Human Immunodeficiency Virus Type 1 Tat ... - Journal of Virology

JOURNAL OF VIROLOGY, July 2004, p. 6846–6854 0022-538X/04/$08.00⫹0 DOI: 10.1128/JVI.78.13.6846–6854.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Vol. 78, No. 13

Human Immunodeficiency Virus Type 1 Tat Increases the Expression of Cleavage and Polyadenylation Specificity Factor 73-Kilodalton Subunit Modulating Cellular and Viral Expression Marco A. Calzado, Rocío Sancho, and Eduardo Mun ˜oz* Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Medicina, Universidad de Co ´rdoba, 14004 Co ´rdoba, Spain Received 12 August 2003/Accepted 20 February 2004

The human immunodeficiency virus type 1 (HIV-1) Tat protein, which is essential for HIV gene expression and viral replication, is known to mediate pleiotropic effects on various cell functions. For instance, Tat protein is able to regulate the rate of transcription of host cellular genes and to interact with the signaling machinery, leading to cellular dysfunction. To study the effect that HIV-1 Tat exerts on the host cell, we identified several genes that were up- or down-regulated in tat-expressing cell lines by using the differential display method. HIV-1 Tat specifically increases the expression of the cleavage and polyadenylation specificity factor (CPSF) 73-kDa subunit (CPSF3) without affecting the expression of the 160- and 100-kDa subunits of the CPSF complex. This complex comprises four subunits and has a key function in the 3ⴕ-end processing of pre-mRNAs by a coordinated interaction with other factors. CPSF3 overexpression experiments and knockdown of the endogenous CPSF3 by mRNA interference have shown that this subunit of the complex is an important regulatory protein for both viral and cellular gene expression. In addition to the known CPSF3 function in RNA polyadenylation, we also present evidence that this protein exerts transcriptional activities by repressing the mdm2 gene promoter. Thus, HIV-1-Tat up-regulation of CPSF3 could represent a novel mechanism by which this virus increases mRNA processing, causing an increase in both cell and viral gene expression. phosphorylation of RNA polymerase II (RNAPII) CTD repeats (19, 64). In addition to its regulatory activity over the HIV-1 LTR, other pleiotropic effects exerted by HIV-1 Tat on the host cell have also been observed (10). The Tat protein has been shown to regulate the rate of transcription of host cellular genes and to interact with the signaling machinery, leading to cellular dysfunction and immunosuppression associated with viral infection (34, 56). Tat can affect the expression of cellular genes, including those for cytokines (4, 48), cycle-related proteins (41, 42, 52), surface receptors such as EDF-1 and TCR/CD3 (5, 62), or chemokine receptors (CCR5,CXCR4) (28, 55). Moreover, recent studies using microarray techniques have demonstrated that this protein down-regulates principally genes involved in differentiation signal control and significantly increases expression of genes necessary for proliferation (host cell transcription and translation machinery), probably to establish an adequate environment to enhance viral replication (13, 22). Interestingly, Izmailova et al. have demonstrated that both HIV-1 infection and adenovirus-mediated Tat overexpression induce similar up-regulation of gene expression and secretion of chemokines in human dendritic cells (31). Virtually all eukaryotic mRNA precursors (pre-mRNAs) must be modified at the 3⬘ end to serve as efficient templates before they are exported to the cytoplasm. The posttranscriptional acquisition of poly(A) tails on the 3⬘ ends of eukaryotic mRNAs is an essential process which promotes transcription termination (7) and transport of the mRNA from the nucleus (29). The poly(A) tail is also important for optimal translation and for regulating mRNA stability (18, 49, 53, 61). PremRNAs are maturated in a two-step reaction: site-specific endonucleolytic cleavage followed by poly(A) addition to the

Human immunodeficiency virus type 1 (HIV-1) is a member of the lentivirus family of retroviruses. In addition to common structural viral proteins present in retroviruses, the HIV-1 genome encodes six unique regulatory or accessory proteins that play a critical role in viral gene expression, transmission, and pathogenesis (15, 17). Among them, the HIV-1 Tat protein has been shown to be essential for the efficient transcription of HIV-1 provirus and viral replication (33, 36). This protein augments levels of viral RNA transcripts by increasing transcriptional initiation and elongation (35, 37, 40, 60). This function is controlled by interplay of viral and host regulatory proteins that interact with cis-acting sequences located in the HIV-1 long terminal repeat (LTR). Cellular transcription factors such as the members of the NF-␬B/Rel family, NF-AT, and Sp1 bind to the HIV-1-LTR core promoter and have been demonstrated to play a role in mediating the transactivating properties of Tat through direct or indirect interaction with this protein (20, 21). However, Tat requires a cis-activating stem-loop RNA structure called the transactivating response element, which is located immediately 3⬘ from the LTR transcription start site (6, 15, 59). Through interaction with the transactivating response element, Tat recruits a cyclin T-associated kinase, CDK9, into the preinitiation transcription complex (3, 19, 60). The recruitment of p-TEFb (cyclin T/cdk9) to the HIV-1 promoter complex is necessary and sufficient to promote transcriptional elongation and the target-specific * Corresponding author. Mailing address: Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Medicina, Universidad de Co ´rdoba, Avda. Menendez Pidal S/N, 14004 Co ´rdoba, Spain. Phone: 34 957218267. Fax: 34 957218229. E-mail: fi1muble @uco.es. 6846

