HIV-1 structural gene expression requires binding of the rev trans-activator to its RNA target sequence

HIV-1 structural gene expression requires binding of the rev trans-activator to its RNA target sequence

Cell, Vol. 60, 675483, February 23, 1990, Copyright 0 1990 by Cell Press HIV-1 Structural Gene Expression of the Rev Trans-Activator to Its RNA Ta...

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Cell, Vol. 60, 675483,

February

23, 1990, Copyright

0 1990 by Cell Press

HIV-1 Structural Gene Expression of the Rev Trans-Activator to Its RNA Target Sequence Michael H. Malim: Laurence S. Tiley: David k McCarn,’ James R. Rusche,t Joachim Hauber,* and Bryan R. Cullen* Howard Hughes Medical Institute Department of Microbiology and Immunology and Department of Medicine Duke University Medical Center Durham, North Carolina 27710 t Repligen Corporation One Kendall Square, Building 700 Cambridge, Massachusetts 02139 $ Sandoz Research Institute Al235 Vienna Austria l

Summary Expression of human immunodeficiency virus type 1 structural proteins requires both the viral Rev fransactivator and its &-acting RNA target sequence, the Rev response element (RRE). The RRE has been mapped to a conserved region of the HIV-1 env gene and is predicted to form a complex, highly stable RNA stem-loop structure. Site-directed mutagenesis was used to define a small subdomain of the RRE, termed stem-loop II, that is essential for biological activity. Gel retardation assays demonstrated that the Rev frans-activator is a sequence-specific RNA binding protein. The RRE stem-loop II subdomain was found to be both necessary and sufficient for the binding of Rev by the RRE. We propose that the HIV-1 Rev fransactivator belongs to a new class of sequence-specific RNA binding proteins characterized by the presence of an arginine-rich binding motif.

Requires Bindin

Although Rev is absolutely required for the cytoplasmic expression of incompletely spliced viral mRNAs, it appears to have little effect on the pattern of HIV-1 RNA expression in the cell nucleus. Indeed, high levels of unspliced viral transcripts may be detected in the nucleus even in the absence of Rev (Malim et al., 1989a; Felber et al., 1989). The retention of these incompletely spliced RNAs within the nucleus appears to be due to the action of cellular factors that are components of the splicing machinery (Chang and Sharp, 1989). The Rev protein is believed to activate the nuclear export of these sequestered mRNA species either by facilitating their access to a cellular RNA transport pathway or by antagonizing their interaction with splicing factors (Rosen et al., 1988; Malim et al., 1989a; Emerman et al., 1989; Felber et al., 1989). Trans-activation of viral structural gene expression by Rev also requires a &-acting RNA target sequence termed the Rev response element, or RRE. The RRE is localized within the env gene of HIV-1 (Rosen et al., 1988; Malim et al., 1989a; Felber et al., 1989) and is predicted to form a highly ordered, and highly significant, RNA secondary structure (Malim et al., 1989a) (Figure 1). Recently, we have reported that the highly structured nature of the RRE is maintained between all known primate immunodeficiency virus variants, in some cases due to the presence of compensatory base changes within predicted RNA stem structures (Malim et al., 1989c; Le et al., 1990). In this paper we have more fully characterized the functional interaction between Rev and the RRE. Mutational analysis of the RRE was used to define subregions of this RNA structure that are critical for in vivo function and, hence, viral replication. We present data demonstrating that the in vivo phenotype of these mutant RREs correlates with their ability to bind the HIV-1 Rev protein in vitro. Results

The pathogenic haman retrovirus human immunodeficiency virus type 1 (HIV-l) displays a marked temporal regulation in the accumulation and expression of the different viral mRNA species (Kim et al., 1989). After infection of an appropriate CD4+ target cell, HIV-l gene expression is initially limited to the fully spliced, ~2 kb class of mRNAs that encode the viral regulatory gene products Tat, Rev, and Nef. Subsequently, viral gene expression undergoes a marked shift, leading to the predominant expression of unspliced (-9 kb) and singly spliced (~4 kb) transcripts that encode the viral structural proteins, including Gag and Env (Kim et al., 1989). Considerable evidence now exists that this shift is mediated by the the viral Rev Pans-activator (reviewed by Cullen and Greene, 1989). Notably, HIV-1 proviruses lacking a functional rev gene are unable to progress from the early (regulatory) to the late (structural) phase of viral gene expression and are therefore replication defective (Feinberg et al., 1986; Sodroski et al., 1986; Terwilliger et al., 1988; Sadaie et al., 1988).

Mutational Analysis of the RRE Owing to the large size of the RRE, our initial approach to the functional dissection of this RNA structure involved the generation of a series of deletion (pa) mutants targeted to the five predicted stem-loop subdomains of the RRE (Figure 1). The mutants were named according to the inclusive extent of the introduced deletion, using the coordinates shown in Figure 1, and are listed in Table 1. RRE mutants were inserted into the genomic HIV-l tat gene expression vector pgTAT, and RRE function was determined using a previously described transient expression assay (Malim et al., 1988). The pgTAT indicator construct contains the two coding exons of the HIV-1 tat gene separated by a large intron derived primarily from the viral env gene (Malim et al., 1988). In the absence of Rev, pgTAT exclusively expresses a fully spliced, cytoplasmic tat mRNA that encodes the 86 amino acid two-exon form of the Tat protein (Figure 2, lane 2). Similarly, all the RRE mutants described in this study also exclusively expressed fully spliced cytoplasmic tat mRNAs in the absence of Rev

Cell 676

Table

Clone

1. Phenotypic

Tested

taTAT

160 STEM-LOOP

60 2ow

C G-210 “A u

-G:cA

IO / AGGAGCU UCCUyGAc

u ,)G,” ziGcGl AA

uGu” uA A A

STEM I

220

2jo

Figure

1. Predicted

Secondary

Structure

of the HIV-l

RRE

The mutationally defined HIV-I RRE coincides with an HIV-1 env gene sequence that has a high probability of forming an RNA secondary structure (Le et al., 1988). We have previously described the derivation of the computer-predicted RRE structure shown here (Malim et al., 1989a). The complete RRE structure can be subdivided into five RNA subdomains here termed stem I (coordinates l-37 and 195-234) stem-loop II (39104). stem-loop Ill (107-124) stem-loop IV (133-161) and stem-loop V (164-189). Stem-loop II can be further subdivided into stem-lcops IIA (39-48,7l-77, and 98-104) IIB (49-70) and IIC (78-96).

