HIV-1 structural gene expression requires the binding of multiple Rev monomers to the viral RRE: Implications for HIV-1 latency

Cell, Vol. 65, 241-246,

April 19, 1991, Copyright

0 1991 by Cell Press

HIV=1 Structural Gene Expression Requires the Binding of Multiple Rev Monomers to the Viral RRE: Implications for HIV-1 Latency Michael H. Maiim and Bryan Ft. Cuilen Howard Hughes Medical institute and Departments of Medicine and Microbiology and Immunology Duke University Medical Center Durham, North Carolina 27710

Expression of the structural proteins of HIV-1 requires the direct interaction of the viral Rev frans-activator with its cis-acting RNA target sequence, the Rev response element or RRE. Here, we demonstrate that this speclflc RNA-binding event is, as expected, mediated by the conserved arginine-rich motif of Rev. However, we also show that amino acid residues located proximal to this basic domain that are crltlcal for in vivo Rev function are dispensable for sequence-specific binding to the RRE. instead, these sequences are required for the multimerixation of Rev on the viral RRE target sequence. The observation that Rev function requires the sequential binding of multiple Rev molecules to the RRE provides a biochemical explanation for the observed threshold effect for Rev function in vivo and suggests a molecular model for the high incidence of latent infection by HIV-l. introduction Human immunodeficiency virus type 1 (HIV-l) encodes two trans-acting nuclear regulatory proteins, termed Tat and Rev, that play essential roles in the HIV-1 replication cycle (reviewed in Cullen and Greene, 1989). The viral Tat protein enhances the expression of sequences linked in cis to the HIV-1 long terminal repeat (LTR) promoter element. The viral Rev protein, in contrast, acts posttranscriptionally to induce the cytoplasmic expression of the incompletely spliced HIV-1 mRNAs that encode the viral structural proteins, including Gag and Env. Rev is believed to activate the nucieocytoplasmic transport of these incompletely spliced transcripts either directly, by facilitating their access to a nuclear RNA transport pathway, or indirectly, by inhibiting their interaction with cellular splicing factors (Feinberg et al., 1986; Chang and Sharp, 1989; Emerman et al., 1989; Felber et al., 1989; Hammarskjold et al., 1989; Malim et al., 1989b). The HIV-1 Rev protein has been shown to bind with high specificity to its viral RNA target sequence, the highly structured Rev response element (RRE), and this direct interaction appears critical for Rev function in vivo (Daly et al., 1989; Zapp and Green, 1989; Cochrane et al., 1990; Heaphy et al., 1990; Malim et al., 1990). Although at least two and possibly as many as eight Rev proteins have been shown to bind to the RRE in vitro (Daly et al., 1989; Heaphy

