Interaction of YB-1 with human immunodeficiency virus type 1 Tat and TAR RNA modulates viral promoter activity

Sameer A. Ansari1, Mahmut Safak1, Gary L. Gallia1, Bassel E. Sawaya1, Shohreh Amini1 and Kamel Khalili1

Center for NeuroVirology and NeuroOncology, MCP Hahnemann University, Broad and Vine, MS #406, Philadelphia, PA 19102, USA 1

Author for correspondence: Kamel Khalili.Fax +1 215 762 3241. e-mail khalili{at}drexel.edu


   Abstract
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Abstract
Introduction
Methods
Results and Discussion
References
 
Transcriptional regulation of the human immunodeficiency virus type 1 (HIV-1) genome is mediated by viral and cellular factors. TAR, an unusual RNA regulatory element with a stem–bulge–loop structure at the 5' ends of all nascent viral transcripts is critical for HIV-1 transcription. TAR is the target for Tat, a viral transcription factor encoded early in the HIV-1 life-cycle and essential for gene expression. Evidence demonstrating the interaction of a cellular ssDNA/RNA binding protein, YB-1, with TAR through a region which is important for Tat interaction is presented. Interestingly, results from protein–protein interaction studies revealed that YB-1 can also form a complex with Tat. Results from mapping experiments suggest that while the region spanning aa 125–203 within YB-1 is essential for its association with TAR, a truncated YB-1 spanning aa 1–125 can weakly bind to Tat. Functionally, overexpression of full-length YB-1 enhanced Tat-induced activation of the HIV-1 minimal promoter containing TAR sequences, whereas mutant YB-1 with no ability to bind to Tat and TAR failed to affect Tat-mediated activation. Expression of mutant YB-1(1–125), which binds to Tat but not RNA, decreased Tat- mediated enhancement of virus transcription. These observations suggest that while full-length YB-1 may function as a facilitator and, by interaction with both Tat and TAR, increase the level of Tat:TAR association, mutant YB-1 with no TAR binding activity, by complexing with Tat, may prevent Tat interaction with TAR. The importance of these findings in light of the proposed mechanism of Tat function is discussed.


   Introduction
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Abstract
Introduction
Methods
Results and Discussion
References
 
Transcription is a crucial process in the human immunodeficiency virus type 1 (HIV-1) life-cycle as it provides the template for synthesis of viral regulatory and structural proteins and results in the generation of new copies of the viral RNA genome (for review see Emerman & Malim, 1998 ). The maximum level of viral gene transcription is accomplished by cooperative interaction of host cellular proteins and viral regulatory factors (for review see Kingsman & Kingsman, 1996 ; Sawaya et al., 1998a , b ; Wu-Baer et al. , 1995 ). Indeed, direct and/or indirect association of the participant cellular and viral proteins with the HIV-1 regulatory sequence which spans through the long terminal repeat (LTR) is the essential and perhaps the first step in this event (for review see Emerman & Malim, 1998 ; Luo et al., 1993 ). Earlier studies from several laboratories have identified several cis-regulatory elements in the viral LTR which are absolutely required for the initiation of viral RNA synthesis (Blair et al., 1996 ; Jones & Peterlin, 1994 ). While the elements positioned upstream from the transcription start site are targets for binding to a variety of distinct DNA binding cellular regulatory proteins, the region downstream from the start site is critical for the regulatory action of the virus-encoded protein, Tat (Cullen, 1995 ; Emerman & Malim, 1998 ). Tat is a small protein that potently transactivates HIV-1 promoter transcription by interacting with TAR, a hairpin-structured RNA target 3' of the transcription start site (nt 1–59). The TAR RNA contains an extensive secondary structure with a stem, a loop and a UCU bulge, which is the site for Tat interaction. Evidently, the structure of the duplex stem and the presence of the loop nucleotides are required for Tat function. Earlier studies have suggested that Tat may interact with TAR through its basic domain RKKRRQRRR amino acids or the ARM (arginine-rich RNA binding motif) (Calnan et al., 1991 ). ARMs are short regions of basic amino acids (8–20 residues) used by various RNA binding proteins to recognize specific RNA hairpins. These motifs are found in bacterial antiterminators, ribosomal proteins, HIV-1 Rev, bovine immunodeficiency virus Tat and the Y-box family of proteins (Tan & Frankel, 1995 ). Several TAR RNA binding proteins have been isolated which probably modulate Tat binding and function through the bulge–loop structure (Sheline et al., 1991 ). We have focused our study on the Y-box member YB-1 and its effect on the Tat:TAR RNA complex.

The Y-box family of proteins is perhaps the family of most evolutionarily conserved nucleic acid binding proteins from bacteria to plants and vertebrates. The highly conserved central nucleic acid binding domain or cold shock domain defined initially in bacteria is a characteristic feature of this family (Wolffe, 1994 ). The term Y-box derives from the initial cloning of the human homologue YB-1 and its ability to bind the cis-regulatory Y-box region of the major histocompatibility complex class II gene promoter (Didier et al., 1988 ). A paradox of high homology between Y-box family members and a wide range of nucleic acid interactions exists, further accentuated by heterogeneity in function. The Y-box proteins show a preference for duplex DNA enriched with pyrimidines and purines on opposite strands (Ozer et al., 1990 ; Sakura et al., 1988 ), specifically sequences containing a reverse CCAAT box motif (Dorn et al., 1987a , b ). These proteins can also interact with postulated H-form (triplex) DNA structures (Kolluri et al. , 1992 ), apurinic ssDNA motifs (Hasegawa et al., 1991 ; Lenz et al., 1990 ) and mRNA (Deschamps et al., 1992 ; Murray et al., 1992 ; Sommerville & Ladomery, 1996 ). Diverse functional roles have been attributed to the Y-box family ranging from prokaryotic cold shock responses to eukaryotic transcription factors, chromatin modification proteins, DNA repair proteins, translational repressors, and RNA packaging proteins (Wolffe, 1994 ).

