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Genetics, Stafford Hall, University of Vermont, Burlington, VT 05405. Phone: (802) 656-8808. ..... L. F. Hall, F. Wong-Staal, and M. S. Reitz, Jr. 1987. Sequence of simian ... Prescott, J., and E. Falck-Pedersen. 1994. Sequence elements ...
JOURNAL OF VIROLOGY, Mar. 1996, p. 1612–1617 0022-538X/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 70, No. 3

A Common Mechanism for the Enhancement of mRNA 39 Processing by U3 Sequences in Two Distantly Related Lentiviruses BRENTON R. GRAVELEY

AND

GREGORY M. GILMARTIN*

Department of Microbiology and Molecular Genetics and the Markey Center for Molecular Genetics, University of Vermont, Burlington, Vermont 05405 Received 19 October 1995/Accepted 5 December 1995

The protein coding regions of all retroviral pre-mRNAs are flanked by a direct repeat of R-U5 sequences. In many retroviruses, the R-U5 repeat contains a complete core poly(A) site composed of a highly conserved AAUAAA hexamer and a GU-rich downstream element. A mechanism that allows for the bypass of the 5* core poly(A) site and the exclusive use of the 3* core poly(A) site must therefore exist. In human immunodeficiency virus type 1 (HIV-1), sequences within the U3 region appear to play a key role in poly(A) site selection. U3 sequences are required for efficient 3* processing at the HIV-1 poly(A) site both in vivo and in vitro. These sequences serve to promote the interaction of cleavage and polyadenylation specificity factor (CPSF) with the core poly(A) site. We have now demonstrated the presence of a functionally analogous 3* processing enhancer within the U3 region of a distantly related lentivirus, equine infectious anemia virus (EIAV). U3 sequences enhanced processing at the EIAV core poly(A) site sevenfold in vitro. The U3 sequences also enhanced the stability of CPSF binding at the core poly(A) site. Optimal processing required the TAR RNA secondary structure that resides within the R region 28 nucleotides upstream of the AAUAAA hexamer. Disruption of TAR reduced processing, while compensatory changes that restored the RNA structure also restored processing to the wild-type level, suggesting a position dependence of the U3-encoded enhancer sequences. Finally, the reciprocal exchange of the EIAV and HIV U3 regions demonstrated the ability of each of these sequences to enhance both 3* processing and the binding of CPSF in the context of the heterologous core poly(A) site. The impact of U3 sequences upon the interaction of CPSF at the core poly(A) site may therefore represent a common strategy for retroviral poly(A) site selection. downstream element 274 nt away (1). The lentivirus subfamily, however, which includes human immunodeficiency virus type 1 (HIV-1), must directly confront the problem of redundant processing sites. The presence of a 39 processing enhancer unique to the 39 end of the viral pre-mRNA appears to be a key component of the mechanism of poly(A) site selection in HIV-1. Efficient processing at the HIV-1 core poly(A) site requires sequences that reside within the U3 region (2, 7, 12, 32). Sequences that serve to enhance 39 processing both in vivo (32) and in vitro (16, 31) influence the interaction of processing factors at the HIV-1 core poly(A) site (16). The binding of cleavage and polyadenylation specificity factor (CPSF), the factor responsible for the initial recognition of the AAUAAA hexamer, was found to be enhanced by HIV-1 U3 sequences (17). CPSF binding is the first step in the assembly of the 39 processing complex. A second factor, CstF, binds in a cooperative manner with CPSF to form a committed processing complex that serves to recruit the processing enzymes to the pre-mRNA (23). Poly(A) polymerase and cleavage factors I and II interact with the CPSF-CstF-RNA ternary complex to form the active processing complex. HIV-1 U3 sequences therefore appear to participate in poly(A) site selection through their ability to promote 39 processing complex assembly. Elements that enhance 39 processing have been detected upstream of the simian virus 40 late (4, 24); adenovirus L3 (26), L1 (10, 11), and L4 (29); hepatitis B virus (28); ground squirrel hepatitis virus (8); and human C2 gene (25) core poly(A) sites. While these upstream elements apparently lack a conserved sequence or structure, they function in an orientation- and position-dependent manner and are generally U rich in composition. In light of the limited sequence conserva-

