(Received for publication, December 4, 1995; and in revised form, February 7, 1996)
From the
Continuous replication of human immunodeficiency virus type 1 requires the expression of the regulatory protein Rev, which binds to the Rev response element (RRE) and up-regulates the cytoplasmic appearance of singly spliced and unspliced mRNA species. It has been demonstrated that the murine protein YL2 interacts with Rev in vivo and modulates the activity of Rev (Luo, Y., Yu, H., and Peterlin, B. M.(1994) J. Virol. 68, 3850-3856). Here we show that the YL2 human homologue, the p32 protein, which co-purifies with alternative splicing factor ASF/SF2, interacts directly with the basic domain of Rev in vitro and that the Rev-p32 complex is resistant to high concentrations of salt or nonionic detergent. Protein footprinting data suggest that Rev interacts specifically with amino acids within the 196-208 region of p32. An analysis of the ternary complex, formed among p32, Rev, and RRE RNA, shows that Rev can bridge the association of p32 and RRE. Furthermore, we demonstrate that exogenously added p32 specifically relieves the inhibition of splicing in vitro exerted by the basic domain of Rev. Our data are consistent with a model in which p32 functions as a link between Rev and the cellular splicing apparatus.
Human immunodeficiency virus type 1 (HIV-1) ()expresses at least five proteins, Gag, Pol, Env, Tat, and
Rev, which are absolutely essential for viral replication. Gag, Pol,
and Env proteins, which are associated with the virion particle, are
encoded by unspliced and singly spliced mRNA species, characteristic
for the late stage of the viral gene expression. Tat and Rev,
translated from multiply spliced HIV-1 mRNAs characteristic for the
early stage of the gene expression, have regulatory roles in the viral
life cycle. Although Tat and Rev share some structural features, their
functions are distinct. Tat binds to the transactivating region, TAR,
located in the 5`-end of the viral transcript and up-regulates the
viral transcription several hundred-fold. Rev acts at the
posttranscriptional level and stimulates the cytoplasmic appearance of
unspliced and singly spliced viral mRNAs (for reviews, see (1, 2, 3) ). Thus, Rev activity partially
suppresses its own synthesis and shifts the HIV-1 replication cycle
from the early to the late phase. The specificity of Rev activity is
mediated through direct binding to an RNA element, the RRE, which is a cis-component of the unspliced and singly spliced mRNAs (4, 5, 6) . The Rev protein is quite small
(116 amino acids), and two main functional domains have been defined by
mutational analysis. A cluster of leucine residues around positions
78-83 constitutes the nuclear export
signal(7, 8, 9, 10) , which
interacts directly with nuclear proteins implicated in RNA
export(11, 12, 13) . A highly basic region at
positions 34-50 is responsible for specific RNA
binding(14, 15, 16, 17, 18) ,
nuclear localization, and contributes to the oligomerization
process(18, 19, 20) . This domain has also
been shown to inhibit the splicing of RRE-containing mRNAs in
vitro(21, 22) . It was recently shown in a yeast
two-hybrid screen that the murine protein, YL2, interacts with the
basic domain of Rev and that overexpression of YL2 potentiates Rev
function in vivo(23) . The human homologue of YL2 is
the p32 protein, which copurifies with the essential splicing factor
ASF/SF2(24) . In this report, we investigate the interaction of
p32 with Rev protein and test its function in a Rev-dependent in
vitro splicing assay. We demonstrate that Rev interacts strongly
with p32 in vitro and map the binding site by protein
footprinting. Together, our data suggest that p32 functions as a
mediator of Rev activity in RNA splicing.