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Tat PROTEIN INCREASES CPSF 73-kDa SUBUNIT EXPRESSION

new 3⬘ end (39, 58, 63). Nuclear poly(A) addition requires cleavage and polyadenylation specificity factor (CPSF), poly(A) polymerase (PAP), cleavage stimulation factor (CstF), and the cis-acting sequence AAUAAA. CPSF is a complex of four polypeptide subunits (160, 100, 73, and 30 kDa) that binds specifically to the AAUAAA signal (32, 38, 47) and is required for both cleavage and poly(A) addition activity. The contact with the mRNA-specific signal is made through the CPSF 160-kDa subunit (CPSF1). Although the roles of the other subunits remain elusive, a possible regulating activity of the 100-kDa (CPSF2) and 73-kDa (CPSF3) CPSF subunits on the complex has been described. Interestingly, a role for the CPSF complex in HIV-1 gene regulation has been suggested, since functional binding sites for this factor are present in the HIV-1 LTR promoter (14, 54). To further study the effect that Tat exerts on host cell, we used the mRNA differential display method and identified several genes that were up- or down-regulated in cells overexpressing Tat ectopically. Among them, we have found that the CPSF3 gene is clearly up-regulated in Tat-expressing cell lines, and we report here on the possible function of this factor in regard to cellular and HIV-1 gene regulation. MATERIALS AND METHODS Cell lines. The cervical carcinoma HeLa cell line and embryonic kidney transformed 293T cells (American Type Culture Collection, Manassas, Va.) were maintained at 37°C with 5% CO2 in exponential growth in Dulbecco’s modified Eagle’s medium (Gibco, Invitrogen Corp., Carlsbad, Calif.) supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, and the antibiotics penicillin and streptomycin (Gibco). The erythroleukemic K562 cell line and the Jhan clone (Jurkat derived) were maintained in complete RPMI 1640 medium (Gibco). The stably transfected cell lines K562-pcDNA3, K562-Tat, and HeLaTat were generated as previously described and maintained in medium containing G418 (200 ␮g/ml) (43). Several clones showing high levels of Tat-dependent HIV LTR activity were pooled to generate the K562-Tat and HeLa-Tat cell lines in order to avoid false results due to clonality. The HeLa-On-Tat cell line was constructed by stable cotransfection of HeLa cells (American Type Culture Collection) according to the Tet-On system kit instructions (BD Clontech, Basingstoke, United Kingdom). Briefly, this cell line contains three plasmids: pTet-On codifies constitutively for rtTA protein, which, in response to doxycycline, becomes active and binds to pBI-EGFP-Tat, which simultaneously expresses the enhanced green fluorescent protein (EGFP) and Tat genes. This vector was cotransfected with pSV-hygro to allow selection of a double-stable Tet-responsive cell line. The HeLa-On-Tat cell line was maintained in Dulbecco’s modified Eagle’s medium (Gibco) in the presence of 100 ␮g of hygromycin (Invitrogen Life Sciences, Invitrogen Corp.) per ml and 100 ␮g of G418 per ml. The Jhan-Tat cell line was obtained from M. Fresno (CBM-UAM, Madrid, Spain). 293T-siCPSF3 cells were generated by transfecting 293T cells with the pSI-CPSF3 and pSV-hygro plasmids by using the Lipofectamine Plus reagent (Invitrogen Life Sciences). Forty-eight hours after transfection, the cells were cloned by limiting dilution in medium containing the antibiotic hygromycin (400 ␮g/ml). Plasmid constructs. We used the pSilencer 1.0-U6 vector (Ambion, Inc., Austin, Tex.) to express small interfering RNA (siRNA) in mammalian cells. The oligonucleotides for the bodies of the siRNAs used were pSI-CPSF1 (5⬘-GCTT CAAGGATGCCAAGCT-3⬘) (GenBank accession no. NM_013291), pSICPSF2 (5⬘-GAATCTGCCCTTTGCTATC-3⬘) (XM_029311), and pSI-CPSF3 (5⬘-GAAGTAGGAAGATCATGTA-3⬘) (NM_016207), and they were confirmed by BLAST research (http://www.ncbi.nlm.nih.gov) to ensure that they did not have significant sequence homology with other genes. pSI-Control plasmids were constructed for each of the silencing plasmids described above, which had the same sequence with three differing nucleotides. The full-length human CPSF3 cDNA sequence was obtained by reverse transcription-PCR (RT-PCR) from HeLa cells and cloned into pcDNA3. The resulting construct was sequenced to ensure that no punctual mutations were introduced during the PCR amplification. The expression plasmid pBI-EGFP-Tat was generated by cloning the Tat gene between the MluI and NheI sites of the pBI-EGFP vector (BD Clontech).