et al., 1990; Malim et al., 1990), the functional significance of this multimeritation has remained uncertain. However, evidence has been presented suggesting the existence of multiple discrete Rev target sites within the RRE (Kjems et al., 1991). During the HIV-1 replication cycle, the viral Tat and Rev proteins are believed to act in concert to produce sequential changes in both the quantity and quality of viral gene expression (Cullen and Greene, 1989; Kim et al., 1989). Shortly after infection, the HIV-1 provirus gives rise to a relatively low level of viral RNA expression that is limited to the multiply spliced, -2 kb transcripts that encode the viral regulatory proteins Tat, Rev, and Nef. The action of the viral Tat trans-activator leads initially to a transient increase in the expression of these 2 kb viral mRNA species. Shortly thereafter, however, HIV-1 mRNA expression switches to the predominant synthesis of the singly spliced (-4 kb) and unspliced (w9 kb) mRNAs that encode the viral structural proteins (Kim et al., 1989). This qualitative shift in HIV-1 mRNA expression is mediated by the viral Rev &ens-activator. Recently, evidence has been presented suggesting that Rev function requires a critical level of intracellular Rev protein expression (Pomerantz et al., 1990). This hypothesis resulted from an analysis of HIV-1 mRNA expression in human cell lines nonproductively infected with HIV-l. In these cells, viral mRNA expression is not only relatively low but, more importantly, consists predominantly of the multiply spliced ~2 kb class of viral mRNAs. Treatment of these cells with agents that stimulate the HIV-1 LTR promoter results in a transition to viral structural gene expression that closely mimics that observed during a lytic replication cycle. It was therefore proposed that latent or nonproductive infection of cells by HIV-1 resulted from the long-term maintenance of a pattern of viral gene expression essentially identical to that observed transiently during the early stages of a productive HIV-1 replication cycle (Pomerantz et al., 1990). It was hypothesized that this phenotype was due to the expression of a subcritical level of Rev protein that in turn reflected a relatively low level of transcription from the HIV-1 LTR promoter in these cells. Although the concept that a threshold level of Rev protein expression is required for Rev function in vivo provides an attractive explanation for the high level of latent infection observed with HIV-1 and other complex retroviruses, no molecular basis for this requirement has been suggested. Here, we describe negative mutants of the viral Rev trans-activator that bind to the RRE with apparently normal affinity. However, these mutants are unable to participate in the subsequent multimerization of Rev on the RRE. We therefore propose that Rev function requires the successive binding of at least two Rev monomers to this &-acting viral RNA target sequence. The conclusion that the functional interaction of Rev with the RRE is not a first order reaction provides a biochemical explanation for the observed threshold effect for Rev function.

Cdl 242

Results The HIV-1 Rev frans-activator is a 116 amino acid phosphoprotein that is localized to the nuclei and, particularly, the nucleoli of expressing cells(Malim et al., 1969a; Felber et al., 1989). The major notable feature of the primary sequence of the Rev protein is an arginine-rich motif, extending from amino acid position 35 to 50, that has been shown to function as a nuclearlnucleolar localization signal (Kubotaet al., 1989; Malim et al., 1989a; Perkinset al., 1989). Extensive mutational analysis has defined at least two additional functional domains within Rev. Mutations introduced between approximately amino acid position 18 and 56 result in a recessive negative phenotype (Malim et al., 1989a; Perkins et al., 1989; Hope et al., 1990). This protein domain includes, but extends both N-and C-terminal to, the sequences required for Rev nuclear/nucleolar localization. A smaller leucine-rich domain, extending from approximately amino acid position 75 to 83, is also essential for Rev function, but mutations within this sequence give rise to a dominant negative phenotype (Malim et al., 1989a; Hope et al., 1990; Mermer et al., 1990; Olsen et al., 1990). We have previously described a series of Rev proteins bearing mutations both within and adjacent to each of these functional domains (Table 1) (Malim et al., 1989a). To determine the biochemical basis of the various phenotypes displayed by these mutants in vivo, we expressed both mutant and wild-type Rev proteins in bacteria as C-terminal fusionswith the enzyme glutathione S-transferase (GST) (Smith and Johnson, 1988). Expression of these Rev proteins as GST fusions provides a number of technical advantages. These include excellent solubility, high yield, and the ability to purify the fusion proteins in a single step under nondenaturing conditions using a glutathioneagarose affinity column. Most importantly, the presence of the GST “tag” has allowed us to examine the ability of the

Table Name ~M3 M4 M5 M6 M7 ME M9 Ml0

1. Description

of HIV-1

Mutation R+D (aa 17) YSN-DDL (aa 23, 25, 26) RR-DL (aa 38, 39) RRRR-DL (aa 41-44) 5X3-i (aa 54-56) ST-+DL (aa 81, 62) SA+DL (aa 67, 88) LE-DL (aa 78, 79)

Rev Mutants Biological Activity

RNA Binding

+

+

- (W

+

- (R)

-

NA

- (R)

-

NA

- (R) +

+ +

+

+

+

+

- (TD)

+

+

Multimerization + -

The derivation and in vivo phenotypic analysis of the indicated Rev mutants have been described (Malim et al., 1989a). The in vitro RNAbinding and multimerization properties of these mutants are described in this paper. R, recessive negative mutant; TD. Pans-dominant negative mutant; NA, not applicable; aa, amino acids.

