Immunodeficiency Virus Type 1 Rev Proteins: Implications for Function

Transcription

1 JOURNAL OF VIROLOGY, Sept. 1992, p X/92/ $02.00/0 Copyright 1992, American Society for Microbiology Vol. 66, No. 9 In Vivo Binding of Wild-Type and Mutant Human Immunodeficiency Virus Type 1 Rev Proteins: Implications for Function SALVATORE J. ARRIGO,1* SHAUN HEAPHY,2 AND JULIA K. HAINES' Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina ,1 and Department of Microbiology, University of Leicester School of Medicine, Leicester, LEI 9HN England2 Received 14 March 1992/Accepted 12 June 1992 The Rev transactivator protein of human immunodeficiency virus type 1 (HIV-1) is required for protein expression from the HIV-1 RNAs which contain a binding site for the Rev protein, termed the Rev-responsive element (RRE). This transactivator acts both at the level of splicing/transport of nuclear RNAs and at the level of translation of cytoplasmic RNAs. We used a monoclonal antibody specific for the HIV-1 Rev protein to immunoprecipitate cellular extracts from HIV-1-infected and -transfected cells. High levels of specific binding of wild-type Rev to the RRE-containing RNAs were found in cytoplasmic, but not nuclear, extracts from these cells. A Rev mutant which lacked both nuclear and cytoplasmic Rev function but retained RNA binding in vivo was generated. This binding was detectable with both nuclear and cytoplasmic extracts. These results verify the existence of direct binding of Rev to HIV-1 RNAs in vivo and conclusively prove that binding of Rev is not sufficient for nuclear or cytoplasmic Rev function. The results also support a direct role for Rev in the nuclear export and translation of HIV-1 RNAs. Human immunodeficiency virus type (HIV-1) encodes a regulatory transactivator, termed Rev, which is essential for viral replication (14, 31). The inability of the virus to replicate without Rev is in part due to a lack of viral structural protein expression (14, 31). The Rev protein appears to act at two levels in allowing the production of structural proteins. It functions at the nuclear level to inhibit splicing or facilitate the nuclear export of HIV-1 RNAs (2, 13, 16, 17, 24, 25), and it functions at the cytoplasmic level to permit the translation of HIV-1 RNAs (1, 4, 7). We have previously demonstrated that cytoplasmic accumulation of the singly spliced Rev-responsive element (RRE)-containing HIV-1 RNAs encoding Vif, Vpr, Env, and Vpu was unaffected by the presence or absence of Rev; however, these RNAs were not translated owing to a defect in polysome formation (1). A cis-acting element found in the env gene of HIV-1, the RRE, has been shown to mediate both of these effects of Rev on viral RNAs (2, 13, 16, 17, 24, 25). The Rev protein localizes to the nucleus of transfected cells, and purified Rev protein binds with high affinity to in vitro-synthesized RRE-containing RNA (6, 10, 15, 18, 27, 28, 30, 33). This binding is dependent on an extensive secondary structure within the RRE. Site-directed mutagenesis of the RRE has localized important structures involved in this binding and has shown that these structures are important in vivo for Rev function (6, 9-12, 19, 20, 22, 27, 29, 32). Site-directed mutagenesis of the Rev protein has delineated domains of Rev important for RNA binding, multimerization, and function (4, 21, 23, 26, 28). These studies have shown that mutagenesis of the functional domain of Rev can result in mutants which retain the in vitro binding properties of wild-type Rev but lack Rev function. These mutants have * Corresponding author. been shown to exert transdominant repression of wild-type Rev protein. The binding of Rev to the RRE in vitro appears to correlate well with the function of Rev in vivo; therefore, the association of Rev with the HIV-1 RRE-containing RNAs in vivo is proposed to be a prerequisite for Rev function. Since Rev localizes to and functions