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J Virol, April 1998, p. 2935-2944, Vol. 72, No. 4
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Functional Analysis of the Human Immunodeficiency
Virus Type 1 Rev Protein Oligomerization Interface
Sarah L.
Thomas,1
Martin
Oft,2
Herbert
Jaksche,1
Georg
Casari,3
Peter
Heger,4
Marika
Dobrovnik,1
Dorian
Bevec,1 and
Joachim
Hauber1,*
Department of Immunology, Novartis Research
Institute, A-1235 Vienna,1 and
Research
Institute of Molecular Pathology, A-1030
Vienna,2 Austria, and
Department of
Biocomputing, EMBL, D-69117 Heidelberg,3 and
Institute for Clinical and Molecular Virology, University
of Erlangen-Nürnberg, D-91054 Erlangen,4
Germany
Received 16 June 1997/Accepted 22 December 1997
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ABSTRACT |
The expression of human immunodeficiency virus type 1 (HIV-1)
structural proteins requires the action of the viral
trans-regulatory protein Rev. Rev is a nuclear shuttle
protein that directly binds to its cis-acting Rev response
element (RRE) RNA target sequence. Subsequent oligomerization of Rev
monomers on the RRE and interaction of Rev with a cellular cofactor(s)
result in the cytoplasmic accumulation of RRE-containing viral mRNAs.
Moreover, Rev by itself is exported from the nucleus to the cytoplasm.
Although it has been demonstrated that Rev multimerization is
critically required for Rev activity and hence for HIV-1 replication,
the number of Rev monomers required to form a
trans-activation-competent complex on the RRE is unknown. Here we report a systematic analysis of the putative multimerization domains within the Rev trans-activator protein. We identify
the amino acid residues which are part of the proposed single
hydrophobic surface patch in the Rev amino terminus that mediates
intermolecular interactions. Furthermore, we show that the expression
of a multimerization-deficient Rev mutant blocks
HIV-1 replication in a trans-dominant (dominant-negative) fashion.
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INTRODUCTION |
The replication of human
immunodeficiency virus type 1 (HIV-1) is characterized by a temporal
pattern of viral mRNA expression. HIV-1 uses alternative splicing
of its primary ~9-kb transcript to generate the various
mRNAs that are necessary for virus production (55,
77). Initially, only fully spliced mRNAs of the ~2-kb class, encoding the regulatory proteins Tat, Nef, and Rev, are expressed (31, 67, 88, 92). While the Tat protein acts as a
transcriptional activator of the viral long terminal repeat (LTR)
promoter, Nef appears to be a regulatory protein that directly affects
viral pathogenesis in infected individuals (for reviews, see references
20 and 21). The subsequent
expression of the ~9-kb and partly spliced ~4-kb classes of viral
mRNAs, encoding the structural proteins Gag, Pol, and Env,
depends on the action of the Rev trans-activator (28,
40, 58). In the absence of Rev, these transcripts are retained in
the cell nucleus (15, 41, 62, 76, 85, 93) and are either
spliced to completion or subjected to degradation. In the presence of
Rev, the incompletely spliced mRNAs accumulate in the
cytoplasm to serve as templates for protein synthesis or as viral
genomes (32, 65, 68). Rev activity is therefore essential
for virus replication (30, 91, 94, 99).
Rev itself is a small phosphoprotein of 116 amino acids (aa) which, at
steady state, accumulates primarily in the nucleoli of the host cell
(14, 16, 22, 27, 43) and has the capacity to shuttle between
the nucleus and the cytoplasm (53, 74, 86, 108). Nuclear
localization is mediated by a short stretch of basic amino acids
characterized by eight arginine residues located near the amino
terminus of Rev (11, 16, 42, 48, 63, 104). In addition, this
same protein domain (positions 33 to 46) is responsible for the binding
of Rev to its cis-acting RNA target sequence, the Rev
response element (RRE) (11, 24, 42, 44, 57, 70, 98, 109,
110), although some more-amino-terminal residues appear to
contribute to binding specificity (26, 50). The RRE itself
is encoded by sequences of the env gene (39, 68,
89) and is therefore present in all unspliced and incompletely spliced viral mRNAs, making them subject to Rev regulation.
A second essential region within Rev is located near the carboxy
terminus and has been mapped to residues 75 to 93 (47, 69, 73,
103, 105). Mutation of the four leucine residues within this
domain results in nonfunctional Rev proteins that retain wild-type
nuclear localization and RRE binding characteristics but also
display a pronounced dominant-negative
(trans-dominant) phenotype (6, 29, 63,
66). Therefore, this domain appears to mediate effector functions
and has been defined as the activation domain. Recently, this
domain was shown to contain a nuclear export signal which, when
conjugated to heterologous proteins, is sufficient to mediate
their transport from the nucleus to the cytoplasm in eukaryotic cells
(33, 75, 106). These findings suggest that the signals for
nuclear RNA export reside in proteins that act as carriers to transport
RNA across the nuclear envelope. At least in Xenopus
oocytes, this viral mRNA transport appears to be independent of any pre-mRNA splicing events (34).