Y-box family members have been shown previously to bind mRNA in Xenopus oocytes (FRGY2) (Murray et al., 1992 ) and rabbit reticulocytes (p50) (Minich et al., 1993 ). These RNA binding proteins store maternal mRNA in ribonucleoprotein (RNP) complexes masking them from the translational machinery. Recently, YB-1 itself was identified as a protein which interacts with glutathione peroxidase transcripts at the selenocysteine insertion sequence located 3' to alternatively read UGA codons (Shen et al., 1998 ). This RNA element forms a stem–loop RNA structure similar to TAR RNA. The structure of the YB-1 protein is divided into three general domains and confers several RNA binding regions. The N terminus is unremarkable, composed of alanine and proline residues possibly involved in mediating protein–protein interactions. The highly conserved central cold shock domain contains basic and aromatic amino acids to attract nucleic acid backbones and associate with DNA or RNA nucleotide bases. In addition, a short RNP-1 motif lies within the cold shock domain, a stretch of eight amino acids characteristic of RNA binding domains (Landsman, 1992 ). The C terminus of YB-1 consists of alternating regions of basic and acidic residues forming a charge-zipper domain. The basic regions interspersed with aromatic amino acids are termed B/A islands and correspond to four ARM regions of YB-1. ARM 1 lies within the first B/A island which is located at the C terminus of the cold shock domain. Hence, the cold shock domain possesses two different RNA binding domains with perhaps distinct RNA specificities (Ladomery & Sommerville, 1994 ).

YB-1 has been isolated by several other groups. DbpA and dbpB (YB-1) were characterized by their interaction with c-erbB-2 gene promoter sequences (Sakura et al., 1988 ). The nearly identical rat EFIA (Ozer et al., 1990 ) was described as a trans-acting factor binding the Y-box of the Rous sarcoma virus (RSV) LTR. Earlier studies from our laboratory have led to the identification of a partial cDNA encoding a truncated protein, HF-1, by screening with the CT-rich lytic control element of the human neurotropic JC virus (JCV) enhancer/promoter (Kerr et al. , 1994 ). HF-1 represents the N-terminal region of YB-1 with the ability to regulate JCV gene transcription. Of note, earlier studies showed that YB-1/dbpB/EFIA acts as a transcription factor to activate several viral promoters such as the RSV, human T- lymphotropic virus 1 and HIV-1 LTR (Kashanchi et al., 1994 ; Sawaya et al., 1998a ). The ability of YB-1 to bind ssDNA and RNA prompted us to examine the ability of this protein to modulate the HIV-1 LTR through the TAR RNA sequence. In this study, we demonstrate that YB-1 binds to TAR and Tat, and that binding to TAR RNA and the Tat protein is specific to the ARM containing regions of both proteins. While co-expression of YB-1 and Tat can enhance the level of Tat activation of the HIV-1 LTR, mutant YB- 1 capable of interacting with Tat, but not with TAR, decreased the level of viral gene transcription in the cells.


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Methods
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{blacksquare} Cell culture and transfection.
U-87MG is a human astrocytic cell line obtained from the ATCC. In general, cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (Life Technologies). All transfections were carried out by the calcium phosphate precipitation method (Graham & van der Eb, 1973 ) with 15 µg total DNA per 60 mm in each transfection mixture, not exceeding 25 µg per 100 mm plate or 15 µg per 60 mm plate. CAT assay was performed 36 h post-transfection with 75 µg extract as described previously (Gorman et al., 1982 ).

{blacksquare} Plasmid constructs.
Glutathione S-transferase (GST)-Tat 86 and its mutant constructs (GST-Tat 72, GST-Tat 48 and GST-Tat {Delta}2/36) were generously provided by John Brady (National Cancer Institute, NIH, Bethesda, MD, USA) and Andrew Rice (Rhim et al., 1994 ). pCDNA3-Tat was constructed by placing the BamHI fragment of GST-Tat 86 into the BamHI site of pCDNA3 (Invitrogen). pGEX2T-YB-1 was provided by Gene MacDonald (Lineberger Cancer Center, University of North Carolina, Chapel Hill, NC, USA). GST- YB-1 and pEBV-His A mutants were created by the use of appropriate DNA restriction enzymes or the DNA fragment or PCR amplification. The full- length YB-1 cDNA (1–318) and all the deletion mutants of YB-1 were cloned in-frame with the pEBV-His A expression vector (Invitrogen). The TAR RNA mutants were generously provided by Ajit Kumar (Rounseville & Kumar, 1992 ). The TM27mt RNA expression plasmid was created by replacing the wild-type cassette with an oligonucleotide expressing the truncated TM27 mutant in reverse orientation.