Pre-mRNA 39 processing is an obligatory step in the biosynthesis of all eukaryotic mRNAs. In mammalian cells, the endonucleolytic cleavage and polyadenylation event that serves to generate the mature 39 end of the message requires a conserved AAUAAA hexamer 10 to 30 nucleotides (nt) upstream of the cleavage site and a highly variable downstream U- or GU-rich element (reviewed in reference 23). Together these two elements compose the core poly(A) site, which appears to be sufficient, at least in some cases, to direct polyadenylation in vivo. Retroviruses face an intriguing dilemma in the 39 processing of their primary transcripts. For many retroviruses, the entire core poly(A) site is encoded within the terminal redundancy of the pre-mRNA, composed of R-U5 sequences. A core poly(A) site must therefore be bypassed when present at the 59 end of the transcript but efficiently utilized when present near the 39 end of the pre-mRNA. Several retroviruses have circumvented this obstacle by placing the AAUAAA hexamer within the U3 region. Therefore, the core poly(A) site is present only once, near the 39 end of the transcript. The cleavage site which forms the 39 boundary of the R region, however, must be within ;30 nt of the hexamer. Rous sarcoma virus and mouse mammary tumor virus accomplish this by reducing the size of the R region (to 21 and 13 nt, respectively) (13, 30). Human T-cell leukemia virus type 1 (HTLV-1) has developed an alternative strategy. HTLV-1 utilizes an RNA stem-loop structure (the Rex response element) to spatially juxtapose the U3-encoded hexamer with the cleavage site and U5-encoded * Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics and the Markey Center for Molecular Genetics, Stafford Hall, University of Vermont, Burlington, VT 05405. Phone: (802) 656-8808. Fax: (802) 656-8749. Electronic mail address: [email protected]. 1612

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FIG. 1. Nucleotide sequence of the E-U3-R-U5 pre-mRNA containing the EIAV 39 poly(A) site. This pre-mRNA contains 58 nt of U3, the entire R region, and 36 nt of U5. The RNA stem-loop structure of TAR encompasses the first 28 nt of the R region (denoted by inverted arrows). The AAUAAA hexamer resides 28 nt downstream of TAR and 16 nt upstream of the cleavage site. The GU-rich downstream element is located within U5 beginning 6 nt downstream of the cleavage site.