(data not shown). In the case of pgTAT, coexpression of Rev results in the cytoplasmic expression of an unspliced fat mRNA that encodes a truncated, one-exon form of Tat (72 amino acids; Figure 2, lane 3). However, deletion of the RRE from the env gene-derived intron present in pgTAT renders this indicator cdnstruct refractory to the action of Rev (Malim et al., 1969a). A representative experiment measuring the ability of the RRE mutants to function as in vivo targets for Rev is shown in Figure 2, and a summary of the data obtained is presented in Table 1. The pgTAT-based vectors lacking RRE stem-loop Ill (AKWl25; Figure 2, lane 7), IV (Al301 162, lane 15), or V (AlWl91, lane 16) retain between 50% and 100% of the Rev responsiveness observed for pgTAT itself. Simultaneous deletion of both stem-loop IV and stem-loop V, in pAl27/l91, did result in a readily detectable, -3fold drop in RRE function, however (Figure 2, lane 14). Nevertheless, these results clearly indicate that sequences between coordinates 106 and 191 are not required for RRE function in vivo. A major feature of the RRE is the long predicted RNA double helix here termed stem I (Figure 1). Mutations that delete either the entire 5’ (Al&t; Figure 2, lane 9) or 3 (A196/234, lane 6) strand of stem I were found to reduce

pAll34 ~A15134 pA41l63 ~A411105 pA5Ol75 pA55/90 pA73l94 pA106/125 PA1271191 pA13Ol162 pA163/191 pAl96/222 pA198/234 Substitutions pM35/41 PM41145 PM47153 pM49/70 pM53/58 pM55l63 pM83/69 pM63l69 + 50155 pM70l77 pM73178

pwm3 pM84/90 pM97l102

Analysis

of HIV-1

RRE Mutants

Stem-loop Region Mutated

Phenotype

wild type

++

I I IIA + IIA + 118 IIB + IIC Ill IV + IV V I I

+ ++ + ++ ++ + *

B B + C C

v

I + IIA IIA IIB IIB IIB IIB 118 118 IIA IIA IIC IIC IIA

+ + ++ ++ ++ ++ f ++ ++ ++ ++ ++ ++

The effect of the indicated RRE mutations on the Rev responsiveness of the pgTAT indicator construct was assayed as described in Figure 2. All mutants expressed exclusively the 86 amino acid Tat in the absence of Rev (data not shown). The relative activity of each RRE mutant was quantitated by scanning the resultant autoradiographs using an LKB Ultrascan XL laser densitometer. Average activity, obtained from several separate experiments, is expressed relative to the parental pgTAT vector: + + , 60%-100% of wild-type activity; + , 20%-50%; f , 5%-20%; - , <5%.

RRE function by between 50% and 90%. Partial deletion of stem I (AW34 or A196/222) resulted in a less severe, ~30% to 60% inhibition of RRE function (Figure 2, lanes 10 and 11). Therefore, although an intact stem I is not essential for RRE function, this structure does markedly enhance the biological activity of the RRE. In contrast to the intermediate phenotypes of the RRE deletion mutants discussed above, deletion of the entire RRE stem-loop II, in pA4VlO5, resulted in the complete loss of RRE function (Figure 2, lane 6). Smaller deletions targeted to the shorter predicted stem-loops IIA, IIB, and IIC demonstrated that any deletion that affected stem-loop IIB abolished detectable RRE function in the pgTAT assay (i.e., A41/63, lane 13; A50/75, lane 4; A55/90, lane 12). In contrast, a deletion that removed only stem-loop IIC reduced RRE activity by only -2-fold (A73/94, lane 5). These results therefore suggest that the structural integrity of stem IIB, and possibly of stem IIA, is essential for RRE function in vivo.

Rev Function 677

Requires

Binding

to Viral RNA

4325.7-

-86aa -72aaTat

1234687 Figure

2. Phenotypic

8 Analysis

of HIV1

9

10

11

12

13

14

16

16

17

18

19

20

21

22

23

RRE Mutants

Mutations of the HIV-I RRE were tested for in vivo function after insertion into the indicator construct pgTAT (Malim et al., 1988). Biological activity was quantitated by transfection into cultures of the monkey cell line COS together with an equimolar amount of the rev expression vector pcREV (except lanes 1 and 2; see below). At 86 hr after tranefection, the cultures were labeled with [ssS]cysteine and then lysed and sub@cted to immunoprecipitation analysis using an anti-Tat antiserum (Malim et al., 1988). Precipitated proteins were resolved by electrophoresis on 14% discontinuous SDS-polyacrylamide gels and visualized by autoradiography. The relative migration of known protein molecular weight markers is shown at left. The 86 amino acid and 72 amino acid forms of Tat migrate at 15.5 kd and 14 kd, respectively, and the level of Rev response is reffected in the level of 72 amino acid Tat obtained (Malim et al., 1989a). All lanes derive from cultures transfected with both a pgTAT-derived vector and pcREV, except for lane 1, where cells were transfected with the negative control vector pBCQ/CMV (Cullen, 1986) and lane 2. where cells were transfected with pgTAT alone.

To define more closely the sequences within RRE stemloop II involved in mediating RRE function, we next constructed a series of scanning substitution or missense (PM) mutations across this sequence element. (The precise nucleotide changes introduced by these mutations are described in Table 2.) Mutations that were targeted to predicted loop sequences within stem-loop II (e.g., M55/ 63, M7Oi77, M73l78, and M84/QO) were found to have little or no effect on RRE function (Figure 2, lanes 17-19). Substitution mutations targeted to RNA stem structures had variable effects. Thus, two mutations predicted to affect a part of stem IIB (M47/53 and M53/58), as well as one mutation predicted to affect stem IIA (I&7/102), had little effect on RRE function, In contrast, two other stem IIA mutations (M35/41 and M41/45) inhibited RRE function by 3- to 4-fold . Table 2. Nucleotide Missense Mutations

Clone

Changes

Present

Table 3. Predicted Effect of Selected Substitution on the Integrity of RRE Stem-Loop IIB

in RRE

Mutated Sequence

Restriction Site

GGAAGCA

CUCGAGU

Xhol

pM41 I45

CACUAU

Smal

pM47153

GGCGCAG

CCCGGG -~ CCAUGGU

pM53158

GCGUCA

CUCGAG

Xhol

PM55163

GUCAAUGAC CGUGAC

CUCCCCGGG _&JAUUU

Smal

pM63/69 pM7Ctl77

GGUACAGG

Asp71 8

pM73i78

ACAGGC

AGGUACCU -~ CCCGGG

Ncol

“CGCAGCGU 76GCGUCGCA A

GCCAGAC

AAGCUUU

Hindlll

AAUUAUU

CCCCGGG

Smal

pM971102

UAUAGU

CCCGGG

Smal

A GU CA

pM63/69

4gCGCAGCGU 7oG UUAUAA U

A

A GU

CA pM63/69

Smal

pM84/9O

are underlined.