{blacksquare} GST fusion proteins and GST affinity chromatography.
GST, GST-Tat 86, GST-YB-1 and their respective mutants were expressed and purified according to the standard procedure described previously (Smith & Johnson, 1988 ). The integrity and purity of all proteins were examined by SDS–PAGE and Coomassie blue staining. In vitro synthesis of proteins was carried out with the TnT-coupled wheat germ extract system (Promega) according to the supplier's protocol. For the GST pull-down assay, 3 µl 35S-labelled protein reaction or 250 µg cellular extract were mixed with 5 µg GST or GST-Tat fusion bound to glutathione–Sepharose beads in 300 µl LB150 buffer [50 mM Tris (pH 7·4), 150 mM NaCl, 5 mM EDTA, 0·1% NP-40, 50 mM NaF and a cocktail of protease inhibitors] or TNN buffer [50 mM Tris (pH 7·4), 150 mM NaCl, 5 mM EDTA, 0·5% NP-40 and protease inhibitors], respectively, at 4 °C for 2 h. After extensive washing, proteins bound to beads were eluted with protein sample buffer and were separated by SDS–PAGE prior to immunoblotting with specific antibodies.

Co-immunoprecipitation was performed by a standard procedure utilizing anti-Tat antibody (Ensoli et al., 1990 ), control mouse sera or anti-His-T7 antibody, as described previously (Wong et al., 1998 ).

{blacksquare} Band-shift assay, Northwestern blotting and RNA UV-crosslinking.
For band-shift assay, the GST moiety of GST-YB-1 was removed upon thrombin treatment as instructed by the supplier (Pharmacia). YB-1 proteins were incubated with 50000 c.p.m. TAR RNA or its respective mutants in RNA binding buffer containing 50 µg/ml poly dI–dC, 50–500 µg/ml tRNA, 5 mM GTP, 12 mM HEPES (pH 7·9), 4 mM Tris (pH 7·4), 60 mM KCl, 5 mM MgCl2, 2·5 mM CaCl2, 0·5% NP-40 and 0·8 mM dithiothreitol at 4 °C for 30 min. Purified Tat protein was kindly provided by Jay Rappaport (MCP Hahnemann University, Philadelphia, PA, USA), for TAR binding experiments with YB-1. Protein:RNA complexes were resolved on a low ionic strength 0·5x TBE (Tris–borate–EDTA) 6% native polyacrylamide gel, dried and visualized by autoradiography.

For Northwestern blot analysis, GST, GST-YB-1 and GST-YB-1 C- terminal deletions were separated by 10% SDS–PAGE, transferred to a supported nitrocellulose membrane and blocked in 10% nonfat dry milk in PBS-T (PBS containing 0·1% Tween 20) at room temperature for 30 min. A parallel SDS–PAGE gel was stained with Coomassie blue. The membrane was rinsed twice with 0·5% nonfat dry milk in PBS-T and subsequently blocked in RNA binding buffer and 0·5% nonfat dry milk for 30 min. Fresh RNA binding buffer, 0·5% nonfat dry milk and 1x106 c.p.m. TAR RNA were added and the membrane was incubated for 4 h with rocking. The membrane was rinsed and washed in the same buffer for 10 min intervals prior to autoradiography.


   Results and Discussion
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Abstract
Introduction
Methods
Results and Discussion
References
 
Interaction of YB-1 and HIV-1 TAR sequence
In previous studies, we have demonstrated that YB-1 can stimulate transcription of the HIV-1 LTR in human astrocytic cells through the upstream promoter sequence spanning the GC-rich motif and the downstream region, corresponding to the TAR sequence (Sawaya et al. , 1998a ). The latter is particularly interesting in light of an earlier study (Shen et al., 1998 ) showing that YB-1 binds the selenocysteine insertion sequence with an RNA secondary structure resembling TAR RNA and contains four ARMs similar to the TAR-specific ARM of HIV-1 Tat (Fig. 1a). Thus, as a first step in studying the regulatory effect of YB-1 on HIV-1 transcription via TAR, we assessed the ability of YB-1 to directly bind TAR RNA. To this end, by utilizing recombinant cDNA clones expressing different regions of YB-1 in a prokaryotic system (Fig. 1a), the GST fusion proteins containing full-length or C-terminal deletions of YB-1 were produced in bacteria and resolved by SDS–PAGE (Fig. 1b, left). The fractionated proteins were transferred to nitrocellulose and the transblot was incubated with a riboprobe containing HIV-1 TAR. As shown in Fig. 1(b) (right), TAR bound to the full-length GST-YB-1 (lane 2), but not to GST alone (lane 1). A C-terminal GST-YB-1 deletion mutant lacking ARMs 3 and 4, GST-YB-1(1–203), was also able to interact with TAR RNA (lane 3). A GST-YB-1 deletion mutant, GST-YB-1(1–125) , which does not contain ARMs 1 and 2, was unable to interact with TAR RNA (lane 4). Similarly, further C-terminal GST-YB-1 mutants are unable to associate with TAR RNA (lanes 4 and 5). These results suggest that the region of YB-1 which is involved in the interaction with TAR RNA resides between aa 125–203. In an alternative approach, we utilized band-shift assays and confirmed that the region between aa 125–203 is important for YB-1:TAR complexation (data not shown). These observations suggest that YB-1 interacts with TAR and that specific regions within YB-1, which encompass ARMs 1 and 2, confer TAR RNA binding ability.



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Fig. 1. Interaction of YB-1 with HIV-1 TAR. (a) Structural organization of YB-1. The N terminus is composed of alanine and proline residues (hatched area), whereas the highly conserved cold shock domain (CSD) contains basic aromatic residues and a short RNP-1 motif. The C terminus consists of alternating basic and acidic residues forming a charge-zipper structure. The basic residues are separated by aromatic residues (grey areas), termed basic/aromatic islands which are also known as the ARMs. (b) Northwestern blot analysis. Approximately 5 µg GST, GST-YB-1 or its mutant variants, as depicted in (a), were resolved by 10% SDS–PAGE and, after transfer to nitrocellulose, incubated with 106 c.p.m. 32P-labelled TAR RNA probe. The left panel shows a Coomassie blue-stained gel and the right panel shows results from Northwestern blot analysis. Arrows indicate GST-YB-1 proteins which interact with the TAR RNA probe. The positions of molecular mass markers (in kDa) are shown on the left.

