Definition of the RRE Binding and Activation Domains of the Caprine Arthritis Encephalitis Virus Rev Protein

VIROLOGY

226, 113–121 (1996) 0633

ARTICLE NO.

SHORT COMMUNICATION Definition of the RRE Binding and Activation Domains of the Caprine Arthritis Encephalitis Virus Rev Protein R. V. SCHOBORG*,1 and J. E. CLEMENTS† *Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614; and †Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Received February 28, 1996; accepted September 16, 1996 Caprine arthritis encephalitis virus (CAEV) is a lentivirus which is closely related by nucleotide sequence and biological properties to visna virus and is more distantly related to the human AIDS virus, HIV-1. Previous studies indicated that the CAEV Rev protein (Rev-C) functions as a trans-activator of mRNA cytoplasmic transport and expression. The function of Rev-C is mediated through an RNA element (RRE-C) present between nucleotides (nt) 7906 and 8110 in the CAEV env gene. In this study, RNA/protein immunoprecipitation experiments were used to demonstrate that Rev-C binds directly to the 204-nt RRE-C in vitro. Competition assays illustrate that this interaction is specific for the positive sense RRE-C RNA. Glutaraldehyde crosslinking studies demonstrate that the wildtype Rev-C protein can also form multimeric complexes in vitro. Deletions or amino acid alterations within the basic domain of Rev-C reduce affinity for the RRE and disrupt assembly of Rev-C multimers in vitro, indicating that this domain is involved in RRE binding and Rev multimer formation. Mutations within the leucine-rich domain of Rev-C do not greatly effect RRE-C binding or self-assembly. However, previous results demonstrate that some leucine-rich domain mutants are unable to trans-activate. These data are consistent with the hypothesis that the leucine domain is the effector domain of Rev-C. q 1996 Academic Press, Inc.

The lentiviruses are a subfamily of the Retroviridae that are characterized by similarities in ultrastructure, genomic organization, and disease progression. These viruses typically cause chronic, multiorgan diseases that are apparent only months to years after initial infection (1). The most extensively studied lentivirus to date is HIV-1 (human immunodeficiency virus 1), which causes immunodeficiency and neurological disease in humans (1). Visna virus, the prototype nonprimate lentivirus, infects sheep and causes a chronic disease state characterized by pneumonitis and progressive demyelination within the central nervous system of the infected animal (2, 3). Caprine arthritis encephalitis virus (CAEV), which is closely related to visna virus, is a lentivirus of goats that causes neurological disease in kids and chronic arthritis in adults (4). Sequence analysis of lentiviral genomes demonstrates that they share similar genomic organization. Like other retroviruses, lentiviral genomes are flanked by long terminal repeats (LTR) and contain the genes for the structural proteins: gag (capsid proteins), pol (reverse transcriptase), and env (envelope glycoproteins). These structural proteins are produced from full length genomic

RNA (gag and pol) and from singly-spliced messages (env) (1). The lentiviruses can be distinguished from other retroviruses by the presence of small open reading frames (ORFs) in their genomes that encode regulatory proteins (Fig. 1A). The location and number of these small regulatory genes vary between lentiviruses; the ovine lentiviruses, CAEV and visna virus, express three different regulatory proteins (5, 6), while the more complex human lentivirus HIV-1 produces at least seven (7, 8). Two of these nonstructural genes, tat and rev, have been found in all lentiviruses studied to date (9–16). Tat and Rev are both translated from small, multiply spliced mRNAs (Fig. 1A) and have been shown to be absolutely required for replication of HIV-1, visna virus, and CAEV in culture (17–19). Examination of HIV, visna virus, and CAEV gene expression at various times after infection has demonstrated that their genes are temporally regulated. Early in infection, the small, multiply spliced mRNAs that encode Tat and Rev predominate. Later during infection, the singly spliced and unspliced RNAs increase with a concomitant decrease in the levels of the multiply spliced RNAs (20, 21; R. Schoborg, unpublished results). Analysis of Rev-deficient clones of HIV-1 and CAEV has indicated that in the absence of the Rev protein, viral gene expression is trapped in the ‘‘early phase’’ in which only the multiply spliced viral mRNAs are transported to the cytoplasm of an infected cell, indicating that Rev is required

1 To whom correspondence and reprint requests should be addressed at Quillen College of Medicine, Department of Microbiology, Box 70579, Johnson City, TN 37614-0579. Fax: (423) 929-5847.