Wild type

Sspl

pM77l83

nucleotides

Mutations

CA

Wild-Type Sequence

PM35141

Mutated

while one missense mutation targeted to stem IIB, termed M63/6Q, reduced RRE function dramatically (Figure 2, lanes 20 and 21). The low biological activity of RRE substitution mutant M63/6Q could reflect the importance of the affected primary sequence or the affected RNA secondary structure. Indeed, the M83/6Q mutation is predicted to have a marked impact on the integrity of stem-loop IIB (Table 3). To test this latter hypothesis, we inserted a second mutation into M63/6Q that is predicted to restore stem-loop IIB (M63/6Q+50/55, Table 3). This compensatory mutation fully restored RRE function (Figure 2, lane 22). This result validates the in vivo existence of stem-loop IIB and suggests that the role of this stem-loop in RRE function is pri-

+ 50155

4gCAAUAUUU 76GUUAUAAA U

A GU AU

pM49/70

4gGCGUCGAC 76CGCAGCUG A

’ CA

Cdl 678

A

123

4567

B

Figure 3. Effect of RRE Mutations on HIV-1 Provirus Gene Expression (A) Cytoplasmic RNA was isolated (Malim et al., 1989a) 80 hr after transfection of COS cell cultures with the indicated wild-type and mutant provirus expression vectors. Northern analysis, using a probe specific for the HIV-l long terminal repeat, was then used to quantitate the levels of the different classes of viral mRNA. (B) COS cell cultures were transfected with vectors that express a wildtype HIV-1 provirus (pHIV-1) or with proviruses mutated in the viral rev gene or RRE, as indicated. Supernatant media were sampled 80 hr after transfection and assayed for secreted p24 Gag protein expression levels (Malim et al., 1989b). The values shown are derived from three independent experiments and are given as the average percentage (2 the indicated standard deviation) of the level obtained with the wildtype proviral expression vector pHIV-1. A secreted atkaline phosphatase gene expression vector (Berger et al., 1988) was cotransfected as an internal control, and values were corrected for the slight variability observed in supernatant levels of this enzyme. (Setting mean secreted alkaline phosphatase activity at 1.00 units, the observed standard deviation was 50.12 and the range 1.20 to 0.79.)

marily structural. lacking in essential replaced the entire

To confirm primary stem-loop

that stem-loop II6 is indeed sequence information, we

IIB with a heterologous sequence predicted to form an RNA stem-loop of similar size and stability (M49/70, Table 3). This extensive RRE substitution mutant was also observed to be essentially fully active in vivo (Figure 2, lane 23).

Effect of RRE Mutations on HIV-1 Pmvirus Gene Expression The results presented in Table 1 suggest that mutations within FIRE stem-loop II can dramatically affect RRE function. To confirm the validity of this observation, we selected five representative RRE mutants for phenotypic analysis within the context of an otherwise intact HIV-l provirus. The effect of these RRE mutants on the pattern of viral gene expression was then monitored by transfection into COS cell cultures (Cullen, 1987) followed by Northern analysis of cytoplasmic viral mRNA species (Figure 3A), and quantitative assay of secreted p24 Gag protein (Figure 38). As previously reported (Muesing et al., 1985; Feinberg et al., 1988) the intact HIV-1 provirus normally expresses three distinct size classes of viral RNA (Figure 3A, lane 2). These are a full-length genomic RNA of -9 kb, a singly spliced RNA class of ~4 kb, and a complex class of multiply spliced RNAs of ~2 kb. In HIV1 ARev proviruses, which lack a functional rev gene, cytoplasmic expression of the 9 kb and 4 kb classes of viral mRNA is no longer detectable (Figure 3A, lane 3) and expression of the viral structural proteins is lost (Figure 38). As predicted, proviruses bearing the RRE mutations A41M05 or A50175 were observed to yield the same pattern of viral RNA expression as HIV-l ARev (Figure 3A, lanes 4 and 5) while a provirus bearing the A183/191 RRE mutation retained an essentially wild-type RNA expression pattern (Figure 3A, lane 8). The RRE substitution mutation M63/89, which resulted in much-reduced activity in the pgTAT assay, also markedly inhibited the cytoplasmic expression of incompletely spliced viral mRNA species (Figure 3A, lane 7). However, a low level of these RNAs could be detected upon prolonged autoradiographic exposure (data not shown). Quantitative analysis of the level of p24 Gag protein released from cultures transfected with the indicated proviral RRE mutants (Figure 38) confirmed the defectiveness of the provirus containing the A41/105 RRE mutant as well as the essentially wild-type phenotype of the A183/191 RRE mutant (Figure 3A). The proviral A1301162 RRE mutant was observed to yield ~50% of the wild-type level of secreted p24 protein while the pM63/69 mutant was found to be -12% active, as predicted (Table 1). Interestingly, analysis of the supernatant media from cultures transfected with a proviral vector bearing the A50175 deletion consistently yielded a low but significant level of secreted p24 Gag protein. This level of activity, ~3% of that observed for the wild-type provirus, could not be detected using the less sensitive pgTAT assay qable 1). In total, these results demonstrate, as predicted, that the phenotypes of proviruses bearing either a defective rev gene or a defective RRE are identical. Biological Activity of RRE Mutants Correlates with In Vitro Rev Binding Having defined sequences within the complete FIRE that are required for the recognition of the element in vivo, we next wished to define’sequences involved in the in vitro interaction of Rev with the RRE. We therefore developed an RNA gel retardation assay (Konarska and Sharp, 1986)