at the level of splicing and transport in the nucleus, Rev should initially interact with these RNAs in the nucleus. The binding of Rev to the RRE-containing RNAs would then either inhibit the splicing or facilitate the nuclear export of these RNAs. However, it is assumed that binding of Rev is not sufficient for function since transdominant mutants of Rev, which retain the in vitro binding and multimerization properties of wild-type Rev but do not retain in vivo function, have been generated (4, 21, 23, 27, 28). Since Rev has an effect on the translational capacity of these RNAs in the cytoplasm, it seemed likely that Rev would continue its association with these RNAs after nuclear export. However, the association of Rev with HIV-1 RNAs in vivo has not been established. Using monoclonal antibodies raised against the Rev protein, we examined the in vivo association of Rev with the HIV-1 RRE-containing RNAs. We demonstrate that in HIV- 1-infected and -transfected cells, Rev is found associated with high levels of RRE-containing RNAs in the cytoplasm. This association is dependent on the presence of Rev and the RRE. The bulk of the wild-type Rev-RRE binding was detected in the cytoplasmic, but not nuclear, fractions of infected and transfected cells. Results with a nonfunctional Rev mutant, which retains RNA binding, indicate that in vivo binding of Rev is not sufficient for function. The detection of nuclear binding with this mutant indicates that the binding of functional Rev to an RNA enhances its export from the nucleus. The continued association of Rev with cytoplasmic RNAs suggests that Rev allows the association of HIV-1 RNAs with the translational machinery. 5569

2 5570 ARRIGO ET AL. MATERIALS AND METHODS Proviral constructs. The construction of CSF/EBV- and ASal/EBV- has been previously described (1). TDBg/EBVwas generated by using polymerase chain reaction (PCR) mutagenesis. The oligonucleotide primers TDBg and M112 were used first to amplify a PCR product containing the desired mutation. The oligonucleotide primers BamS and CR2 were used to amplify an overlapping PCR product. The gel-purified PCR products were then amplified with BamS and M112 oligonucleotide primers. The PCR product was digested with BamHI and XhoI and subcloned into pbluescript (Stratagene). This fragment was then isolated from pbluescript and cloned at these unique sites in the proviral construct. This reconstructed a full-length provirus containing the desired mutation. The sequences of these oligonucleotide primers are as follows: TDBg, 5'-GCTACCACCGCTTGAGAGATCTAC TCTFGAC-3'; M112, 5'-GCITACITTGTGATTGCTCCATG- 3'; BamS, 5'-CACTCCTCAGGAGGGGATCC-3'; and CR2, 5'-CTCTCAAGCGGTGGTAGCTG-3'. Tissue culture, infections, and electroporation. Peripheral blood lymphocytes were prepared by using lymphocyte separation medium (Organon Teknika). These were maintained for 3 days in RPMI 1640 medium supplemented with 20% fetal calf serum (GIBCO or Whittacre) and 0.5 mg of phytohemagglutinin (Sigma) per ml. Nonadherent cells were infected with HIV-1 culture supematants in the presence of 20,ug of Polybrene per ml for 1 to 2 h. Cells were pelleted by low-speed centrifugation and resuspended in RPMI 1640 medium supplemented with 20% fetal calf serum and 5 U of human recombinant interleukin-2 (GIBCO) per ml. The 729 B-cell line was maintained in Iscove's medium with 10% fetal calf serum. Cells were electro- supplemented porated as previously described (2). The wild-type, Revmutant, and RRE- mutant DNAs refer to constructs previously described as pykjrcsf/ebv-, ASal/EBV-, and ABX/EBV-, respectively (1). Cellular fractionation and immunoprecipitation. Nuclear and cytoplasmic fractions were prepared as previously described (2). Nuclear fractions were prepared for immunoprecipitation by being treated in a mini-beadbeater (Biospec Products) with 0.1-mm glass beads for 2 min. The supernatants were centrifuged