Proc. Natil. Acad. Sci. USA Vol. 88, pp. 683-687, February 1991 Biochemistry
Structural analysis of the interaction between the human immunodeficiency virus Rev protein and the Rev response element (baculovirus/expression system/RNA-protein interaction/RNA stem-loop)
J0RGEN KJEMS, MYLES BROWN*, DAVID D. CHANGt,
AND
PHILLIP A. SHARP
Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139
Contributed by Phillip A. Sharp, October 18, 1990
The specific interaction between a defined ABSTRACT structural element of the human immunodeficiency virus mRNA (RRE, the Rev response element) and the virus-encoded protein Rev has been implicated in the regulation of the export of unspliced or singly spliced mRNA from the nucleus to the cytoplasm. Rev protein was expressed and purified from insect cells using the baculovirus expression system. Chemical and RNase probes were used to analyze the structure of the RRE and the regions involved in Rev binding. Increased reactivity to single-strand-specific probes of nucleotides in two helical domains indicates that Rev binding induces conformational changes in the RRE. Binding of Rev to the RRE primarily protects helical segments and adjacent nucleotides in domain II. A Rev unit binding site is proposed that consists of a six-base-pair helical segment and three adjacent nucleotides. The data also suggest that multiple Rev proteins bind to repeated structural elements of the RRE.
The human retroviruses have proven to be an excellent system to study the complex regulation of gene expression in mammalian cells. Human immunodeficiency virus type 1 (HIV-1), which is implicated in the pathogenesis of acquired immunodeficiency syndrome (AIDS) (1, 2), has at least two novel genetic regulatory mechanisms. These are mediated by the virus-encoded regulatory proteins Rev and Tat, which are essential for viral replication (3). Whereas Tat appears to act on transcriptional initiation and elongation (3), Rev functions posttranscriptionally. Rev is necessary for the production of viral structural proteins (4, 5) through regulation of the cytoplasmic appearance of unspliced or singly spliced viral mRNA (6-8). Rev functions either directly or indirectly at the level of splicing (5, 6, 9, 10). Rev action requires the presence of a cis-acting RNA target sequence termed the Rev response element (RRE), which is located in the envelope gene of HIV-1 (8, 11-13). The minimal sequence required for full Rev response in vivo has been mapped to a 234-nucleotide region that has the potential of forming four stem-loops (I, III, IV, and V) and one branched stem-loop (IIA, IIB, and IIC) secondary structure (8). Moreover, stem-loops III, IV, and V can be deleted without abolishing the Rev response in vivo (11, 14, 15). Physical interaction between purified Rev and RRE has been demonstrated in vitro (16-23). Use of a gel retardation approach has shown that Rev binds to a minimal structure containing the branched stem-loop IIA-C (19, 22). However, the precise localization of Rev binding has not been determined. To approach this question we have investigated the relative accessibility of individual nucleotides within the RRE to a variety of chemicals and RNases in the presence or absence of Rev. These data test the proposed structural model of the RRE directly and define the binding site of Rev. In addition, we have found conformational The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
changes in the RNA upon Rev binding, which may have important implications for Rev function.