various Rev mutants to multimerize with the wild-type 116 amino acid Rev protein during in vitro binding assays with the RRE (see below). We have previously used gel retardation analysis to investigate the in vitro interaction of recombinant Rev protein with a radiolabeled probe containing the complete RRE (Malim et al., 1990). These results, which are reproduced in Figure 1 A, demonstrated the formation of at least two specific Rev-RRE complexes in vitro. At low levels of Rev protein a rapidly migrating complex (complex Cl’) is predominantly detected (Figure 1 A, lane 6), while at higher levels a second, more slowly migrating complex (complex C2’) is observed (Figure 1A, lanes 4 and 5). Both of these Rev-RRE complexes migrate more rapidly than an RRE RNA dimer (our unpublished data; Kjems et al., 1991), and the C2’ complex must therefore result from multimerization of Rev protein on the RRE. Addition of further Rev protein gives rise to a series of more slowly migrating and increasingly indistinct protein-RNA complexes. At saturating concentrations of Rev, binding to nonspecific RNA probes becomes significant, and these bands merge into a diffuse, highly retarded band of uncertain specificity(Figure 1A, lane 2). A direct comparision of the RRE-binding properties displayed by increasing levels of the 116 amino acid wild-type Rev and GST-Rev proteins reveals very similar patterns of in vitro protein-RNA complex formation. In particular, GST-Rev also gives rise to a single predominant proteinRRE complex, here termed Cl, at low protein levels (Figure lA, lanes 10 and 11) and to a second, more slowly migrating complex, termed C2, at higher protein levels (Figure 1A, lane 9). As predicted by the greater molecular mass of the GST-Rev protein, complexes formed between GST-Rev and the RRE were observed to migrate significantly more slowly than those formed between Rev and the RRE. As expected, equivalent levels of the GST protein itself produced no detectable protein-RNA complexes (Figure 1A, lanes 12 and 13). Both complexes formed between GST-Rev and the RRE were efficiently competed by the addition of unlabeled RRE RNA but not by the addition of 55 rRNA, thus confirming the specificity of this interaction (Figure lB, lanes 2-4). In addition, the GSTRev fusion protein was observed to be functional in vivo, as demonstrated by the ability to rescue a Rev-deficient HIV-1 proviral construct in cotransfection experiments (data not shown). Having demonstrated that the GST-Rev fusion protein is indeed functionally comparable to the wild-type Rev protein, we next examined the ability of a series of mutant Rev proteins (described in Table 1) to interact with the RRE in vitro when expressed as GST fusion proteins (Figure 2A). This experiment revealed three distinct in vitro phenotypes. The wild-type GST-Rev protein, three mutants that displayed normal activity in vivo, and the frans-dominant negative GST-Ml0 mutant all bound the RRE efficiently and gave rise to both the predicted Cl and C2 complexes (Figure 2A, lanes 3, 4, and 9-l 1). In contrast, the GSTM5 and M6 proteins, which are mutated in the Rev arginine-rich motif, failed to detectably bind the RRE probe (Figure 2A, lanes 6 and 7). Of particular interest were the

Role of HIV-1 Rev Multimerization 243

Figure

1. Comparison

of the ME-Binding

Properties

of the Wild-Type

Rev and GST-Rev

Proteins

(A) A constant level of a labeled 252 nucleotide RNA probe containing the full-length HIV-t RRE was incubated with increasing levels of recombinant Rev protein (lanes 2-S) or GST-Rev protein (lanes 7-13). Binding of the probe was visualized as slower migration upon electrophoresis through a native polyacrylamide gel. Both the Rev and GST-Rev proteins give rise to distinct faster migrating (Cl’ and Cl) and slower migrating (C2’ and C2) protein-RNA complexes. The GST protein itself was unable to bind the RRE probe (lanes 12 and 13). (6) The ability of 200 ng of added unlabeled RRE RNA or added 5S rRNA to compete with the RRE for binding by the indicated GST fusion proteins was analyzed.