Rev Function 679

Requires

Binding

to Viral RNA

A 12

3

4

5

6

7

8

9

101112

I-

Co~r$$;ed

--Input

Probe

\M

SPECIFIC COMPETITOR

NON-SPECIFIC COMPETITOR

6 12

3

4

12

3

4

5

6

7

8

910111213

C 5

6

7

8

9101112

1

Co;~;;;ed

-Input

Stem-loop Sense Figure

4. Gel Retardation

II

Analysis

Stem-loop Anti-sense of Rev-RRE

Probe

II

Binding

that permitted the visualization of the specific interaction of recombinant Rev protein with a synthetic, radiolabeled RNA probe containing the intact RRE (see Experimental Procedures for details). Incubation of pure Rev protein with the RRE probe was observed to result in the appearance of two distinct protein-RNA complexes (Figure 4A, lane 2). That both protein-RNA complexes were formed with Rev protein was confirmed by the enhanced retardation of these complexes after incubation with an affinitypurified anti-Rev antibody (data not shown). Addition of a specific competitor, i.e., unlabeled RRE transcripts, to the reaction resulted in a concentration-dependent reduction in the formation of both Rev-RRE complexes (Figure 4A, lanes 3-7). Interestingly, low levels of unlabeled RRE preferentially competed the more slowly migrating complex, such that loss of the faster migrating Rev-RRE complex was not observed until titration of the slower Rev-RRE complex was essentially complete. In contrast, inclusion of up to 200 ng of a nonspecific competitor RNA, in this case Escherichia coli 5S ribosomal RNA, had no significant effect on the level of interaction of Rev with the RRE probe (Figure 4A, lanes 8-12). We next examined the ability of heterologous structured RNA species and phenotypically defined mutant RRE molecules to compete with the wild-type RRE for binding to Rev. This experiment (Figure 48) demonstrated that tRNA (lane 3) or 16s and 23s rRNA (lane 5) derived from E. coli are also poor competitors of Rev-RRE binding, In addition, in vitro synthesized RNAs derived from the highly structured poliovirus untranslated mRNA leader region (Nomoto et al., 1982) (Figure 4B, lane 6), or containing the HIV-l trans-activation response (TAR) stem-loop element (Feng and Holland, 1988) (Figure 48, lane 7) were also unable to compete effectively. Other heterologous RNAs that lacked the ability to compete with the RRE for Rev binding included a synthetic RNA containing the mutationally defined target sequence for the Rex trans-activator of human T cell leukemia virus type I (Hanly et al., 1989) and total cytoplasmic RNA (i.e., primarily rRNA) isolated from primate cells (data not shown). This latter observation may be of interest in light of the reported nucleolar localization of the Rev protein in expressing cells (Felber et al., 1989; Malim et al., 19896). Unlabeled mutant RRE competitor RNAs were observed to give two distinct phenotypes in this competitive RNA binding analysis. The RRE deletion mutants A130/ 162 and A163/191, which retain considerable in vivo biological activity (Table 1, Figure 3B), also retained the ability to compete for Rev binding in vitro (Figure 48, lanes 11

In Vitro

(A) A constant level of a labeled 252 nucleotide synthetic RNA probe containing the intact HIV-1 RRE was incubated with 27 ng of purified Rev protein as described in Experimental Procedures. Binding of the RNA probe by Rev was visualized as slower migration upon electrophoresis through a native polyacrylamide gel (lane 2). The labeled RRE probe was also incubated with Rev in the presence of increasing levels of either unlabeled RRE transcript (lanes 3-7) or E. coli 5s ribosomal RNA (lanes 6-12). Only the specific RNA competitor was found to yield a dose-dependent inhibition in Rev-RRE complex formation. (9) The ability of the indicated natural (lanes 3-5) or synthetic (lanes

6-13) RNA species to compete with the wild-type RRE for binding to Rev in vitro was analyzed. A constant level (299 ng) of each unlabeled competitor RNA was preincubated with 27 ng of Rev protein prior to addition of the labeled RRE probe. The effect of this preincubation on the level of Rev-RRE complex formation was then determined by gel retardation analysis. Polio 5’ UTR = poliovirus 5’ untranslated region. (C)The indicated amounts of unlabeled synthetic RNA transcripts containing the entire RRE stem-loop II (coordinates 39-194, Figure 1) in either the sense orientation (lanes 3-7) or antisense orientation (lanes 8-12) were analyzed for their ability to compete with the full-length RRE for binding to Rev in vitro.

Cdl 680

and 12). In contrast, the inactive RRE deletion mutant A41/105, as well as FIRE mutants A50175 and M6!3/69, which retain detectable but low in vivo biological activity (Figure 3B), were unable to effectively compete with the wild-type RRE for Rev binding in vitro (Figure 48, lanes 9, 10, and 13). We therefore conclude that the Rev protein is a sequence-specific RNA binding protein that displays a marked affinity for its mutationally defined RNA target sequence, the RRE. We also conclude that the in vivo phenotype of mutations of the RRE correlates with, and is almost certainly determined by, the effect of the mutation on the ability of the RRE to bind Rev. Stem-Loop II of the RRE Is Sufficient for Rev Binding The mutational analysis of the RRE presented above suggested that stem-loop II was the only RRE subdomain absolutely required for biological activity. However, these data also demonstrated that deletion of other RRE sequences could result in a significant reduction in biological activity (Table 1). One possible explanation for these observations is that stem-loop II contains the actual binding site for the Rev trans-activator while the remainder of the RRE primarily functions to facilitate this interaction in vivo. A prediction of this hypothesis is that, while RRE stem-loop II might display reduced biological activity in the absence of the remainder of the RRE, it should nevertheless retain significant in vitro Rev binding activity as an isolated RNA stem-loop structure. To test this hypothesis, the entire stem-loop II (coordinates 39 to 164) was precisely excised from the RRE using the polymerase chain reaction (Mullis and Faloona, 1967) and inserted into a bidirectional RNA transcription vector. “Sense” and “antisense” RNA transcripts of stemloop II were then assayed for their ability to compete with the wild-type RRE for binding to Rev (Figure 4C). It has been shown that RRE function is orientation dependent (Malim et al., 1969a); the antisense transcript therefore serves as a negative control. This experiment clearly showed that sense, but not the antisense, transcripts of RRE stem-loop II are effective competitive inhibitors of the interaction between Rev and the full-length RRE probe. Indeed, transcripts containing only stem-loop II of the RRE appeared to compete for Rev binding to the full-length RRE probe as well as, if not better than, the full-length RRE itself (compare Figures 4A and 4C). The ability of the sense stem-loop II RNA transcript to bind specifically to Rev in vitro was confirmed by gel retardation analysis using a radiolabeled stem-loop II RNA probe (data not shown). Therefore, these results demonstrate that RRE stem-loop II is both necessary and sufficient for Rev binding in vitro. Discussion The interaction between the HIV-1 Rev trans-activator and its &-acting RNA target site, the Rev response element (RRE), is essential for the activation of viral structural gene expression and, hence, viral replication (reviewed by