MATERIALS AND METHODS Construction of Recombinant Baculovirus Expressing HIV Rev. The Stu I-HindIII fragment of the plasmid pcREV (24) containing the Rev coding region was cloned into the BamHI site of the baculovirus expression vector pVL941 (25) by standard techniques. The resulting plasmid, pVREV, was cotransfected with wild-type baculovirus (Autographa californica nuclear polyhedrosis virus, AcNPV) DNA into Sf9 insect cells and a recombinant baculovirus expressing Rev was plaque-purified (26). Purification of Recombinant HIV Rev from Infected Insect Cells. Large-scale infections of insect cells were performed as described (27). Infected cells were harvested 48-72 hr postinfection and washed twice with phosphate-buffered saline. The cell pellet was suspended in lysis buffer [20 mM Hepes, pH 7.9/140 mM NaCI/1.5 mM MgCl2/1% (vol/vol) Nonidet P-40/1 mM phenylmethylsulfonyl fluoride/1 mM dithiothreitol (DTT)J and incubated on ice for 5 min. Nuclei were prepared by centrifugation through a 25% (wt/vol) sucrose cushion in lysis buffer. The nuclei were then suspended and treated with DNase 1 (40 ,ug/ml) in 20 mM Hepes, pH 7.9/100 mM NaCI/5 mM MgCl2/1 mM DTT for 30 min at room temperature. Nucleoplasm containing the majority of the HIV Rev protein was prepared from the DNase I-treated nuclei after centrifugation to remove the insoluble nuclear residue. This fraction was then applied to a Pharmacia FPLC Mono S column and eluted with a linear gradient of NaCl in 20 mM Hepes, pH 7.9/2.5 mM MgCI2/0.5 mM EDTA/10% (vol/vol) glycerol/1 mM DTT. The peak of Rev protein eluted from the Mono S column was >90% pure as analyzed by SDS/17.5% PAGE followed by staining with Coomassie brilliant blue. Preparation of RRE Transcripts and Rev-RRE Complexes. The plasmid pRRE, which contains region 7768-7991 of the HXB-3 isolate of HIV-1 (28), was constructed by cloning a blunted Kpn I-Sac I fragment ofpM7+ (29) into a blunted Kpn I-Pst I fragment of pBS(+) (Stratagene). RRE RNA was prepared by run-off transcription in vitro using EcoRI-digested pRRE plasmid, and antisense RRE RNA was prepared using HindIll-digested pRRE plasmid by standard procedures (Promega). To prepare the Rev-RRE complexes, 2 ,ug of RRE RNA was renatured by incubation at 80'C for 5 min in 40 ,tl of renaturation buffer (10 mM Hepes, pH 7.2/100 mM KCI) followed by slow cooling to 370C. Addition of Mg2+ to the renaturation buffer did not significantly alter the results. Abbreviations: DEP, diethyl pyrocarbonate; DMS, dimethyl sulfate; DTT, dithiothreitol; RRE, Rev response element. *Present address: Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115. tPresent address: Brigham and Women's Hospital, 75 Francis Street,
Boston, MA 02115. 683
684
S*L- e_s
Biochemistry: Kjems et al.
Proc. Natl. Acad. Sci. USA 88 (1991) antisense
sense
CO~~~~~~~~0
o. _' ccau 0) 0 o _ ,
richia coli tRNA at 0.2 mg/ml) that had been preincubated with 1-8 Al of Rev (1 mg/ml in Rev storage buffer, 1 M NaCI/20 mM Hepes, pH 7.9/2.5 mM MgCl2/0.5 mM EDTA/ 10% glycerol/1 mM D1T) or with the same volume of Rev storage buffer in the absence of Rev. After a 10-min incubation at 0C the sample was divided into eight 20-pAl aliquots, and reaction mixtures that received chemicals were diluted by adding 180 pl of Rev binding buffer (without tRNA). Samples for the gel mobility-retardation assay were prepared in the same manner except that complexes were formed using 1-5 ng of renatured and 32P-labeled RRE RNA in 10 pA of Rev binding buffer in the presence of 0-300 ng of Rev protein. The complexes were resolved in a 4% acrylamide gel (39:1 acrylamide/N,N'-methylenebisacrylamide weight ratio; 50 mM Tris/glycine, pH 8.9) run at 12 V/cm. Chemical and RNase Probing and Primer Extension Analysis of Modified RNA. Twenty microliters of renatured RNA or RNA-protein complex was digested by addition of 1 pl of RNase T1 (20 units/ml), RNase T2 (50 units/ml), or RNase CV (50 units/ml), followed by incubation for 20 min at 00C. For the chemical reactions, 200 pl of renatured RNA or RNA-protein complex was incubated with 1 pl of dimethyl sulfate (DMS), 5 1.l of diethylpyrocarbonate (DEP), or 10 ,p of kethoxal at 0C for 20, 30, or 90 min, respectively. The reactions were stopped and the mixtures were deproteinized by standard protocols (30). Modified nucleotides and RNase cleavage sites were detected by primer extension (31) using two oligonucleotide
a,
2x RRE
-4 R~~~~ RE
RRE
41114 1
2
3
5
4
6
8
7
9
10 11 12
FIG. 1. Gel mobility-shift assay of complex formation between Rev and RRE. The indicated amount (ng) of recombinant Rev was incubated with 2 ng of body-labeled RRE RNA probe (lanes 1-6) or antisense RRE RNA probe (lanes 7-12). RRE indicates the position of free monomer probe, and 2x RRE indicates a faint band that corresponds to the homodimer of the RRE.