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The vector pNL4-3.Luc.R-E (National Institutes of Health AIDS Research and Reference Reagent program; catalogue number 3418) contains the firefly luciferase gene inserted into the pNL4-3 nef gene. This proviral construct expresses luciferase activity as a marker of viral gene expression. The plasmid HIV-LTRLuc, harboring the HIV LTR promoter (LAV1 Bru strain) followed by the luciferase gene, has been described previously (30). The plasmid HTLV-Luc, containing the human T-cell leukemia virus (HTLV) type 1 LTR promoter driving the luciferase reporter gene, was previously described (24). The expression vector pSV-Hygro, harboring the bacterial hygromycin phosphotransferase gene under the control of the simian virus 40 promoter, has been described elsewhere (11). The plasmids pGL3-Waf1-Luc, containing 2.4 kb of the human waf1/p21 promoter and upstream region; pGL3-Bax-Luc, containing a SmaI-SacI fragment of the human Bax promoter (position ⫺687 to ⫺318); and pGL3mdm2-Luc, containing the P2 intronic mouse mdm2 promoter, were previously described (1, 16, 45). Differential display-PCR. Total RNA was prepared from K562-pcDNA3 and K562-Tat cells by the lithium chloride-urea method. Isolated total RNA was treated with DNase I (Invitrogen Life Sciences) and analyzed on a 1% agarose gel for quality and quantity before use. Differential display was performed according to the recommendations of the manufacturer (Beckman Coulter, Fullerton, Calif.), using Hieroglyph mRNA profile kit (Genomix Corporation, Foster, Calif.). Amplicons were resolved by electrophoresis on 4.5% denaturing polyacrylamide gels for 3 h 30 min at 50°C and 1,500 V. Modifications to the manufacturer’s procedure were as follows: instead of labeling the arbitrary primers, the anchored primers used in the PCR amplification were end labeled with [␥-33P]dATP (NEN, Zaventen, Belgium) by using T4 polynucleotide kinase (Invitrogen Life Sciences). Cloning, sequencing, and identification of cDNAs. Bands showing a change of expression were reamplified, purified by using the Concert kit (Invitrogen Life Sciences), cloned into the pGEM-Teasy vector (Promega, Madison, Wis.), and sequenced. The cDNA fragments were compared with the sequence data banks by using the BLAST algorithms and the resources at the National Center for Biotechnology Information. RT-PCR amplification. Retrotranscription of mRNA into cDNA was performed in a 20-␮l reaction mixture according to the SuperScript II RNase H⫺ reverse transcriptase (Invitrogen Life Sciences) protocol, using 0.5 ␮g of oligo(dT)12-18 primer (Invitrogen Life Sciences) for 5 ␮g of mRNA. RT-PCR amplification was performed in a 50-␮l PCR mixture containing 0.5 to 2 ␮l of the retrotranscription mixture, 1⫻ PCR buffer, 1.5 mM MgCl2, 200 ␮M deoxynucleoside triphosphates, a 10 ␮M concentration of each of 5⬘ and 3⬘ primer, and 2.5 U of recombinant Taq DNA polymerase (Invitrogen Life Sciences). The mixtures were amplified in a MultiGene cycler IR system (Labnet Co., Woodbridge, N.J.). The primers used were as follows: ␤-actin antisense primer, 5⬘-G CAACTAAGTCATAGTCCGC-3⬘; ␤-actin sense primer, 5⬘-CTGTCTGGCGG CACCACCAT-3⬘; CPSF1 sense primer, 5⬘-CTCTTTCGCTCCATTCCAC-3⬘; CPSF1 antisense primer, 5⬘-TCCTTCTCCTCGCCAGTCA-3⬘; CPSF2 sense primer, 5⬘-GATCAGATTTGGAGGACTAA-3⬘; CPSF2 antisense primer, 5⬘-A GAATGATTGAGTTTTTAGG-3⬘; CPSF3 sense primer, 5⬘-AATGGCTGGCA AACCCTTCTAATG-3⬘; and CPSF3 antisense primer, 5⬘-CATCGTCTTCACT TCCCTCTTCACA-3⬘. The amplification profile consisted of an initial denaturation for 2 min at 95°C and then 20 to 35 cycles of 30 s at 95°C, annealing for 30 s at 55°C (CPSF1, CPSF3, and ␤-actin) or at 52°C (CPSF2), and elongation for 1 min at 72°C. A final extension for 10 min was carried out at 72°C. The expected sizes of the amplicons were 249 bp for CPSF1, 314 bp for CPSF2, 291 bp for CPSF3, and 232 bp for ␤-actin. PCR products were electrophoresed on a 1% (wt/vol) agarose gel and detected by UV visualization. Real-time PCR quantification. For the quantitative real-time RT-PCR analysis of gene expression, the iCycler (Bio-Rad, Hercules, Calif.) system was used. Total RNA was isolated and retrotranscribed as described above. Equal amounts of cDNA were used in triplicate and amplified with the specific primers. The PCR mixture consisted of 25 ␮l containing 1⫻ PCR buffer, 1.5 mM MgCl2, 200 ␮M deoxynucleoside triphosphates, a 10 ␮M concentration of each sequencespecific primer, 3 ␮l of SYBR Green I (diluted 1:15,000), 2 ␮l of transcribed cDNA, and 0.5 U of recombinant Taq DNA polymerase (Invitrogen Life Sciences). Amplification efficiencies were validated and normalized against ␤-actin, and fold increases were calculated by using the comparative threshold cycle method for quantitation. The amplification profiles were the same as described for semiquantitative RT-PCR amplification. Transient transfections and luciferase assays. The transfections were performed with Lipofectamine Plus reagent (Invitrogen Life Sciences) according to the manufacturer’s recommendations for 48 h with the indicated plasmids. The cells were then lysed in 25 mM Tris-phosphate (pH 7.8)–8 mM MgCl2–1 mM dithiothreitol–1% Triton X-100–7% glycerol. Luciferase activity was measured