tion among poly(A) sites, upstream elements may play a general role in poly(A) site definition. In order to understand the nature of retroviral poly(A) site selection, we have compared the 39 processing strategies developed by two distantly related lentiviruses, HIV-1 and equine infectious anemia virus (EIAV). In vitro analysis revealed an enhancement of both CPSF binding and in vitro processing at the EIAV core poly(A) site by U3 sequences. The interchangeability of the HIV-1 and EIAV 39 processing enhancers suggests the use of a common strategy for poly(A) site selection. MATERIALS AND METHODS RNA substrate preparation. pE-U3-R-U5 was generated by PCR amplification of an EIAV proviral clone (22) (a gift of S. Tronick) with primer 1 (59CCTTGATGCATTTGTGACGCGTTAAGTTCC-39) and primer 2 (59-GACA GAATTCTGTAGGATCTCGAACAG-39). The PCR product was digested with NsiI and EcoRI and cloned into pGEM-9Zf(2). pE-R-U5 was generated by PCR amplification of the EIAV proviral clone (22) with primer 2 and primer 3 (59-ACTCAGATTCTGCGG-39). The PCR product was treated with T4 DNA polymerase, digested with EcoRI, and cloned into pGEM-9Zf(2) at EcoRI and SacI. The clones pE-TAR1 and pE-TAR2 were generated by PCR amplification with primers 1 and 2 from pE-STEM(19/24) and pE-STEM(8/13-19/24) (5) (a gift of D. Derse), respectively, and cloned into pGEM-9Zf(2). To generate the clones in which the U3 sequences were exchanged [pE-U3/H(R-U5) and pHU3/E(R-U5)], the EIAV and HIV-1 long terminal repeats were cloned into pALTER-1 (Promega). Site-directed mutagenesis was performed as described by the manufacturer to generate an SmaI site at the U3-R boundary for each long terminal repeat. The U3 sequences were swapped by joining the U3 and R fragments at the SmaI site. All clones were sequenced by using Sequenase v2.0 (United States Biochemical Corp.). Transcription templates encoding full-length pre-mRNAs were generated by digestion with EcoRI. Precleaved templates containing the R region of EIAV were generated from the corresponding full-length clones by PCR amplification with primer 4 (59-TGAGTAGAGAATTATATTTA-39) and either the T7 or the SP6 primer. Templates encoding precleaved RNAs containing the R region of HIV-1 were generated from the corresponding full-length template by digestion with XhoI. Capped transcripts were synthesized with either SP6 or T7 RNA polymerase (Epicenter Technologies) in the presence of 15 mM GTP, 0.4 mM [a-32P]GTP, and 500 mM m7GpppG (Pharmacia) and gel purified prior to use. Nuclear extract preparation and fractionation. Nuclear extracts were prepared as described by Ru ¨egsegger et al. (27). CPSF was fractionated from HeLa nuclear extracts by DEAE-Sepharose, S-Sepharose, and Blue-Sepharose chromatography as previously described (18). In vitro mRNA 3* processing. Cleavage reaction mixtures contained 20 fmol of 32 P-labeled pre-mRNA, 10% (vol/vol) HeLa cell nuclear extract, 1% polyvinylalcohol, 0.5 mg of tRNA, 0.5 mM 39-deoxy ATP (39-dATP) 6.4% glycerol, 30 mM Tris-Cl (pH 7.8), 40 mM MgCl2, 0.1 mM EDTA, 20 mM (NH4)2SO4, 12.5 mM dithiothreitol, 12.5 mM phenylmethylsulfonyl fluoride, 0.02 mg of pepstatin per ml, and 0.01 mg of leupeptin per ml in 25 ml. Reaction mixtures were assembled on ice and incubated at 308C for the times indicated in the figure legends. Reactions were stopped by the addition of ETS (10 mM EDTA, 1 mM Tris-Cl [pH 7.8], 0.5% sodium dodecyl sulfate) and proteinase K, and mixtures were incubated at 378C for 10 min. The reaction mixtures were sequentially extracted with phenol and phenol-chloroform, ethanol precipitated, and analyzed on 10% (19:1) polyacrylamide–7 M urea gels. Processing complex analysis. CPSF-RNA complex stability was determined as follows. CPSF-RNA complexes were formed on 32P-labeled precleaved RNAs in reaction mixtures containing 1% polyvinylalcohol, 1 mg of tRNA, 8 ml of BlueSepharose CPSF fraction, 80 mM KCl, 16 mM HEPES (N-2-hydroxyethylpipera-

zine-N9-2-ethanesulfonic acid; pH 7.9), 8% glycerol, 0.16 mM EDTA, 0.08 mM phenylmethylsulfonyl fluoride, and 0.16 mM dithiothreitol. After 10 min of incubation at 308C, a 5-ml aliquot was removed, treated with heparin to 5 mg/ml at 08C, and designated the time 0 sample. A 100-fold molar excess of unlabeled HIV-1 use/CPS pre-mRNA [containing the wild-type HIV-1 poly(A) site (17)] was added, and the sample was returned to 308C. Aliquots (5.25 ml) were removed at the times indicated below and treated with heparin to 5 mg/ml at 08C. The reaction mixtures were electrophoresed on nondenaturing 3% (80:1) polyacrylamide gels at 48C for 1.5 h at 300 V.

RESULTS The role of U3 sequences in processing at the EIAV core poly(A) site. Lentiviruses contain a complete core poly(A) site within the R-U5 repeat flanking the viral pre-mRNA. The 59 core poly(A) site must therefore be bypassed in favor of the exclusive utilization of the 39 core poly(A) site. It has previously been shown that poly(A) site selection in HIV-1 involves the enhancement of processing by sequences unique to the 39 end of the pre-mRNA. HIV-1 U3 sequences were found to enhance 39 processing both in vivo (32) and in vitro (16, 31). In order to gain further insight into the role of U3 sequences in retroviral poly(A) site selection, we have investigated the function of these sequences in EIAV, a lentivirus distantly related to HIV-1. The impact of U3 sequences on processing at the EIAV core poly(A) site was examined in vitro. The EIAV long terminal repeat sequences encompassing the poly(A) site are illustrated in Fig. 1 (22). The pre-mRNA corresponding to the EIAV 39 poly(A) site used in these studies (E-U3-R-U5) contained 58 nt of U3, the entire R region, and 36 nt of U5 flanked by NsiI and EcoRI sites to facilitate cloning. A second RNA (E-R-U5), which encompassed the 59 core poly(A) site, was also generated. The E-R-U5 pre-mRNA contains only R-U5 sequences and is precisely colinear with the 59 end of the viral mRNA (22). The first 28 nt of R form the RNA structure termed TAR, which constitutes the binding site for the virus-encoded transactivator protein Tat (5). The AAUAAA hexamer of the core poly(A) site resides 28 nt downstream of the TAR element, and the cleavage site is situated 16 nt downstream of the hexamer. U5 contains the second component of the core poly(A) site, the GU-rich downstream element, 6 nt downstream of the cleavage site. Capped pre-mRNAs were synthesized with [a-32P]GTP and gel purified. Equal numbers of moles of each pre-mRNA were processed in a HeLa cell nuclear extract in the presence of 39 dATP. The cleavage products generated upon processing of both the E-U3-R-U5 and E-R-U5 pre-mRNAs are shown in Fig. 2A. (Note that the cleavage products of the E-R-U5 premRNA were detectable only after an extended exposure of the film.) Generation of the observed cleavage products was dependent upon the AAUAAA hexamer, and the identities of the 59 and 39 cleavage products were confirmed by using 39-