Twenty microliters of the renatured RNA mixture was added to 120 ,ul of Rev binding buffer (50 mM Hepes, pH 7.9/150 mM KCl/2 mM MgCI2/0.5 mM EDTA/1 mM DlT with Esche-
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FIG. 2. Enzymatic and chemical footprints of the RRE in the absence or presence of Rev. Chemical reagents and nucleases were chosen to facilitate probing ofall nucleotides. The single-strand-specific reagents were DMS (Merck), which modifies adenosines (N-i) and, more slowly, cytidines (N-3); DEP (Eastman Kodak), which modifies adenosines (N-7); kethoxal (Keth; Sigma), which modifies guanosines (N-i and N on C-2); RNase T1 (Pharmacia), which cleaves the RNA backbone 3' to guanosines; and RNase T2 (Sankyo), which preferentially cleaves 3' to adenosines. RNase CV (Pharmacia) is specific for double-stranded RNA. DMS also reacts at guanosines (N-7) within regular helical regions, but these modifications cannot be detected by the primer extension method (32). Accessibility was monitored by primer extension using RNaseor chemical-treated samples as template. The primer RRE P2 was used to assay region 1-120 (A) and primer RRE P1 was used to assay region 120-204 (B). The Rev-RRE complex was formed using 2 ,&g of RRE RNA and 6 Ag of Rev (molar ratio; 1:15). The accessibility of the region downstream from position 204 was not determined, since the primer RRE P1 binds to this region. Nucleotides 224-233 were not present in the RNA transcript used in this analysis, and thus data covering the base-pairing region 1-15 has not been included. RNases and chemicals are indicated above the lanes; Cont, control reactions that were treated as the RNase samples but in the absence of RNase. The A, G, C, and U tracks refer to the RNA sequence and were generated by dideoxynucleotide sequencing of the untreated RRE RNA. Presence (+) or absence (-) of Rev is indicated. Numbers at right are nucleotide positions corresponding to Fig. 3.
primers, RRE P1 and RRE P2, complementary to positions 200-221 and 134-155 of the RRE RNA, respectively. Endlabeled primer (0.2 pmol) was annealed to one-fifth of the modified RNA complex ("0.2 pmol) and extended by reverse transcriptase (30). The reverse transcriptase pauses or stops at the nucleotide preceding a modified base and transcribes the nucleotide directly preceding an RNase cut (30). The cDNAs were resolved in denaturing 6% polyacrylamide gels containing 8 M urea, 100 mM Tris/borate (pH 8.3), and 1 mM EDTA. An RNA marker sequence was obtained by using the same conditions as for primer extension but including 0.5 pmol of untreated RRE RNA as template and adding a dideoxynucleoside triphosphate (1 ,ul of 1 mM ddATP, 1 mM ddTTP, 0.5 mM ddCTP, or 0.5 mM ddGTP) to each of the sequencing reaction mixtures.