Cullen and Greene, 1969). Here, we report a series of experiments designed to dissect more fully the interaction between Rev and the RRE. Initially, mutational analysis of the RRE, combined with the use of in vivo assays of RRE function, was used to identify a subdomain of the RREstem-loop II-that is absolutely required for biological activity. RNA gel shift assays were then used to demonstrate the specific binding of purified Rev protein to the ARE in vitro. This assay also revealed that an intact RRE stemloop II was both necessary and sufficient for this specific protein-RNA binding event to occur. We therefore propose that the essential in vivo role of RRE stem-loop II is explained by the identification of this RRE subdomain as the binding site for the viral Rev trans-activator. In evaluating these results, it is important to consider that mutations targeted to a particular stem-loop of the RRE could have significant effects on other segments of this RNAstructure. Nevertheless, the gel retardation analysis presented in Figure 4 does demonstrate that RRE stem-loop II transcripts retain full Rev binding capacity in the absence of the remainder of the RRE. In contrast, fulllength RRE transcripts bearing small substitution or dels tion mutations in stem-loop II fail to bind Rev effectively in vitro. Consequently, we believe that the Rev binding site in the RRE is likely to be entirely contained within stemloop II. To date, the precise localization of the Rev binding site within stem-loop II has proven elusive. Thus, the mutational analysis presented in Table 1 demonstrates that stem-loop IIC is dispensable for RRE function and suggests that the role of stem-loop IIB, while essential, is an entirely structural one. In addition, missense mutations designed to scan across stem-loop II failed to detect any fully negative mutations, although several RRE mutants with low or partial activity were obtained (Table 1). One possible explanation for this result is that there might be more than one site for Rev binding within stem-loop II. This kind of functional redundancy is frequently seen for both DNA and RNA response elements. For example, the HIV-1 long terminal repeat contains two independent DNA binding sites for the transcription factor NFKB and three sites for Spl (reviewed by Cullen and Greene, 1969), while the related retrovirus HIV-2 has been shown to contain two independent RNA target sequences for the viral Tat fransactivator (Fenrick et al., 1969). The hypothesis that the RRE contains two independent Rev binding sites could also explain the observation of two distinct Rev-RRE complexes in gel retardation assays (Figure 4). However, complete resolution of this question must clearly await the precise biochemical mapping of the nucleotides involved in the Rev-RRE interaction. Although the data presented in this paper demonstrate that RRE stem-loop II is both sufficient for the in vitro interaction with Rev and necessary for in vivo biological activity, it is equally clear that the remainder of the RRE also plays a critical role in mediating the effective fransactivation of viral structural gene expression by Rev. Thus, deletion mutations of the RRE that do not impact directly on stem-loop II can significantly reduce in vivo RRE func-

2;;

Function

Requires

Binding

to Viral RNA

tion (e.g., pAll34, pA127/191, and pA1981234; see Table 1 and Figure 1). Several possible explanations for the enhanced in vivo activity of the complete RRE exist. For example,. it is possible that the appropriate formation of the stem-loop II structure is facilitated in vivo by its context within the complete FIRE. It is also possible that the complete RRE structure, and particularly stem I, might be essential for the effective presentation of a much smaller Rev binding sequence present in stem-loop II. It is perhaps reasonable to propose that the central location of the RRE within the large RNA genome of HIV-l might otherwise result in the obstruction of the Rev binding site by flanking viral RNA sequences.

The HIV-1 Rev Tram-Activator as an RNA Binding Protein It has become increasingly clear that sequence-specific interactions between proteins and RNA molecules are involved in a wide range of cellular regulatory processes (reviewed by Mattaj, 1989; Bandziulis et al., 1989). These include pre-mRNA splicing (Black et al., 1985; Grabowski et al., 1985; Query et al., 1989) polyadenylation (Hashimoto and Steitz, 1986; Skolnik-David et al., 1987) transcription termination (Lazinski et al., 1989) mRNA translation (Rouault et al., 1988) and even, in the case of the HIV-1 Tat trans-activator, initiation of transcription (Sharp and Marciniak, 1989). Protein-RNA interactions are also believed to be important in developmental regulation (Mattaj, 1989; Bandziulis et al., 1989). For example, the sex determination pathway in Drosophila appears to be regulated entirely at the level of sex-specific alternative splicing (Boggs et al., 1987; Nagoshi et al., 1988). Many of the RNA binding proteins identified thus far contain a highly conserved RNA binding motif termed the RNP consensus sequence (Query et al., 1989; Bandziulis et al., 1989). We have previously noted the absence of any similar sequence within the Rev protein (Malim et al., 1989b). The phenotypic analysis of a series of mutants of the Rev &ins-activator had, however, led us to propose that a highly argin?e-rich sequence present within Rev was likely to function as part of a sequence-specific RNA binding domain (Malim et al., 1989b). Remarkably, this sequence closely resembles a novel RNA recognition consensus sequence-termed the arginine-rich motif-subsequently proposed by Lazinski and co-workers (1989) based on their studies of transcription antitermination in bacteriophages. The apparent conservation of this arginine-rich RNA binding consensus sequence over such a large evolutionary distance suggests that this motif, like the RNP consensus, may mediate a wide range of protein-RNA interactions. The regulation of HIV-1 structural gene expression by the viral Rev rmrrs-activator may therefore emerge as an important model system for the study of posttranscriptional gene regulation in eukaryotic cells. Very recently, two other reports have detailed the use of somewhat different experimental approaches to obtain evidence for the specific in vitro interaction of Rev with the RRE (Daly et al., 1989; Zapp and Green, 1989). These