to remove debris. Then 100 to 200 U of RNasin (Promega Biotec) was added to each extract, and the extracts were aliquoted into the appropriate number of tubes. All tubes contained 40 to 50,ul of a 50% slurry of protein A-Sepharose (Pharmacia) in Nonidet P-40 lysis buffer and 5 p,g of rabbit anti-mouse immunoglobulin G (18). Control immunoprecipitations contained 2,ug of anti-act (a cell surface antigen) or 1,ug of anti-albumin antibodies. Specific immunoprecipitations of Rev-bound RNAs contained 10 to 100 pl of NR4/3C4.22 anti-rev monoclonal antibody culture supernatant (18). The samples were incubated with rocking at 40C overnight. The immunoprecipitate was prepared by low-speed centrifugation and four washes of the pellet with Nonidet P-40 lysis buffer. Transfected cells were radiolabeled as previously described (1). Proteins were immunoprecipitated as above by using pooled serum from patients with AIDS (1). RNA preparation and RNA PCR analysis. RNA was prepared from the immunoprecipitates and immunosupernatants as previously described (2). Yeast trna (40 p,g) was added as carrier to each of the immunoprecipitates. RNA PCR analysis of RNAs was performed as previously described (3). RESULTS J. VIROL. Rev is bound in vivo to high levels of HIV-1 RNA. Since Rev has an effect at the cytoplasmic level on the translation of RRE-containing RNAs, we expected that Rev binding to these RNAs should be detectable in the cytoplasmic fraction of HIV-1-infected cells. All the RRE-containing RNAs should be capable of binding Rev. These RNAs include the gag/pol, vif, vpr, and envlvpu2 RNAs. In contrast, the tat/rev RNA is an HIV-1 RNA that does not contain the RRE and should not bind Rev. Therefore, we initially investigated Rev-RRE binding by using cytoplasmic extracts from phytohemagglutinin-stimulated peripheral blood lymphocytes infected with HIV-1. The cytoplasmic extracts from two separate infections of peripheral blood lymphocytes were aliquoted and analyzed in duplicate. Monoclonal antibodies generated against the carboxyl terminus of Rev were used in the immunoprecipitation analysis of these extracts. In this assay, the antibodies are used to immunoprecipitate Rev protein, a fraction of which might be bound to RNAs through the RRE. If the carboxyl terminus of the bound Rev protein were accessible to the antibody and binding of the antibody did not perturb the binding of Rev to the RNA, specific immunoprecipitation of Rev and the bound RNA should be detected. RNA prepared from the immunoprecipitate was analyzed by using a quantitative RNA PCR procedure (1-3, 5) to determine the specific immunoprecipitation of RREcontaining RNAs (Fig. 1A). High levels of the RRE-containing vif and vpr HIV-1 RNAs were found in the immunoprecipitate only in the presence of anti-rev antibodies and not with the control antibodies. Therefore, it appeared that the anti-rev monoclonal antibodies were able to recognize Rev bound to RRE-containing RNAs in the cytoplasm of infected cells. The binding of the antibodies did not perturb the association of Rev with the RRE-containing RNAs. To determine the specificity of the immunoprecipitation and to determine the level of HIV-1 RNAs associated with Rev, we used anti-rev antibodies in another experiment to immunoprecipitate a cytoplasmic extract of an HIV-1-infected peripheral blood lymphocyte culture. In this experiment, RNA was prepared from both immunoprecipitate and immunosupernatant fractions. The amount of total RNA prepared from each fraction was analyzed by agarose gel electrophoresis (Fig. 1B). Although high levels of 28S and 18S rrnas could be detected in the immunosupernatant, no rrnas were detected in the immunoprecipitate. An HIV-1 RNA which does not contain the RRE, tat/rev, was examined by quantitative RNA PCR (Fig. 1B). This RNA was almost undetectable in the immunoprecipitate and was present at extremely high levels in the immunosupernatant. We