It is therefore expected that the Rev activation domain provides the
site of interaction with host cell proteins that are involved in
nuclear-cytoplasmic translocation. Indeed, various cellular proteins
which appear to be specific binding partners of the Rev activation
domain have been described; these include nucleoporin-like protein
hRIP/Rab (10, 36), eukaryotic initiation factor 5A (eIF-5A)
(90), and nuclear export factor CRM1, which appears to be a
general nuclear export signal receptor (35, 37, 81, 95). For
eIF-5A, nonfunctional mutants with a block in Rev
trans-activation and therefore HIV-1 replication in human T
cells (7, 52) were recently described, demonstrating a novel
way to interfere with the viral life cycle: by inhibition of
Rev-mediated nuclear export.
In addition, an important and often overlooked aspect of Rev with
respect to biological activity is its ability to form multimers. It has
been suggested that this multimerization event is responsible for
a certain threshold level of intracellular Rev proteins and that it is
this threshold level which must be overcome in order to establish a
productive HIV-1 infection, thereby regulating viral latency (83,
84). Rev has a very high tendency to aggregate in solution
(24, 54, 79, 107, 109) and has been reported to form
homomultimeric complexes in vivo even in the absence of RRE RNA
(9, 49, 59, 80, 96, 110). However, with respect to
Rev-mediated viral RNA export and hence Rev biological activity, the
formation of a Rev multimer on the RRE has been shown to be more
critical (17, 18, 25, 51, 61, 64, 102, 111). Random
mutational analysis of the rev gene revealed that the
residues within Rev that participate in these intermolecular
interactions reside in the amino terminus of the protein, in regions
flanking the basic domain responsible for RNA binding and nuclear
localization (61, 64). However, little is known about their
precise localization and how many Rev molecules form the biologically
active complex on the RRE, since as many as 12 Rev proteins have been
shown to bind to the RRE in vitro (25, 56, 71, 110, 111).
This study was undertaken to characterize the regions in HIV-1 Rev
which mediate the formation of homo-oligomeric complexes on the RRE in
more detail.
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MATERIALS AND METHODS |
Molecular clones.
Expression plasmids pcRev and pcTat encode
cDNA copies of the rev and tat genes,
respectively, of isolate HXB-3 (67). Expression vectors
encoding Rev mutants RevM4, RevM5, RevM6, RevM8, RevM10, RevM16,
RevM23, and RevTC5 have been described in detail elsewhere (4, 63,
100). In transfection experiments, the vector pBC12/CMV was used
to maintain constant input DNA levels and pBC12/CMV/IL-2 served as a
negative control plasmid (19). Constructs pBC12/CMV/
Gal (90) and pBC12/RSV/SEAP (5) have been used for
internal control of transfection efficiencies. pDM128/CMV is a
well-established Rev-responsive reporter construct encoding the
bacterial chloramphenicol acetyltransferase (CAT) gene (48,
69). Construct pSLIIB/CAT contains an HIV-1 LTR promoter in which
the trans-activation response element (TAR) sequence has
been replaced by the RRE-derived stem-loop IIB (SLIIB) high-affinity
Rev binding site (101). Plasmids in which the tat
gene is fused to the rev gene (pcTat/Rev), encoding the
fusion proteins Tat-Rev, Tat-RevM4, Tat-RevM5, and Tat-RevM10, have
been described elsewhere (61). Plasmid pGEX-Rev is a
bacterial expression plasmid that expresses Rev fused to the carboxy
terminus of glutathione S-transferase (GST) (7).
pGEM/RRE is an in vitro transcription vector that contains the RRE
(64).
Oligonucleotide-directed mutagenesis with a bacteriophage M13
mutagenesis system (United States Biochemicals, Cleveland, Ohio) was
used to introduce targeted nucleotide substitutions encoding amino acid
alterations into the rev gene of pcRev. All introduced mutations were confirmed by DNA sequence determination with Sequenase 2.0 (United States Biochemicals). Variants of pGEX-Rev and pcTat/Rev possessing mutated rev genes were generated by exchange of
the wild-type gene for the respective mutated gene by PCR technology. Finally, the coding regions of the constructs were confirmed by DNA
sequence determination as described above. Protein expression of all
mutants that were generated in the course of this study was confirmed
by specific immunoprecipitation analysis with radiolabelled protein
extracts from transiently transfected COS cells.
Cell cultures, transfections, and assays.
COS and HeLa cells
were maintained and transfected with DEAE-dextran and chloroquine or
calcium phosphate as previously described (101). Rev
trans-activation was investigated by cotransfection of
2.5 × 105 COS cells with 375 ng of pDM128/CMV DNA and
75 ng of pBC12/CMV/Gal DNA, together with 250 ng of either pcREV
(positive control), pBC12/CMV/IL-2 (negative control), or mutant Rev
expression plasmid. At ~60 h posttransfection, cell lysates were
prepared and the levels of -galactosidase activity were measured as
described previously (101). These values were subsequently
used to determine the amount of cell extract to be assayed for CAT by
an enzyme-linked immunosorbent assay (ELISA) (Boehringer GmbH,
Mannheim, Germany).