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Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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FIG. 1. Organization of the CAEV genome and amino acid sequence of the visna virus and CAEV Rev proteins. (A) Organization of the CAEV genome and map of the Rev-C expressing cDNA clone. The open reading frames (ORFs) and LTRs of CAEV are represented by open boxes; the identity of each is indicated. The exons of the Rev-C are represented by thick solid lines; introns are indicated by dashed lines. Splice donors are indicated by SD; splice acceptor sites by SA. Numbering (bps) is with respect to the CAEV RNA genome with nucleotide (nt) / 1 being the RNA cap site. (B) Amino acid sequence comparison of the visna virus and CAEV Rev proteins. The amino acids have been aligned for best fit. Amino acid identity is indicated by a vertical line; amino acid similarity is denoted by single or double dots. The basic and leucine-rich domains are shown in boxes. The C-terminal peptide used for production of the Rev-C specific antibodies is underlined with a dark bar (15, 19).

for cytoplasmic transport of lentiviral structural gene mRNAs (19, 22, 23). The lentiviral mRNA processing/transport pathway requires at least two distinct elements in order to function: (1) the Rev protein and (2) a discrete, structured RNA element called the Rev Response Element (RRE). The Rev protein functions in trans by binding to the RRE; the RRE is present in the target RNA (23–25). Two domains of the Rev protein are required for function (Fig. 1A); these are the basic and the leucine-rich domains. Mutation of basic amino acid residues (especially arginines) in the HIV-1 Rev basic domain has severe affects upon its biologic function; in addition, in vitro RRE binding and multimerization activities of these mutants are often impaired (26–30). The nuclear and nucleolar localization of many of these mutants is also diminished (29, 30). Similar mutations within CAEV Rev (Rev-C) also decrease trans-activating activity, alter subcellular localization, and eliminate function during viral infection (15, 19). The other essential domain of Rev is the activation (or leucine-rich) domain. Deletion or mutation of the leucines in this domain inactivate the HIV, visna, and CAEV Rev proteins (15, 26, 30, 31). Mutation of two leucine residues at positions 112 and 114 (Fig. 1B) in the activation domain greatly decreases the ability of the visna Rev protein (Rev-V) to trans-activate a visna virus RRE (RRE-V) con-

taining reporter construct (30). In addition, certain leucine domain mutants of HIV and visna virus Rev can act as dominant negative repressors of wildtype Rev function (26, 30). Recent studies have indicated that the leucine domain of Rev interacts with several cellular proteins (32–34). This interaction is thought to direct the nuclear Rev/ viral RNA complex to a nonmessenger RNA cytoplasmic transport pathway and, therefore, mediate Revdependent transport of RRE containing RNAs to the cytoplasm of the host cell (32–35). Another critical component of the Rev response system is the RRE. The RREs of HIV, visna virus, and CAEV are RNA stem-loop structures that are located in the env ORF of each virus (36–40). Comparison of the env sequences of CAEV and visna virus followed by minimal energy folding secondary structure analysis reveals a potential RNA stem-loop structure, similar to that of the HIV RRE, between nucleotide (nt) 7924 and nt 8125 of visna virus and between nt 7906 and nt 8108 of the CAEV genome (6, 40). Even though the predicted secondary structures of these regions are almost identical, they share no clear sequence similarity (6). These regions of visna virus and CAEV have been functionally defined as the target for their cognate Rev proteins using cotransfection assays (11, 15, 30). In vivo analysis has indicated that both HIV-1 and visna