estimate that less than 1% of this RNA was nonspecifically immunoprecipitated in this experiment. In contrast, analysis of RRE-containing RNAs demonstrated that high levels (10 to 50%) of vpr, env/vpu2, and full-length gag/pol RNAs were specifically immunoprecipitated in this experiment. Thus, binding of Rev to RNAs appeared to be specific for RNAs which contained the RRE. These results demonstrated that Rev remains associated with a high proportion of the RRE-containing RNAs after transport of these RNAs from the nucleus to the cytoplasm. Thus these results indicated that the binding of Rev to these RNAs was a prolonged association and not merely a transient event, restricted to the nucleus. If the antibodies were binding to Rev which was bound to the RRE, the immunoprecipitation of RNAs should be specific for RRE-containing RNAs and should occur only in

3 VOL. 66, 1992 A INF 1 INF2 NF 1 INF 2 u. REV (. ACT -- vpr -_N-- vif B > _trna E-.i-- 28S rrna -..-W--18S rrna i - * *2- ---tat/rev v.1 "O...-* p *0S -* i envlvpu 2 CL Un a -.s-gagpol FIG. 1. Detection of Rev bound to HIV-1 RNAs in vivo. (A) A total of 3 x i07 peripheral blood lymphocytes stimulated for 3 days with phytohemagglutinin were infected with HIV-lJR csf (7,ug of p24). Two identical infections (INF 1 and INF 2) were performed. At day 2 postinfection, the cells were harvested and cytoplasmic extracts were prepared. Immunoprecipitations were performed in duplicate with either anti-rev or control (anti-act) antibodies. RNA was prepared from the immunoprecipitates, and RNA PCR analysis was performed with oligonucleotide primers specific for HIV-1 vif and vpr RNAs as previously described (3). (B) Infections, harvests, and immunoprecipitations were performed essentially as in panel A. RNA was prepared from both the immunosupernatant (IMMSUP) and the immunoprecipitate (IMMPPT). Of the recovered RNA, 15%o was analyzed on a 1% agarose gel stained with ethidium bromide. RNA PCR analysis of HIV-1 RNAs was performed as in panel A. RNA standards (STDS) were made by sequential dilutions of RNA from transfected or infected cells. the presence of both an intact RRE and Rev. To determine whether these criteria were met, we transfected a B-lymphoblastoid cell line with a wild-type proviral construct, an RRE mutant, and a Rev mutant. The RRE mutant has been previously characterized in terms of its ability to still produce a functional Rev protein and truncated RNA species, deleted for the RRE (1, 2). The Rev mutant is a truncation of Rev which removes the activation domain and the antibody recognition site. Our previous results have shown that in this lymphoid cell type, the level of cytoplasmic HIV-1 fulllength gag/pol RNA was decreased with both of these mutants (1, 2). In addition, the level of tat/rev RNA, which does not contain the RRE, was concomitantly increased with the mutants; however, the levels of the RRE-containing vif, vpr, and env/vpu2 RNAs were not affected by the absence of Rev or the RRE (1). Therefore we expected that, in this experiment, these previous results should be reflected in the amount of nonspecific immunoprecipitation, since nonspecific immunoprecipitation should be independent of the presence of Rev or the RRE and constant within a given experiment. Cytoplasmic extracts from these cells were prepared and immunoprecipitated with anti-rev antibodies or control antibodies. RNA extracted from the immunoprecipitates was subjected to RNA PCR analysis (Fig. 2). Analysis of tat/rev RNA, which does not contain the RRE, C: C: C C: c: a:< a:cc IN VIVO Rev BINDING 5571 a ft w --*- tat/rev * 4o. * _ -m-a gag/pol w * -- < vif - - -* -- I--- --*-env/vpu2 a: Z cn co i: < 06 I FIG. 2. Detection of specific Rev binding requires Rev, the RRE, and anti-rev monoclonal antibodies. A total of 2 x 107 cells of a B-lymphoblastoid cell line (line 729) were electroporated with 100,g of the indicated DNA. At 