Dominant-negative inhibition of Rev function was investigated by
cotransfection of 2.5 × 105 COS cells with 250 ng of
HIV-1
rev proviral DNA (87) and 250 ng of
pBC12/RSV/SEAP DNA (transfection efficiency control), together with 125 ng of pcRev and 1.25 µg of mutant Rev expression plasmid. Total input
DNA was kept constant in all transfections by inclusion of the parental
vector pBC12/CMV. At ~60 h posttransfection, cell supernatants were
assayed for secreted alkaline phosphatase (SEAP) (5) and p24
Gag protein synthesis.
In vivo RNA binding of Tat-Rev fusion proteins was assessed by
cotransfecting 2.5 × 10
5 HeLa cells with 2.5 µg of
pSLIIB/CAT reporter plasmid, 1.5 µg
of pBC12/CMV/
Gal plasmid, and
2.5 µg of either pcTat/Rev (positive
control), pcTat/RevM5 (negative
control), or pcTat/mutant Rev
expression vector. At 42 h
posttransfection, cell lysates were
prepared and the levels of
-galactosidase activity were determined.
These values were then used
to adjust the amount of protein extract
to be assayed in a radioactive
CAT assay (
101).
The multimerization capacity of Rev proteins was assessed by
cotransfection of 2.5 × 10
5 HeLa cells with 1.5 µg
of pSLIIB/CAT reporter plasmid, 1.5 µg
of internal control plasmid
(pBC12/CMV/
Gal), 1.5 µg of pcTat/RevM5,
and 1.5 µg of either
pcRev (positive control), pcRevM5 (negative
control), or mutant Rev
expression plasmid. At 42 h posttransfection,
cell lysates were
prepared for determination of
-galactosidase
and CAT activities.
Purification of GST-Rev fusion proteins and in vitro RRE
binding.
Wild-type Rev and mutant Rev were expressed as
carboxy-terminal fusions to GST in Escherichia coli BL21.
The fusion proteins were purified from crude cell lysates by affinity
chromatography on glutathione-Sepharose 4B according to the
manufacturer's protocol (Pharmacia Biotech, Vienna, Austria). Eluted
protein fractions were analyzed by Rev-specific Western analysis,
pooled, concentrated by ultrafiltration with a PM10 filter device
(Amicon Inc., Beverly, Mass.), and stored at 
70°C. The final
protein concentrations were determined by the method of Bradford
(12). RNA binding assays with a radiolabelled 252-nucleotide
RRE probe were performed as previously described (25),
except that RNA-protein complexes were resolved on 6% polyacrylamide
gels.
Retrovirus-mediated gene transfer and HIV-1 challenge
experiments.
The retroviral vectors were constructed by inserting
PCR-generated rev genes into the single XhoI site
of retroviral vector pBC140 (6). The rev
sequences were subsequently confirmed by DNA sequencing as described
above. The amphotropic GP + envAm12 (Am12) packaging cell
line (72) was transfected with the retroviral DNAs,
and CD4+ human CEM cells were subsequently inoculated with
virus-containing infectious Am12 supernatants and selected for
neomycin-resistant populations as described previously (7,
8). RNA expression of the transgene in clonal populations of the
transduced CEM cells was characterized as described previously
(6).
CD4
+ rev-transduced CEM cells (2.0 × 10
6) were infected with 2,000 tissue culture infective
doses (TCID) of HIV-1 strain SF2
(
60) at an ambient
temperature for 2 h. Subsequently, the cells
were washed three
times, transferred into fresh RPMI 1640 medium
containing 10% fetal
bovine serum, and incubated at 37°C. On days
4, 7, and 11 of the
incubation period, HIV-1 replication, measured
by p24 Gag protein
synthesis with an antigen capture assay (ELISA),
and cell counts,
measured with a Coulter device, were determined.
| |
RESULTS |
Biophysical measurements obtained with circular dichroism and
fluorescence spectra provided the initial insights into the structure
of the HIV-1 Rev protein (1). These data indicated that most
of the amino-terminal half of Rev (residues 1 to 61-66), which
contains the RNA binding-nuclear localization domain (98) as
well as the putative multimerization domains (61, 64), is in
an 
-helical conformation. In particular, this region appears to form
a helix-loop-helix motif via intramolecular hydrophobic contacts
between two helices that are separated by a stretch of proline-rich
residues (1). Additional functional data obtained from
mutagenesis studies (100) refined this structural model of
the Rev amino terminus and allowed the identification of specific hydrophobic amino acids that are exposed on the surface of Rev and that
form a surface for potential intermolecular interactions (Fig.
1A). Interestingly, the previously
described nonfunctional Rev mutants RevM4 and RevM7 (63),
which are unable to form multimers on their RRE RNA target in vitro
(64), map to this region of the Rev protein (Fig. 1B). Using
these data as a starting point, we set out to investigate the sequence
requirements for HIV-1 Rev-specific multimer formation in more detail.
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FIG. 1.
Location of the putative HIV-1 Rev multimerization
domains. (A) Model of the Rev amino terminus, redrawn from Thomas et
al. (100), shows the helix-loop-helix motif of Rev residues
1 to 60. It is evident that the two putative multimerization domains
form a single exposed hydrophobic surface patch which presumably
constitutes an oligomerization interface. Amino acids are colored as
follows: basic residues (Arg, Lys), blue; acidic residues (Asp, Glu),
red; residues analyzed in this study, yellow; and residues identified
in this study as important for intermolecular interactions, purple. (B)
Diagrammatic representation of the 116-aa Rev
trans-activator protein showing the functional domains and
the regions mutagenized. The two protein regions that form a putative
multimerization surface are marked as domains MI and MII. The amino
acid sequences of both regions systematically mutagenized in this study
are shown as expanded sections. Residues potentially involved in
multimerization and previously identified by examination of the refined
structural model of Rev (100) are highlighted by shaded
boxes.