The co-precipitation assays among GST-p32, Rev,
and radioactive RRE RNA or IIB RNA were performed by mixing 1 µl of
p32 storage buffer, containing 100-600 ng of GST-p32 (or 600 ng
of GST as a control) and 1 µl of Rev storage buffer, containing
0-300 ng of Rev (or 0-100 ng of Rev-(34-50)) with 4
10
cpm (approximately 2 ng) of body-labeled,
renatured RRE or IIB RNA in 8 µl of Rev binding buffer (10 mM HEPES/KOH, pH 7.9, 100 mM KCl, 2 mM MgCl
, 0.5 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 0.5 units/µl RNasin (Promega),
50 ng/µl E. coli tRNA) containing 0.2% Tween 20 and 0.2
µg/µl bovine serum albumin, followed by incubation at 4 °C
for 1 h. Twenty µl of a 50% slurry of glutathione-Sepharose beads
equilibrated in Rev binding buffer (including Tween 20 and bovine serum
albumin) were added to each binding reaction, and incubation was
continued with rocking at 4 °C for 1 h. Beads were washed five
times in Rev binding buffer (with Tween 20 and without bovine serum
albumin) and mixed with 100 µl of RNA elution buffer (0.3 M NaCH
COO, 1 mM EDTA, 50 ng/µl E. coli tRNA), and bound RNA was eluted by extraction with phenol and
phenol/chloroform followed by ethanol precipitation. The RNA pellet was
resuspended in 80% formamide, incubated at 95 °C for 3 min, and
analyzed on 4% (RRE) or 8% (IIB) denaturing polyacrylamide gel followed
by autoradiography.
The protein band shift assay was performed by mixing 1 µl of Rev storage buffer, containing 0-300 ng of Rev or 0-100 ng of Rev-(34-50), with 9 µl of Rev binding buffer containing 100 ng of labeled GST-p32-K protein. After a 15-min incubation on ice, the reactions were loaded on a 4% native polyacrylamide gel, containing 50 mM Tris borate, pH 8.3, 1 mM EDTA and run at 4 °C, followed by autoradiography. In some experiments the binding was performed in co-precipitation buffer, containing 10% glycerol and 50 ng/µl E. coli tRNA.
Figure 1:
In vitro interaction between
p32 and Rev. A, a Coomassie-stained protein gel showing the
ability of Rev to co-precipitate with immobilized GST-p32 protein. Two
µg of GST-p32 and 0.45 µg of Rev protein (1:1 molar
concentration) were mixed with 2 µg of bovine serum albumin and
used as input for affinity selection on glutathione-Sepharose beads (lane 5). The proteins retained on the beads after binding and
washings in buffers containing the denoted concentrations of NaCl (lanes 8-12) or nonionic detergent Tween 20 (lanes
13-17) were analyzed. Leaving out the GST-p32 protein (lane 6) or replacing it by GST (lane 7) essentially
eliminated Rev binding. GST, GST-p32, and Rev protein were loaded in lanes 2, 3, and 4, respectively, as markers.
Sizes of marker proteins (lane 1) are indicated. B,
autoradiogram of a native polyacrylamide gel showing the protein band
shift analysis of p32-Rev complexes. One hundred ng of radioactively
labeled GST-p32-K protein (corresponding to a concentration of 0.2
µM) was incubated with the indicated concentrations of Rev
protein and analyzed by native gel electrophoresis. The major discrete
bands, corresponding to free GST-p32-K and GST-p32-KRev complex,
are indicated. C, protein band shift analysis similar to that
in panel B. The complexes formed between GST-p32-K
(concentration of 0.2 µM) and the indicated concentration
of Rev-(34-50) were analyzed in the absence (lanes
2-5) or presence of Rev (lanes 7-11). Lanes 1 and 6 are control lanes showing GST-p32-K
alone. The asterisks in panels B and C indicate a complex that appeared in variable amounts depending on
the p32 preparation. We suspect that it originates from the binding of
Rev to a truncated p32 lacking the GST part, which is a common
degradation product from GST-p32 (T. Ø. Tange, T. H. Jensen, and
J. Kjems, unpublished observation).
The
interaction between p32 and Rev was also investigated using a protein
mobility shift assay. The GST-p32-K fusion protein was labeled at the
C-terminally positioned HMK site, and its ability to form complexes
with Rev in a native gel electrophoresis assay was investigated (Fig. 1B). In the presence of a 2-fold molar excess of
Rev to p32 fusion protein, approximately half of the p32 was shifted to
a major slower migrating band (Fig. 1B, lane
4). The absence of any major higher order complexes at maximum Rev
concentration (Fig. 1B, lane 7) suggests that
Rev has only one major binding site on p32 and that Rev oligomerization
is limited or not detectable in this assay. No differences in the Rev
binding potential were observed when using GST-p32 or p32-K in the
binding reaction, implying that the GST and HMK tags did not interfere
with Rev interaction (data not shown). The binding of GST-p32-K protein
to a synthetic peptide covering only the basic domain of Rev
(Rev-(34-50)) was also investigated (Fig. 1C).