2 days posttransfection, cytoplasmic extracts were prepared and immunoprecipitations were performed essentially as in Fig. 1. Anti-human albumin antibodies were used as a negative control. RNA was prepared from the immunoprecipitates and analyzed by RNA PCR as in Fig. 1. revealed little or no difference between immunoprecipitations with anti-rev or control antibodies of any of the constructs which were tested. This analysis required a long exposure time to enhance the detection of the low levels of nonspecific immunoprecipitation. As expected, the level of this RNA was elevated compared with the wild-type level in all samples from the mutant transfections. Analysis of fulllength gag/pol RNA revealed that specific immunoprecipitation of this RRE-containing RNA was detectable only in the transfections with the wild-type proviral construct. As expected, the level of full-length gag/pol RNA was decreased in the mutant samples compared with wild-type levels. The vif and vpr RNAs both showed specific immunoprecipitation only when an intact RRE and Rev were present in the transfected construct. The level of these RNAs was similar in the wild-type control sample, the mutant control samples, and the mutant anti-rev samples, indicating that the nonspecific immunoprecipitation was independent of the presence of Rev or the RRE and the antibody used. These data indicate that, in vivo, Rev is specifically bound to RNAs which contain an intact RRE. The RRE mutant produces a functional Rev protein, but no increase in nonspecific immunoprecipitation was seen with this mutant in the presence of anti-rev antibodies, eliminating the possibility of nonspecific interaction of Rev with RNAs. Consequently, the specific immunoprecipitation of RRE-containing HIV-1 RNAs from infected-cell extracts represents a biological association of Rev with HIV-1 RNAs in vivo which is absolutely dependent on the presence of Rev and the RRE. Rev is bound to RNAs in the cytoplasm but not in the nucleus. Since Rev exhibits an effect at the nuclear level (splicing and transport) and has been shown to localize to the nucleus (2, 8, 13, 15, 16, 17, 24, 25, 30), it is generally accepted that Rev must initially interact with the RREcontaining RNAs in the nucleus. Therefore, we attempted to

4 5572 ARRIGO ET AL. J. VIROL. IMMPPT IMMSUP Wild-Type Rev C: e C E E E >. D ) > > n > < Ec: <: Er <: a:r Er.: >. 2 ON IP - -W i. v.e a# w 4 _. env vpu2 nuci cyto nucl cyto -* vif FIG. 3. Rev-RRE binding is cytoplasmic and not nuclear. HIV- 1-infected cells (as in Fig. 1) were harvested, and nuclear (nucl) and cytoplasmic (cyto) extracts were prepared. The fractions were then immunoprecipitated, and RNA was prepared from the immunoprecipitates (IMMPPT) and immunosupernatants (IMMSUP). This RNA was subjected to RNA PCR analysis as in Fig. 1. detect Rev binding to RNAs in the nuclear extracts of infected cells. HIV-1 infected cells were separated into nuclear and cytoplasmic fractions and immunoprecipitated with anti-rev or control antibodies. RNA was prepared from the immunoprecipitate and immunosupematant fractions and subjected to RNA PCR analysis (Fig. 3). High levels of the RRE-containing env/vpu2 and vif RNAs were specifically immunoprecipitated from the cytoplasmic fraction of these cells. However, little or no Rev binding was detected in the nuclear fraction. This was reproducibly observed with cells infected with wild-type virus or transfected with the wildtype proviral construct. These results indicated that the bulk of the Rev-RRE binding, in cells infected or transfected with wild-type HIV-1, is in the cytoplasm. A mutant which lacks Rev function. Owing to the nuclear localization of the Rev protein, it was initially expected that Rev binding to RRE-containing RNAs should be detectable in the nucleus. Since high levels of cytoplasmic binding and little or no nuclear binding were observed with the wild-type Rev