Coordinate effects of human immunodeficiency virus type 1 protein Tat and cellular protein Pur{alpha} on DNA replication initiated at the JC virus origin

Dianne C. Daniel1, Margaret J. Wortman1, Robin J. Schiller1, Hong Liu1, Li Gan1, Jonathan S. Mellen1, Chun-F. Chang2, Gary L. Gallia2, Jay Rappaport2, Kamel Khalili2 and Edward M. Johnson1

Department of Pathology, Department of Molecular Biology and Biochemistry and the D. H. Ruttenberg Cancer Center, Box 1194, Mount Sinai School of Medicine, New York, NY 10029, USA1
Center for Neurovirology and Cancer Biology, Temple University, Bio-Life Sciences Building, 1900 N. 12th Street, Philadelphia, PA 19122, USA2

Author for correspondence: Edward M. Johnson. Fax +1 212 534 7491. e-mail edward.johnson{at}mssm.edu


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
JC virus (JCV) causes progressive multifocal leukoencephalopathy, a demyelinating disease in brains of individuals with AIDS. Previous work has shown that the Tat protein, encoded by human immunodeficiency virus type 1 (HIV-1), can interact with cellular protein Pur{alpha} to enhance both TAR-dependent HIV-1 transcription and JCV late gene transcription. Tat has been shown to activate JCV transcription through interaction with Pur{alpha}, which binds to promoter sequence elements near the JCV origin of replication. DNA footprinting has shown that Pur{alpha} and large T-antigen cooperatively interact at several binding sites in the origin and transcriptional control region. Overexpression of Pur{alpha} inhibits replication initiated at the JCV origin by T-antigen. In transfected glial cells Tat reversed this inhibition and enhanced DNA replication. In an in vitro replication system maximal activation by Tat, more than sixfold the levels achieved with T-antigen alone, was achieved in the presence of Pur{alpha}. Effects of mutant Tat proteins on both activation of replication and binding to Pur{alpha} have revealed that Cys22 exerts a conformational effect that affects both activities. The origin of an archetypal strain of JCV was less susceptible to activation of replication by Tat relative to the rearranged Mad-1 strain. These results have revealed a previously undocumented role for Tat in DNA replication and have indicated a regulatory role for JCV origin auxiliary sequences in replication and activation by Tat.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
The circular DNA papovavirus JC (JCV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a demyelinating disease resulting from destruction of infected oligodendrocytes in the brain (for review see Berger & Major, 1999 ). Although more than 90% of adults demonstrate antibodies to JCV, only a small percentage of immunosuppressed people develop PML. With the onset of the AIDS epidemic PML has become prominent, and it is now found in approximately 4% of all AIDS cases (Berger & Major, 1999 ). Since the incidence of PML in AIDS is particularly high (Berger et al., 1987 ; Stoner et al., 1986 ), the hypothesis has been put forth that human immunodeficiency virus type 1 (HIV-1) may directly influence infection by JCV (Krachmarov et al., 1996 ; Tada et al., 1990 ). HIV-1 infects primarily microglial cells and astrocytes in the brain (Bagasra et al., 1996 ; Tornatore et al., 1994 ), while JCV infects primarily oligodendrocytes (ZuRhein & Chou, 1965 ). There is no evidence for coinfection of cells by the two viruses. Local, severe HIV encephalitis has previously been observed in PML lesions (Vazeux et al., 1990 ), highlighting the close proximity of cells infected by the two viruses. Although productive HIV infection is not always associated with PML lesions, it is known that HIV-1 can infect astrocytes in a limited fashion, and that early proteins, including Tat, may be produced in the absence of virus particles (Shahabuddin et al., 1996 ). The strains of JCV identified in the brains of individuals with PML are distinct from an archetypal strain frequently detected in the blood or urine of individuals without disease. While multiple JCV strains have now been detected in PML tissue, the patterns of difference from the archetypal strain are similar, all involving rearrangements in the late promoter side of the origin of replication. Little is known about the apparent reactivation of JCV in the brain in AIDS patients, but the observed sequence rearrangements may well play a prominent role. As does its kindred papovavirus, SV40, JCV relies on its virally encoded large T-antigen for initiation of replication. JCV T-antigen is highly homologous to the SV40 counterpart, and primary T-antigen binding sites in both viruses are also similar. Research has identified flanking sequences critical for initiation at the JCV origin. In particular a repeated pentanucleotide, AGGGA, at the late side of the origin of the Mad-1 strain of JCV is critical for maximal replication initiated by the JCV T-antigen (Chang et al., 1994 ; Lynch & Frisque, 1991 ). It has been reported that this element exists in a non-B DNA configuration (Amirhaeri et al., 1988 ). The pentanucleotide, with its G triplet repeats, is bound avidly by Pur{alpha} (Chen et al., 1995 ). Both pentanucleotide repeats are adjacent to an A–T tract, also critical for initiation (Lynch & Frisque, 1990 ). This arrangement, while repeated twice at the Mad-1 origin, is absent from that of the archetypal strain.