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with an Autolumat LB 953 (EG&G Berthold). The background obtained with the lysis buffer was subtracted from each experimental value, and all experiments were repeated at least three times. Western blots. Total cell extracts were obtained by incubating the cells in lysis buffer (20 mM HEPES [pH 8.0], 0.35 M NaCl, 0.1 mM EGTA, 0.5 mM EDTA, 1 mM MgCl2, 20% glycerol, 1 mM dithiothreitol, 1 ␮g of leupeptin per ml, 0.5 ␮g of pepstatin per ml, 0.5 ␮g of apronitin per ml, and 1 mM phenylmethylsulfonyl fluoride) containing 1% NP-40. Cells were incubated for 15 min in ice, and cellular proteins were obtained by centrifugation at 10,000 ⫻ g for 10 min. Protein concentrations were determined by the Bradford assay (Bio-Rad), and 20 ␮g of proteins was boiled in Laemmli buffer and electrophoresed in sodium dodecyl sulfate–7.5% polyacrylamide gels. Separated proteins were transferred to nitrocellulose membranes (0.5 A at 100 V; 4°C) for 1 h. The blots were blocked in Tris-buffered saline solution containing 0.1% Tween 20 and 5% nonfat dry milk overnight at 4°C, and immunodetection of specific proteins was carried out with the primary antibodies by using an ECL system (Amersham, Little Chalfont, United Kingdom). Antitubulin monoclonal antibody was purchased from Sigma Co. (St. Louis, Mo.), and rabbit anti-CPSF3 was a gift from D. Bentley (University of Colorado Health Center, Denver). Scanning electron microscopy. The cells were cultured, collected over polycarbonate membranes, washed twice with phosphate-buffered saline, fixed with fixative solution (2% glutaraldehyde in 0.1 M phosphate buffer) for 1 h, and postfixed with 1% osmium tetroxide for 2 h. Following dehydration with ethanol, membranes were sputter coated with a gold layer of 1.5 to 2 nm. The preparations were examined under a Jeol JSM 6300 microscope.

RESULTS HIV-1 Tat protein increases CPSF3 expression. To investigate the gene expression profiles of cellular genes regulated by the HIV-1 Tat protein, we generated stably tat-transfected cell lines. Initially, we compared the different gene expression patterns in K562-pcDNA3 and K562-Tat cells by RT-differential display in 20% of the total mRNA. Amplified cDNAs were selected on the basis of a clear difference in expression and later were cloned into a T/A vector and sequenced. The sequences obtained were identified by BLAST searching of the National Center for Biotechnology Information database, and from among the identified genes we selected a cDNA of 632 bp which was highly up-regulated in K562-Tat cells (Fig. 1A). This sequence was found to be 100% homologous to the human CPSF 73-kDa subunit (CPSF3 [GenBank accession no. NM_016207]; 2,286 bp). To further demonstrate that the CPSF3 gene was up-regulated in tat-transfected cells, we performed semiquantitative RT-PCR in the K562-Tat, HeLa-Tat, and Jhan-Tat cell lines. The functionality of the Tat protein in the stable cell lines was routinely verified by transient transfections with the HIV-1-LTR-Luc plasmid (43), and we found that although Tat was always functional in the three cell lines, different Tat-mediated LTR-transactivation indices were obtained with each cell type (data not shown). The primers used to detect CPSF3 mRNA by RT-PCR were designed by using the original cDNA sequence isolated by differential display method. Figure 1B shows an increased expression of CPSF3 in three tat-transfected cell lines compared to their parental control and that ␤-actin gene expression was not modified by this viral protein. From these data, it is clear that CPSF3 upregulation by HIV-1 Tat overexpression is not cell type specific. Since the product of this gene plays a critical role in the correct processing of the mRNAs, we investigated the function of this protein in cellular and viral gene expression. CPSF3 mRNA is specifically up-regulated by HIV-1-Tat. CPSF is a complex formed by four subunits, among which the 160-kDa subunit (CPSF1) has been reported to be directly

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FIG. 1. HIV-1 Tat protein increases the expression of CPSF3. (A) Identification of a 632-bp gene fragment up-regulated in K562-Tat cells. To carry out the differential display assays, we used four similar samples of the same cell to minimize artifactual amplification. Thus, two different extractions of mRNA (first extraction, lanes 1 and 3; second extraction, lanes 2 and 4) were subjected to two different RT-PCRs (lanes 1 and 2 and lanes 3 and 4). A differentially amplified 632-bp fragment (arrow) separated on 4.5% denaturing polyacrylamide gels was identified by autoradiography, excised from the gel, reamplified, cloned, and sequenced. (B) Tat increases the expression of CPSF3 in tat-transfected cells. Total RNA was extracted from HeLa (lanes 1), HeLa-Tat (lanes 2), Jhan (lanes 3), Jhan-Tat (lanes 4), K562 (lanes 5), and K562-Tat (lanes 6) cells, and the expression of CPSF3 and ␤-actin was detected by RT-PCR.

implicated in the contact with the mRNA (57). While the role of the CPSF2 and -3 subunits seems to be the regulation of CPSF activity in mRNA processing, the function of the CPSF4 subunit is a subject of controversy (38). For this reason, we focused our study on the mRNA expression of the CPSF1 and -2 genes in tat-transfected cells. RT-PCR experiments revealed that neither CPSF-1 nor CPSF-2 mRNA expression was upregulated in K562-Tat and HeLa-Tat cells compared to the parental control cells (Fig. 2A). By means of real-time kinetic RT-PCR experiments, the fold increase in the expression level of each of these three CPSF mRNAs was determined by comparing their expression in K562-Tat and HeLa-Tat cells to that in their respective parental cell lines. As expected, the CPSF3 gene had the greatest increase in expression both in the K562 cell line (6-fold) and in the HeLa cell line (3.8-folds) compared to the other two genes (Fig. 2B). To analyze the possible regulation of CPSF3, we evaluated whether CPSF3 mRNA could be regulated by other stimuli, such as tumor necrosis factor alpha (TNF-␣,) phorbol myristate acetate (PMA), or the transactivator protein X of hepatitis B virus (HBx), and we did not find any change in the expression of CPSF3 (data not shown). Taken together, our results suggest that HIV-1 Tat regulates CPSF3 gene expression by a specific pathway different from those activated by TNF-␣, PMA, or the HBx protein. CPSF3 protein expression is regulated by HIV-1 Tat. Next, we were interested in studying whether the increase in the mRNA expression of CPSF3 regulated by the Tat protein induces a significant effect on the steady-state levels of CPSF3 protein. To evaluate this, we carried out Western blot experiments with both the K562-Tat and HeLa-Tat cell lines compared with their respective parental cell lines by using a specific antibody that recognizes CPSF3 (44). As shown in Fig. 3A,