protein, it seemed likely that this subcellular localization might be correlated with the involvement of Rev in the nuclear export of HIV-1 RNAs. If this were the case, a nonfunctional Rev which retained RNA-binding capacity should be readily detected bound to RRE-containing RNAs in the nucleus. Such a mutant would also provide direct evidence that nuclear Rev binding exists and that the binding of Rev to an RNA in vivo is not sufficient for Rev function. Dissection of the Rev protein has elucidated RNA binding, protein multimerization, nuclear localization, and activation domains. Although the binding, multimerization, and localization domains overlap, the activation domain appears to be separate. Mutants have been generated with mutations in this domain and shown to retain nuclear localization and in vitro binding and multimerization but to be nonfunctional (4, 21, 23, 26, 28). A point mutant, TDBg/EBV-, in which the conserved leucine at amino acid 81 of Rev is changed to a serine, was generated (Fig. 4). This mutation did not alter the amino acid sequence of the overlapping Env reading frame. This leucine had been previously mutated to an alanine, abolishing Rev function in fibroblast cells (26). The TDBg/ EBV- mutant preserved the RNA-binding, protein multimerization, nuclear localization, and antibody-binding domains of the wild-type Rev protein GlnLeuPrP roleugluargleuthrleuasp CAGCUACCACCGCUUGAGAGACUUACUCUUGAC... TDBg Mutant Rev GlnLeuProProLeuGluArgSerThrLeuAsp......CAGCUACCACCGCUUGAGAGAUCUACUCUUGAC... FIG. 4. RNA and protein sequences of wild-type and mutant Rev constructs. The amino acid sequence and corresponding nucleotide sequence of Rev between residues 74 and 84 is shown. Changes in nucleotide and amino acid sequences between the wild type and mutant are underlined. To determine whether the TDBg/EBV- mutant was defective for Rev function, we transfected it into lymphoid cells in parallel with previously characterized wild-type and Rev mutant constructs (1). The cells were radiolabeled, and HIV-1 proteins were immunoprecipitated and analyzed by polyacrylamide gel electrophoresis. The results are shown in Fig. 5A. The wild-type construct, CSF/EBV-, produced high levels of p24 Gag, p55 Gag precursor, and gpl20/160 Env. The ASal/EBV- Rev mutant did not produce detectable levels of these proteins but, rather, produced elevated levels of Nef compared with the wild type. These results are consistent with our previously published results with these constructs (1). The TDBg/EBV- mutant produced a protein profile indistinguishable from that of ASal/EBV-, indicating that the TDBg/EBV- mutant was exhibiting a lack of structural protein expression and an increase in Nef production characteristic of a Rev- mutant. The TDBg/EBV- mutant was also analyzed for cytoplasmic RNA production. Mutant and wild-type constructs were transfected into lymphoid cells, and cytoplasmic RNA was prepared 48 h posttransfection. This RNA was subjected to quantitative RNA PCR analysis to determine the level of specific HIV-1 RNAs (Fig. SB). The levels of unspliced gag/pol RNA produced by both Rev mutants were reduced and the levels of spliced tat/rev RNA produced by both Rev mutants were concomitantly increased compared with wildtype levels. The levels of env/vpu2 RNA were similar in all constructs. These results were in agreement with our previous results (1) and demonstrated that the nuclear export and splicing function of Rev was abolished in the TDBg/EBVmutant. Although the level of cytoplasmic env/vpu2 RNA was similar to that produced by wild type, no Env protein was detectable with this mutant. These results demonstrated that this mutant was deficient in both the nuclear export/ splicing and translation functions of Rev. Rev binding is not sufficient for function. Although the TDBg/EBV- mutant produced a nonfunctional Rev, the mutant protein retained the RNA-binding, protein multimerization, nuclear localization, and antibody-binding domains of the wild-type Rev protein. Functionally impaired mutants similar to the TDBg/EBV- mutant have been shown to retain the binding properties of wild-type Rev in in vitro binding assays; however, it has not been demonstrated whether this binding occurs in vivo. To determine whether the mutant Rev protein was capable of binding in vivo to