A variety of cellular proteins have been identified which bind to Tat (Desai et al., 1991 ; Jeang et al., 1993 ; Kashanchi et al., 1994 ; Ohana et al., 1993 ; Taylor et al., 1994 ; Yu et al., 1995 ), including cyclin T1, the activator of Cdk9, the PITALRE kinase capable of phosphorylating the C-terminal domain of RNA polymerase II (Mancebo et al., 1997 ; Wei et al., 1998 ). In addition, Tat binds the ubiquitous cellular single-stranded DNA- and RNA-binding protein Pur{alpha}. Pur{alpha} has been observed to bind the HIV-1 TAR RNA element, at a site distinct from that at which Tat binds the element, and to activate HIV-1 transcription in a TAR-dependent manner (Chepenik et al., 1998 ). Recently Tat has been colocalized with Pur{alpha} in nuclei of cultured human glial cells constitutively producing both proteins (Wortman et al., 2000 ). Tat has been shown to activate transcription at the major late promoter of JCV through its interaction with Pur{alpha} (Chen et al., 1995 ). Tat does not itself bind to JCV DNA, but Tat and Pur{alpha} together bind to PUR elements and synergistically activate transcription (Krachmarov et al., 1996 ). These PUR elements are located in and near the JCV origin of DNA replication, where Pur{alpha} and large T-antigen interact to influence binding of both proteins (Chen et al., 1995 ). Tat can freely traverse cell membranes and enter adjacent cells in a capacity to alter gene activity (Ensoli et al., 1990 , 1993 ; Ezhevsky et al., 1997 ; Frankel & Pabo, 1988 ; Hofman et al., 1993 ; Schwarze et al., 1999 ). We have asked here whether the Tat–Pur{alpha}–DNA interaction could affect not only gene transcription but DNA replication as well. Results have demonstrated that Tat stimulates replication initiated at the JCV origin both in vitro and in vivo, displaying a heretofore unknown activity of this pathogenic protein.


   Methods
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Introduction
Methods
Results
Discussion
References
 
{blacksquare} JCV DNA replication in transfected human glial cells.
Plates of 1·5x106 U-87MG human astrocytic glial cells were transfected with 3 µg of plasmid pBLCAT3-Mad1L, containing a 388 bp segment from the JCV Mad-1 strain comprising the origin of replication, as described previously (Chang et al., 1996 ). Alternatively, transfection was performed with a control plasmid, pBLCAT3, with no insert. The following additional plasmids were cotransfected as indicated: pJCT, expressing JCV large T-antigen; pCMV-Pur{alpha}, expressing the cellular protein Pur{alpha}; and pTat, expressing the HIV Tat protein. All transfected plates were balanced to the same concentration of total plasmid DNA using respective empty vectors. At 72 h after transfection, plasmid DNA was recovered by the method of Hirt (1967) as previously modified (Johnson & Jelinek, 1986 ). This method involves extraction of total cellular DNA in SDS and selective removal of chromosomal DNA by precipitation with NaCl. Recovered DNA was treated with restriction endonucleases SacI and DpnI, and subjected to agarose gel electrophoresis as described previously (Chang et al., 1996 ). DpnI-resistant bands were detected in gel blots by hybridization to the origin insert labelled with [32P]phosphate. Intensities of fully DpnI-resistant SacI bands, representing replicated DNA, were obtained using Imagequant 1.2 and analysed using Photoshop 5.0.