RESULTS Rev Forms Multiple Complexes with the RRE. Recombinant Rev purified from insect cells (see Materials and Methods) and bound to 32P-labeled RRE RNA (240 nucleotides). The resulting complexes were analyzed by gel retardation (Fig. 1). Multiple bands that migrated slower than the free RRE RNA were observed, suggesting that multiple Rev proteins can bind to the RRE. At very high concentrations of Rev (==1000fold molar excess of Rev to RRE), only a smear of high molecular weight material was observed. This was probably the result of additional nonspecific binding of Rev. In contrast, Rev bound to antisense RRE with a 100-fold lower affinity (Fig. 1). Renaturation of the sense RRE always produced some RRE homodimer complexes, probably
A
caused by intermolecular base pairing. Interestingly, Rev showed the same binding affinity and formed a similar pattern ofcomplexes with the RRE homodimer as with the monomer. Probing the Secondary Structure of RRE. The structure of the RRE in the absence of protein was investigated by monitoring the accessibility of nucleotides to chemical and RNase probes (Fig. 2). The results are summarized on the putative secondary structure in Fig. 3A. The single-strandspecific probes (DMS, DEP, kethoxal, RNase T1, and RNase T2) reacted mainly with the loops of the proposed structure, while the double-strand-specific probe (RNase CV) cleaved the RNA mainly in the double-stranded regions (see legend to Fig. 2). All of the terminal loops, except loop IV, were hypersensitive to single-strand-specific probes, whereas the internal loops were less reactive. Direct structural analysis, however, revealed subtle differences from the computergenerated RRE secondary structure (33). The accessibility of region 133-136 and 158-161 to kethoxal and RNases T1 and T2 suggests that this region is not involved in regular base pairing. Furthermore, the relative inaccessibility of region 72-75 in loop IIA and 190-194 in loop I suggests that these regions are involved in secondary or tertiary structure. Rev Binds Primarily to Positions Within the Helix II Domain of the RRE. Nucleotide accessibilities throughout the RRE were compared for both the Rev-RRE complex and the RNA alone (Fig. 2). The data are summarized in Table 1 for positions where Rev induced altered nucleotide reactivities and are illustrated on the RRE secondary structure in Fig. 3B. Pronounced Rev protection was observed around U20-U21 in helix I, within the 3' strand of helices IIA and IIB, within both
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685
Proc. Natl. Acad. Sci. USA 88 (1991)
Biochemistry: Kjems et al.
A G G A GC UU U CC U C
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FIG. 3. Putative secondary structure of RRE showing the chemical and RNase reactivities of the free RRE RNA (A) and the changes caused by Rev binding (B). (A) Accessibilities to the various reagents are indicated by the symbols. One, two, and three symbols indicate weak, medium strong, and strong accessibility, respectively, based on visual inspection from several experiments. Nucleotides and helices are numbered according to Malim et al. (8). Bold arrows indicate nonspecific termination or pausing sites in the primer extension analysis of untreated RNA. A few specific RNase T1 cleavages were observed at nucleotides other than guanosine. This may reflect impurity of the enzyme or an unusual activity of RNase T1 on this RNA. (B) Summary of the altered reactivities upon Rev binding. Bold and thin circles denote nucleotides where the reactivity varied >2-fold or 2-fold or
Structural analysis of the interaction between the human immunodeficiency virus Rev protein and the Rev response element (baculovirus/expression system/RNA-protein interaction/RNA stem-loop)
J0RGEN KJEMS, MYLES BROWN*, DAVID D. CHANGt,
AND
PHILLIP A. SHARP
Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139
Contributed by Phillip A. Sharp, October 18, 1990
The specific interaction between a defined ABSTRACT structural element of the human immunodeficiency virus mRNA (RRE, the Rev response element) and the virus-encoded protein Rev has been implicated in the regulation of the export of unspliced or singly spliced mRNA from the nucleus to the cytoplasm. Rev protein was expressed and purified from insect cells using the baculovirus expression system. Chemical and RNase probes were used to analyze the structure of the RRE and the regions involved in Rev binding. Increased reactivity to single-strand-specific probes of nucleotides in two helical domains indicates that Rev binding induces conformational changes in the RRE. Binding of Rev to the RRE primarily protects helical segments and adjacent nucleotides in domain II. A Rev unit binding site is proposed that consists of a six-base-pair helical segment and three adjacent nucleotides. The data also suggest that multiple Rev proteins bind to repeated structural elements of the RRE.
The human retroviruses have proven to be an excellent system to study the complex regulation of gene expression in mammalian cells. Human immunodeficiency virus type 1 (HIV-1), which is implicated in the pathogenesis of acquired immunodeficiency syndrome (AIDS) (1, 2), has at least two novel genetic regulatory mechanisms. These are mediated by the virus-encoded regulatory proteins Rev and Tat, which are essential for viral replication (3). Whereas Tat appears to act on transcriptional initiation and elongation (3), Rev functions posttranscriptionally. Rev is necessary for the production of viral structural proteins (4, 5) through regulation of the cytoplasmic appearance of unspliced or singly spliced viral mRNA (6-8). Rev functions either directly or indirectly at the level of splicing (5, 6, 9, 10). Rev action requires the presence of a cis-acting RNA target sequence termed the Rev response element (RRE), which is located in the envelope gene of HIV-1 (8, 11-13). The minimal sequence required for full Rev response in vivo has been mapped to a 234-nucleotide region that has the potential of forming four stem-loops (I, III, IV, and V) and one branched stem-loop (IIA, IIB, and IIC) secondary structure (8). Moreover, stem-loops III, IV, and V can be deleted without abolishing the Rev response in vivo (11, 14, 15). Physical interaction between purified Rev and RRE has been demonstrated in vitro (16-23). Use of a gel retardation approach has shown that Rev binds to a minimal structure containing the branched stem-loop IIA-C (19, 22). However, the precise localization of Rev binding has not been determined. To approach this question we have investigated the relative accessibility of individual nucleotides within the RRE to a variety of chemicals and RNases in the presence or absence of Rev. These data test the proposed structural model of the RRE directly and define the binding site of Rev. In addition, we have found conformational The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
changes in the RNA upon Rev binding, which may have important implications for Rev function.