5 VOL. 66, 1992 IN VIVO Rev BINDING 5573 A 2a1 a) c E _ > c E : > CL co > > tl -1 m cc w w 0u0m >- *-s---gaglpol 200i _ ^ CL g. B -a : _- gpl m u-0 vif -a 0 0 -_- env/vpu 2 _ p55 so_ - -.*- tat/rev a co > >D >- 0 m _4 - Net _-. p24 *- gag/poi so -4 tat/rev o w onvlvpu 2 FIG. 5. Effect of the Rev mutation on protein and cytoplasmic RNA expression. (A) Lymphoid cells were transfected with the indicated constructs. At 48 h posttransfection, cells were radiolabeled with 35S translabel. Cytoplasmic lysates were immunoprecipitated with pooled serum from AIDS patients and analyzed by polyacrylamide gel electrophoresis. (B) Lymphoid cells were transfected with the indicated constructs. At 48 h posttransfection, cytoplasmic RNA was prepared and subjected to RNA PCR analysis for the indicated RNAs. RRE-containing RNAs, we transfected lymphoid cells with the TDBg/EBV- mutant. Nuclear and cytoplasmic extracts were prepared and immunoprecipitated with anti-rev or control antibodies. RNA was prepared from the immunoprecipitates and analyzed by RNA PCR for specific HIV-1 RNAs (Fig. 6). No specific immunoprecipitation of tat/rev RNA was seen. In contrast, gag/pol, env/vpu2, and vif RNAs were all specifically immunoprecipitated by the anti- Rev antibodies, indicating high levels of binding of the mutant Rev protein to the RRE-containing RNAs. As opnuci cyto FIG. 6. Mutant Rev is bound to HIV-1 RNAs in the nucleus. Lymphoid cells were transfected with TDBg/EBV-. At 48 h posttransfection, cells were harvested and nuclear (nucl) and cytoplasmic (cyto) extracts were prepared. The fractions were immunoprecipitated, and RNA was prepared from the immunoprecipitates. The RNA was subjected to RNA PCR analysis as in Fig. 1. posed to results with the wild-type protein, binding was readily detected in the nucleus as well as in the cytoplasm. This nuclear binding was reproducibly seen in nuclear fractions from all experiments performed with this mutant. These results show that the binding of the mutant Rev to RRE-containing RNAs is both nuclear and cytoplasmic and that this binding is not sufficient for either nuclear or cytoplasmic Rev function. DISCUSSION Using monoclonal antibodies generated against the Rev protein, we have analyzed the association of Rev with HIV-1 RNAs in vivo. We have demonstrated that these antibodies can be used to specifically immunoprecipitate HIV-1 RNAs which contain the RRE, depending on the presence of Rev, the RRE, and anti-rev antibodies. These RNAs include those encoding Gag, Pol, Vif, Vpr, Vpu, and Env. These results indicate that a high level of the RRE-containing RNAs is associated with Rev in cytoplasmic extracts from infected and transfected cells and that Rev is capable of a prolonged association with the RRE-containing RNAs. We conclude from these observations that Rev is capable of maintaining its association with the RRE-containing RNAs after their transport from the nuclear to the cytoplasmic fraction. Since these cytoplasmic RNAs are incapable of translation in the absence of Rev (1), these results suggest a direct role for Rev in this process. The detection of both nuclear and cytoplasmic binding with the TDBg/EBV- mutant has several important implications. First, the binding of Rev to RRE-containing RNAs in vivo is not sufficient for either the nuclear export/splicing or translational role of Rev. Second, the presence of nuclear binding with the TDBg mutant and its absence with wildtype Rev indicates a direct role for Rev in the nuclear export of HIV-1 RNAs.