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FIG. 2. U3 sequences enhance 39 processing of the EIAV core poly(A) site in vitro. (A) Cleavage products of pre-mRNAs containing the EIAV core poly(A) site. Twenty femtomoles of each uniformly 32P-labeled EIAV premRNA corresponding to either the 59 (E-R-U5) or the 39 (E-U3-R-U5) poly(A) site was incubated in HeLa cell nuclear extract in the presence of 39-dATP for 10 min at 308C. The reaction products were resolved on a denaturing 10% polyacrylamide gel. Detection of the 39 products required a longer exposure of the gel than did that of the 59 products. (B) Kinetics of EIAV poly(A) site 39 processing. Cleavage reactions were performed in HeLa cell nuclear extract for the times indicated, and the products were resolved on a denaturing 10% polyacrylamide gel. The 39 products were quantitated with a Bio-Rad phosphorimager. The y axis is in arbitrary phosphor density units (PD Units).

end-labeled pre-mRNAs (data not shown). The kinetics of processing are illustrated in Fig. 2B, for which the 39 products were quantitated on a Bio-Rad phosphorimager. The 39 products of the two RNAs were identical in sequence and specific activity, thereby allowing their direct comparison. At 15 min, there was a sevenfold enhancement in the processing efficiency of the E-U3-R-U5 pre-mRNA over that of the E-R-U5 premRNA. The EIAV U3 region therefore contains sequences that enhance 39 processing at the EIAV core poly(A) site in vitro. The HIV-1 39 processing enhancer has previously been shown to function in a position-dependent manner (16). The RNA secondary structure of HIV-1 TAR is required to spatially juxtapose the upstream element and the core poly(A) site. To examine the role of TAR in EIAV poly(A) site function, we have used a TAR disruption mutation previously characterized in vivo along with a compensatory mutation that restored both the RNA secondary structure and the in vivo function of TAR (Fig. 3A) (5). Disruption of the TAR stemloop structure would be expected to effectively increase the distance between the EIAV core poly(A) site and U3 sequences by ;28 nt. As illustrated in Fig. 3B, disruption of the RNA structure of the EIAV TAR element reduced processing by twofold with respect to the wild-type pre-mRNA (compare lanes 1 and 2). Restoration of the RNA structure of TAR by the introduction of a compensatory mutation restored processing to the wildtype level (Fig. 3B, lane 3). These results suggest that as with HIV-1, the EIAV U3 sequences serve to enhance 39 processing in a position-dependent manner. U3 sequences enhance the stability of CPSF binding. The enhancement of mRNA 39 processing by the EIAV U3 region suggests that these sequences may function in a manner similar to that of the HIV-1 U3 region. In HIV-1, U3 sequences