Addition of a 2-fold molar concentration of Rev-(34-50) to p32
fusion protein resulted in a slower migrating complex (Fig. 1C, lane 3). At larger concentrations of
Rev-(34-50) a second complex appeared, which may represent
binding of multiple Rev-(34-50)s to a single p32 fusion molecule (Fig. 1C, lane 5). The relatively large shift,
caused by the binding of a 17-amino acid peptide to a 518-amino acid
large p32 fusion protein, may be explained by the high positive charge
of the peptide. The binding of Rev-(34-50) to p32 fusion protein
was also investigated in a competition assay in the presence of intact
Rev protein (Fig. 1C, lanes 6-11).
Rev-(34-50) efficiently competed with Rev for p32 binding, and
comparable levels of p32Rev-(34-50) and p32-Rev complexes
were formed in the presence of similar molar concentrations of
Rev-(34-50) to Rev (Fig. 1C, lanes 9 and 10). This result implies that Rev-(34-50) and intact Rev
protein bind with comparable affinities to p32.
Figure 2: Investigating the p32-Rev interaction in the context of RRE. A, autoradiogram of a denaturing polyacrylamide gel showing the amount of radioactively labeled RRE RNA that was retained on glutathione-Sepharose beads in the presence of increasing amounts of Rev and GST-p32 proteins (lanes 6-11) or GST as a control (lanes 3-5). GST-p32 protein, at the indicated concentration, was incubated with increasing concentrations of Rev protein and a fixed amount of radioactively labeled RRE RNA. The total input of RRE probe in each lane is shown in lane 1. B, experiment similar to that in panel A except that Rev was replaced with Rev-(34-50) at different concentrations. C, RNA band shift analysis of the effect of GST-p32 on Rev-RRE complex formation. Two ng of radioactively labeled RRE RNA was complexed with Rev, at the indicated concentrations and challenged with increasing concentrations of GST-p32 protein or GST protein as a control, according to the scheme above. The position of the free RRE probe is indicated.
Formation of the
GST-p32Rev
RRE complex was also analyzed by a gel mobility
shift assay (Fig. 2C). Rev forms multiple complexes
with RRE in this type of assay (Fig. 2C, lanes 5 and 10)(29) . The GST-p32 protein, by itself,
exhibited no measurable affinity for the RRE (Fig. 2C, lanes 1-3). The addition of increasing amounts of
GST-p32 protein together with a constant amount of Rev protein
gradually inhibited formation of the larger Rev-RRE complexes (Fig. 2C, lanes 6-8 and 11-13). This effect was specific to GST-p32 and was not
observed with GST alone (Fig. 2C, lanes 9 and 14) or with human TATA-box binding protein (data not shown).
Changing the order of addition of p32 and RNA to Rev protein or
replacing the RRE probe with a single high affinity Rev binding site
(IIB), gave a similar result (data not shown). Surprisingly, we were
not able to observe any novel complexes that could represent the
ternary complexes among GST-p32, Rev, and RRE or IIB, predicted from
the co-precipitation experiment (Fig. 2A). This
suggests that such complexes are unstable under gel electrophoresis
conditions.
Figure 3: Footprinting the binding site of Rev protein on p32. A, autoradiogram of a protein gel showing the protein footprint of Rev on p32. One hundred ng of C-terminally labeled p32-K protein was digested with Glu-C proteinase at two different concentrations in the presence of 1 µg of Rev protein (approximately 18 times molar excess) or the same amount of bovine serum albumin, indicated by + and -, respectively. C denotes the control lane in which Glu-C proteinase was omitted. Glu-C cleavages, which were specifically inhibited or enhanced by the Rev protein, are marked with solid and open arrows, respectively. The indicated Glu-C-specific cleavage sites were putatively identified on the basis of their relative mobility of products from other specific proteinases and from interpolations on a graph indicating the relationship between the logarithm of the mass of radioactive C-terminal fragment produced by proteolytic cleavage and migration of the corresponding band on the gel (data not shown). B, amino acid sequence of pro-p32. Glu-C-specific cleavages, which are specifically inhibited or enhanced by the Rev protein, are marked with solid and open arrows, respectively. Dotted lines indicate that the exact identification of glutamic acids in this region was uncertain, and underlining marks a region that is relatively inaccessible to all tested proteinases. Glutamic acids are denoted by boldface E, and amino acid numbering is done according to the pro-p32. The N termini of pro-p32 and the processed form of p32 are indicated by horizontal arrows.