 
In the next series of studies, we determined the specificity of YB-1 binding to TAR and identified a region within TAR which is important for its interaction with YB-1. In this study, we utilized several TAR RNA mutants in band-shift assays (Fig. 2a). As shown previously (Cullen, 1995 ), the association of Tat with TAR is mediated through the UCU bulge located near the loop structure of TAR. Results from RNA binding studies revealed a strong association of wild-type TAR and a truncated mutant with an intact stem–bulge–loop RNA structure (TM27) (Fig. 2b, lanes 2 and 8). Extremely weak binding was observed with TAR RNA mutants lacking the bulge structure (TM12) and with nucleotides mutated in both the bulge and loop structures (TM26) (lanes 4 and 6, respectively). A non- specific RNA loop structure with TAR nucleotides in reverse orientation (TM27mt) exhibited no binding activity to YB-1 (lane 10). These observations suggest that maximum binding of YB-1 to TAR requires, in addition to a stem and loop structure, the UCU bulge region. In the next study, we examined the ability of YB-1 and Tat to bind to the TAR RNA probe. The addition of purified Tat to the TAR probe caused the formation of a Tat:TAR complex with a fast electrophoretic mobility and a much larger complex (Tat*:TAR) which represents Tat aggregates (Fig. 2 c, lane 2). A distinct band corresponding to YB-1:TAR complexes was observed in a reaction containing TAR and purified YB-1 (Fig. 2c, lane 3). The addition of increasing amounts of Tat to the YB-1 binding reaction caused a noticeable decrease in the intensity of the bands corresponding to YB- 1:TAR complexes (compare lane 3 with lanes 4–6). Interestingly, an increasing amount of Tat in this reaction showed no enhancement in the level of Tat:TAR complex, although the intensity of the Tat*:TAR aggregate was increased. As the binding reaction was performed in the presence of excess probe, the observed decrease in the level of YB- 1:TAR complex upon addition of Tat implies a potential interplay between Tat and YB-1. However, one cannot exclude the possibility that only a small fraction of RNA may be in a conformation required for protein interactions and that Tat and YB-1 may compete for RNA binding.



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Fig. 2. YB-1 binding to TAR RNA mutant and the effect of Tat on YB-1 RNA binding activity. (a) Computer generated secondary structure of the wild-type TAR RNA and its mutant variants (kindly provided by A. Kumar) used as RNA probes in binding studies. ( b) Approximately 250 ng purified full-length YB-1 was incubated with 50000 c.p.m. 32P-labelled wild-type RNA or its mutant variants as indicated. RNA:YB-1 complex was resolved on a 0·5x TBE 6% native PAGE and visualized by autoradiography. (c) Approximately 50 ng purified YB-1 was incubated with TAR probe alone (lane 3) or in the presence of increasing amounts of Tat (25, 50 and 100 ng; lanes 4, 5 and 6, respectively). Lane 2 shows a binding reaction containing 50 ng Tat plus RNA probe. Positions of the TAR probe, Tat:TAR and YB-1:TAR complexes are shown. Tat*:TAR points to the aggregate Tat and TAR probes.

 
Interaction of YB-1 with Tat
To investigate whether YB-1 and Tat can interact with each other, in vitro translated (IVT) 35S-labelled Tat and YB-1 were incubated with anti-Tat antibody alone or together, and the immunocomplexes were analysed by SDS–PAGE. Fig. 3(a) illustrates the position of Tat and YB- 1 in gel analysis of 1/10 of input in vitro synthesized Tat (lane 1) and YB-1 (lane 2) used in the immunoprecipitation reaction. For clarity, the shorter exposure of the region of the gel where Tat co- migrated is shown at the bottom of Fig. 3(a). As shown in Fig. 3(a), anti-Tat antibody reacts with the in vitro synthesized Tat, but not YB-1, as expected (compare lane 3 with lane 4). Interestingly, anti-Tat antibody was able to co-immunoprecipitate YB-1 in a reaction containing Tat plus YB-1 (lane 5). To further demonstrate the association of YB-1 and Tat in cell extract, glial cells were transfected with the YB-1 expression plasmid pEBV-His-YB-1 and/or the Tat expression plasmid pCDNA3-Tat. The expression of YB-1 and Tat and the level of their association were determined by immunoprecipitation/Western blot analysis. For the detection of YB-1, anti-T7-tagged antibody was utilized to recognize the His-YB-1 fusion protein. Fig. 3(b and c ) illustrates the detection of YB-1 and Tat, respectively, in extracts from cells transfected with the corresponding plasmids by Western blot analysis (lane 2) or immunoprecipitation/Western blot analysis (lanes 3 and 4, respectively). Examination of YB-1:Tat interaction by co-immunoprecipitation/Western blot analysis revealed the detection of Tat in the immunocomplex pulled down by anti-T7 antibody suggesting that YB-1 may be associated with Tat in the transfected cells (Fig. 3d, compare lane 5 with lane 6).