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virus Rev mediate their activity by binding to their cognate RREs (23, 30, 40). In addition, both HIV and visna Rev proteins have been shown to interact with their respective RREs in vitro (39–41). Since we previously localized the RRE-C to nt 7906–8110 using functional assays, we decided to test the ability of Rev-C and RRE-C to interact specifically in vitro using an RNA binding/immunoprecipitation assay (Fig. 2A). The RRE-C (nt 7906 to 8110) was PCR amplified using specific oligonucleotides and cloned into the unique KpnI site of the plasmid pGEM 7Z (Promega). This construct can be in vitro transcribed to produce both positive (/) and negative (0) polarity RRE-C transcripts. Human actin (nt 150 to nt 254; (42)), visna tat (nt 5652 to 5944), visna env (nt 6486 to nt 6277), and CAEV rev (CAEV nt 6012 to 6122 and 8514 to 8802) sequences were also cloned into this vector for use as negative controls. 32P-labeled RRE-C RNAs were generated by in vitro transcription of linearized plasmids (43). 35 S-labeled Rev-C protein was in vitro translated (15, 19), reacted with labeled target RNA, and precipitated with specific anti-Rev-C peptide antibody as described in the legend to Fig. 2. The RRE-C (/) probe/Rev-C protein complex was specifically precipitated by anti-Rev-C sera only in the presence of the Rev protein (Fig. 2A, lane 3). The antisense RRE-C transcript, RRE-C (0), was not precipitated (Fig. 2A, lane 4), indicating that Rev-C does not interact with negative polarity RRE-C 7906-8110 transcripts in these assays. This result is consistent with the observation that RRE-C function is orientation dependent; only the sense (coding) orientation mediates Rev function in

FIG. 2. (A) Immunoprecipitation of Rev-C/RRE-C complexes. Wildtype Rev-C was tested for its ability to specifically interact in vitro with RRE-C using an RNA immunoprecipitation assay. In this experiment, [35S]methionine-labeled Rev-C was translated in vitro and then mixed with various 32P-labeled, in vitro-transcribed RNAs. Protein/RNA binding reactions were assembled by combining 2 to 6 ml of in vitro-translated protein, 2 to 4 ml (200,000 to 500,000 cpm) of 32P-labeled RNA, 1 ml RNAsin (Promega), 1 ml 100 mM PMSF (phenylmethylsulfonyl flouride, Sigma Chemicals), 25 ml 21 RNA binding buffer (1.3% Nonidet P-40, 0.3 M NaCl, 0.02 M Tris, pH 7.5, 0.003 M MgCl2 ), and ddH2O to bring the reaction volume up to 50 ml. After incubation at room temperature for 30 min, 342 ml 11 RNA binding buffer and 4 ml of affinity-purified anti-Rev-C antisera were added to each reaction. The reactions were incubated at 47 for 1 h. Protein/RNA complexes were precipitated by addition of Protein A–Sepharose beads (Pharmacia), incubation at 47 for 1 h with gentle shaking, followed by centrifugation at 12,000g for 1 min at 47. The pellets were washed six times in cold 11 RNA binding buffer and resuspended in 100 ml of sequencing dye. Samples were

boiled, quenched, and electrophoresed on 6% acrylamide/urea/TBE gels. Lanes 1 and 6 are reactions containing mock in vitro-translated protein (IVT); lanes 2, 3, 4, 7, and 8 contain Rev-C IVT. Lanes 1, 2, and 3 are reactions containing 32P-labeled, sense strand RRE-C (RRE-C(/)); lane 4 is a reaction with 32P-labeled, antisense strand RRE-C (RREC(0)). Lanes 6, 7, and 8 are RNA IPs of a mixture of five different 32Plabeled RNAs. Lanes 1, 3, 4, 6, and 8 are precipitated with anti-Rev-C sera; lanes 2 and 7 are preimmune precipitated. Lane 5 contains the unprecipitated probe mix so that the relative positions of each RNA on the gel can be seen. The expected sizes of the transcripts are as follows: visna env (244 nt); visna tat (323 nt); human actin (144 nt); RevCwildtype (505 nt). Lane M contains 32P-labeled single-stranded DNA markers, the size of which (in nucleotides) is shown to the left. The identity of each RNA is indicated to the right. (B) RNA/protein immunoprecipitation control assays. 35S-labeled Rev-C (lanes 2, 3, 4, 5, 6, and 7) or mock (lane 1) in vitro translates were combined with 1.0 mg unlabeled RNA (lane 2, no RNA; lane 3, RRE-C (0) RNA; and lane 4, RRE-C (/) RNA) and were precipitated with anti-Rev-C antibody (lanes 1–4). Lanes 5, 6, and 7 contain sequential immunoprecipitations in which 35S-labeled Rev-C was combined with 1.0 mg of unlabeled RREC (/) RNA and precipitated with Rev-C-specific sera (lane 5). The supernatant from this precipitation was then again subjected to antiRev-C immunoprecipitation (lane 6). The supernatant from the second precipitation was then immunoprecipitated a third time (lane 7). All samples were immunoprecipitated using the RNA IP protocol and electrophoresed on a 15% SDS–PAGE protein gel as described (15, 19). Lane M contains protein molecular weight markers. The location of RevC is indicated to the right; the sizes of the molecular weight markers in kDa are shown to the left.