MATERIALS AND METHODS Construction of Recombinant Baculovirus Expressing HIV Rev. The Stu I-HindIII fragment of the plasmid pcREV (24) containing the Rev coding region was cloned into the BamHI site of the baculovirus expression vector pVL941 (25) by standard techniques. The resulting plasmid, pVREV, was cotransfected with wild-type baculovirus (Autographa californica nuclear polyhedrosis virus, AcNPV) DNA into Sf9 insect cells and a recombinant baculovirus expressing Rev was plaque-purified (26). Purification of Recombinant HIV Rev from Infected Insect Cells. Large-scale infections of insect cells were performed as described (27). Infected cells were harvested 48-72 hr postinfection and washed twice with phosphate-buffered saline. The cell pellet was suspended in lysis buffer [20 mM Hepes, pH 7.9/140 mM NaCI/1.5 mM MgCl2/1% (vol/vol) Nonidet P-40/1 mM phenylmethylsulfonyl fluoride/1 mM dithiothreitol (DTT)J and incubated on ice for 5 min. Nuclei were prepared by centrifugation through a 25% (wt/vol) sucrose cushion in lysis buffer. The nuclei were then suspended and treated with DNase 1 (40 ,ug/ml) in 20 mM Hepes, pH 7.9/100 mM NaCI/5 mM MgCl2/1 mM DTT for 30 min at room temperature. Nucleoplasm containing the majority of the HIV Rev protein was prepared from the DNase I-treated nuclei after centrifugation to remove the insoluble nuclear residue. This fraction was then applied to a Pharmacia FPLC Mono S column and eluted with a linear gradient of NaCl in 20 mM Hepes, pH 7.9/2.5 mM MgCI2/0.5 mM EDTA/10% (vol/vol) glycerol/1 mM DTT. The peak of Rev protein eluted from the Mono S column was >90% pure as analyzed by SDS/17.5% PAGE followed by staining with Coomassie brilliant blue. Preparation of RRE Transcripts and Rev-RRE Complexes. The plasmid pRRE, which contains region 7768-7991 of the HXB-3 isolate of HIV-1 (28), was constructed by cloning a blunted Kpn I-Sac I fragment ofpM7+ (29) into a blunted Kpn I-Pst I fragment of pBS(+) (Stratagene). RRE RNA was prepared by run-off transcription in vitro using EcoRI-digested pRRE plasmid, and antisense RRE RNA was prepared using HindIll-digested pRRE plasmid by standard procedures (Promega). To prepare the Rev-RRE complexes, 2 ,ug of RRE RNA was renatured by incubation at 80'C for 5 min in 40 ,tl of renaturation buffer (10 mM Hepes, pH 7.2/100 mM KCI) followed by slow cooling to 370C. Addition of Mg2+ to the renaturation buffer did not significantly alter the results. Abbreviations: DEP, diethyl pyrocarbonate; DMS, dimethyl sulfate; DTT, dithiothreitol; RRE, Rev response element. *Present address: Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115. tPresent address: Brigham and Women's Hospital, 75 Francis Street,
Boston, MA 02115. 683
684
S*L- e_s
Biochemistry: Kjems et al.