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Mutational analysis of the putative Rev multimerization
domains.
As outlined in Fig. 1B, the two regions of the Rev
protein (here termed domains MI and MII) that create a potential
multimerization surface were extensively mutagenized by introducing
scanning missense mutations (Table 1).
All mutant genes generated appeared to express protein at
levels comparable to that of the wild-type rev gene, as tested by Rev-specific immunoprecipitation analysis with
protein extracts of transiently transfected COS cells (data not shown). In order to test the functionality of these Rev mutants, we used the
Rev-responsive reporter construct pDM128/CMV. This reporter plasmid
contains the gene encoding CAT and the RRE target sequence of Rev, both
of which are positioned between HIV-1 splice sites and are under
the control of the cytomegalovirus immediate-early promoter
(48, 69). RNA produced by pDM128/CMV therefore has a single intron containing the CAT gene and the RRE sequence, which is
removed when the RNA is spliced. As HIV-1 splice sites are
inefficiently recognized in primate cells (13),
significant levels of both spliced and unspliced pDM128/CMV-specific
mRNAs accumulate in the cell nucleus. However, in the
presence of functional Rev protein, the unspliced message is exported
to the cytoplasm, resulting in high levels of CAT expression.
Therefore, by cotransfection of pDM128/CMV and the rev
vector in question into COS cells, we were able to monitor Rev
trans-activation.
Figure
2 summarizes the results of the
functional analysis of Rev domains MI and MII. These and all other
transfection experiments
in this study were internally controlled for
various transfection
efficiencies by inclusion of the constitutive
control vector pBC12/CMV/
Gal
(
90). All CAT values are
expressed as a percentage of wild-type
Rev activity (set arbitrarily to
100%) and were corrected for
background (mock) activity. The scanning
functional analysis of
the two putative multimerization domains
revealed a wide range
of functional phenotypes, ranging from wild-type
activity through
partial activity to complete nonfunctionality (Fig.
2). The detection
of mutants with partial activity indicated a
moderately important
functional role for the mutated residues. However,
a series of
mutants showed 10% or less wild-type
trans-activation activity
and were essentially completely
nonfunctional. Evaluation of all
mutant phenotypes obtained allowed us
to identify amino acid residues
Ala
15, Val
16,
Leu
18, Ile
19, and Leu
22 in domain
MI as being critical for Rev function and therefore
potentially
involved in multimer formation (
1,
100). Residues
Ile
52, Ile
59, and Leu
60 appear to
be the functionally important residues in domain MII.
The lack of
biological activity seen with mutant RevM23, which
was included as a
control in these experiments, resulted from
its inability to localize
to the cell nucleus (
4). Upon analysis
of the data obtained,
the important residues identified by functional
dissection of domains
MI and MII correlated well with the residues
previously identified by
examination of a refined Rev model (
100)
as components of
the exposed hydrophobic patches which form the
potential
multimerization interfaces of Rev (Fig.
1B).
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FIG. 2.
Biological activities of HIV-1 Rev mutant proteins. (A)
Scanning mutagenesis of putative multimerization domain MI. Rev
trans-activation capacity was determined by cotransfection
of COS cell monolayers with the Rev-responsive reporter plasmid
pDM128/CMV, the various mutant Rev expression plasmids (indicated on
the x axis; see also Table 1), and the constitutive internal
control vector pBC12/CMV/Gal. Data are expressed as a percentage of
wild-type (WT) Rev activity (set to 100%), and the error bars
represent the standard deviations of four independent experiments. All
CAT values were adjusted for transfection efficiency by determining the
level of -galactosidase in each culture and were corrected for
background (mock) activity. IL-2, negative control plasmid
pBC12/CMV/IL-2; WT, wild-type Rev (pcRev). (B) Scanning mutagenesis of
putative multimerization domain MII. Details are as for panel A.
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RNA binding and multimerization characteristics of
nonfunctional Rev mutants.
Use of the pDM128/CMV-based
trans-activation assay allowed nonfunctional Rev
proteins to be identified but provided no insight into the reason for
their aberrant function. Therefore, the ability of selected mutants
identified above to bind to and multimerize on the RRE target RNA was
investigated.
The RRE binding characteristics of Rev mutants RevSLT4, RevSLT21 to
RevSLT23, RevSLT26, RevSLT40, and RevTC5 (Table
1) were
first assessed
in vitro by RNA gel retardation analysis. For this,
the respective
proteins were expressed and purified in the context
of fusions to GST
and then analyzed in combination with an in
vitro-transcribed RRE RNA
probe (
42) (Fig.
3). Despite
the fact
that GST may itself form homodimers, GST-Rev fusion proteins
have
been used to successfully identify in vitro
multimerization-deficient
Rev mutants (
64), provided that
the addition of increasing amounts
of GST-Rev to a preformed initial
complex does not cause higher-order
complexes. Control experiments
confirmed that the addition of
increasing amounts of Rev wild-type
protein to the binding reaction
mixture resulted in the successive
appearance of RNA-protein complexes
with slower mobilities in
nondenaturing gel electrophoresis (C1
and C2 in Fig.