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cotransfection assays (15). In addition, mutational analysis of the HIV RRE has shown that maintenance of the correct secondary structure in mutant RREs is critical for maintaining Rev binding activity and, hence, Rev responsiveness (38, 44, 45). Since a negative sense RRE-C RNA would have a completely different sequence and secondary structure than the positive polarity RRE-C, it is not surprising that the RRE-C (0) fails to bind Rev-C in vitro (Fig. 2A) or to mediate Rev function in vivo (15). In Fig. 2A, lanes 6 through 8, Rev-C was incubated with a mixture of different RNAs (human actin, visna tat, visna env, CAEV rev, and RRE-C (/)). Anti-Rev-C sera precipitated only complexes of Rev-C and RRE-C (/) RNA (Fig. 2A, lane 8). These data demonstrate that the Rev protein interacts specifically with its cognate RRE in vitro and suggest, when considered with the previously published cotransfection experiments, that Rev-C mediates its effect by interacting with the RRE (15). Several additional control experiments were performed to confirm that the RNA immunoprecipitation assay is a quantitative measure of Rev-C/RRE-C interaction. In Fig. 2B, 35S-labeled, in vitro-translated Rev-C protein was combined with excess (1 mg/reaction) unlabeled RRE-C (/) RNA (lane 4), unlabeled RRE-C (0) RNA (lane 3), or no RNA (lane 2) and was then immunoprecipitated. Addition of RRE-C (/) does not change the amount of Rev-C protein precipitated in the assay (lane 4) relative to that precipitated in the presence of RRE-C (0) or in the absence of RNA, demonstrating that RRE-C binding does not effect the ability of the antisera to immunoprecipitate the Rev-C protein. In addition, Rev-C was combined with excess unlabeled RRE-C (/) RNA and subjected to multiple rounds of immunoprecipitation. Greater than 95% of the Rev-C protein present was precipitated after the first addition of antisera (compare lane 5 to 6 and 7). These data indicate that the precipitations are being performed in antibody excess and that the Rev-C/ RRE-C complexes are being quantitatively precipitated from these reactions. As an independent confirmation that the observed in vitro interaction between Rev-C and RRE-C is specific, competition experiments were carried out (Fig. 3). In these studies, various amounts of unlabeled competitor RNAs were added to reactions that contained a constant amount of both 32P-labeled RRE-C (/) RNA and Rev-C protein. Addition of increasing amounts of unlabeled RRE-C (/) competitor significantly reduces precipitation of 32P-labeled RRE-C (/) RNA (Fig. 3, lanes 1 through 6); inclusion of 25-fold excess unlabeled RRE-C (/) almost completely eliminates 32P-RRE-C precipitation compared to the control (lanes 1 and 7). Addition of similar quantities of CAEV rev (Fig. 3, lanes 8–13) and RRE-C (0) (lanes 15–20) competitor RNAs has no effect on the Rev-C/RREC (/) interaction. Dose response Rev-C/RRE-C binding experiments indicate that the amount of RRE-C precipitated is directly proportional to the quantity of Rev-C or