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FIG. 2. HIV-1 Tat protein specifically up-regulates the 73-kDa subunit of the CPSF complex in both K562 and HeLa cells. (A) Total RNA was extracted from K562-pcDNA3, K562-Tat, HeLa, and HeLa-Tat cells, and the expression of three CPSF subunits was studied by semiquantitative RT-PCR. ␤-Actin amplification was carried out in parallel as a control. (B) Quantitative real-time RT-PCR of CPSF subunit expression in K562-pcDNA3, K562-Tat, HeLa, and HeLa-Tat cells. Amplification was normalized against ␤-actin gene expression, and fold increases were calculated by using the comparative threshold cycle method for quantification. The results shown are representative of those from three experiments. Error bars indicate standard deviations.

CPSF3 protein levels were significantly up-regulated in both K562-Tat and HeLa-Tat cells compared to their parental cell lines, with ␣-tubulin protein expression used as a control. To rule out the possibility that the increase in the Tat-induced

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CPSF3 protein level was due to a change in the rate of protein stabilization, we performed experiments to measure the halflife of CPSF3 protein in both HeLa and HeLa-Tat cells. Thus, HeLa and HeLa-Tat cells were treated with cycloheximide (10 ␮g/ml) for the indicated times, and the steady-state levels of the CPSF3 protein were studied by immunoblotting. As shown in Fig. 3B, no significant differences in the half-life of the CPSF3 protein were found in HeLa-Tat cells compared to the parental cell line. Next, we studied the up-regulation of CPSF3 protein in an inducible model of HIV-Tat by using the HeLaOn-Tat cell line, in which Tat protein is induced in response to doxycycline. As shown in Fig. 3C, HeLa-On-Tat cells stimulated with doxycycline showed a clear increase in CPSF3 expression that was clearly more evident after 12 h of stimulation. ␣-Tubulin protein expression was used as a control for the protein levels. To study the functionality of HIV-Tat protein, HeLa-On-Tat cells were transiently transfected with the HIVLTR-Luc plasmid, and after 48 h the cells were stimulated with doxycycline for the indicated times. As shown in Fig. 3D, doxycycline treatment resulted in a time-dependent induction of the Tat-dependent HIV-LTR promoter activity. Effects of CPSF3 on viral and cellular gene expression. In the light of the previous results, we were interested in studying the effect of CPSF3 protein overexpression on viral and cellular gene expression. To analyze this, 293T and HeLa cells were transiently transfected with the pNL4-3.Luc.R-E plasmid alone or in combination with the expression vector encoding CPSF3 (pcDNA3-CPSF3). As shown in Fig. 4, pNL4-3.Luc.R-E luciferase activity as a marker of viral gene expression was highly activated by CPSF3 overexpression in both HeLa and 293T cells. Similarly, overexpression of CPSF3 also increased luciferase activity in cells cotransfected with a plasmid containing

FIG. 3. CPSF3 protein induction by HIV-1 Tat. (A) Total cell extracts were obtained from K562-pcDNA3, K562-Tat, HeLa, and HeLa-Tat cells, and CPSF3 protein expression was detected by Western blotting. (B) Cellular proteins were obtained from cycloheximide (CHX)-treated HeLa and HeLa-Tat cells at the indicated times, and the levels of CPSF3 protein were analyzed by Western blotting. (C) HeLa-On-Tat cells were stimulated with doxycycline (Dox) (1 ␮g/ml) for 1, 3, 6, and 12 h; total proteins were extracted; and the expression of CPSF3 was identified by immunoblotting. In all the cases the same membranes were reblotted with a monoclonal antibody recognizing ␣-tubulin. (D) HIV-LTR induction in HeLa-On-Tat cells by doxycycline. The cells were transfected with the HIV-LTR-Luc plasmid and 24 h later were stimulated with doxycycline as indicated. After this time, the cells were lysed and the luciferase activity was measured as described in Material and Methods. The results shown are representative of those from three experiments. RLU, relative light units. Error bars indicate standard deviations.

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FIG. 5. Specific inhibition of CPSF3 mRNA expression by siRNA. 293T cells were transiently transfected with either the pSI-CPSF3 plasmid (lanes 2) or the pSI-Control plasmid (lanes 1). Forty-eight hours later, total RNA was extracted and CPSF3 expression was studied by RT-PCR, using the ␤-actin gene expression as a control. The change in expression was analyzed by using image analysis software (Kodak digital science 1D, version 2.01) and expressed as relative units. Results from one representative experiment out of four independent experiments are shown. Error bars indicate standard deviations. FIG. 4. Effects of CPSF3 overexpression on viral and cellular gene expression. 293T and HeLa cells were transiently transfected with the pNL4-3.Luc.R-E, p21-Luc, or mdm2-Luc plasmid along with either the expression vector encoding CPSF3 (pcDNA3-CPSF3) (gray bars) or the empty pcDNA3 plasmid (black bars). After 48 h of transfection, luciferase activity was assayed. The results show mean fold induction ⫾ standard deviation from three different experiments.