A 1

B 2

3

4

5

6

7

0

Q

1011

1

-

2

3

4

5

6

78QlO

c2

cc1

-1

\\\ Added

Figure

2. Analysis

of the RRE-Binding

Properties

of a Series

of Mutant

GST-Ml0

Added

GST-M4

Rev Proteins

(A) Equal amounts (1 U or 330 ng) of each indicated GST fusion protein were incubated with the full-length radiolabeled HIV-l RRE probe. The pattern of protein-RNA complex formation was then visualized by gel electrophoresis followed by autoradiography. The GST-M4 fusion protein displayed a slightly reduced electrophorectic mobility relative to other GST-Rev proteins both on native gels, as a complex with the RRE (lane 5) and as a denatured protein on SDS-polyacrylamide gels (data not shown). (6) This protein titration experiment was performed as described in Figure 1 A, using increasing levels of the GST-Ml0 or GST-M4 fusion proteins.

Cell

244

plex (Figure 2B, lane 8). As discussed above, this diffuse, highly retarded complex appears unlikely to be specific. The data presented in Figure 2 suggest that the M4 and M7 mutations are within a protein domain that is required for multimerization of Rev on the RRE but dispensable for the initial Rev-RRE interaction. We therefore next wished to address two further issues pertinent to an understanding of the Rev-RRE interaction: Was the initial “Cl” complex detected upon incubation of Rev with the RRE formed by the binding of a Rev monomer? Would the M4 mutation be able to form a mixed multimer on the RRE with the wild-type Rev protein? To address these questions, we took advantage of the fact that the wild-type Rev protein and the GST-Rev fusion proteins gave rise to proteinRNA complexes of distinctly different gel mobilities (Figure 1A). We therefore analyzed the pattern of protein-RNA complexes formed after incubation of constant amounts of both the RRE probe and of selected GST-Rev fusion proteins with increasing amounts of the wild-type Rev protein (Figure 3). If the initial Cl complex was formed by binding of more than one Rev protein to the RRE, then we predicted that this experiment would detect a novel retarded band(s) with a mobility intermediate between the Cl and Cl’signals. Conversely, if the predicted interaction of more than one Rev molecule with the RRE was actually equivalent to the C2 complex, then we should detect a novel retarded band with a mobility intermediate between the Cl and C2 signals As previously shown (Figure 1A), incubation of increasing levels of Rev with the RRE probe resulted in the formation of two discrete Rev-RRE complexes when analyzed with wild-type Rev (Cl ‘and C2’; Figure 3, lanes 2-4) or with the GST-Rev or GST-Ml0 proteins (Cl and C2; Figure 3, lanes 5 and 13) and a single complex when analyzed with the GST-M4 protein (Cl; Figure 3, lane 9). Addition of increasing levels of wild-type Rev to a reaction mixture containing a constant level of the GST-Rev protein resulted in the decreased formation of the C2 complex and the appearance of a novel complex, with a mobility intermediate between the Cl and C2 complexes, which is here termed C?(Figure 3, compare lanes 5 and 8). An identical