reports appear, in general, data presented here. Experimental

Procedures

Construction

of Molecular

to be in agreement

with the

Clones

Oligonucleotide-directed mutagenesis (Taylor et al., 1985) of the previously defined HIV-1 RRE (Malim et al., 1989a) was performed using a bacteriophage Ml3 mutagenesis system (Amersham Corp., Arlington Heights, IL). To facilitate the recloning of mutated RREs into the two indicator plasmids pgTAT and pHIV-1, the Sall-BamHI subfragment of the HXB-3 isolate of HIV-l (sequence coordinates 5367-8053; Ratner et al., 1985) was used as the mutagenesis substrate. Initially a series of deletions (PA) were generated within the RRE (see Table 1). Each mutation was designed to remove specific segments (either stemloops or regions of stem I) of the RRE structure and to insert a unique Smal site (5’~CCCGGG-3’) at the site of the deletion. The extent of each deletion is described in Table I, utilizing the RRE coordinates illustrated in Figure 1. These numbers refer to the 5’-most and 3’-most nucleotides that are mutated in each construct. The mutated RREs were introduced into the previously described pgTAT indicator plasmid (Malim et al., 1988) as Sall-Hindlll (sequence coordinate 7719) fragments The pcREV and pBCl2/CMV vectors have been described elsewhere (Malim et al., 1988; Cullen, 1966). The pM vectors contain a series of missense mutations that scan stem-loop II of the RRE. Each mutation was designed to introduce a novel restriction enzyme site and to introduce neither frameshift nor nonsense mutations into the env open reading frame. The precise sequences of the various pM mutants are shown in Tables 2 and 3. The coordinates refer to the 5’-most and 3’-most mutated nucleotides. respectively. As with the deletion mutations, these mutated RREs were introduced into pgTAT as Sall-Hindlll fragments. All RRE deletion and missense mutations were confirmed by DNA sequence analysis prior to phenotypic analysis. The wild-type HIV-l proviral expression vector pHIV-1 was constructed by insertion of a 13.5 kb Xbal fragment, containing the entire HXB-3 provirus and some flanking cellular sequences (Shaw et al., 1984) into the unique polylinker Xbal site of the replicating COS cell vector pCV1. pCV1 contains a truncated SV40 origin of replication and was derived by insertion of a polylinker sequence between the Ncol and EcoRl sites of the pBC12BI vector (Cullen, 1987). The Revdeficient derivative pHIV-1 ARev was obtained by cleavage of pHIV-1 at the unique rev gene BamHl site, followed by filling in with Klenow poly merase and blunt end ligation. This frameshift mutation has previously been shown to disrupt fev gene function (Malim et al., 1988; Feinberg et al., 1986). Selected mutated RREs were introduced into pHIV-1 as Sall-BamHI fragments. The vectors used for synthesis of in vitro transcripts were all derivatives of pGEM3i!f(+) (Promega Corp., Madison, WI). This vector contains a polylinker sequence sandwiched between the promoters for T7 RNA polymerase and SP6 RNA polymerase. The wild-type RRE and selected mutations thereof were isolated as Styl-Sau3Al fragments (coordinates 7359-7566 for the wild-type RRE), filled in with Klenow polymerase, and then ligated into the Smal site of pGEM3Zf(+). In each case the recombinant plasmid predicted to generate sense transcripts with T7 RNA polymerase was retained for in vitro transcription reactions. Wild-type RRE transcripts were synthesized with T7 RNA polymerase after linearization with Xbal and were predicted to be 252 nucleotides in length. The polymerase chain reaction (Mullis and Faloona, 1987) was used to excise the complete RRE stem-loop II from the RRE. The primers used also inserted Sphl sites (S’-GCATGC-3’) at each side of the base of stem IIA, thus resulting in a predicted 4 bp extension of this stem. The stem-loop II fragment was isolated after cleavage with Sphl and inserted in both orientations into the Sphl site of the pGEM3Zf(+) polylinker. Both sense and antisense stem-loop II transcripts were prepared using SP6 polymerase (predicted size, 91 nucleotides). Poliovirus 5’ untranslated region transcripts were synthesized as T7 transcripts using a pGEM3Zf(+)-based vector containing the 509 bp SauSAl-EcoRV leader fragment from the Lansing strain of poliovirus type 2 (La Monica et al., 1986) inserted between the BamHl and Hincll

Cdl 682

sites present in the vector polylinker. HIV-1 TAR element RNA transcripts were synthesized using a construct in which the SP6 promoter was precisely aligned with the site of transcription initiation in HIV-l. Vector linearization at an HIV-l-derived Hindlll site allowed the production of 78 nucleotide run-off transcripts containing the complete TAR element.

50 mM Tris-HCI, 50 mM glycine (pH 8.8) as the running buffer. Gels were prerun for 20 min at 12 V/cm at 4OC prior to the direct loading of binding reactions, and electrophoresis was allowed to proceed for a further 3X hr at constant voltage. The gels were then dried down and visualized by autoradiography.

Acknowledgments Ceil Cuiture

and Transfection

COS cells were maintained as previously COS cell cultures were transfected using quine (Cullen, 1987).

immunoprecipitation

described (Cullen, 1986). DEAE-dextran and chloro-

Analysis

Functional assays of RRE mutations introduced into the indicator construct pgTAT were performed by immunoprecipitation of transfected COS cell cultures after labeling with IssS]cysteine (Malim et al., 1988). The rabbit polyclonal anti-Tat antiserum used in these analyses has been described (Hauber et al., 1987).

The authors thank Pamela Brown for technical assistance, Kathy Theisen and Richard Randall for oligonucleotide synthesis, and Sharon Goodwin for secretarial assistance. This work was funded by the Howard Hughes Medical Institute and by Nationarlnstitutes of Health grant Al-28233 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

Analysis

of HIV-1 Prwirus

The level of viral replication in COS cell cultures transfected with the wild-type HIV-1 proviral expression vector pHiV-1. or with derivatives mutated within the proviral revgene or the RRE, was quantitated using an enzyme-linked immunosorbent assay for secreted p24 Gag expression (Du Pent-NEN Inc., Billerica, MA). The RRE is located within the HIV-l env gene, and mutations of this sequence are therefore also predicted to affect the env gene product. However, it has been shown that the release of Gag protein from expressing cells does not require Env expression (Gheysen et al., 1989). Ail HIV-l provirus rescue experiments were internally controlled by cotransfection with the secreted alkaline phosphatase gene expression vector pBCXXMV/SEAP (Bergsr et al., 1988; Maiim et al., 1989b). In some experiments, COS cell cultures were harvested for preparation of cytoplasmic RNA immediately after sampling of the supernatant media for p24 and secreted alkaline phosphatase expression levels (Malim et al., 1989b). Northern analysis of these RNA samples was performed as described, using a probe specific for the HIV-I long terminal repeat (Malim et al., 1988).