HTLV-1-Luc, p21-Luc, Bax-Luc, and mdm2-Luc) alone or in combination with the expression vector encoding siRNA for CPSF3. As shown in Fig. 6, the inhibition of endogenous CPSF3 impaired the luciferase activity in both cell types. Since the amounts of pSI-CPSF3 transfected in the two cell lines

the luciferase gene driven by the p21WAF1 gene promoter. Interestingly, CPSF3 overexpression, in both HeLa and 293T cells, resulted in a decrease in the luciferase gene expression driven by the P2 intronic mdm2 promoter. From these results, it can be deduced that CPSF3 overexpression can differentially regulate cellular and viral gene expression. To further confirm the role of CPSF3 in both viral and cellular gene expression, we constructed several silencer plasmids for CPSF mRNA interference. We designed one vector which directs the synthesis of the same 21-bp double-stranded CPSF3 target sequence (pSICPSF3). 293T cells were transiently transfected with the pSICPSF3 plasmid and its corresponding pSI-Control plasmid. Forty-eight hours later, total RNA was extracted and CPSF3 expression was studied by RT-PCR, using the ␤-actin gene expression as a control. As shown in Fig. 5, in cells transfected with the pSI-CPSF3 plasmid the expression of the endogenous CPSF3 mRNA was markedly reduced. In our hands the efficiency of transient transfections with Lipofectamine in 293T cells is around 60%, which suggests that the inhibition of endogenous CPSF3 mRNA by pSI-CPSF3 should be clearly higher in the cell population where the silencer has been introduced. The specificity of the pSI-CPSF3 vector was verified by means of transient transfections in 293T cells with p-GFP plasmid alone or in combination with either pSI-CPSF3 or pSI-GFP. GFP expression was tested by flow cytometry, and while pSI-GFP was able to reduce the expression of GFP, no changes in the fluorescent protein levels with the pSI-CPSF3 vector were observed (data not shown). Next, we examined the effects of CPSF3 silencing on viral and cellular gene expression. 293T and HeLa cells were transiently cotransfected with different plasmids (pNL4-3.Luc.R-E,

FIG. 6. Effects of CPSF3 silencing on viral and cellular gene expression. 293T and HeLa cells were transiently cotransfected with the indicated luciferase reporter vectors and either the pSI-CPSF3 plasmid (gray bars) or the pSI-Control plasmid (black bars) (silencer/target plasmid ratio of 10:1; 1 ␮g of DNA total). After 48 h of transfection, luciferase activity was assayed. Each transfection was assayed in triplicate, and the results show mean percent activation ⫾ standard deviation.

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FIG. 7. Long-term inhibition of CPSF3 subunit expression. (A) Total RNA was isolated from either 293T-siControl (lanes 1) or 293T-siCPSF3 cells (lanes 2), and the expression of CPSF1, -2, and -3 was analyzed by RT-PCR, using ␤-actin gene expression as a control. (B) Phenotype changes in 293T-siCPSF3 clones were monitored by light (A and B) and scanning electron (C and D) microscopy in comparison to the parental controls. Corresponding images were taken at the same exposure. Magnifications, ⫻400 (light microscopy [LM]) and ⫻1,600 (scanning electron microscopy [SEM]).

were similar, the different percentages of pNL4-3.Luc.R-E inhibition in 293T and HeLa could be due to the higher expression of CPSF3 mRNA detected in the 293T cell line. As expected, and unlike the case for the other analyzed promoters (Fig. 6A), the effect of cotransfection with the pSI-CPSF3 and mdm2-Luc plasmids showed a different pattern, with a clear increase in the luciferase activity in 293T cells but no modification in HeLa cells. It is assumable that CPSF3 exerts counteracting effects on luciferase activity induced by the mdm2 promoter. First, this factor could repress the transcriptional activity of the promoter, and second, it could increase the polyadenylation of luciferase gene. Together, these results indicate that CPSF3 plays a crucial role in correct viral and cellular gene expression, although the siRNA-induced silencing of CPSF3 appears to impair HIV-1 expression more significantly, at least in HeLa cells. Stable silencing of CPSF3 induces a phenotype change in 293T cells. Stable expression of siRNAs mediates persistent suppression of gene expression, allowing the analysis of lossof-function phenotypes which develop over longer periods of time. In the light of the previous results, we were interested in studying the role that the CPSF complex has in cellular growth. Thus, 293T-siCPSF3 cells were generated as indicated in Materials and Methods (pSI-CPSF1 and -2 transfectants were not viable in long-term cultures). RT-PCR analyses showed a significant repression of CPSF3 mRNA without an effect on the steady-state mRNAs for the other two subunits (CPSF1 and -2) (Fig. 7A). Although the 293T-siCPSF3 clones were viable, the rate of growth was extremely low, and cell division took approximately 48 h, compared to 12 h in control cells. Moreover, important phenotypic changes were induced by the repression of CPSF3 in 293T cells, which were strongly contracted and rounded up, with diminished contacts with the