Proc. Natl. Acad. Sci. USA 88 (1991) antisense
sense
CO~~~~~~~~0
o. _' ccau 0) 0 o _ ,
richia coli tRNA at 0.2 mg/ml) that had been preincubated with 1-8 Al of Rev (1 mg/ml in Rev storage buffer, 1 M NaCI/20 mM Hepes, pH 7.9/2.5 mM MgCl2/0.5 mM EDTA/ 10% glycerol/1 mM D1T) or with the same volume of Rev storage buffer in the absence of Rev. After a 10-min incubation at 0C the sample was divided into eight 20-pAl aliquots, and reaction mixtures that received chemicals were diluted by adding 180 pl of Rev binding buffer (without tRNA). Samples for the gel mobility-retardation assay were prepared in the same manner except that complexes were formed using 1-5 ng of renatured and 32P-labeled RRE RNA in 10 pA of Rev binding buffer in the presence of 0-300 ng of Rev protein. The complexes were resolved in a 4% acrylamide gel (39:1 acrylamide/N,N'-methylenebisacrylamide weight ratio; 50 mM Tris/glycine, pH 8.9) run at 12 V/cm. Chemical and RNase Probing and Primer Extension Analysis of Modified RNA. Twenty microliters of renatured RNA or RNA-protein complex was digested by addition of 1 pl of RNase T1 (20 units/ml), RNase T2 (50 units/ml), or RNase CV (50 units/ml), followed by incubation for 20 min at 00C. For the chemical reactions, 200 pl of renatured RNA or RNA-protein complex was incubated with 1 pl of dimethyl sulfate (DMS), 5 1.l of diethylpyrocarbonate (DEP), or 10 ,p of kethoxal at 0C for 20, 30, or 90 min, respectively. The reactions were stopped and the mixtures were deproteinized by standard protocols (30). Modified nucleotides and RNase cleavage sites were detected by primer extension (31) using two oligonucleotide
a,
2x RRE
-4 R~~~~ RE
RRE
41114 1
2
3
5
4
6
8
7
9
10 11 12
FIG. 1. Gel mobility-shift assay of complex formation between Rev and RRE. The indicated amount (ng) of recombinant Rev was incubated with 2 ng of body-labeled RRE RNA probe (lanes 1-6) or antisense RRE RNA probe (lanes 7-12). RRE indicates the position of free monomer probe, and 2x RRE indicates a faint band that corresponds to the homodimer of the RRE.
Twenty microliters of the renatured RNA mixture was added to 120 ,ul of Rev binding buffer (50 mM Hepes, pH 7.9/150 mM KCl/2 mM MgCI2/0.5 mM EDTA/1 mM DlT with Esche-
A
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_
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_
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.
,:
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--
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Se
- 4*.
--
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a i
-
a-
w
FIG. 2. Enzymatic and chemical footprints of the RRE in the absence or presence of Rev. Chemical reagents and nucleases were chosen to facilitate probing ofall nucleotides. The single-strand-specific reagents were DMS (Merck), which modifies adenosines (N-i) and, more slowly, cytidines (N-3); DEP (Eastman Kodak), which modifies adenosines (N-7); kethoxal (Keth; Sigma), which modifies guanosines (N-i and N on C-2); RNase T1 (Pharmacia), which cleaves the RNA backbone 3' to guanosines; and RNase T2 (Sankyo), which preferentially cleaves 3' to adenosines. RNase CV (Pharmacia) is specific for double-stranded RNA. DMS also reacts at guanosines (N-7) within regular helical regions, but these modifications cannot be detected by the primer extension method (32). Accessibility was monitored by primer extension using RNaseor chemical-treated samples as template. The primer RRE P2 was used to assay region 1-120 (A) and primer RRE P1 was used to assay region 120-204 (B). The Rev-RRE complex was formed using 2 ,&g of RRE RNA and 6 Ag of Rev (molar ratio; 1:15). The accessibility of the region downstream from position 204 was not determined, since the primer RRE P1 binds to this region. Nucleotides 224-233 were not present in the RNA transcript used in this analysis, and thus data covering the base-pairing region 1-15 has not been included. RNases and chemicals are indicated above the lanes; Cont, control reactions that were treated as the RNase samples but in the absence of RNase. The A, G, C, and U tracks refer to the RNA sequence and were generated by dideoxynucleotide sequencing of the untreated RRE RNA. Presence (+) or absence (-) of Rev is indicated. Numbers at right are nucleotide positions corresponding to Fig. 3.