M4 and M7 mutations, which efficiently bound to the FIRE to give the Cl complex but which did not give rise to any detectable C2 complex under these conditions (Figure 2A, lanes 5 and 8). All the retarded complexes formed between the RRE probe and the various mutant GST-Rev proteins, including GST-M4 and GST-M7, could be competed by the addition of excess unlabeled RRE RNA, but not by excess 5s rRNA (Figure 16 and data not shown), and these complexes therefore represent specific proteinRNA interactions. The results presented in Figure 2A suggested that the M4 and M7 Rev mutants retained the ability to bind the RRE but were unable to multimerize on the RRE after the initial binding event had occurred. To confirm that this result did not simply reflect a low level of input protein, we next compared the ability of increasing levels of the GSTMl0 and GST-M4 proteins to bind to a constant level of the RRE probe in vitro (Figure 28). The GST-Ml0 protein, like the GST-Rev protein (Figure 1A), was observed to form the Cl complex at low levels of protein (Figure 28, lane 5) and gave rise to increasing levels of the slower migrating C2 complex as the level of protein was increased (Figure 28, lanes 2-4). At very high protein levels, the GST-Ml0 protein, like the GST-Rev and Rev proteins (Figure 1 A), gave rise to an indistinct and highly retarded complex of uncertain specificity (Figure 28, lane 1). At low levels of protein, the GST-M4 protein gave rise to a level of the Cl complex comparable to that observed for GSTMl 0 (Figure 28, compare lanes 5 and 10). No C2 complex was detected, however, with the GST-M4 protein even at protein concentrations that retarded almost all the available RRE probe (Figure 2B, lanes 7-9). Notably, under conditions in which the GST-Ml0 and GST-M4 proteins each retarded ~75% of the input probe, the GST-Ml0 protein gave rise to predominantly the more slowly migrating C2 protein-RNA complex, while the complex formed between the RRE and the GST-M4 protein was exclusively of the more rapidly migrating, Cl type (Figure 28, compare lanes 3 and 8). Only at the highest protein concentration tested did the GST-M4 protein give rise to a band that migrated more slowly than the Cl com-

12345676

9

10

11

12

13

14

15 16

c2C2'Cl

Figure 3. Analysis of Mixed Multimer Formation by Wild-Type and Mutant Rev Proteins on an HIV-1 RRE Probe

-c2 -C2'

-

-Cl

C2'-

-C2'

Cl’-

-Cl’

I-

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i \ I t I I I i *o "r 3 *, *o 5 '2 3 33 75 5 GST-M4

GST-Ml0

Equal amounts (113 U) of the GST-Rev, GSTM4, and GST-Ml0 proteins were incubated with 0, l/9, l/3 or 1 U of the wild-type recombinant Rev protein for 10 min prior to addition of a constant level of the radiolabeled RRE probe. As a control, the wild-type Rev protein was also preincubated in the absence of any GST fusion protein (lanes l-4). The different protein-RNA complexes formed under these conditions were then resolved by gel electrophoresis.

Role of HIV-1

Rev Multimerization

245

pattern of RNA-protein complex formation was observed using the GST-Ml0 fusion protein in combination with wild-type Rev (Figure 3, compare lanes 13 and 16). In neither case was a novel band detected with a mobility faster than that observed for the Cl complex. We therefore suggest that the novel C2” complex is likely to represent a ternary complex comprising the FIRE probe, a wild-type Rev molecule, and a GST-Rev or GST-Ml0 molecule. In contrast to GST-Rev and GST-MlO, addition of increasing levels of wild-type Rev protein to a incubation mixture containing a constant level of the GST-M4 protein failed to result in the formation of any novel protein-RNA complexes (Figure 3, compare lanes 9 and 12). Instead, a slight reduction in the level of the Cl complex was obsewed. We therefore conclude that the GST-M4 protein is unable to form multimers on the RRE in combination with either itself or with wild-type Rev protein. Discussion The in vitro analysis of the Rev-RRE interaction presented here, combined with previously published mutational analyses of in vivo Rev function (Kubota et al., 1989; Malim et al., 1989a; Perkins et al., 1989; Hope et al., 1990; Mermer et al., 1990), allow us to propose a detailed domain structure for the HIV-1 Rev trens-activator (Figure 4). The Rev arginine-rich motif, extending from approximately amino acid position 35 to 50, appears to serve as both a nuclear/ nucieolar localization signal and as the specific RNAbinding domain of Rev. Flanking this sequence both N-terminally and C-terminally are protein sequences essential for the subsequent multimerization of Revon the viral RRE target sequence (Figure 4). A final functional domain, extending from approximately position 75 to 83, plays no detectable role in either RNA binding or multimerization

/

2”

4”

6p

“p

100 ,

116 ,

.~.~.~.~.~.~_~.~_~.~.~.‘.‘.‘.‘.‘.’.’. .~.~.~.~,~.~.~.~.~.~.~.‘.‘.‘.‘.‘.’.’.