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Fig. 3. Interaction of YB-1 and Tat. (a) {alpha}Tat antibody (5 µl) was incubated with 3 µl IVT 35S-labelled Tat reaction (lane 3), 6 µl IVT 35 S-labelled YB-1 (lane 4) or 12 µl of both reactions (lane 5) in LB150 buffer at 4 °C for 4 h. Immune complexes were isolated with protein A–Sepharose after extensive washing and resolved by 15% SDS–PAGE followed by fluorography. Lanes 1 and 2 show 1/10 of input IVT 35S-labelled Tat and IVT 35 S-labelled YB-1 used in co-immunoprecipitation (lane 5), respectively. A shorter exposure of bands corresponding to IVT 35S- labelled Tat is shown in the bottom panel to delineate it from IVT background shown by an asterisk. (b) Approximately 500 µg glial cell extract transfected with YB-1 expression plasmid was incubated with normal mouse serum (NMS; lane 3) or anti-His- T7 ({alpha}T7; lane 4) and the resulting complexes were analysed by Western blot analysis. In lanes 1 and 2, 50 µg protein from untransfected and YB-1 transfected cells, respectively, were analysed. ( c) Immunoprecipitation and Western blot analysis of glial cells transfected with Tat expression plasmid. A similar procedure as described in (b) was adapted. (d) Protein extracts from cells transfected with YB-1 and Tat expression plasmids were incubated with NMS or {alpha}T7; the immunocomplexes were analysed by Western blot utilizing anti-Tat antibody. The position of the Tat protein is shown by an arrow.

 
To further investigate the interaction of YB-1 and Tat, we mapped the YB-1 interaction domain within the Tat protein utilizing GST pull- down assays with IVT 35S-labelled YB-1 and protein extracts from cells transfected with pEBV-His-YB-1. Results shown in Fig. 4(b) indicate that the IVT YB-1 can be associated with full-length Tat (Tat 86) and its mutant variant Tat 72, but not mutant Tat 48 (compare lanes 3 and 4 with lane 5). Similar results were observed with extracts from cells transfected with pEBV- His-YB-1. As shown in Fig. 4(c), a band corresponding to YB-1 was detected in eluates from GST-Tat 86, but not from GST-Tat 48, suggesting that the C-terminal region of Tat which contains an ARM (Fig. 4 a) is important for YB- 1:Tat interaction. Results from Northwestern analysis of GST-Tat 86, GST-Tat 72 and GST-Tat 48 utilizing a TAR RNA probe further reiterated the importance of the C-terminal ARM of Tat for its interaction with TAR (data not shown). These data suggest that the C terminus of Tat is important for YB-1 and TAR RNA interaction. In a reciprocal study, we mapped the Tat binding region within the YB-1 protein with a GST pull- down assay. IVT full-length Tat (Tat 86) was used for its ability to bind to GST, GST-YB-1(1–318), and the various mutants that remove the C-terminal region of YB-1, as illustrated in Fig. 1(a). The association of Tat was decreased in the presence of mutant YB-1 containing residues 1–125 and abrogated upon deletion of the region between aa 75–125 (Fig. 5a, compare lane 5 with lane 6). Examination of the N-terminal mutants of YB-1 for their binding to Tat revealed that the mutant proteins encompassing aa 204–318 and 250–318 are unable to interact with Tat while the region aa 125–318 may weakly interact with Tat (data not shown). Thus, it is evident that the maximum level of YB-1 interaction with Tat requires the region aa 75–203, which encompasses both the cold shock domain and ARMs 1 and 2, known to be responsible for the ability of YB-1 to bind the RNA structure. Fig. 5(b) summarizes the results from YB-1 binding experiments to Tat and TAR.



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Fig. 4. Mapping the Tat domain required for YB-1 interaction. (a) Schematic illustration of HIV-1 Tat depicting the various regions of Tat (top) and the position of the mutant Tat used in this study (bottom). (b) Approximately 5 µg GST (lane 1), GST-Tat 86 (lane 2) or its mutant variants as indicated above the lanes (lanes 4–6) immobilized on glutathione–Sepharose beads were incubated with 3 µl IVT 35S-labelled YB-1 reaction mixture. The bound proteins were eluted and analysed by SDS–PAGE followed by autoradiography. Lane 1 shows 1/10 of input in vitro synthesized YB-1. The arrow indicates the position of YB-1. (c) GST, GST-Tat 86 or GST-Tat 48 beads were incubated with 250 µg glial cell extract transfected with YB-1 expression plasmid. After extensive washing of the beads, proteins attached to the GST-Tat Sepharose were eluted and analysed by SDS–PAGE followed by Western blot analysis utilizing anti-T7 antibody. In lanes 1 and 2, 25 µg untransfected and transfected cells, respectively, were directly loaded onto the gel.

 


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Fig. 5. Mapping the YB-1 domain involved in Tat interaction. (a) GST, GST-YB-1 full-length (1–318) and the various YB-1 mutants (as shown at the top of each lane) immobilized on Sepharose beads were incubated with 3 µl IVT 35S- labelled Tat protein. The bound Tat protein was eluted from the beads and analysed by SDS–PAGE and autoradiography. Lane 1 shows 1/10 of input in vitro reaction mixture. (b) Summary of YB- 1 binding to Tat and TAR.