Figure 4: p32 relieves the specific inhibition of Rev-(34-50) on splicing in vitro. A, autoradiogram of splicing products. Splicing reactions, containing two substrates, PIP/B1/RRE transcript (containing the RRE) and PIP7.A (internal control lacking the RRE), were incubated under splicing conditions without nuclear extract (NE) (lane 1), or with nuclear extract in the presence of the indicated amount of Rev-(34-50) peptide, and GST-p32 protein (lanes 2-7) or GST protein as a control (lanes 8 and 9). The identities of the splicing products are indicated schematically. RRE is indicated by a black box; open boxes and thin lines correspond to the exons and the introns, respectively. The splicing products for each of the constructs include intact pre-mRNAs, lariat introns, and intermediate lariats composed of intron and 3`-exon. Ligated exons and 5`-exons migrated near the bottom of the gel and are not shown. B, a graphic representation of the RRE-specific inhibitory effect of Rev-(34-50) on splicing shown in panel A. The numbers indicated below correspond to lane numbers in panel A. Black bars and cross-hatched bars indicate the absence and presence of Rev-(34-50), respectively. The level of specific inhibition of splicing was calculated as follows. The yield of intron lariat products, containing the RRE was divided by the yield of intron lariat products from the control substrate without RRE. The numbers were normalized to 1 in the absence of Rev-(34-50) and GST-p32.
Rev plays a major mechanistic role in switching the viral expression pattern from multiply spliced viral mRNAs to singly spliced and unspliced mRNA in the cytoplasm. In addition to the viral RNA target, the RRE, Rev interacts with cellular components in order for the mRNA to escape from the default splicing and transport pathways utilized by other mRNAs. It has recently been shown that a number of nucleoporin-like proteins interact specifically with a leucine-rich motif in Rev, constituting the nuclear export domain, and that this interaction is crucial for efficient transport of the mRNAs to the cytoplasm(9, 10, 11, 12, 13) . In this report, we characterize a very stable in vitro interaction among another functionally important region of Rev, the basic domain, and the human ASF/SF2-associated p32 protein. p32 is an acidic protein, rich in glutamic and aspartic acid residues, which raises the possibility that p32 interacts with the highly basic region of Rev protein through unspecific ionic interactions. Although this possibility is difficult to disprove, particularly since simple ionic interactions often play an important role in molecular recognition, several of our observations suggest that the molecular recognition is specific and biologically relevant: (a) the p32-Rev complex was resistant to salt concentrations up to 750 mM, which destabilizes ionic interactions, (b) the p32-Rev complex appeared as a single major band on a native gel over a large titration range, and (c) only one short stretch of glutamic acids, out of several highly acidic regions in p32, was specifically protected by Rev. Furthermore, in vivo studies have shown that transient expression of the murine homologue of p32, YL2, potentiated the function of Rev up to 4-fold and that antisense YL2 transcripts abolished Rev function(23, 31) .
Since both p32 and the RRE interact with the basic domain of Rev, one may suspect that simultaneous binding of p32 and RRE to the Rev protein is impaired. The observation that Rev was capable of bridging p32 to the RRE in a solution binding assay, therefore suggests that Rev forms oligomers in such a manner that distinct basic regions can interact with the RRE and p32 independently. However, in the RNA mobility shift assay we found that p32 competed with RRE for Rev binding, and we were unable to detect any ternary complex implying a simultaneous association of p32, Rev, and RRE. We suspect that a reason for this discrepancy is an instability of the Rev-Rev interactions under the applied electrophoresis conditions. In favor of this interpretation is the previously reported differences between solution binding and gel shift assays. Whereas Rev tends to form RNA independent oligomers in solution to a variable extent, when analyzed by gel filtration or chemical cross-linking (18, 19, 32, 33) only a single complex is seen between Rev and a high affinity binding site (IIB RNA), using native gel electrophoresis(16, 34, 35, 36) .