3A). These
complexes were not detected when mutant
RevM6 was used (Fig.
3B); this
mutant is characterized by an internal
deletion in the RNA binding
domain and therefore serves as a negative
control for RRE recognition
(
63,
64). Inspection of the data
revealed that nonfunctional
Rev mutants RevSLT22, RevTC5, and
RevSLT40 retained their in vitro RRE
binding capacity (Fig.
3E,
H, and I, respectively). However, all of
these proteins lacked
the ability to bind to the RRE in a cooperative
manner, a result
which is indicated by the formation of a single
retarded complex
(C1), even at the highest protein concentrations
tested. An intermediate
result was observed with RevSLT26 protein
in these experiments
(Fig.
3G). RevSLT26 clearly bound to the RRE and
formed the initial
C1 complex. However, at higher protein
concentrations, no distinct
multimeric complexes comparable to those
seen with the wild-type
protein were formed. This result may indicate
that RevSLT26 is
severely impaired in this in vitro multimerization
assay.
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FIG. 3.
In vitro RRE binding analysis of wild-type and mutant
GST-Rev proteins. A constant level of a 32P-labelled RRE
RNA probe was incubated with increasing amounts (30 ng to 2 µg, lanes
1 to 7) of wild-type Rev (RevWT) (A) or the indicated Rev mutant fusion
proteins (B to I) and then subjected to RNA gel mobility shift
analysis. In each case, lane 8 contained free (unbound) RRE RNA. The
various distinct complexes formed upon Rev binding to the RRE RNA probe
(C1 and C2) are indicated to the right of each panel.
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It was recently shown that Rev mutants that are multimerization
deficient in vitro do in fact form multimers in vivo (
61,
64). The reason for this obvious discrepancy is not yet known.
Nevertheless, in order to characterize the selected nonfunctional
Rev
mutant proteins as fully as possible with respect to RRE binding
and
multimerization capabilities, we also tested them in vivo.
The primary high-affinity Rev binding site within the RRE was
previously assigned to a region known as SLIIB (
3,
18,
51,
82,
102). The identification of this site allowed the
development of
a reporter gene-based in vivo system to measure
the binding capacity
(
101) and, moreover, the multimerization
capacity of Rev on
its SLIIB RNA target site (
61).
As depicted in Fig.
4A, the pSLIIB/CAT
reporter construct used for these assays contains the CAT gene under
the transcriptional
control of the HIV-1 LTR promoter. The wild-type
TAR element,
which is the promoter-proximal RNA target sequence of the
HIV-1
Tat transcriptional
trans-activator, is replaced by a
29-nucleotide
RNA sequence encoding SLIIB of the RRE (
101).
This promoter is
only activated by Tat-Rev fusion proteins and is not
responsive
to Tat or Rev alone. Thus, the quantity of CAT produced
gives
an indication of the RNA binding ability of a given Rev mutant
tested in the context of a Tat-Rev fusion protein.
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FIG. 4.
RNA binding and multimerization activities of
nonfunctional Rev mutant proteins in a mammalian cell culture system.
(A) Diagrammatic representation of the pSLIIB/CAT reporter system.
Activation of a chimeric HIV-1 LTR promoter in which the TAR sequence
was replaced by the RRE SLIIB element (101) allowed the
monitoring of in vivo RRE SLIIB RNA binding and multimer formation by
Rev (see the text for further details). (B) In vivo RNA binding
activity of nonfunctional Rev mutants. HeLa cells were cotransfected
with reporter construct pSLIIB/CAT, the various Tat-Rev (TR) expression
plasmids (indicated on the x axis), and constitutive
internal control plasmid pBC12/CMV/Gal. pBC12/CMV/IL-2 (IL-2),
pcTat, pcRev, and pcTat/Rev (Tat/Rev) were included as controls. At
42 h posttransfection, protein extracts were prepared to assay for
-galactosidase and CAT levels. Data are expressed as a percentage of
wild-type (WT) Rev activity (set to 100%) and were corrected for
background (mock) activity. (C) Analysis of Rev multimerization in
vivo. The ability of the selected nonfunctional Rev mutants to form
multimers was determined by cotransfection of HeLa cells with reporter
construct pSLIIB/CAT, pcTat/RevM5 (an RNA binding-negative mutant), the
various mutant Rev expression plasmids (indicated on the x
axis), and constitutive internal control plasmid pBC12/CMV/Gal.
pcRev (RevWT) and pcTat/RevM5 (Tat/RevM5) alone were included as
negative controls. At 42 h posttransfection, levels of
-galactosidase and CAT were determined as described above. Data are
expressed as a percentage of wild-type (WT) Rev activity (set to
100%). Error bars represent the standard deviations of four
independent experiments.
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As expected, cotransfection of HeLa cells with an unrelated control
plasmid expressing human interleukin-2 (IL-2) or with
expression
vectors encoding HIV-1 Tat or Rev did not result in
trans-activation of the pSLIIB/CAT reporter construct (Fig.
4B).