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interact directly with CPSF, the factor responsible for recognition of the AAUAAA hexamer, to stabilize its binding at the core poly(A) site (17). We therefore investigated the impact of EIAV U3 sequences on CPSF binding. In order to examine the effect of U3 sequences on the binding of CPSF at the AAUAAA hexamer, we have used precleaved RNAs [RNAs that extend only to the site of poly(A) addition]. 32P-labeled RNAs that contained U3 sequences (E-U3-R) or lacked U3 sequences (E-R) were incubated with extensively purified CPSF for 10 min at 308C. An aliquot was removed (designated the time 0 sample), treated with heparin, and placed on ice. A 100-fold molar excess of an unlabeled pre-mRNA containing the wild-type HIV-1 poly(A) site (use/CPS) (16) was added to the remainder of the reaction mixture to sequester any CPSF that dissociated from the 32Plabeled RNA. Aliquots were removed at 1, 2, 4, 6, 8, and 10 min following the addition of the competitor, treated with heparin, and placed on ice. The CPSF-RNA complexes were then resolved by electrophoresis on a native 3% (80:1) polyacrylamide gel at 48C. As illustrated in Fig. 4, the presence of U3 sequences stabilized the CPSF-RNA complex. Phosphorimager quantitation of the data shown in Fig. 4 revealed that the half-life of the CPSF-RNA complex formed on E-U3-R RNA was 4 min whereas that of the complex formed on E-R RNA was less than 30 s. These differences in CPSF binding were reflected in the efficiency of both cleavage (Fig. 2) and poly(A) addition (data not shown). Functional equivalence of HIV-1 and EIAV U3 sequences.

FIG. 3. Formation of the TAR element is required for efficient processing at the EIAV 39 core poly(A) site in vitro. (A) Schematic depiction of the premRNAs containing mutations within the TAR element. These pre-mRNAs were derived from in vivo-characterized viral mutants (5) containing mutations that disrupted [pE-STEM(19/24)] and restored [pE-STEM(8/13-19/24)] the base pairing potential of the TAR element. E-U3-R-U5 contains the wild-type EIAV sequence. E-TAR1 contains a 6-nt substitution that disrupts the TAR RNA stem-loop. E-TAR2 contains six additional nucleotide substitutions that restore the base pairing potential of the TAR element. dse, downstream element. (B) In vitro processing of pre-mRNAs containing mutations within the TAR element. Twenty femtomoles of each uniformly 32P-labeled pre-mRNA was incubated in HeLa cell nuclear extract in the presence of 39-dATP for 10 min at 308C. The reaction products were resolved on a denaturing 10% polyacrylamide gel. Detection of the 39 products required a longer exposure of the gel than did that of the 59 products.

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stability of CPSF binding at the HIV-1 core poly(A) site (Fig. 6, lanes 11 to 25). In the absence of U3 sequences, the half-life of the CPSF-RNA complex at the HIV-1 core poly(A) site was ;45 s. Addition of EIAV or HIV-1 U3 sequences increased the half-life of the CPSF-RNA complexes to $6 min. The HIV-1 and EIAV 39 processing enhancers are therefore functionally equivalent and interchangeable in vitro. The function of the HIV-1 and EIAV 39 processing enhancers is not restricted to their cognate poly(A) sites; rather, they can function to enhance processing at a heterologous poly(A) site. DISCUSSION

FIG. 4. U3 sequences stabilize the binding of CPSF at the EIAV core poly(A) site. Uniformly 32P-labeled E-U3-R or E-R precleaved RNAs were incubated with extensively purified CPSF (see Materials and Methods) for 10 min at 308C. An aliquot was removed at time 0, treated with heparin, and placed on ice. To the remainder of the reaction mixture a 100-fold molar excess of unlabeled HIV-1 use/CPS pre-mRNA [containing the wild-type HIV-1 poly(A) site (16)] was added, and the incubation was continued at 308C. At 1, 2, 4, 6, 8, and 10 min, aliquots were removed, treated with heparin, and placed on ice. The protein-RNA complexes were resolved on a nondenaturing 3% (80:1) polyacrylamide gel at 48C. The CPSF-RNA complexes were quantitated with a Bio-Rad phosphorimager.