A number of other proteins have been found to interact with p32. Originally, p32 was characterized as being a component of the ASF/SF2 splicing activity purified from HeLa cells(24) . Subsequently, it was shown that p32 was dispensable for the general splicing activity, although the possibility cannot be ruled out that p32 has a more specialized role in splicing(37) . Recent evidence suggests that p32 also interacts with the HIV-1 Tat protein(38, 39, 40) . In one report, a Tat-binding protein (TAP) was isolated on the basis of Tat affinity chromatography, and the sequence turned out to be identical to p32 except for a few amino acids in the N-terminal precursor segment(39) . The same group also found that TAP (p32) interacts with the C terminus of TFIIB, and it was suggested that TAP (p32) may function as a cellular co-activator that bridges Tat to the general transcription machinery(38) . The significance of the TAP (p32)-Tat interaction was substantiated by a two-hybrid analysis, in vitro binding studies, and a demonstration implying that TAP (p32) was able to cooperate with Tat to synergistically stimulate transcription(38, 39) . The region involved in Tat binding was mapped to amino acids corresponding to 247-282 in p32, which is outside the region where we see protection by Rev (amino acids 196-208). Also, it was found that TAP (p32) primarily interacted with a 17-amino acid conserved core segment of the Tat activation domain, whereas the basic domain of Tat was dispensable and by itself unable to bind(38) . Together, these data suggest that Tat and Rev may interact with p32 in different ways and raises the possibility that p32 plays multiple roles in HIV-1 replication.
Several lines of evidence suggest that the basic domain of Rev plays a direct functional role other than RNA binding, nuclear localization, and protein oligomerization. First, it has been demonstrated that, even when multiple Rev molecules were tethered to the mRNA through heterogeneous RNA binding sites, the basic domain was not dispensable for Rev function(41, 42) . Secondly, the basic domain alone can specifically inhibit splicing of RRE containing transcripts in vitro, suggesting a role in RNA splicing(21) . The binding potential of the basic domain of Rev for p32 and the RRE suggests that p32 is a cellular co-factor for Rev. The association of the p32 protein with ASF/SF2, which binds in a cooperative fashion with U1 snRNP to the 5`-splice site, theoretically places Rev and p32 on the same mRNA. It is therefore likely that Rev, sequestered on the RRE, is able to interact with p32 and directly influence the splicing process (Fig. 5). In this report, we show that equimolar amounts of GST-p32 and Rev-(34-50) diminished the inhibitory effect of Rev-(34-50) on splicing. A possible explanation for this antagonizing effect of p32 in this assay may be that exogenously added p32 squelches the functional interaction between Rev peptide and endogenous p32, associated with ASF/SF2, and thereby inhibits Rev function. An alternative explanation, which cannot be excluded, is that p32 may disrupt the binding of Rev-(34-50) to the RRE, thereby relieving the inhibition of splicing.
Figure 5: Putative functional model for the p32-Rev interaction. Rev protein, bound to the RRE, interacts with the p32 protein associated with ASF/SF2 at the 5`-splice site. This interaction could stabilize the interaction of U1 snRNP with the 5`-splice site and inhibit assembly of functional spliceosomes. The arrested complex may subsequently function as a substrate for Rev-mediated nuclear export.
A prediction from the model shown in Fig. 5would be that Rev may increase the stability of the U1 snRNP interaction with the 5`-splice site and thereby inhibit the subsequent displacement of U1 snRNP, which is necessary for U4/U6.U5 tri-snRNP to enter the spliceosome(43) . In support of this model, it has previously been shown that Rev specifically increases the amount of U1 snRNP in prespliceosomes formed on RRE containing mRNAs and efficiently blocks the entry of the U4/U6.U5 tri-snRNP(22) . Furthermore, in vivo experiments have demonstrated that Rev regulation of a construct, containing an intron, requires that the 5`-splice site be recognized by U1 snRNP and probably also ASF/SF2(44, 45, 46) .
In conclusion, Rev most likely functions both at the level of splicing and at the level of transport, through the basic domain and the nuclear export signal, respectively. Further investigations of the functional roles of the interaction between p32 and the basic domain of Rev and the association of nucleoporin-like proteins with the nuclear export signal will be crucial for understanding Rev function and provide a valuable tool to study the functional interplay between splicing and transport in general.