ACTIVATION MULTIMERIZATION

Figure 4. Proposed Domain Structure of the HIV-1 Rev Protein Three distinct functional domains located within the 116 amino acid Rev protein are identified (see text for details). The leucine-rich activation domain (amino acids 75 to 93) plays no role in RRE binding or Rev multimerization but is essential for in vivo function. The argininerich motif (amino acids 35-50) appears to be both necessary and sufficient for Rev nucleadnucleolar localization and specific binding to the RRE. Sequences flanking the arginine-rich motif between approximately amino acid 19 and 59 are essential for multimertzation of Rev on the RRE target sequence. The Rev protein sequences located between approximately amino acid 59 and 75 may primarily serve to position appropriately these flanking functional domains, as this region appears highly tolerant of m&sense mutations but not of deletion mutations (Hope et al., 199Q Malim et al., unpublished data). The N-and C-terminal Rev sequences indicated by stippling are dispensable for in vivo Rev function (Malim et al., 1999a).

yet is essential for in vivo Rev activity. It therefore appears probable that this sequence interacts with a cellular protein(s) importantly involved in the mechanism of action of the Rev trans-activator and can therefore be defined as the Rev activation domain. We and others have reported that Rev proteins mutated in this activation domain display a trensdominant negative phenotype in vivo (Table 1) (Malim et al., 1989a; Hope et al., 1990; Mermer et al., 1990). We had therefore previously suggested that these mutant proteins might efficiently compete with wild-type Rev for binding to the viral RRE sequence but would then be unable to activate viral structural gene expression. The results presented here raise the possibility that these mutant Rev proteins might also act by forming inactive mixed muitimers with wild-type Rev protein on the viral RRE target sequence. The observation that the M4 and M7 Rev mutants are able to compete with wild-type Rev for binding to the RRE, yet are relatively ineffective inhibitors of Rev function in vivo (Malim et al., lSSSa), suggests that the ability to multimerize with wild-type Rev protein may be an essential requirement for an effective transdominant negative Rev mutant. The data presented in this paper demonstrate that the arginine-rich motif present in Rev serves as the RNA sequence-specific recognition domain. This observation therefore confirms the hypothesis that Rev belongs to the novel arginine-rich class of RNA-binding proteins that also includes the HIV-1 Tat &ens-activator (Dingwall et al., 1989; Lazinski et al., 1989,; Maiim et al., 1989a, 1990). These results also suggest that the Rev protein initially binds to the RRE as a monomer. Subsequently, additional Rev monomers bind to the RRE in a process mediated by protein sequences that flank the arginine-rich motif but that appear to play no direct role in sequence-specific binding to the RNA (Figures 2A and 4). We therefore suggest that these sequences, which are here identified by the M4 and M7 Rev mutations, facilitate the specific interaction of Rev protein monomers on the viral RRE target sequence. This multimerization event, which appears critical for in vivo Rev activity, may be required for the formation of a functional Rev activation domain and/or for the interaction of this domain with its currently unknown cellular target. A rather different hypothesis has emerged from the recent work of Olsen et al. (1990). These authors have presented data suggesting that Rev multimer formation is a prerequisite for RRE binding and, more specifically, that “formation of the Rev RNA-binding site is dependent on muitimerization.” The sequences required for muitimerization-and hence RNA binding-were proposed to extend from approximately amino acid position 28 to position 57 within Rev, i.e., extensively overlap with the sequences here proposed to facilitate multimerization after binding has occurred. Of note, Rev proteins mutated at residues 28 to 31 or residues 55 to 57 were proposed to be unable to bind the RRE in vitro. In contrast, we report here that similar mutants (M4 and M7; Table 1) retain the ability to bind the RRE. Moreover, recent data demonstrate that a synthetic peptide consisting of residues 34 to 50 of the HIV-1 Rev protein, i.e., that do not extend beyond the arginine-rich motif of Rev,