The similarity of EIAV and HIV-1 U3 sequences in the enhancement of 39 processing suggests that these U3 sequences may serve similar roles in poly(A) site selection. To address the functional equivalence of the EIAV and HIV-1 U3 sequences, we exchanged these elements and assayed their abilities to enhance 39 processing of the heterologous core poly(A) site. To facilitate the construction of the chimeric pre-mRNAs, an SmaI site was introduced at the U3-R boundary of each long terminal repeat by site-directed mutagenesis. This allowed the precise reciprocal exchange of the HIV-1 and EIAV U3 regions. The chimeric pre-mRNAs were tested for the ability of the upstream sequences to enhance 39 processing of the heterologous core poly(A) sites. Cleavage assays were performed in a HeLa cell nuclear extract in the presence of 39dATP. The introduction of the SmaI site alone had little impact on the enhancement of processing by either the EIAV or the HIV-1 U3 regions in the context of their cognate core poly(A) site (Fig. 5; compare lanes 1 and 2 and lanes 4 and 5). Furthermore, in each case, the HIV-1 and EIAV U3 sequences enhanced the 39 processing of the heterologous poly(A) site (Fig. 5, lanes 3 and 6). The HIV-1 U3 sequences enhanced processing of the EIAV core poly(A) site to 96% of the level of EIAV U3 sequences. Addition of EIAV U3 sequences enhanced processing of the HIV-1 core poly(A) site to 76% of the level of the HIV-1 U3 sequences. We then examined the ability of each U3 region to promote the stable binding of CPSF in the context of the heterologous core poly(A) site. Figure 6 shows the results of an analysis of CPSF-RNA complex stability on the chimeric RNAs. A comparison of complexes formed on precleaved RNAs containing the EIAV R region and either the cognate (E-U3-R) or the heterologous (H-U3/E-R) U3 region shows that both stabilized the CPSF-RNA complex (Fig. 6, lanes 1 to 10). The HIV-1 U3 sequences stabilized CPSF to a greater extent than the EIAV U3 sequences did (half-lives of 6 and 4 min, respectively). Both the EIAV and HIV-1 U3 sequences also served to enhance the

Many retroviruses face what appears to be an enormous obstacle in the synthesis of a mature viral mRNA. The terminally redundant R-U5 sequences that flank the pre-mRNA house the elements of the core poly(A) site. As a consequence of such a genomic architecture, a core poly(A) site must be bypassed at the 59 end of the pre-mRNA and an identical site must be efficiently utilized at the 39 end. The results described in this report suggest that the U3 region of EIAV may participate in poly(A) site selection through the enhancement of processing at the 39 core poly(A) site. The U3 sequences of EIAV enhanced 39 processing in vitro and stabilized the binding of CPSF at the core poly(A) site. Optimal activity of this 39 processing enhancer was dependent upon its juxtaposition with the core poly(A) site by the RNA secondary structure of TAR. Finally, the EIAV and HIV-1 39 processing enhancers, although having little sequence similarity, were shown to be

FIG. 5. Impact of the reciprocal exchange of EIAV and HIV-1 U3 sequences on 39 processing. Twenty femtomoles of each uniformly 32P-labeled pre-mRNA was incubated in HeLa cell nuclear extract in the presence of 39-dATP for 10 min at 308C. (A) Cleavage products of pre-mRNAs containing the EIAV core poly(A) site. E-R-U5, EIAV 59 core poly(A) site; E-U3*-R-U5, EIAV 39 poly(A) site. The pre-mRNA H-U3*/E-(R-U5) contained a substitution of HIV-1 U3 sequences for those of EIAV. Asterisks denote the presence of an SmaI site at the U3-R boundary that was introduced to enable the exchange of U3 sequences. (B) Cleavage products of pre-mRNAs containing the HIV-1 core poly(A) site. H-R-U5, HIV-1 59 core poly(A) site; H-U3*-R-U5, HIV-1 39 poly(A) site. The pre-mRNA E-U3*/H-(R-U5) contained a substitution of EIAV U3 sequences for those of HIV-1. For both panel A and panel B the detection of the 39 products required a longer exposure of the gel than did that of the 59 products.

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FIG. 6. Impact of the reciprocal exchange of EIAV and HIV-1 U3 sequences on CPSF binding. Uniformly 32P-labeled pre-mRNAs were incubated with extensively purified CPSF (see Materials and Methods) for 10 min at 308C. An aliquot was removed at time 0, treated with heparin, and placed on ice. To the remainder of the reaction mixture a 100-fold molar excess of unlabeled HIV-1 use/CPS pre-mRNA [containing the wild-type HIV-1 poly(A) site (16)] was added, and the incubation was continued at 308C. At 1, 2, 4, and 6 min, aliquots were removed, treated with heparin, and placed on ice. The protein-RNA complexes were resolved on nondenaturing 3% (80:1) polyacrylamide gels at 48C. (A) CPSF-RNA complexes formed on precleaved RNAs containing the EIAV R region. The E-U3*-R RNA contained the EIAV U3 region, while the H-U3*/ E-R RNA contained the HIV-1 U3 region. Asterisks denote the presence of an SmaI site at the U3-R boundary that was introduced to enable the exchange of U3 sequences. (B) CPSF-RNA complexes formed on precleaved RNAs containing the HIV-1 R region. The H-R RNA contained only the HIV-1 R region. The H-U3*-R RNA contained the HIV-1 U3 region, while the E-U3*/H-R RNA contained the EIAV U3 region.