However, significant CAT activity was detected when an expression
plasmid encoding a Tat-Rev fusion protein was included in the
transfection. This promoter activation was not detected when the
SLIIB
binding-incompetent Tat-RevM5 mutant protein (
61) was
used
in these experiments. The data obtained clearly demonstrated
that all
of our nonfunctional Rev mutant proteins bound to RRE
SLIIB in vivo,
although mutant RevSLT4 appeared to have impaired
activity in this
assay (Fig.
4B). In general, the RNA binding
data obtained in this in
vivo assay correlated well with the data
from the RNA gel retardation
assays presented above.
Next, the ability of the mutants to multimerize in vivo was tested with
a similar assay that also used the pSLIIB/CAT reporter
construct.
The principle of this assay is explained diagrammatically
in Fig.
4A.
The assay relies on the ability of the test mutant
to rescue the
nonbinding phenotype of another mutant, Tat-RevM5,
by the formation of
a Rev-Rev multimer (
61). The Tat portion
of the nonbinding
RNA fusion protein is thus brought into close
proximity with the LTR
promoter, resulting in
trans-activation.
Figure
4C shows the
activities of the nonfunctional Rev mutants
in this assay. Negative
controls of wild-type Rev or Tat-RevM5
alone were inactive in
these experiments. Likewise, when Tat-RevM5
was assayed with
RevM5, only background levels of activity were
measured. Control
mutants RevM4 and RevM10 (
63) gave the phenotypes
that had
been previously described (
61), while the test mutants
resulted in a variety of different phenotypes. RevSLT22 multimerized
with essentially wild-type efficiency. RevSLT4, RevSLT21, and
RevTC5
showed a partial phenotype. Rev mutants RevSLT23, RevSLT26,
and
RevSLT40 as well as the control RevSLT30 all multimerized
with
approximately 20% wild-type activity, and we therefore considered
these mutants multimerization defective in this artificial mammalian
cell culture system. Interestingly, testing of multimerization
capacity
resulted in contradictory data for some of these nonfunctional
Rev
mutants. For example, no in vitro multimer formation was detected
with
RevSLT22 and RevTC5 (Fig.
3E and H). In contrast, both proteins
clearly
displayed multimerization activity in the mammalian cell
culture system
(Fig.
4C). With RevSLT23, multimerization activity
was detected in
vitro but not in vivo (compare Fig.
3F and Fig.
4C). However, the same
result was already reported in earlier
studies. In particular, mutant
RevM4 was originally proposed to
be a prototype
multimerization-deficient Rev protein due to its
multimerization
deficiency in RNA gel retardation experiments
(
64). A
subsequent study, however, showed that RevM4 multimerizes
about 40% as
effectively as wild-type Rev in the in vivo assay
(
61),
although there is no good explanation for this discrepancy.
One
explanation might be that the interaction of mutant proteins
is
measured in the in vitro assay, while the in vivo assay reflects
the
ability of a mutant Rev protein to interact with the wild-type
protein.
Nevertheless, considering both in vitro and in vivo data
generated in
this study, two mutant proteins, namely, RevSLT26
and RevSLT40,
appeared to be inactive due to an inherent inability
to form functional
multimeric complexes.
trans-Dominant inhibition of HIV-1 replication.
It
was previously shown that Rev multimerization is required for Rev
activity (17, 18, 25, 51, 61, 64, 102, 111). Therefore, true
multimerization-deficient mutants should be able to block Rev
function in a dominant-negative manner. In theory, these Rev mutants,
when present in trans, should act as competitive inhibitors
of the wild-type protein by sequestering RRE-containing viral RNAs.
To test this hypothesis, we first investigated the effects of RevSLT26
and RevSLT40 on Rev
trans-activation using a provirus
rescue assay. As described before (
87), cotransfection of
COS
cells with an HXB-2-derived Rev-deficient proviral DNA
(HIV-1
rev)
and pcRev resulted in the Rev-dependent
accumulation of p24 Gag
antigen in COS cell supernatants (Fig.
5). As expected, the additional
coexpression (10-fold excess) of wild-type Rev protein
increased
the release of p24 Gag into the supernatants. However,
the expression
of RevSLT26 or RevSLT40 protein clearly correlated
with the inhibition
of wild-type Rev function. Obviously, the strong
inhibitory effect
seen with the RevM10 protein, which is characterized
by a mutation
in the Rev activation domain and has been described so
far as
the most powerful
trans-dominant Rev protein
(
69), could not
be reached by the RevSLT26 or RevSLT40
protein in this transient
assay. Notwithstanding this result, these
experiments indicated
that both RevSLT26 and RevSLT40 might have a
moderate dominant-negative
potential, with the inhibitory effect of
RevSLT40 appearing to
be more pronounced (Fig.
5).
View larger version (9K):
[in this window]
[in a new window]
|
FIG. 5.
Competitive inhibition of Rev function. COS cell
cultures were cotransfected with the HXB-2-derived Rev-deficient
proviral DNA HIV-1rev, pBC12/RSV/SEAP (internal control
vector), and pBC12/CMV/IL-2 (negative), pcRev (positive), or pcRev plus
a 10-fold excess of expression vector encoding wild-type (WT) Rev,
RevSLT26, RevSLT40, or RevM10. Total input DNA was maintained at a
constant level by inclusion of parental vector pBC12/CMV. All p24 Gag
antigen values were adjusted for transfection efficiency by determining
the level of SEAP (5) in each culture and were corrected for
background (mock) activity. Error bars represent the standard
deviations of three independent experiments.
|
|
To test the RevSLT40 phenotype in a more rigorous way, we investigated
the ability of RevSLT40 to inhibit HIV-1 replication.