RRE-C added to the reaction, which is indicative of a specific, dose-dependent reaction between Rev-C and the RRE (data not shown). Taken together these data indicate that the observed reaction is specific and the immunoprecipitation method provides accurate quantitative estimates of in vitro Rev/RRE interaction. Comparison of visna virus Rev (Rev-V) and CAEV Rev amino acid sequences indicates that they share very little homology except in two regions of their second exon (Fig. 1B). Mutational analysis of Rev-V has demonstrated that these domains have the same functions as in HIV Rev (30). To determine the importance of these domains to Rev-C function, mutations were constructed by polymerase chain reaction (PCR) amplification with overlapping oligonucleotides (Fig. 4A; 15, 19). Alteration of RR to DL in the basic domain of Rev-C (pCMVRev-CRR-DL ) reduces trans-activating activity 20-fold (Fig. 4A; 15); deletion of the entire basic region (pCMVRev-Cdelta basic ) abrogates Rev-C trans-activation (M. J. Saltarelli, personal communication). Both Rev-CRR-DL and Rev-Cdelta basic bind significantly less RRE-C RNA than does wildtype Rev-C (Fig. 4B, lanes 3 and 4) in RNA IP assays. Quantitation of six independent RRE-C binding assays indicates that Rev-Cdelta basic and RR-DL bind 24- and 15-fold less RRE-C, respectively, than does wildtype Rev-C (P value of õ 0.005; Fig. 5A). These data demonstrate that the Rev-C basic domain is required for efficient RRE-C binding in vitro, similar to results obtained by mutational analysis of the visna virus Rev protein (30). The leucine domain mutants pCMVRev-Cdelta leu-rich and Rev-CLL-FV show greater than 40-fold reduced trans-activating activity in previous studies (Fig. 4A; 15). However, analysis of the RRE-C binding phenotype of Rev-C leucine domain mutants indicates that the leucine domain has little influence on in vitro interaction with the RRE (Fig. 4B, lanes 5,6, and 7). Two leucine domain mutants, Rev-CEE-DD and Rev-Cdelta leu-rich , bind the RRE as well as wildtype Rev-C in this assay (Figs. 4B and 5A). Interestingly enough, the Rev-CLL-FV mutant has a small, but significant (P value of õ 0.05) and reproducible, deficiency in RRE-C binding compared to wildtype Rev-C (Fig. 5A). It is possible that substitution of FV for LL at amino acid positions 100 and 102 of Rev-C causes a conformational change in the protein that renders it less able to bind to the RRE. However, this small reduction in RRE binding probably does not significantly contribute to this mutant’s trans-activation defect, since Rev-Cdelta leu-rich has full RRE-C binding activity but is equally deficient as a trans-activator (Fig. 4A; 15). Further experimentation is necessary to determine exactly how much the leucine-rich domain contributes to RRE binding and whether the small reduction in RRE binding contributes to the massive loss of trans-activating activity caused by the leucine domain LL-FV mutation. The Rev protein of HIV has been shown to form homomultimers in vitro; in addition, the ability to multimerize

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FIG. 3. Competition assays with unlabeled RRE-C (/) and irrelevant RNAs. RNA immunoprecipitation assays were performed as described in Fig. 2 except that constant amounts of Rev-C and 32P-RRE-C (/) were incubated with increasing amounts of unlabeled competitor RNAs. The unlabeled competitors used (RRE-C (/), RRE-C (0), or Rev-C RNAs) are indicated above each panel. The competitor/32P-RRE-C ratio is shown above each lane (lanes 1, 8, and 15 are 25:1; lanes 2, 9, and 16 are 5:1; lanes 3, 10, and 17 are 1:1; lanes 4, 11, and 18 are 0.2:1; lanes 5, 12, and 19 are 0.04:1; lanes 6, 13, and 20 are 0.008:1; lanes 7, 14, and 21 contain no competitor RNA). Lane M contains 32P-labeled single-stranded DNA markers; the size of each is shown to the left. The position of the RRE-C RNA is indicated to the right.

is thought to be required for in vivo function (25, 28). Therefore, we used glutaraldehyde crosslinking assays to examine formation of Rev-C multimers in vitro (46). S35-labeled wildtype Rev-C was crosslinked, immunoprecipitated, and electrophoresed; these experiments revealed the presence of a labeled protein band that is expected size (38 kDa) of a Rev-C homodimer (Fig. 4C, lane 2). Glutaraldehyde-crosslinked samples were also electrophoresed without prior immunoprecipitation; the results obtained were identical to those of immunoprecipitated samples (data not shown). Faint, higher-molecular-weight, crosslinked products can also be seen when crosslinked Rev-C protein is electrophoresed on longer (30 cm) SDS–PAGE gels (data not shown), indicating that Rev-C can form higher-order multimers as well. These higher-molecular-weight complexes differ in size from each other by approximately 19 kDa, exactly what would be expected if they are Rev-C multimers. These highermolecular-weight forms appear only when Rev-C, and not mock, in vitro translates are crosslinked; in addition, they are specifically immunoprecipitated by anti-Rev-C sera (Fig. 4C, lanes 1 and 2). Therefore, it is likely that they are indeed multimers of Rev-C. However, it is possible that Rev-C monomers are being nonspecifically crosslinked to some other 19-kDa protein present in the in vitro translates. As a control for nonspecific protein