functionally equivalent. The reciprocal exchange of the EIAV and HIV-1 U3 regions demonstrated that these sequences were capable of enhancing both 39 processing and CPSF binding in the context of the heterologous core poly(A) site. We propose that the utilization of a processing enhancer unique to the 39 end of the viral pre-mRNA represents a common strategy for poly(A) site selection in lentiviruses. In the absence of an enhancer, both the EIAV and HIV-1 core poly(A) sites are very inefficient (Fig. 5). The inefficiency of the HIV-1 core poly(A) site has been shown to be a consequence of the suboptimal sequence context within which the AAU AAA hexamer resides (17). In the absence of U3 sequences, CPSF is unable to stably interact with the AAUAAA hexamer (17). Recognition of the AAUAAA hexamer by CPSF is the first event in the assembly of a 39 processing complex and appears to be a key determinant of poly(A) site efficiency. The U3 sequences of both EIAV and HIV-1 serve to stabilize the binding of CPSF at the core poly(A) site and enhance the efficiency of processing (17). The impact of the sequence context of the EIAV hexamer on processing efficiency is currently under investigation. The inefficiency of the core poly(A) site may be only one component of the mechanism that allows the occlusion of this

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site at the 59 end of the pre-mRNA. Utilization of the ground squirrel hepatitis virus, spleen necrosis virus, and HIV-1 core poly(A) sites has been shown to be sensitive to the proximity of the pre-mRNA 59 end (7, 8, 21, 33). Although the mechanism by which promoter proximity influences 39 processing remains entirely unclear, the inefficiency of the lentiviral core poly(A) sites may render them sensitive to such effects and thus ensure that the 59 core poly(A) site is not utilized. mRNA 39 processing has been shown to be influenced by the presence of RNA secondary structures. An RNA structure is required for the function of the HTLV-1 poly(A) site, in which the AAUAAA hexamer and downstream element are separated by 274 nt. Poly(A) site function requires that the two elements of the core poly(A) site be spatially juxtaposed by the formation of the Rex response element RNA stem-loop structure (1). The TAR RNA stem-loop structures of HIV-1 and EIAV appear to participate in mRNA 39 processing by juxtaposing the U3 sequences and the core poly(A) site (16; also this report). In the lentiviruses HIV-2, simian immunodeficiency virus, and bovine immunodeficiency virus, the AAU AAA hexamer resides 150, 153, and 86 nt downstream of the U3-R boundary, respectively (3, 6, 15, 20). Extensive RNA structures that may be capable of juxtaposing the U3 sequences and the core poly(A) site are predicted to form in these intervening regions (3, 14). The participation of U3 sequences in the 39 processing of these viral mRNAs is currently under investigation. The problem of poly(A) site selection is not restricted to the retroviruses, but it must be faced by the processing machinery on a regular basis. Sixteen percent of all mRNAs contain the sequence AAUAAA within their protein coding region (9), and a far greater percentage contain this sequence within an intron. Since the location of AAUAAA is not restricted to poly(A) sites, a highly variable U- or GU-rich downstream element is unlikely to be sufficient to define an authentic poly(A) site. Additional information is almost certainly required. The use of mRNA 39 processing enhancers may represent a general strategy for the definition of an authentic poly(A) site. This strategy for poly(A) site definition appears to have been exploited by retroviruses as a solution to the problem of redundant core poly(A) sites. The recent identification of a nonviral mRNA 39 processing enhancer within the human C2 gene supports the ubiquitous nature of these elements (25). It will be interesting to determine the mechanism by which the upstream sequences of the C2 poly(A) site operate. The identification of functionally equivalent mRNA 39 processing enhancers within the HIV-1 and EIAV U3 regions provides a means for dissecting the mechanism by which these elements act to stabilize the CPSF-RNA complex. Previous work has demonstrated a direct interaction between CPSF and the HIV-1 mRNA 39 processing enhancer (17). An examination of the sequences encompassing the HIV-1 mRNA 39 processing enhancer (nt 9571 to 9588 [32]) and the EIAV U3 region (nt 8054 to 8114 [22]) revealed high uridine contents in both regions (47 and 42%, respectively). Mutagenesis of a U5 segment within the EIAV U3 region, however, had no effect on the efficiency of 39 processing or CPSF binding (19a). More recent work suggests that mRNA 39 processing enhancers play a structural role in promoting the accessibility of the AAU AAA hexamer to CPSF (19). Having demonstrated the functional equivalence of two very dissimilar retroviral sequences, we are now in a position to address the relative contributions of sequence contacts and higher-order RNA structure to poly(A) site selection.