substratum (Fig. 7B). Cell cycle analysis was evaluated by flow cytometry, and no great change in the cell cycle was observed (data not shown). To further confirm that the inhibitory effects of the pSI-CPSF3 plasmid were not due to an artifact during the transient-transfection experiments, we transfected both 293T-siControl and 293T-siCPSF3 cells with the plasmids pNL43.Luc.R-E, HTLV-1-Luc, p21-Luc, Bax-Luc, and mdm2-Luc, and we found that stable silencing of CPSF3 mRNA also led to results similar to those obtained with transient transfections (data not shown). Viral or cellular gene expression is inhibited by the silencing of the CPSF1 and -2 subunits. It has been suggested that the subunits of the CPSF complex may play different roles in the cleavage and polyadenylation activities (32). To determine the roles that CPSF1 and -2 subunits play in viral and cellular gene expression, we designed expression vectors encoding specific siRNAs for the CPSF1 and CPSF2 subunits (pSI-CPSF1 and pSI-CPSF2, respectively). Again, 293T cells were transiently cotransfected with the indicated plasmids (pNL4-3.Luc.R-E, HTLV-Luc, p21-Luc, and mdm2-Luc), along with either each CPSF subunit silencer plasmid or the pSI-Control plasmid. Figure 8 shows that RNA interference and silencing of both CPSF1 and CPSF2 resulted in the inhibition of the luciferase activity, which was significantly greater than that observed after silencing the CPSF3 subunit. In the case of cotransfection with the mdm2-Luc plasmid, CPSF1 and -2 silencing equally resulted in the inhibition of the luciferase activity, whereas CPSF3 silencing produced an increase in this activity. These results, together with those obtained in the stable-silencing experiments with the different CPSF subunits, may suggest different functional roles for these three subunits of the CPSF complex in the control of both cleavage and polyadenylation activities and gene expression.

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FIG. 8. Effects of CPSF subunit specific inhibition on viral and cellular gene expression. 293T cells were transiently cotransfected with the indicated plasmids along with either each CPSF subunit silencer plasmid (pSI-CPSF1 [dark gray bars], pSI-CPSF2 [light gray bars], or pSI-CPSF3 [white bars]) or the pSI-Control plasmid (black bars) (silencer/target plasmid ratio of 10:1; 1 ␮g of DNA total). The luciferase activity was measured and expressed as percent activation. Values are means ⫾ standard deviations from three independent experiments.

DISCUSSION In the last few years, the role of HIV-1 Tat protein in the expression of host cellular genes and in the signaling transduction pathways has been the subject of intense research using different approaches. Recent studies have demonstrated that this protein significantly increases the expression of genes whose products are involved in the transcription and translation machinery (13). The biological results obtained by experimental approaches in which the HIV tat gene is controlled by viral promoters such as those of cytomegalovirus or simian virus 40 and then the HIV-1 Tat protein is overexpressed are usually difficult to extrapolate to the biological effects of Tat under the physiological conditions of natural HIV-1 infections. However, it has been shown that in human primary dendritic cells both HIV-1 and adenovirus Tat infection resulted in similar increases in Tat mRNA expression, thereby showing that in a more physiological model of viral infection, high levels of Tat protein expression can also be induced (31). In this report, we have provided evidence that CPSF 73-kDa subunit (CPSF3) expression is up-regulated specifically by the HIV-1 Tat protein in three different cell lines and also in a Tat-inducible cell line. Also, knockdown of the endogenous CPSF3 by RNA interference, together with gene overexpression experiments, have shown that CPSF3 is an important regulatory protein for both viral and cellular gene expression. Thus, HIV-1-Tat up-regulation of CPSF3 could represent a novel mechanism by which this virus establishes an adequate cellular environment to enhance viral replication. We found that CPSF3 is not regulated by other stimuli, such as TNF-␣, PMA, or the HBx protein. This result implies that Tat may regulate CPSF3 gene expression by a specific pathway that probably takes place at the transcriptional level by interacting with non-cell type-specific transcription factors modulating its activity. Interestingly, the CPSF3 proximal promoter contains several consensus sites for transcription factors such as CREB and c-Rel, which have been demonstrated to interact physically

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or functionally with the HIV Tat protein (2, 23). Experiments with the CPSF3 promoter are in progress to elucidate this point. Alternatively, Tat may affect the mRNA stability of CPSF3, but, taking into account the high homology between the three subunits of the CPSF complex and the fact that Tat does not induce the expression of CPSF1 and -2, it is unlikely that the high CPSF3 mRNA expression observed in tat-expressing cells is due to mRNA stabilization. The 3⬘ ends of eukaryotic mRNAs are generated by endonucleolytic cleavage and polyadenylation. In mammals, the multisubunit CPSF plays a central role in both steps of the processing reaction. Although the precise role of the CPSF3 subunit is not completely clarified, a function for this protein in mammalian cells can be inferred from experiments performed with yeast. Significant studies have shown that both the mRNA maturation process and the involved protein complexes in yeast are highly similar to those in mammals (39). In this regard, the equivalent to the mammalian CPSF3 protein was identified as a subunit (Ysh1/Brr5) of yeast polyadenylation factor I (PFI). Disruption of the open reading frame of this protein is lethal, which demonstrates that this gene is essential for viability in yeast (32). Similarly, studies by means of protein mutation and extract depletion by immunoprecipitation have demonstrated its role in both the cleavage and the polyadenylation of mRNA in vivo and in vitro (9). The association of PFI with Pap1p (PAP) confers on the former a possible modulating role in cleavage and polyadenylation activities in yeast (50). The fact that CPSF, together with its equivalent PFI, are among the best conserved components of the 3⬘-end-processing machinery in these highly divergent organisms indicates their key function in the mRNA maturation process. The modifications of pre-RNAs during their maturation process are central to the correct development of many processes, such as transcription, splicing, nucleocytoplasmic transport, and translation. Among these modifications, the most rate-limiting step is that of 3⬘-end processing, including cleavage and poly(A) addition. Since polyadenylation is a prerequisite for HIV-1 RNA nucleocytoplasmic transport and 3⬘-end processing is necessary for nuclear export (8, 29), the data in this study suggest a model in which Tat protein increases CPSF3 expression to favor mRNA processing and its translation in the cytoplasm, thereby causing an increase in both cell and viral gene expression. However, our results suggest that CPSF3 is more relevant for viral gene expression than for cellular gene expression. In this regard, some authors have demonstrated that an excessive polyadenylation activity could impair the nucleocytoplasmic transport of de novo synthesized cellular mRNAs (8, 25). Nevertheless, an increase in this activity would not equally affect the poly(A) tails of the viral mRNA during viral replication (46). Therefore, through CPSF3 up-regulation the HIV-1 Tat protein could preferentially increase the amount of cytoplasmic viral RNAs exported from the nucleus, at the expense of cellular RNAs. Interestingly, Vpr, another HIV-1 regulatory protein, has been shown to modulate the activity of poly(A) polymerase, thus enhancing the polyadenylation processes in order to optimize HIV-1 replication and contribute to HIV-1 pathogenesis (46). In addition to its role in mRNA 3⬘-end processing, there is increasing evidence that the CPSF complex also plays a key function in the transcription initiation of genes specifically