Cell 246

retain the ability to bind specifically to the HIV-1 RRE in vitro (A. Frankel and P. Sharp, personal communication). This observation clearly demonstrates that sequences outside the Rev arginine-rich motif are dispensable for sequence-specific RNA binding and must therefore fulfill a different essential function. Our results indicate that this function is multimerization of Rev on the viral RRE target sequence (Daly et al., 1989; Heaphy et al., 1990; Malim et al., 1990; Kjems et al., 1991). The observed multimerization of Rev in the absence of the RRE target sequence (Olsen et al., 1990; Nalin et al., 1990) may therefore represent the detection of a relatively low affinity protein-protein interaction when compared with multimerization on the RRE. We note that others have reported that purified, biologically active preparations of Rev protein display a high tendency to aggregate when examined at neutral pH (Daly et al., 1990). Implications for HIV-i Latency As noted in the Introduction, persuasive evidence has been presented suggesting that Rev function isessentially absent when expression of Rev protein is low (Pomerantz et al., 1990). It has therefore been proposed that the relationship between the level of Rev function and protein expression is distinctly nonlinear and, more specifically, that there is a threshold level below which the Rev protein fails to function effectively (Pomerantz et al., 1990). The concept that a critical level of Rev expression is essential for a productive viral infection provides an attractive explanation for the high level of nonproductive or latent HIV-1 infection observed in vivo (Fauci, 1988). Under conditions in which the level of transcription of the HIV-1 genome is low, due to the absence of cellular factors required either directly for proviral transcription or indirectly for trans-activation by Tat (Nabel and Baltimore, 1987; Jones, 1989; Jakobovits et al., 1990; Marciniak et al., lQQO), this level of regulation would prevent the premature expression of the viral structural proteins. An untimely progression to the late, structural phase of the HIV-1 replication cycle might result either directly or indirectly(due to immunological surveillance) in the death of the HIV-l-infected cell prior to the release of a significant level of progeny virions and might therefore be highly deleterious to the efficient spread of HIV-1 in the infected host. Conversely, if viral structural gene expression were to occur only in cells capable of maintaining a high level of proviral transcription, this should facilitate the production of a high level of progeny virions from each infected cell prior to cell death. In this context, it is of interest to note that several other retroviruses that display a high tendency to establish latent or nonproductive infections in vivo, including visna virus and the human T cell leukemia virus, have also been shown to encode a Rev-like regulatory protein (Gendelman et al., 1986; Hidaka et al., 1988; Tiley et al., 1990). It therefore appears possible that a shared pattern of gene regulation in these complex retroviruses might underlie a similar pattern of in vivo pathogenesis. In this paper, we have presented evidence indicating that the interaction of a single Rev protein molecule with

the RRE is insufficient for Rev activity. Instead, Rev function requires the binding of one or more additional Rev monomers to the RRE in a process mediated by a specific interaction between the bound and incoming Rev protein molecules. The Rev sequences required for this proteinprotein interaction are here shown to be distinct from those directly involved in initial binding to the RRE. Because Rev function requires the sequential binding of at least two, and possibly more, Rev monomers to the RRE, this interaction may be viewed as a second or higher order biochemical reaction. The level of Rev activity in vivo would therefore be expected to be proportional to the second or higher power of the concentration of Rev within the cell. Therefore, low levels of Rev protein would be predicted to be ineffective in inducing significant levels of HIV-1 structural gene expression. We therefore propose that a requirement for multimerization of Rev on the RRE is directly responsible for the observed threshold effect for Rev function in vivo. Experimental