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LENTIVIRUS mRNA 39 PROCESSING ACKNOWLEDGMENTS

We thank Steven Tronick and David Derse for providing plasmids; Ursula Ru ¨egsegger and Walter Keller for communicating the nuclear extract protocol prior to publication; and Elizabeth Fleming and Sean Flaherty for preparation of the nuclear extracts, stimulating discussions, and critical reading of the manuscript. This work was supported by grants from the Lucille P. Markey Charitable Trust [NSF-VT EPSCoR (RII-8610679)] and from the National Institutes of Health (GM46624) to G.M.G. REFERENCES 1. Ahmed, Y. F., G. M. Gilmartin, S. M. Hanly, J. R. Nevins, and W. C. Greene. 1991. The HTLV-1 Rex response element mediates a novel form of mRNA polyadenylation. Cell 64:727–737. 2. Brown, P. H., L. S. Tiley, and B. R. Cullen. 1991. Efficient polyadenylation within the human immunodeficiency virus type 1 long terminal repeat requires flanking U3-specific sequences. J. Virol. 65:3340–3343. 3. Carpenter, S., S. A. Nadin-Davis, Y. Wannemuehler, and J. A. Roth. 1993. Identification of transactivation-response sequences in the long terminal repeat of bovine immunodeficiency-like virus. J. Virol. 67:4399–4403. 4. Carswell, S., and J. C. Alwine. 1989. Efficiency of utilization of the simian virus 40 late polyadenylation site: effects of upstream sequences. Mol. Cell. Biol. 9:4248–4258. 5. Carvalho, M., and D. Derse. 1991. Mutational analysis of the equine infectious anemia virus Tat-responsive element. J. Virol. 65:3468–3474. 6. Chakrabarti, L., M. Guyader, M. Alizon, M. D. Daniel, R. C. Desrosiers, P. Tiollais, and P. Sonigo. 1987. Sequence of simian immunodeficiency virus from macaque and its relationship to other human and simian retroviruses. Nature (London) 328:543–547. 7. Cherrington, J., and D. Ganem. 1992. Regulation of polyadenylation in human immunodeficiency virus (HIV): contributions of promoter proximity and upstream sequences. EMBO J. 11:1513–1524. 8. Cherrington, J., R. Russnak, and D. Ganem. 1992. Upstream sequences and cap proximity in the regulation of polyadenylation in ground squirrel hepatitis virus. J. Virol. 66:7589–7596. 9. Day, I. N. M. 1992. Analysis of the 59-AAUAAA motif and its flanking sequence in human RNA: relevance to cDNA library sorting. Gene 110:245– 249. 10. DeZazzo, J. D., E. Falck-Pedersen, and M. J. Imperiale. 1991. Sequences regulating temporal poly(A) site switching in the adenovirus major late transcription unit. Mol. Cell. Biol. 11:5977–5984. 11. DeZazzo, J. D., and M. J. Imperiale. 1989. Sequences upstream of AAU AAA influence poly(A) site selection in a complex transcription unit. Mol. Cell. Biol. 9:4951–4961. 12. DeZazzo, J. D., J. E. Kilpatrick, and M. J. Imperiale. 1991. Involvement of long terminal repeat U3 sequences overlapping the transcription control region in human immunodeficiency virus type 1 mRNA 39 end formation. Mol. Cell. Biol. 11:1624–1630. 13. Donehower, L. A., A. L. Huang, and G. L. Hager. 1980. Regulatory and coding potential of the mouse mammary tumor virus long terminal redundancy. J. Virol. 37:226–238. 14. Feng, S., and E. C. Holland. 1988. HIV-1 tat trans-activation requires the loop sequence within tar. Nature (London) 334:165–167. 15. Franchini, G., C. Gurgo, H.-G. Guo, R. C. Gallo, E. Collalti, K. A. Fargnoli,

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