 
Functional interaction of YB-1 and Tat on HIV-1 LTR promoter activity
The ability of YB-1 to interact with Tat and its target RNA sequence within TAR prompted us to examine the cooperative action of Tat and YB- 1 on HIV-1 promoter transcription. To further focus our attention to the TAR region of LTR, a reporter plasmid containing the CAT gene under the control of a minimal upstream regulatory sequence of LTR and the TAR region (nt -49 to +80, with respect to the transcription start site at +1) was utilized. Transcriptional activation of the reporter plasmid (p49/80-CAT) by HIV-1 Tat in the absence and presence of YB-1 was determined by co-transfection of human astrocytic U-87MG cells. In this experiment, the ability of full-length YB-1 [YB-1(1–318)], the mutant variant with binding ability to Tat and TAR [YB-1(1–203)], a mutant variant which showed reduced binding activity to Tat and no binding ability to TAR [YB-1(1–125)], and a mutant with no binding capacity to Tat and TAR [YB-1(1–37)], was examined for basal and Tat- mediated transcription of the minimal LTR promoter. As shown in Fig. 6, ectopic expression of full-length YB- 1 increased the basal transcription of the LTR in the transfected cells. Furthermore, at higher DNA concentrations, full-length YB-1 was able to increase the level of transcriptional activation of the LTR by Tat (Fig. 6a, compare lane 4 with lane 6). Expression of mutant YB-1(1–203), which binds to Tat and TAR in the transfected cells, caused an increase in basal and Tat- induced transcription of the LTR (Fig. 6b, compare lanes 1 and 3, and lanes 4 and 5). Mutant variant YB-1(1–125), which showed reduced binding activity to Tat but extremely weak, if any, binding to TAR, marginally increased the basal activity of the LTR and exhibited no stimulatory effect on Tat-mediated transcription of the LTR (Fig. 6c, compare lanes 1 and 3, and lanes 4 and 5). Mutant YB-1(1–37) with no ability to bind to Tat and TAR had no effect on basal and Tat-induced transcription of the viral promoter (Fig. 6d). Under similar conditions, the LTR reporter construct with no functional TAR region (nt -49 to +23) was not responsive to either Tat or full-length YB-1 (data not shown). Altogether, these observations suggest that to exert its positive effect on Tat function, YB-1 requires its Tat and TAR binding domains. Evidently, removal of the TAR binding domain, which abrogates the ability of YB-1 to interact with TAR but retains its Tat-binding activity, has a negative effect on Tat transactivation. This inhibition may result from sequestration of Tat by mutant YB-1 that, in turn, prevents Tat:TAR interaction and/or formation of a transcriptionally inactive complex containing mutant YB- 1, Tat and TAR. Under any scenario, one may question whether or not mutant YB-1 with Tat binding activity can be utilized to impair Tat function. This is an important notion as a large body of studies have ascribed an important pathogenic role for Tat during the course of the disease. Tat stimulates HIV-1 replication and alters expression of several regulatory cellular genes, including cytokines and immunomodulators, and activates other opportunistic infectious viral genes to exert a global effect on the pathogenesis of AIDS (van Baalen et al., 1997 ; Sanhadji et al., 1997 ). Therefore, the development of molecular therapeutics against Tat may have a dual beneficial effect by inhibiting the HIV-1 lytic cycle and by blocking the de-regulatory/stimulatory effects on cellular and viral genes.



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Fig. 6. Effect of YB-1 on Tat activation of the HIV-1 promoter. Approximately 1 µg reporter CAT construct containing the LTR sequence from -49 to +80 (transcription start site at +1) was introduced into glial cells either alone (lane 1) or together with various amounts of Tat and YB-1 expression plasmids as indicated in each part. In (a), full-length YB-1 was utilized whereas in ( b)–(d), YB-1 deletion mutants containing aa 1–203, 1–125 and 1–37, respectively, were utilized. The mean fold transactivation is shown for each condition as determined by scintillation counting of multiple experiments relative to the basal activity of one shown in lane 1.

 
In summary, results from our previous studies have revealed that YB- 1 can up-regulate the minimal promoter of HIV-1 through a Tat- responsive TAR element (Sawaya et al., 1998a ). Similarly to Tat, YB-1 specifically binds TAR RNA and its binding site spans the TAR bulge structure which is important for the Tat:TAR interaction. Interestingly, YB-1 also binds to Tat through a region which partially contains the amino acid residues important for its association with TAR.

Several TAR RNA binding proteins have been previously isolated and were shown to modulate HIV-1 LTR transcription by localizing to the loop and bulge region of TAR such as TRP-1, TRP-2 and BBP (bulge binding protein) (Baker et al., 1994 ; Wu et al. , 1991 ). In fact, efficient transcriptional elongation by RNAPII is mediated by recruitment of Tat and these cellular co- factors to TAR RNA (Wu-Baer et al., 1995 ). It is possible that binding to RNA is required by cellular co-factors to conformationally interact with the Tat transcriptional complex or vice versa. For example, the TAK (Tat-associated kinase) interacts with Tat to phosphorylate the RNAPII C-terminal domain and its cyclin T-CDK9 subunit confers novel TAR loop sequence specificity required by Tat (Wei et al., 1998 ; Yang et al., 1997 ). The results in this study provide evidence for the stimulatory effect of YB-1 on Tat transactivation. Although this effect may be mediated through the interaction of Tat with YB-1, however, the possibility exists that YB-1 may affect LTR activity by other mechanisms, i.e. influencing cellular gene expression. Currently, studies are in progress to evaluate the effect of YB-1 and its mutant variants on cyclin T and its partner kinase, CDK9, in the presence and absence of the TAR RNA sequence.


   Acknowledgments
 
We wish to thank past and present members of the Center for NeuroVirology and NeuroOncology for their support and sharing of reagents. We also wish to thank Cynthia Schriver for editorial assistance and preparation of the manuscript. This work was made possible by grants awarded by NIH to K.K. and S.A.


