thereafter with EMCV (A) or VSV
(B). Virus was harvested 18 h later and titrated on indicator
cells as indicated under ``Experimental
Procedures.''
IFN treatment of
the pcDNAIneo cells resulted in a dose-dependent reduction in EMCV
yield (Fig. 9A). The antiviral activity of IFN at
concentrations as high as 1000 units/ml was significantly reduced in
H2ABP/pcDNAIneo cells (Fig. 9A), in keeping with a
reduced level of 2-5ApCp binding by RNase L. In contrast, IFN
protection from VSV challenge was similar in the H2ABP/pcDNAIneo cells
and in control pcDNAIneo cells (Fig. 9B). These results
indicate that RLI inhibits the IFN activation of RNase L pathway in
vivo as well. Moreover they confirm that the 2-5A system is
a potent inhibitor of EMCV replication and that anti-VSV effect of IFN
must predominantly be mediated by another pathway
(6) .
-Zap library was prepared from Daudi cells with no IFN
induction, (ii) the filters were screened without guanidine
denaturation of proteins, (iii) 2-5ApCp was prepared from natural
2-5A rather than from bromo-substitued 2-5A (the two have
different affinities for RNase L, 38).
RLI mRNAs Are Not Regulated by IFN
The RLI cDNA
hybridizes with two mRNAs of 3.5 and 2.8 kb (Fig. 1) which differ
in their 3`-untranslated regions. The biological significance of these
two mRNAs has not been investigated. Several putative AUUUA instability
sequences (39) are present in their 3`-untranslated region, perhaps
indicating differences in their post-transcriptional regulation. Their
half-lives in HeLa cells following actinomycin-D treatment are,
however, similar.
units/ml) IFN
does not regulate RLI mRNAs abundance. This contrasts with the fact
that IFN regulates the 2-5A pathway with a large increase in the
level of 2-5A synthetases and a 3-fold increase in RNase L
mRNA
(22) . The ratio between the activator (2-5A), RNase
L, and the inhibitor will therefore be shifted toward activation after
IFN treatment. Further investigations are, however, required to confirm
if these two proteins always display opposite regulation in
circumstances under which RNase L activity is modified.
Consensus Sequences in RLI cDNA
The cDNA of RLI
exhibits a few interesting features. RLI contains two phosphate-binding
loop (P-loop) motifs between amino acids 110-117, and
379-386 (Fig. 1B). These P-loop motifs are
conserved in most adenine and guanine nucleotide-binding proteins, such
as adenylate kinase, RecA protein, ras oncogene product p21,
heterodimeric G proteins, elongation factors, or proteins involved in
active transport (see Ref. 35 for a review).
CX
CX
C
ferredoxin-like sequence between the amino acids 55 and 66
(Fig. 1B). This ferredoxin-like sequence is found in
proteins with very differ(
)ent functions and in particular in
proteins which interact with nucleic acids such as bacterial
endonuclease III. Kuo et al.(1992) suggested that endonuclease
III is the prototype of a new class of iron-sulfur proteins wherein the
primary rôle of the iron-sulfur cluster is to position conserved
basic residues for interaction with the phosphate backbone of the DNA
substrate
(40) . The iron-sulfur cluster plays a rôle
analogous to the zinc atom in ``zinc-finger''
proteins
(41) . This cysteine-rich region in RLI could be
involved in binding of RLI with nucleic acids. Alternatively, this
cysteine-rich region could mediate the formation of heterodimers with
RNase L. We indeed demonstrated here that RNase L can be
immunoprecipitated in association with RLI (Fig. 6). Finally, it
is worth mentioning here that RNase L behaves as a high molecular
weight complex in non-denaturing conditions
(19) .
Inhibition of RNase L Activity by RLI Is Due to the
Association of the Two Proteins
The inhibition of RNase L
activity by RLI is not due to the degradation of 2-5A. RLI does
not increase the degradation rate of 2-5ApCp in a reticulocyte
extract (Fig. 7) in the conditions where an inhibition of
2-5A binding is observed (Fig. 4).
Figure 10:
Hypothetical scheme of RNase L activation
by 2-5A and its inhibition by RLI.
The human Daudi cell RLI is active on
RNase L from other human cells (HeLa) or from other species (rabbit) in
keeping with the finding by Zhou et al.(1993) that 2DR is a
well conserved protein (22). Expression of RLI Inhibits the 2-5A/RNase L Pathway in Intact
Cells-The inhibitor of RNase L is also active in intact cells
(Fig. 9). Its overexpression in HeLa cells partly antagonizes the
antiviral effect of IFN against EMCV but not VSV as
expected
(6) . This confirms and extends previous studies showing
that the 2-5A system is an important component of the antiviral
activity of IFNs on picornaviruses. The overexpression of a cDNA coding
for the 40 kDa form of 2-5A-synthetase largely reduces
picornavirus yield
(42, 43, 44) .
Biological Role of RLI
Hassel et
al.(1993) have recently confirmed the central role played by RNase
L and the 2-5A system in the antiviral and in the
antiproliferative effects of IFNs, and they have postulated its
implication in the control of mRNA stability
(6) . 2-5A and
RNase L are known to vary in various physiological conditions as IFN
treatment, cell growth arrest, or hormone status. Several authors (see
Ref. 24 for a review) have underlined the importance of nucleases in
the regulation of mRNA stability and consequently in gene regulation.
As cited in the introduction, the 2-5A system is a well
characterized system of RNA degradation whose activation was believed
to be controlled mainly by 2-5A synthetase(s) and 2-5A. RLI
might constitute an important additional element for the
down-regulation of the 2-5A pathway. RI or RAI for
ribonuclease/angiogenin inhibitor was, to our knowledge, the only other
known RNase inhibitor cloned
(47, 48) . RI belongs to a
highly diversified protein superfamily with a common repetitive module.
RLI shows no sequence homology with this inhibitor which is not
surprising since RNase L is very different from placental ribonuclease
and angiogenin. It is of note, however, that both RNase inhibitors are
very rich in leucine residues and in thiol groups.
/EMBL Data Bank with accession number(s) X74987 and
X76388.
Zap library, to Dr. Ara
Hovanessian (Institut Pasteur, Paris) for the gift of Hu
/
IFN, to Catherine Tissot (IGMM, Montpellier) for the gift of the 15-kDa
cDNA, to Dr. Sylvie Huck (IGMM, Montpellier) for assistance in PCR
technics, and Dr. Ian Robbins (IGMM, Montpellier) for revising this
manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.