6 5574 ARRIGO ET AL. J. VIROL. Rev Nuclear Export' FIG. 7. Model for bimodal Rev function. Rev would be translated from an RNA which is not dependent on Rev for its expression. Rev would then migrate to the nucleus, where it would bind to RRE-containing RNAs and promote their export from the nucleus. Rev would remain bound to the RNA during this process. In the cytoplasm, Rev would allow the association of the RNA with the translational machinery. RRE RRE 0~~~~~~~-.%. Translation / ~ ~~~~ Structural Proteins Downloaded from The detection of wild-type Rev binding in the cytoplasm, but not in the nucleus (although a large amount of RREcontaining RNAs is presumably available as substrate in the nucleus), of infected and transfected cells cannot completely exclude a role for Rev in the inhibition of splicing of RRE-containing RNAs. However, the results are not strictly in agreement with what might be expected from a simple block to splicing by Rev. If the role of Rev were simply to inhibit splicing, one might expect to find a high level of Rev binding with the wild type in the nuclear fraction. The Rev mutant would be expected to have a lower level of nuclear Rev binding as a result of an increase in the removal of the RRE and the bound Rev protein by splicing. It is formally a possibility that Rev inhibits the splicing of HIV-1 RNAs and that these RNAs are concomitantly exported from the nucleus; however, the simplest interpretation of the data leads to model in which the binding of Rev to an RRE-containing RNA in the nucleus enhances the nuclear export of that RNA (Fig. 7). In the cytoplasm, Rev would allow the interaction of that RNA with the translational machinery. By interacting with the RRE-containing RNAs in the nucleus and continuing its association in the cytoplasm, Rev could provide both nuclear and cytoplasmic functions. In the absence of Rev and its associated enhancement of nuclear export, complete splicing of precursor RNA would be augmented. In the absence of Rev, unspliced (at a reduced level) and singly spliced HIV-1 RNAs would still accumulate in the cytoplasm through an alternate (slower) transport pathway. Without Rev, these RNAs would be incapable of associating with the translational machinery. Thus Rev would be capable of a bimodal effect on nuclear export and translation of RRE-containing RNAs. More research is required to analyze the precise logistics processes involved. of Rev-RRE binding and the cellular ACKNOWLEDGMENTS We thank A. Lowe and S. Green, Laboratory for Molecular Biology, Cambridge, England, for production of the antibody NR4/ 3C4.22. We also thank Irvin S. Y. Chen, in whose laboratory some of the basic concepts for this study evolved, and M. Schmidt, P. Arnaud, and K. Arrigo for helpful discussions. S.H. is a Medical Research Council Senior AIDS Research Fellow. This work was supported in part by grant RG from the American Foundation for AIDS Research (to S.J.A.). REFERENCES 1. Arrigo, S. J., and I. S. Y. Chen Rev is necessary for translation but not cytoplasmic accumulation of HIV-1 vif, vpr, and env/vpu-2 RNAs. Genes Dev. 5: Arrgo, S. J., S. Weitsman, J. D. Rosenblatt, and I. S. Chen Analysis of rev gene function on human immunodeficiency virus type 1 replication in lymphoid cells by using a quantitative polymerase chain reaction method. J. Virol. 63: Arrigo, S. J., S. Weitsman, J. A. Zack, and I. S. Chen Characterization and expression of novel singly spliced RNA species of human immunodeficiency virus type 1. J. Virol. 64: Benko, D. M., S. Schwartz, G. N. Paviakis, and B. K. Felber A novel human immunodeficiency virus type 1 protein, tev, shares sequences with tat, env, and rev proteins. J. Virol. 64: Cann, A. J., J. A. Zack, A. S. Go, S. J. Arrigo, Y. Koyanagi, P. L. Green, Y. Koyanagi, S. Pang, and I. S. Chen Human immunodeficiency virus type 1 T-cell tropism is determined by events prior to provirus formation. J. Virol. 64: Cochrane, A. W., C. H. Chen, and C. A. Rosen Specific on December 18, 2018 by guest

7 VOL. 66, 1992 interaction of the human immunodeficiency virus Rev protein with a structured region in the env mrna. Proc. Natl. Acad. Sci. USA 87: Cochrane, A. W., K. S. Jones, S. Beidas, P. J. Dillon, A. M. Skalka, and C. A. Rosen Identification and characterization of intragenic sequences which repress human immunodeficiency virus structural gene expression. J. Virol. 65: Cullen, B. R., J. Hauber, K. Campbell, J. G. Sodroski, W. A. Haseltine, and C. A. Rosen Subcellular localization of the human immunodeficiency virus trans-acting art gene product. J. Virol. 62: Daefler, S., M. E. Klotman, and S. F. Wong Transactivating rev protein of the human immunodeficiency virus 1 interacts directly and specifically with its target RNA. Proc. Natl. Acad. Sci. USA 87: Daly, T. J., K. S. Cook, G. S. Gray, T. E. Maione, and J. R. Rusche Specific binding of HIV-1 recombinant Rev protein to the Rev-responsive element in vitro. Nature (London) 342: Dayton, E. T., D. M. Powell, and A. I. 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