primers, RRE P1 and RRE P2, complementary to positions 200-221 and 134-155 of the RRE RNA, respectively. Endlabeled primer (0.2 pmol) was annealed to one-fifth of the modified RNA complex ("0.2 pmol) and extended by reverse transcriptase (30). The reverse transcriptase pauses or stops at the nucleotide preceding a modified base and transcribes the nucleotide directly preceding an RNase cut (30). The cDNAs were resolved in denaturing 6% polyacrylamide gels containing 8 M urea, 100 mM Tris/borate (pH 8.3), and 1 mM EDTA. An RNA marker sequence was obtained by using the same conditions as for primer extension but including 0.5 pmol of untreated RRE RNA as template and adding a dideoxynucleoside triphosphate (1 ,ul of 1 mM ddATP, 1 mM ddTTP, 0.5 mM ddCTP, or 0.5 mM ddGTP) to each of the sequencing reaction mixtures.
RESULTS Rev Forms Multiple Complexes with the RRE. Recombinant Rev purified from insect cells (see Materials and Methods) and bound to 32P-labeled RRE RNA (240 nucleotides). The resulting complexes were analyzed by gel retardation (Fig. 1). Multiple bands that migrated slower than the free RRE RNA were observed, suggesting that multiple Rev proteins can bind to the RRE. At very high concentrations of Rev (==1000fold molar excess of Rev to RRE), only a smear of high molecular weight material was observed. This was probably the result of additional nonspecific binding of Rev. In contrast, Rev bound to antisense RRE with a 100-fold lower affinity (Fig. 1). Renaturation of the sense RRE always produced some RRE homodimer complexes, probably
A
caused by intermolecular base pairing. Interestingly, Rev showed the same binding affinity and formed a similar pattern ofcomplexes with the RRE homodimer as with the monomer. Probing the Secondary Structure of RRE. The structure of the RRE in the absence of protein was investigated by monitoring the accessibility of nucleotides to chemical and RNase probes (Fig. 2). The results are summarized on the putative secondary structure in Fig. 3A. The single-strandspecific probes (DMS, DEP, kethoxal, RNase T1, and RNase T2) reacted mainly with the loops of the proposed structure, while the double-strand-specific probe (RNase CV) cleaved the RNA mainly in the double-stranded regions (see legend to Fig. 2). All of the terminal loops, except loop IV, were hypersensitive to single-strand-specific probes, whereas the internal loops were less reactive. Direct structural analysis, however, revealed subtle differences from the computergenerated RRE secondary structure (33). The accessibility of region 133-136 and 158-161 to kethoxal and RNases T1 and T2 suggests that this region is not involved in regular base pairing. Furthermore, the relative inaccessibility of region 72-75 in loop IIA and 190-194 in loop I suggests that these regions are involved in secondary or tertiary structure. Rev Binds Primarily to Positions Within the Helix II Domain of the RRE. Nucleotide accessibilities throughout the RRE were compared for both the Rev-RRE complex and the RNA alone (Fig. 2). The data are summarized in Table 1 for positions where Rev induced altered nucleotide reactivities and are illustrated on the RRE secondary structure in Fig. 3B. Pronounced Rev protection was observed around U20-U21 in helix I, within the 3' strand of helices IIA and IIB, within both
B 40
A
G A
140
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A
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150
130
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170 GUUCUG! AU G CA G GGC C G A A 94 G
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RNase CV Unspecific
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A U C C U C G AC
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685
Proc. Natl. Acad. Sci. USA 88 (1991)
Biochemistry: Kjems et al.
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Ac
FIG. 3. Putative secondary structure of RRE showing the chemical and RNase reactivities of the free RRE RNA (A) and the changes caused by Rev binding (B). (A) Accessibilities to the various reagents are indicated by the symbols. One, two, and three symbols indicate weak, medium strong, and strong accessibility, respectively, based on visual inspection from several experiments. Nucleotides and helices are numbered according to Malim et al. (8). Bold arrows indicate nonspecific termination or pausing sites in the primer extension analysis of untreated RNA. A few specific RNase T1 cleavages were observed at nucleotides other than guanosine. This may reflect impurity of the enzyme or an unusual activity of RNase T1 on this RNA. (B) Summary of the altered reactivities upon Rev binding. Bold and thin circles denote nucleotides where the reactivity varied >2-fold or 2-fold or