crosslinking, unlabeled Rev-C in vitro translate was mixed with 35S-labeled, in vitro-translated visna Tat (TatV; an irrelevant protein which does not interact with RevC), crosslinked, and immunoprecipitated with anti-visna Tat sera. No labeled complexes of any kind were precipitated in these experiments, indicating that Rev-C and Tat-V cannot be randomly crosslinked to each other under these reaction conditions (data not shown). These results suggest that the higher-molecular-weight forms seen in crosslinked samples are not due to random crosslinking of 35S-labeled Rev-C to other proteins in the rabbit reticulocyte lysate. These studies therefore demonstrate that Rev-C can self-associate and form multimers in vitro in the absence of its cognate RRE. Since homomultimer formation of the HIV Rev protein maps to the basic domain (28), we examined the capacity of each Rev-C mutant to form multimers (Fig. 4C). All of the mutants were able to form multimeric complexes in vitro; however, the Rev-Cdelta basic mutant was significantly deficient in multimer formation compared to the rest of the mutants and wildtype Rev-C. Determination of the monomer/dimer ratios of each mutant (Fig. 5B) clearly illustrates the difference between Rev-Cdelta basic and the other Rev-C proteins. The fact that the Rev-Cdelta basic crosslinking reactions produce fewer multimeric complexes (relative to the number of monomers present) than

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FIG. 5. Quantitative comparison of mutant Rev-C protein RNA binding and multimerization phenotypes. Six independent experiments of the types shown in Figs. 4B and 4C were executed; the results are presented in graphical form in A and B. (A) Quantitation of Rev-C wildtype and mutant RNA immunoprecipitation experiments. The quantity of RRE-C or Rev-C precipitated was determined directly from RNA IP or SDS–PAGE gels using a Molecular Dynamics Phosphorimager. Amounts of protein/RNA were calculated by subtracting the volume obtained in the same region of a control immunoprecipitation lane from the volume of a particular protein/RNA band. The quantity of RNA bound by the Rev-C mutants (and wildtype Rev-C) was normalized to the quantity of protein precipitated in matched control protein immune precipitates (Fig. 4B) so that RNA binding affinities of the different proteins could be directly compared. The mean amount of RRE-C bound by each mutant (and wildtype) is indicated by the height of the appropriate vertical column; standard errors of the mean for the values obtained for each set of experiments are shown by the error bars. The identity of each Rev-C protein used is shown along the X-axis. The Y-axis is in arbitrary volume units. (B) Quantitation of Rev-C wildtype and mutant crosslinking experiments. Monomer and multimer forms were quantitated by phosphorimager analysis; the amount of each present was normalized according to the number of methionines present in each. The amount of ‘‘dimer’’ present in each lane was calculated by addition of the quantities of all detectable multimeric forms present in a lane. In most cases, dimer was the predominant multimer form present. Monomer/dimer ratios were calculated for each experiment; the mean values and the standard errors are shown in the graph as described in A. The identity of each Rev-C protein used is indicated along the X-axis. SEM (standard error of the mean) was calculated using Minitab. T tests were used to determine whether the differences between the mean activities of different mutants were significant. Bar graphs were made using Cricket Graph.

does wildtype indicates that the basic domain is required for efficient multimer formation in vitro. None of the leucine domain mutants are significantly impaired in multimer formation compared to wildtype Rev-C (Figs. 4C and 5B), which indicates that the leucine domain proba-

bly has little or no functional role during Rev-C in vitro self-assembly. As discussed above, both Rev-CRR-DL and Rev-Cdelta basic mutants are essentially nonfunctional as trans-activators (15). In addition, examination of the subcellular localiza-