in Vitro Transcrlption

December

5, 1989; revised

January

8, 1990

Replication

and RNA Binding

Analyses

In vitro run-off transcription reactions were performed with linearized plasmid template using standard protocols (Promega Corp., Madison, WI). To synthesize radiilabeled transcripts, [a-32P]UTP (3000 Ci/mmol) was included in the synthesis reaction. Full-length transcripts were purified on denaturing poiyacrylamide gels by standard procedures prior to their inclusion in binding assays. The unlabeled RNAs used in the competition studies and the [a-32P]UTP-labeled RNAs used as probes were diluted in 50 mM NaCi. 0.25 mg/ml bovine serum albumin (DNAase and RNAase free; Boehringer Mannheim Biochemicals), heated at 80°C for 5 min, and annealed by cooling at 3pC for 20 min prior to their inclusion in binding reactions. E. coli ribosomal RNAs were purchased from Boehringer Mannheim Biochemicais. Pure recombinant HIV-l Rev protein, produced in E. coli, was obtained from Repligen Corporation and Sandoz Research Institute. The purification and biophysical characterization of this protein have been described elsewhere (Daly et al., 1989). The protocol developed for the analysis of Rev binding to the RRE is based on the method devised by Singh et al. (1988) for the study of DNA-protein interactions. Optimum incubation conditions were determined experimentalfy (data not shown) and comprised: 10 mM HEPES (pH Z6), 20 mM NaCI, 150 mM KCI, 2 mM MgCIz, 0.5 mM EGTA, 10% glycerol, 1 mM dithiothreitol, 7.5 &ml bovine serum albumin, 1W U/ml RNase guard (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ). A standard RNA binding experiment was performed by mixing 27 ng of purified Rev protein with 500 ng Of E. coli tRNA in binding buffer (usually 7 pi) on ice for 10 min; this preincubation served to minimize the nonspecific association of Rev with RNA. If required, RNA competitors (usually 200 ng in 2 pl) were added and the reactions incubated for a further 10 min on ice. Finally, the labeled probe (approx. 20,000 cpm in 1 rJ) was added and the reaction maintained on ice for a further 10 min. Increasing this incubation period had no effect on the level of complex formation (data not shown). The Rev-RNA protein complexes wera resolved by electrophoresis through 4% nondenaturing polyacrylamide (acrylamide to bisacrylamide ratio of 79:l) gels supplemented with 3% glycerol using

Bandziulis, R. J., Swanson, M. S., and Dreyfuss, G. (1989). RNAbinding proteins as developmental regulators. Genes Dev. $431-437. Berger, J., Hauber, J., Hauber. R., Geiger, R., and Cullen, B. R. (1988). Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. Gene 66, i-10. Black, D. L., Chabot, B., and Steitz, J. A. (1985). U2 as well as Ul small nuclear ribonucleoproteins are involved in pre-mRNA splicing. Cell 42, 737-750. Boggs, R. T., Gregor, P. idriss, S., Belote, J. M., and McKeown, M. (1987). Regulation of sexual differentiation in D. melanogaster via alternative splicing of RNA from the transformer gene. Cell 50, 739-747. Chang, D. D., and Sharp, P A. (1989). Regulation by HIV Rev depends upon recognition of splice sites. Cell 59, 789-795. Cullen, B. R. (1986). Tins-activation occurs via a bimodal mechanism.

of human immunodeficiencyvirus Cell 46, 973-982.

Cullen, B. R. (1987). Use of eukaryotic expression technology in the functional analysis of cloned genes. Meth. Enzymol. 152, 884-704. Cullen, 8. R., and Greene, W. C. (1989). ing HIV-I replication. Cell 58, 423-428.

Regulatory

pathways

govern-

Daly, T. J., Cook, K. S., Gray, G. S., Maione, T. E., and Rusche, J. R. (1989). Specific binding of HIV-I recombinant Rev protein to the Revresponsive element in vitro. Nature 342, 816-819. Emerman, M., Vazeux, R., and Peden, K. (1989). The revgene of the human immunodeficiency virus affects envelope-specific localization. Cell 57, 1155-1165.

product RNA

Feinberg, M. B., Jarrett, R. F., Aldovini, A., Gallo, R. C., and WongStaai, F. (1986). HTLV-III expression and production involve complex regulation at the levels of splicing and translation of viral RNA. Cell 46, 807-817. Felber. B. K., Hadzopoulou-Cladaras, M., Cladaras, C., Copeland, T., and Paviakis, G. N. (1989). Rev protein of human immunodeficiencyvirus type 1 affects the stability and transport of the viral mRNA. Proc. Natl. Acad. Sci. USA 86, 1496-1499. Feng, S., and Holland, E. C. (1988). HIV-1 tat trens-activation the loop sequence within tar. Nature 334, 185-167.

requires

Fenrick, R., Malim. M. H.. Hauber, J., Le, S.-Y., Maizel, J. V., and Cullen, B. R. (1989). Functional analysis of the Tat trans-activator of human immunodeficiency virus type 2. J. Virol. 63. X106-5012. Gheysen, D., Jacobs, E., de Thines, D., and De Wilde, M. precursor Pr5v virus-like rus-infected insect cells. Cell

Foresta, F., Thiriart, C., Francotte, M., (1989). Assembly and release of HIV-I particles from recombinant bacuiovi59, 103112.

Grabowski, P. J., Seiler, S. R., and Sharp, I? A. (1985). A multicomponent complex is involved in the splicing of messenger RNA precursors. Cell 42, 345-353 Hanly. S. M., Rimsky, L. T., Malim, M. H.. Kim, J. H., Hauber, J., Due Dodon, M., Le, S.-Y, Maizel, J. V., Cullen, B. R., and Greene, W. C.

Rev Function 683

Requires

(1989). Comparative regulatory proteins 1534-1544.

Binding

to Viral RNA

analysis of the HTLV-1 Rex and HIV-l Rev transand their RNA response elements. Genes Dev. 3,

the human 2071-2075.

immunodeficiency

virus.

Proc.

Natl. Acad.

Sci. USA 85,

Hashimoto. C.. and Steitz, J. A. (1986). A small nuclear ribonucleoprotein associates with the AAUAAA polyadenylation signal in vitro. Cell 45, 581-591.

Rouault, T A., Hentze, M. W., Caughman, S. W., Harford, J. B., and Klausner, R. D. (1988). Binding of a cytosolic protein to the ironresponsive element of human ferritin messenger RNA. Science 241, 1207-1210.