   References
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
Baker, B. , Muckenthaler, M. , Vives, E. , Blanchard, A. , Braddock, M. , Nacken, W. , Kingsman, A. J. & Kingsman, S. M. (1994). Identification of a novel HIV-1 TAR RNA bulge binding protein. Nucleic Acids Research 22, 3365-3372 .[Abstract]

Blair, W. S. , Fridell, R. A. & Cullen, B. R. (1996). Synergistic enhancement of both initiation and elongation by acidic transcription activation domains. EMBO Journal 15, 1658-1665 .[Abstract]

Calnan, B. J. , Biancalana, S. , Hudson, D. & Frankel, A. D. (1991). Analysis of arginine- rich peptides from the HIV Tat protein reveals unusual features of RNA–protein recognition. Genes & Development 5, 201-210.[Abstract]

Cullen, B. R. (1995). Regulation of HIV gene expression. AIDS 9 (suppl. A), 19–32.[Medline]

Deschamps, S. , Viel, A. , Garrigos, M. , Denis, H. & le Maire, M. (1992). mRNP4, a major mRNA-binding protein from Xenopus oocytes is identical to transcription factor FRG Y2. Journal of Biological Chemistry 267, 13799-13802 .[Abstract/Free Full Text]

Didier, D. , Schiffenbauer, J. , Woulfe, S. , Zacheis, M. & Schwartz, B. (1988). Characterization of the cDNA encoding a protein binding to the major histocompatibility complex class II Y box. Proceedings of the National Academy of Sciences, USA 85, 7322-7326 .[Abstract]

Dorn, A. , Bollekens, J. , Staub, A. , Benoist, C. & Mathis, D. (1987a). A multiplicity of CCAAT box-binding proteins. Cell 50, 863 -872.[Medline]

Dorn, A. , Durand, B. , Marfing, C. , Le Meur, M. , Benoist, C. & Mathis, D. (1987b). Conserved major histocompatibility complex class II boxes--X and Y--are transcriptional control elements and specifically bind nuclear proteins. Proceedings of the National Academy of Sciences, USA 84, 6249 -6253.[Abstract]

Emerman, M. & Malim, M. H. (1998). HIV-1 regulatory/accessory genes: key to unraveling viral and host cell biology. Science 280, 1880-1884 .[Abstract/Free Full Text]

Ensoli, B. , Barillari, G. , Salahuddin, S. Z. , Gallo, R. C. & Wong-Staal, F. (1990). Tat protein of HIV-1 stimulates growth of cells derived from Kaposi's sarcoma lesions of AIDS patients. Nature 345, 84-86.[Medline]

Gorman, C. M. , Moffat, L. F. & Howard, B. H. (1982). Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Molecular and Cellular Biology 2, 1044-1051.[Medline]

Graham, F. L. & van der Eb, A. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456-467.[Medline]

Hasegawa, S. L. , Doetsch, P. W. , Hamilton, K. K. , Martin, A. M. , Okenquist, S. A. , Lenz, J. & Boss, J. M. (1991). DNA binding properties of YB-1 and dbpA: binding to double-stranded, single-stranded, and abasic site containing DNAs. Nucleic Acids Research 19, 4915-4920 .[Abstract]

Jones, K. A. & Peterlin, B. M. (1994). Control of RNA initiation and elongation at the HIV-1 promoter. Annual Review of Biochemistry 63, 717-743.[Medline]

Kashanchi, F. , Duvall, J. F. , Dittmer, J. , Mireskandari, A. , Reid, R. L. , Gitlin, S. D. & Brady, J. N. (1994). Involvement of transcription factor YB-1 in human T-cell lymphotropic virus type I basal gene expression. Journal of Virology 68, 561-565.[Abstract]

Kerr, D. , Chang, C. F. , Chen, N. , Gallia, G. , Raj, G. , Schwartz, B. & Khalili, K. (1994). Transcription of a human neurotropic virus promoter in glial cells: effect of YB-1 on expression of the JC virus late gene. Journal of Virology 68, 7637-7643 .[Abstract]

Kingsman, S. M. & Kingsman, A. J. (1996). The regulation of human immunodeficiency virus type 1 gene expression. European Journal of Biochemistry 240, 491-507.[Abstract]

Kolluri, R. , Torrey, T. A. & Kinniburgh, A. J. (1992). A CT promoter element binding protein: definition of a double-strand and a novel single-strand DNA binding motif. Nucleic Acids Research 20, 111-116.[Abstract]

Ladomery, M. & Sommerville, J. (1994). Binding of Y-box proteins to RNA: involvement of different protein domains. Nucleic Acids Research 22, 5582-5589 .[Abstract]

Landsman, D. (1992). RNP-1, an RNA-binding motif is conserved in the DNA-binding cold shock domain. Nucleic Acids Research 20, 2861-2864 .[Abstract]

Lenz, J. , Okenquist, S. A. , LoSardo, J. E. , Hamilton, K. K. & Doetsch, P. W. (1990). Identification of a mammalian nuclear factor and human cDNA-encoded proteins that recognize DNA containing apurinic sites. Proceedings of the National Academy of Sciences, USA 87, 3396-3400 .[Abstract]

Luo, Y. , Madore, S. J. , Parslow, T. G. , Cullen, B. R. & Peterlin, B. M. (1993). Functional analysis of interactions between Tat and the trans-activation response element of human immunodeficiency virus type 1 in cells. Journal of Virology 67, 5617-5622 .[Abstract]

Minich, W. B. , Maidebura, I. P. & Ovchinnikov, L. P. (1993). Purification and characterization of the major 50-kDa repressor protein from cytoplasmic mRNP of rabbit reticulocytes. European Journal of Biochemistry 212, 633-638.[Abstract]

Murray, M. T. , Schiller, D. L. & Franke, W. W. (1992). Sequence analysis of cytoplasmic mRNA-binding proteins of Xenopus oocytes identifies a family of RNA-binding proteins. Proceedings of the National Academy of Sciences, USA 89, 11-15.[Abstract]