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regulated by RNAPII (26). It has been shown that RNAPII transcription and mRNA maturation are tightly coupled processes (51). Transcription initiation by RNAPII requires the assembly of initiation factors to form a preinitiation complex. CPSF is brought to the preinitiation complex by TFIID, and after the start of the transcription, it is transferred to the elongating polymerase and bound to CTD for correct 3⬘-end processing (12, 27). Interestingly, functional binding sites for CPSF present in the HIV-1 LTR promoter (positions ⫹71 to ⫹76) have also suggested a role for this factor in HIV-1 gene transcription (54). So far, no DNA binding activities have been specifically described for the CPSF3 subunit in cellular gene promoters, but the results presented here indicate that CPSF3 functions as a gene transcription repressor, at least for the mdm2 promoter, and since this subunit does not contain any consensus DNA binding domain, it is possible that CPSF3 interacts with other nuclear factors to form a negative regulatory complex. HIV-1 has evolved various means to perturb the cell cycle to optimize the cellular conditions in favor of its own replication. The facts that HIV-1 Tat induces an increase in CPSF3 expression and that this protein plays a crucial role not only in mRNA processing but also in transcriptional repression of the mdm2 gene promoter suggest a novel mechanism by which the HIV-1 virus modulates cellular factors required for optimal viral replication. ACKNOWLEDGMENTS This work was supported by MCyT grant SAF2001-0037-C04-02, FIPSE grant 36391/03, and EU grant QLK3-CT-2000-00463 to E.M. We thank D. Bentley (University of Colorado Health Center) for rabbit anti-CPSF3 and M. Fresno (CBM-UAM, Madrid, Spain) for Jhan-Tat cells. We thank Carmen Cabrero-Doncel for assistance with the manuscript. REFERENCES 1. Barak, Y., E. Gottlieb, T. Juven-Gershon, and M. Oren. 1994. Regulation of mdm2 expression by p53: alternative promoters produce transcripts with nonidentical translation potential. Genes Dev. 8:1739–1749. 2. Benkirane, M., R. F. Chun, H. Xiao, V. V. Ogryzko, B. H. Howard, Y. Nakatani, and K. T. Jeang. 1998. Activation of integrated provirus requires histone acetyltransferase: p300 and P/CAF are coactivators for HIV-1 Tat. J. Biol. Chem. 273:24898–24905. 3. Bieniasz, P. D., T. A. Grdina, H. P. Bogerd, and B. R. Cullen. 1998. Recruitment of a protein complex containing Tat and cyclin T1 to TAR governs the species specificity of HIV-1 Tat. EMBO J. 17:7056–7065. 4. Buonaguro, L., G. Barillari, H. K. Chang, C. A. Bohan, V. Kao, R. Morgan, R. C. Gallo, and B. Ensoli. 1992. Effects of the human immunodeficiency virus type 1 Tat protein on the expression of inflammatory cytokines. J. Virol. 66:7159–7167. 5. Caldwell, R. L., B. S. Egan, and V. L. Shepherd. 2000. HIV-1 Tat represses transcription from the mannose receptor promoter. J. Immunol. 165:7035– 7041. 6. Calnan, B. J., B. Tidor, S. Biancalana, D. Hudson, and A. D. Frankel. 1991. Arginine-mediated RNA recognition: the arginine fork. Science 252:1167– 1171. 7. Colgan, D. F., and J. L. Manley. 1997. Mechanism and regulation of mRNA polyadenylation. Genes Dev. 11:2755–2766. 8. Custodio, N., M. Carmo-Fonseca, F. Geraghty, H. S. Pereira, F. Grosveld, and M. Antoniou. 1999. Inefficient processing impairs release of RNA from the site of transcription. EMBO J. 18:2855–2866. 9. Chanfreau, G., S. M. Noble, and C. Guthrie. 1996. Essential yeast protein with unexpected similarity to subunits of mammalian cleavage and polyadenylation specificity factor (CPSF). Science 274:1511–1514. 10. Chang, H. K., R. C. Gallo, and B. Ensoli. 1995. Regulation of cellular gene expression and function by the human immunodeficiency virus type 1 Tat protein. J. Biomed. Sci. 2:189–202. 11. Chirillo, P., M. Falco, P. L. Puri, M. Artini, C. Balsano, M. Levrero, and G. Natoli. 1996. Hepatitis B virus pX activates NF-␬B-dependent transcription through a Raf-independent pathway. J. Virol. 70:641–646.

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