Hauber, J., Perkins, A., Haimer, E. P, and Cullen, 8. R. (1987). Tramactivation of human immunodeficiency virus gene expression is mediated by nuclear events. Proc. Natl. Acad. Sci. USA 84, 6364-6368.

Sadaie, M. R., Benter, T., and Wong-Staal, F. (1988). Site-directed mutagenesis of two bans-regulatory genes (tat-Ill, trs) of HIV-l. Science 239, 910-914.

Kim, S., Bym, R., Groopman, J., and Baltimore, D. (1989). Temporal aspects of DNA and RNA synthesis during human immunodeficiency virus infection: evidence for differential gene expression. J. Virol. 63, 3708-3713.

Sharp, P A., and Marciniak, Cell 59, 229-230.

Konarska, M. M., and Sharp, F! A. (1986). Electrophoretic separation of complexes involved in the splicing of precursors to mRNAs. Cell 46, 845-855. La Monica, N., Meriam, C., and Racaniello, V R. (1986). Mapping of sequences required for mouse neurovirulence of poliovirus type 2 Lansing. J. Virol. 57, 515-525. Lazinski, D.. Grzadzielska, E., and Das, A. (1989). Sequence-specific recognition of RNA hairpins by bacteriophage antiterminators requires a conserved arginine-rich motif. Cell 59, 207-218. Le, S.-Y.., Chen. J.-H., Braun, M. J., Gonda, M. A., and Maizel. J. V (1988). Stability of RNA stem-loop structure and distribution of nonrandom structure in the human immunodeficiency virus (HIV-l). Nucl. Acids Res. r6, 5153-5168. Le. S.-Y., Malim, M. H., Cullen, B. R., and Maizel, J. V (1990). A highly conserved RNA folding region coincident with the Rev response element of primate immunodeficiency viruses. Nucl. Acids Res.. in press. Malim, M. H., Hauber, J., Fenrick, R., and Cullen, B. R. (1988). nodeficiency virus rev bans-activator modulates the expression viral regulatory genes. Nature 335, 181-183.

Immuof the

Malim. M. H.. Hauber, J., Le, S.-Y., Mabel, J. V, and Cullen, 8. R. (1989a). The HIV-1 rev &arm-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature 338.254-257. Malim, M. H., B5hnlein, S., Hauber, J., and Cullen, B. R. (1989b). Functional dissection of the HIV-l Rev rrens-activator-derivation of a transdominant repressor of Rev function. Cell 58, 205-214. Malim, M. H.. Biihnlein. S., Fenrick, R., Le, S.-Y., Maizel, J. V, and Cullen, B. R. (1989c). Functional comparison of the Rev trarrs-activators encoded by different primate immunodeficiency virus species. Proc. Natl. Acad. Sci. USA 86, 8222-8226. Mattaj, I. W. (1989). A binding consensus: splicing, snRNPs, and sex. Cell 57, 1-3.

RNA-protein

interactions

in

Muesing, M. A., Smith, D. H., Cabradilla, C. D., Benton, C. V, Lasky, L. A., and Capon, D. J. (1985). Nucleic acid structure and expression of the human AlDSllymbhadenopathy retrovirus. Nature 313,450-458. Mullis, K. B., and Faloona, F. A. (1987). Specific vitro via a polymerase catalyzed chain reaction. 335-350.

synthesis of DNA in Meth. Enzymol. 255,

Nagoshi, R. N.. McKeown, M., Burtis, K. C., Belote, J. M., and Baker, 8. S. (1988). The control of alternative splicing at genes regulating sexual differentiation of D. melanogaster. Cell 53, 229-236. Nomoto. A., Omata, T., Toyoda, H., Kuge, S., Horie, H., Kataoka, Y., Genba, Y.. Nakano, Y., and Imura, N. (1982). Complete nucleotide sequence of the attenuated poliovirus Sabin 1 strain genome. Proc. Natl. Acad. Sci. USA 79, 5793-5797. Query, C. C., Bentley, R. C.. and Keene, J. D. (1989). A common RNA recognition motif identified within a defined Ul RNA binding domain of the 70K Ul snRNP protein. Cell 57, 89-101. Ratner, L., Haseltine, W., Patarca, R., Livak, K. J., Starcich, B., Josephs, S. F, Doran, E. R., Rafalski, J. A., Whitehorn, E. A., Baumeister, K., Ivanoff. L., Petteway, S. R., Jr., Pearson, M. L., Lautenberger. J. A., Papas, T. S.. Ghrayeb, J.. Chang, N. T., Gallo, R. C., and Wong-Staal, F. (1985). Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature 373, 277-284. Rosen, C. A., Terwilliger, W. A. (1988). lntragenic

E., Dayton, A., Sodroski, J. G., and Haseltine. c&acting aft gene-responsive sequences of

R. A. (1989). HIV TAR: an RNA enhancer?

Shaw, G. M., Hahn, B. H., Arya, S. K., Groopman, J. E., Gallo, R. C., and WongStaal, F. (1984). Molecular characterization of human T-cell leukemia (lymphotropic) virus type Ill in the acquired immune deficiency syndrome. Science 226, 1165-l 17l. Singh, H., Sen, R., Baltimore, factor that binds to a conserved trol elements of immunoglobulin Skolnik-David, retie separation 672-682.

D., and Sharp, F! A. (1986). A nuclear sequence motif in transcriptional congenes. Nature 379, 154-158.

H., Moore, C. L., and Sharp, l? A. (1987). Electrophoof polyadenylation-specific complexes. Genes Dev. 7,

Sodroski, J., Goh, W. C., Rosen, C., Dayton, A., Terwilliger, E., and Haseltine, W. (1986). A second post-transcriptional bans-activator gene required for HTLV-III replication. Nature 327, 412-417. Taylor, J. W., Ott, J., and E&stein. F. (1985). The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucl. Acids Res. 73, 8765-8785. Terwilliger, E., Burghoff, R., Sia, R., Sodroski, J., Haseltine, W., and Rosen, C. (1988). The art gene product of human immunodeficiency virus is required for replication. J. Virol. 62, 655-658. Zapp, M. L., and Green, M. R. (1989). Sequence-specific by the HIV-l Rev protein. Nature 342, 714~7t6. Note

Added

RNA binding

In Proof

A recently reported structural analysis of the RRE (Dayton et al., Science 246, 1625-1629. 1989) fully confirms the predicted secondary structure of the RRE presented in Figure 1. In addition, these authors have observed that the RRE stem-loops here termed I and II are particularly critical for in vivo RRE function.