Ozer, J. , Faber, M. , Chalkley, R. & Sealy, L. (1990). Isolation and characterization of a cDNA clone for the CCAAT transcription factor EFIA reveals a novel structural motif. Journal of Biological Chemistry 265, 22143-22152 .[Abstract/Free Full Text]

Rhim, H. , Echetebu, C. O. , Herrmann, C. H. & Rice, A. P. (1994). Wild-type and mutant HIV-1 and HIV-2 Tat proteins expressed in Escherichia coli as fusions with glutathione S-transferase. Journal of Acquired Immune Deficiency Syndromes 7, 1116-1121.[Medline]

Rounseville, M. P. & Kumar, A. (1992). Binding of a host cell nuclear protein to the stem region of human immunodeficiency virus type 1 trans- activation-responsive RNA. Journal of Virology 66, 1688-1694 .[Abstract]

Sakura, H. , Maekawa, T. , Imamoto, F. , Yasuda, K. & Ishii, S. (1988). Two human genes isolated by a novel method encode DNA-binding proteins containing a common region of homology. Gene 73, 499-507.[Medline]

Sanhadji, K. , Leissner, P. , Firouzi, R. , Pelloquin, F. , Kehrli, L. , Marigliano, M. , Calenda, V. , Ottman, M. , Tardy, J. C. , Mehtali, M. & Touraine, J. L. (1997). Experimental gene therapy: the transfer of Tat-inducible interferon genes protects human cells against HIV-1 challenge in vitro and in vivo in severe combined immunodeficient mice. AIDS 11, 977-986.[Medline]

Sawaya, B. E. , Khalili, K. & Amini, S. (1998a). Transcription of the human immunodeficiency virus type 1 (HIV-1) promoter in central nervous system cells: effect of YB-1 on expression of the HIV-1 long terminal repeat. Journal of General Virology 79, 239 -246.[Abstract]

Sawaya, B. E. , Khalili, K. , Mercer, W. E. , Denisova, L. & Amini, S. (1998b). Cooperative actions of HIV-1 Vpr and p53 modulate viral gene transcription. Journal of Biological Chemistry 273, 20052 -20057.[Abstract/Free Full Text]

Sheline, C. T. , Milocco, L. H. & Jones, K. A. (1991). Two distinct nuclear transcription factors recognize loop and bulge residues of the HIV-1 TAR RNA hairpin. Genes & Development 5, 2508-2520.[Abstract]

Shen, Q. , Wu, R. , Leonard, J. L. & Newburger, P. E. (1998). Identification and molecular cloning of a human selenocysteine insertion sequence-binding protein. A bifunctional role for DNA-binding protein B. Journal of Biological Chemistry 273, 5443-5446 .[Abstract/Free Full Text]

Smith, D. B. & Johnson, K. S. (1988). Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67, 31-40.[Medline]

Sommerville, J. & Ladomery, M. (1996). Masking of mRNA by Y-box proteins. FASEB Journal 10, 435-443.[Abstract/Free Full Text]

Tan, R. & Frankel, A. D. (1995). Structural variety of arginine-rich RNA-binding peptides. Proceedings of the National Academy of Sciences, USA 92, 5282-5286 .[Abstract]

van Baalen, C. A. , Pontesilli, O. , Huisman, R. C. , Geretti, A. M. , Klein, M. R. , de Wolf, F. , Miedema, F. , Gruters, R. A. & Osterhaus, A. D. M. E. (1997). Human immunodeficiency virus type 1 Rev- and Tat-specific cytotoxic T lymphocyte frequencies inversely correlate with rapid progression to AIDS. Journal of General Virology 78, 1913-1918 .[Abstract]

Wei, P. , Garber, M. E. , Fang, S. M. , Fischer, W. H. & Jones, K. A. (1998). A novel CDK9- associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA. Cell 92, 451-462.[Medline]

Wolffe, A. P. (1994). Structural and functional properties of the evolutionarily ancient Y-box family of nucleic acid binding proteins. Bioessays 16, 245-251.[Medline]

Wong, M. W. , Henry, R. W. , Ma, B. , Kobayashi, R. , Klages, N. , Matthias, P. , Strubin, M. & Hernandez, N. (1998). The large subunit of basal transcription factor SNAPc is a myb domain protein that interacts with Oct-1. Molecular and Cellular Biology 18, 368-377.[Abstract/Free Full Text]

Wu, F. , Garcia, J. , Sigman, D. & Gaynor, R. (1991). tat regulates binding of the human immunodeficiency virus trans-activating region RNA loop-binding protein TRP-185. Genes & Development 5, 2128-2140.[Abstract]

Wu-Baer, F. , Sigman, D. & Gaynor, R. B. (1995). Specific binding of RNA polymerase II to the human immunodeficiency virus trans-activating region RNA is regulated by cellular cofactors and Tat. Proceedings of the National Academy of Sciences, USA 92, 7153-7157 .[Abstract]

Yang, X. , Gold, M. O. , Tang, D. N. , Lewis, D. E. , Aguilar-Cordova, E. , Rice, A. P. & Herrmann, C. H. (1997). TAK, an HIV Tat- associated kinase, is a member of the cyclin-dependent family of protein kinases and is induced by activation of peripheral blood lymphocytes and differentiation of promonocytic cell lines. Proceedings of the National Academy of Sciences, USA 94, 12331-12336 .[Abstract/Free Full Text]

Received 6 January 1999; accepted 17 June 1999.