Procedures

Construction of Molecular Clones The construction and phenotypic analysis of the wild-type (pcRev) and mutant Rev expression vectors M3, M4, M5, M6, M7, M8, M9, and Ml0 (Table 1) have been described (Malim et al., 1989a). The coding sequences of these Rev genes were excised by the polymerase chain reaction (Mullis and Faloona, 1987) and ligated into the unique BamHi site of the isopropyl-b-o-thiogaiactopyranoside (IPTG) inducible GST Escherichia coli expression vector pGEX-PT (Smith and Johnson, 1988; purchased from Pharmacia LKB Biotechnology). Dideoxynucleotide sequencing confirmed that the GST and Rev coding se quences were translationally in frame with each other and could therefore express the predicted GST-Rev fusion proteins. Purification of GST-Rev Fuslon Proteins The GST-Rev (wild-type and mutant) expression vectors were transformed into the /on protease and ompTouter membrane proteasedeficient E. coli strain BL21 (Grodberg and Dunn, 1988). For purification of the fusion proteins, E. coli cultures were grown at 37OC in ampicillin medium to an ODem of 0.6-0.8 and induced with 0.1 mM IPTG for 90 min. The cells were harvested at 4OC and resuspended in 9.5 ml of 25% sucrose, 50 mM Tris-HCI (pH 8.0), 1 mM EDTA, and 0.25 mfvl phenyimethyisulfonyl fluoride. Lysozyme (15 mg) was added, and the ceils were incubated at 25’C for 5 min. The suspension was adjusted to 10 mM MgCI,. 1 mM MnCI? prior to the addition of DNAase I (US Biochemical Corp.) to 10 vglml and subsequent incubation at 37’C for 15 min. The ceils were lysed by adjustment to 1 x phosphatebuffered saline, 1% Tween-20, l%Triton X-100, and 1OmM dithiothreitoi followed by vigorous vortexing. The lysate was then cleared by centrifugation at 15,000 x g for 20 min at 4’C. GST or GST-Rev was purified from the lysate by passage over a glutathione-Sepharose 48 (Pharmacia LKB Biotechnology) affinity column, followed by elution with 10 mM giutathione (Sigma) in 50 mM Tris-HCI (pH 8.0). 0.25 mM phenylmethylsuifonyi fluoride. The eluate was adjusted to 10% glycerol, 10 mM HEPES-NaOH (pH 7.8). 50 mM NaCi, 10 mM KCI. 0.5 mM EGTA, 2 mM dithiothreitol, 2 @ml leupeptin (Boehringer Mannheim Biochemicals), and 3 pg/ml aprotinin (Boehringer Mannheim Biochemicais), concentrated in acentricon 10microconcentrator (Amicon), and stored at - 70°C. Protein concentrations were determined by the method of Bradford (1978). The purity and integrity of the purified GST-Rev fusions were assessed by SDS-poiyacryiamide gel eiectrophoresis followed by Coomassie blue staining. All preparations were observed to yield essentially equivalent levels of the predicted -40 kd full-length fusion protein (data not shown). The 116 amino acid form of HIV-1 Rev protein purified from E. coli was a gift of Repiigen Corporation and Sandoz Research Institute and has been described previously (Daly et al., 1989; Malim et al., 1990).

Role of HIV-1 Rev Multimerization 247

In Vitro Tnnscrlption

and RNA-Blnding

Analyses

The synthesis and purification of unlabeled and [aewP]UTP-labeled RNAs used in the RNA-binding assays were performed using standard protocols (Promega Corp.). The plasmid templates used for the synthe sis of the full-length RRE transcript have been described previously (Malim et al., 1990). Rev-RRE complexes were assembled and visualized using the gel retardation protocol described by Malim et al. (1990). The amount of protein used in each assay was determined empirically such that 1 U of the wild-type or GST-Rev protein bound between 30% and 70% of the input radiolabeled RNA probe. Specifically, 1 U corresponds to 27 ng of purified 116 amino acid Rev and 330 ng of partially purified GST or GST-Rev (wild type and mutant). In the mixed multimers experiment (Figure 3), l/3 U of GST fusion protein was equilibrated with 1, l/3, 119 or zero U of wild-type, 116 amino acid Rev for 10 min prior to the addition of radiolabeled RRE and subsequent electrophoretic separation. However, it was noted that experiments in which the 116 amino acid Rev was the last component added to the binding reaction yielded the same results (data not shown).

Acknowledgments The authors wish to thank A. Frankel for communication of results prior to publication, H. Bogerd for helpful discussions, and S. Goodwin for secretarial assistance. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advaflissmenr in accordance with 1.5 USC Section 1734 solely to indicate this fact. Received

December

17, 1990; revised

February

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G. to of

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