For this,
retroviral vector pBC140 (
6) was used for
retrovirus-mediated
gene transfer of various
rev genes into
human CEM T cells. Transduced,
neomycin-resistant CD4
+ CEM
cell clones were characterized for transgene expression by
Northern
analysis (data not shown) and subsequently inoculated
with 2,000 TCID
of replication-competent HIV-1 strain SF2 (
60).
The numbers
of cells in the cultures were determined on days 0,
4, 7, and 11 postinfection, and p24 Gag protein levels were assayed
as a measure of
HIV-1 replication (Table
2). In all
cases, infected
cells proliferated at the same rate as uninfected
control cells
(data not shown), indicating that constitutive expression
of the
mutant
rev genes was not toxic for the cells. As
shown in Table
2, CEM cells transduced with pBC140 alone provided no
protection
against HIV-1 infection. Likewise, significant levels
of p24 Gag
antigen were measured in the culture containing CEM clone
RevM4,
a mutant previously described to be multimerization defective
in
vitro (
61,
64) and serving as a control in these
experiments.
The two CEM cell lines expressing Rev mutant
RevSLT40 (RevSLT40/1
and RevSLT40/2; Table
2), which were
considered to be multimerization
deficient in vitro and in vivo, gave
low levels of p24 Gag antigen
when supernatants were assayed at day 4, 7, or 11 postinfection.
RevM10, serving as a positive control for the
trans-dominant phenotype,
strongly inhibited virus
replication, as has been reported previously
(
6,
63,
66).
| |
DISCUSSION |
The rev gene of HIV-1 encodes a key activity for the
complex regulation of viral gene expression. Rev acts early in the
HIV-1 replication cycle to bring about the export from the nucleus to the cytoplasm of incompletely spliced and unspliced viral
mRNAs encoding the viral structural proteins (reviewed in
references 23 and 46). Numerous
independent studies have determined a number of specific requirements
for full Rev activity, resulting in a frequently updated model of Rev
trans-activation. The protein must be localized in the
correct subcellular compartment (the nucleus), must be able to bind
directly and specifically to its RRE RNA target sequence, and, after
multimerization by cooperative binding of further Rev molecules, must
interact via its activation domain with one or more cellular cofactors.
These cofactors, including nucleoporin-like proteins such as hRIP/Rab
(10, 36, 97), CRM1 (35, 37, 81, 95), and nuclear
eIF-5A (7, 90), are thought to mediate the subsequent
nuclear export of Rev (reviewed in reference 38).
Rev was recently shown to be a shuttling protein (53, 74, 86,
108); therefore, it is thought that after Rev enters the
cytoplasm and dissociates from the RRE, it then returns to the nucleus
to transport further viral messages.
To date, these aspects of Rev function have been addressed mostly
separately and not in a complete model system. Therefore, a number of
important questions still remain unanswered. For example, the amino
acid residues in Rev which mediate Rev multimer formation have not been
fully characterized, and no true multimerization-deficient Rev mutants
(defined as inactive in the in vitro and in vivo assays) have been
identified.
However, the capacity of Rev to form multimers and the clear
requirement of multimer formation for Rev function have placed this
viral trans-regulatory protein at the center of models which describe the regulation of viral latency. It has been shown that some
cell lines which are nonproductively infected with HIV-1 are
characterized by low Rev protein levels (84). Reaching a critical intracellular threshold level of Rev protein obviously correlates with Rev function and allows virus production
(83). Thus, the transition from a quiescent (latent) state
to a productive state of infection appears to be controlled in these
cells at least in part by the ability of Rev to multimerize
(64).
Advances in the determination of the structure of Rev have allowed the
development of a refined structural model of the amino terminus of the
protein (1, 100). This model has provided the first
indication that the Rev multimerization interface, although generated
by two separate protein regions, may actually form a single interaction
area in the functional protein structure (Fig. 1A). Interestingly, the
previously described Rev mutants RevM4 and RevM7, which appear to be
defective in their ability to multimerize in vitro (64), map
to these regions. The detailed functional analysis of these regions
carried out in this study has allowed the amino acid residues that are
critical for Rev activity to be identified (Fig. 2). Of note, these
experimentally defined residues coincide perfectly with the amino acids
which have been suggested by the structural model of the Rev amino
terminus to form the Rev dimerization interface (1, 100).
Testing the nonfunctional Rev mutants generated in this study in both
in vitro and in vivo multimerization assay systems revealed conflicting
multimerization activities (e.g., RevSLT22 and RevSLT23 [Fig. 3 and
4]). Such inconsistency among phenotypes obtained with two different
assay systems was previously reported for the RevM4 protein (61,
64) and led to some confusion as to what the precise requirements
for Rev multimer formation are. However, these data also emphasized
that a combination of both in vitro and in vivo assays for Rev multimer
formation is needed in order to identify true multimerization-deficient
Rev mutants.