FIG. 4. In vivo and in vitro functional analysis of Rev-C basic and leucine-rich domain mutants. (A) Sequence of Rev-C mutants. Asterisks below the sequence of each mutant denote locations of amino acid mutations. The trans-activating activity of each mutant is shown to the right as fold activation; these data are from previous studies (15). (B) In vitro analysis of the RRE binding phenotype of mutant Rev-C proteins. RNA IP analysis was carried out as described using wildtype or mutant Rev-C IVTs and 32P-labeled RRE-C (/) in lanes 1 through 7 (or 32P-labeled RRE-C (0) in lane 8). The Rev-C wildtype and mutant in vitro translates used in each reaction are shown above the appropriate lane. Matched control immunoprecipitations of the proteins used in the RNA IP reactions (identical reactions with no RNA added) were run on an SDS–PAGE gel; this gel is shown below the RNA IP gel. The controls demonstrate that the lack of RRE precipitated by Rev-CRR-DL and Rev-Cdelta basic is not due to absence of protein in the reactions. Lane M contains either single-stranded DNA or protein size markers as appropriate; the sizes in nucleotides or kDa are shown to the left. The locations of RRE-C and Rev-C mutant proteins are indicated to the right. (C) In vitro multimer formation by the Rev-C mutants. In vitro-translated Rev-C wildtype and mutant proteins were tested for their ability to form multimers in the absence of RRE-C using a glutaraldehyde crosslinking assay (46). After crosslinking, Rev-C IVTs were immunoprecipitated using Rev-C-specific sera and electrophoresed on denaturing PAGE gels (15, 19). Lanes 1 through 7 are crosslinking reactions of Rev-C mutants in the same order as in the RNA IP assay above. The location of RevC monomer and dimer forms is indicated to the right; the sizes of the protein standards in lane M are denoted to the left.

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tion of each mutant in transfected Cos cells by immunofluorescence has revealed that both basic domain mutants fail to localize to the nucleus and nucleolus of the cell, while wildtype Rev-C strongly localizes to these areas (15; R. Schoborg, unpublished results). Rev-CRR-DL and delta basic are also severely deficient in RRE-C binding activity (Figs. 4B and 5A); however, only Rev-Cdelta basic has impaired ability to assemble into multimers in vitro (Figs. 4C and 5B). These data suggest that although both RRE binding and self-assembly of Rev-C require sequences in the basic domain, the sequences mediating these two functions are to some degree separable. Since insertion of the Rev-CRR-DL mutation into the infectious clone of CAEV renders the virus replication incompetent, these data also suggest that the RRE binding and/or the nuclear/nucleolar localization functions of Rev-C are required to support productive viral replication in culture (19). Unlike the basic domain mutants, all three leucine domain mutants are identical to wildtype Rev-C in their subcellular localization (15; R. Schoborg, unpublished results) and ability to form multimers in vitro (Figs. 4C and 5B); they also have RRE-C binding phenotypes similar, or identical, to that of wildtype Rev-C. Also, Rev-CLL-FV and delta leu-rich are severely disfunctional as trans-activators (15). These data indicate that the leucine domain is required for the trans-activation function of Rev-C but not for RRE binding, multimerization, or nuclear/nucleolar localization. Therefore, by analogy with both HIV and visna Rev, it is likely that the leucine-rich domain of Rev-C is the activation domain of the molecule. Some activation domain mutants of HIV and visna Rev can act as dominant negative suppressors of Rev function in cotransfection assays (26, 30). The ability of the Rev-C leucinerich domain mutants to function as dominant negative repressors of Rev-C function has not yet been evaluated; however, future studies examining the ability of these mutants to inhibit CAEV infection are planned. The studies presented here clearly demonstrate that the functional organization of CAEV Rev is similar to that of all other Rev proteins that have been studied (24). The basic domain of Rev-C mediates RRE binding, Rev multimerization, and nuclear localization, while the leucine-rich domain mediates effector function of the protein. When these data are considered along with results obtained from earlier studies, it becomes clear that function of Rev-C during the viral replication is also similar to the Rev proteins of other lentiviruses (15, 19). The fact that the different lentiviruses, which infect different cell types in different host species, have such a conserved mechanism of genetic regulation implies that this type of posttranscriptional gene regulation may be used to regulate cellular genes as well. Hopefully, continued examination of lentiviral Rev function in various cell types may allow us to begin examination of uninfected cells for the existence of similar gene regulatory circuits.

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ACKNOWLEDGMENTS We thank Lynn Lindstrom for excellent technical assistance, Lillia Holmes for assistance with the statistical analysis, and Drs. Lucy Carruth, Jody Pyper, and Jens Kurth for helpful discussion. This work was supported by grants from the National Institutes of Health (T32 A107394; NS 23039; AI 28748) and by East Tennessee State University RDC Grants 2-25330 and 95-012.

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