Analysis of mutants generated in this study allowed dissection of the
RevM4 mutant (63) (YSN to DDL at aa positions 23, 25, and
26) phenotype. Individual mutations in the component residues revealed
no functional importance for these residues with respect to
multimerization. It therefore seems likely that the partial multimerization deficiency phenotype seen for RevM4 is the result of a
localized structural change which is due to the introduction of three
mutations in close proximity and which consequently affects the helix in this region. Likewise, it appears that the isoleucine residue
at position 55, rather than the residues on either side (Ser54 and Ser56) that are deleted in RevM7
(63), is the functionally important residue which produces
the multimerization deficiency phenotype and that it is the highly
localized structural disruption caused by the mutation in this region
which is responsible for the complete loss of Rev function.
Despite efficiently binding to its RNA target in vitro, nonfunctional
mutant RevSLT4 appeared to be at least partially active in RNA binding
and multimerization in vivo. Thus, some defect other than one in the
capacity to multimerize might have been responsible for its
nonfunctionality. We were, however, able to attribute the functional
inactivity of mutants RevSLT26 and RevSLT40 to their impaired ability
to form Rev multimers. The phenotypes of these two mutants identified
amino acid residues Leu22, Ile59, and
Leu60 as being essential for multimerization. Obviously,
this result is in agreement with the structural models of the Rev amino
terminus which suggested that these hydrophobic residues
(Leu22, Ile59, and Leu60) are
exposed and presumably create a single intermolecular oligomerization surface (1, 100).
A number of trans-dominant Rev mutants have been identified,
but so far the best characterized appears to be RevM10 (2, 6, 29,
63, 66, 69). The basis of the trans-dominant phenotype
of an activation domain mutant such as RevM10 is thought to be an
inherent inability to interact with a cellular cofactor. Therefore, by
extension of the same idea, a multimerization-deficient mutant such as
RevSLT26 or RevSLT40 should also show a dominant-negative phenotype.
This was indeed found to be the case in a transient transfection assay
carried out to investigate the trans-dominant phenotypes of
RevSLT26 and RevSLT40 (Fig. 5). Dominant-negative phenotypes were
observed for RevSLT40 and, to a lesser degree, for RevSLT26, although
the inhibitory effects were not as great as those seen for RevM10. The
molecular basis for the trans-dominant phenotypes of
multimerization-deficient Rev mutants such as RevSLT26 and RevSLT40 is
likely to be an ability to bind to the RRE but not form the
homomultimeric complex which is required either to allow Rev to assume
the correct conformation for interaction of one or more cellular
cofactors with the activation domain or to allow the formation of a
multicomponent binding site to which these cofactors then bind. Hence,
by sequestering all available RRE-containing RNA,
multimerization-deficient Rev mutants should be able to competitively
inhibit wild-type Rev function.
The single most trans-dominant multimerization-deficient
mutant identified (RevSLT40) in the transient assay was then tested for
its ability to inhibit virus replication. Upon challenge of an
RevSLT40-transduced human T-lymphocyte cell line with high doses of
replication-competent HIV-1 strain SF2, significant inhibition of virus
replication was seen during the experiments (up to 11 days
postinfection). Of note, these virus challenge experiments were
designed to achieve maximal virus replication. The virus titers used
(2,000 TCID) are comparable to those typically seen in the peripheral
blood mononuclear cells of patients suffering from full-blown AIDS
(45). Clearly, the HIV-1 inhibitory level seen with
multimerization-deficient mutant RevSLT40 did not reach the level seen
with activation domain mutant RevM10 (Table 2). However, this result is
almost to be expected since, as mentioned above, a
multimerization-deficient mutant is only able to compete with wild-type
Rev for RRE binding, while an activation domain mutant should, at least
in theory, also be able to enter preexisting Rev complexes, thereby
rendering them inactive. Therefore, activation domain mutants such as
RevM10 are still the trans-dominant reagents of choice for
the clinical development of anti-HIV somatic gene therapies
(78). Although the trans-dominant
multimerization-deficient mutant RevSLT40 will, for this reason, most
likely not be used in these types of gene intervention strategies, the
detailed characterization in this study of the regions in Rev which are
required for protein dimerization and the generation of true
multimerization-deficient mutants such as RevSLT40 will provide the
tools to allow further analysis of the biologically active multimeric
Rev complex in the future. It is obvious that the detailed
determination of how many Rev monomers form the
trans-activation-competent complex will have consequences
for cofactor interaction and therefore Rev-mediated nuclear export.
| |
ACKNOWLEDGMENTS |
We thank Lotte Hofer and Johannes Pertl for excellent technical
assistance and Thomas Baumruker for helpful discussions during the
course of this study.
This work was supported in part by a grant from the Deutsche
Forschungsgemeinschaft (to M.O. and P.H.; SFB 466).
| |
FOOTNOTES |
*
Corresponding author. Present address: Institute for
Clinical and Molecular Virology, University of Erlangen-Nürnberg,
Schlossgarten 4, D-91054 Erlangen, Germany. Phone: 49-9131-85 6182. Fax: 49-9131-85 2101. E-mail:
jmhauber{at}viro.med.uni-erlangen.de
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J Virol, April 1998, p. 2935-2944, Vol. 72, No. 4
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