{blacksquare} JCV DNA replication in vitro.
Plasmid DNA used for templates in the replication reaction were prepared without exposure to phenol, ethidium or UV light to minimize nicking and allow for high yields of highly supercoiled DNA. Plasmids were propagated in E. coli strain XL-1 Blue (Stratagene). After alkaline lysis, plasmid DNA was isolated using the Qiagen Maxi Prep kit. Plasmids used consisted of >95% supercoiled DNA. Plasmids, described in detail in the text, were pBLCAT3-Mad-1L, containing the Mad-1 strain origin of replication, pJCV archetype, containing the archetypal origin in the same vector as that for the Mad-1 origin, and pBLCAT3 with no insert. Replication in vitro of plasmid DNA was carried out in 25 µl reaction vols containing 30 mM HEPES buffer, pH 7·5, 7 mM MgCl2, 4 mM ATP, 100 µM each of dATP, dGTP, dCTP, TTP, 50 µM each of GTP, CTP, UTP, 40 mM phosphocreatine, 0·625 units of creatine phosphokinase, 400 ng of plasmid DNA, 9·0 µl HeLa cell extract (CHIMERx, 240 µg protein, added last to begin the reaction) and 1·0 µCi of [{alpha}-32P]dCTP (New England Nuclear; 3000 Ci/mmol). JCV T-antigen was prepared from extracts of Sf9 cells infected with baculovirus vector bearing the T-antigen gene and purified by immunoaffinity chromatography. In certain experiments SV40 T-antigen (CHIMERx) was substituted with no noticeable effect. Glutathione S-transferase (GST)–Pur{alpha} and GST–Tat were purified as previously described (Johnson et al., 1995 ). After 2·5 h at 37 °C, reactions were stopped by addition of 200 µl of 10 mM EDTA with 2·0 µg yeast tRNA. After three extractions with phenol–chloroform:isoamyl alcohol (50:49:1) and two extractions with ice-cold diethyl ether, DNA was precipitated with 3 vols of 95% ethanol, 0·2 M sodium acetate. DNA was redissolved for treatment with restriction endonucleases HindIII and DpnI and subjected to electrophoresis on a 1·4% agarose gel (Daniel & Johnson, 1989 ). The gel was dried, and radioactivity was detected using a Molecular Dynamics Phosphor 860 phosphorimager. Quantitative comparison of band intensities was performed as described above. Incorporation of 32P into a labelled reference band was determined by scintillation spectrometry. Comparison of band intensities with this reference band were used to calculate [32P]dCMP incorporation.

{blacksquare} Binding of Tat or Tat mutant proteins to Pur{alpha}.
HIV-1 Tat proteins were bacterially produced as GST fusion proteins and coupled to glutathione–agarose beads. Beads coupled to equimolar amounts of Tat or each of the Tat mutants were reacted with a 20-fold excess of Pur{alpha} (2x10-7 M), derived by thrombin cleavage of GST–Pur{alpha}. GST–Tat is not cleaved by thrombin in the binding buffer (50 mM Tris–HCl, pH 7·4, 150 mM NaCl, 50 mM NaF, 5·0 mM EDTA, 0·1% Nonidet P-40, 1·0 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin). After binding for 30 min and washing with binding buffer (Johnson et al., 1995 ), proteins were extracted from beads in SDS sample buffer, subjected to SDS–PAGE on a 10% gel, blotted to an Immobilon P membrane and probed with anti-Pur{alpha} monoclonal antibody 9C12. Detection was with the Pierce SuperSignal Enhancer system.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Enhancement by Tat of replication initiated at the JCV origin in human glial cells and reversal of an inhibitory effect of Pur{alpha}
Previous studies had demonstrated a pronounced inhibitory effect of Pur{alpha} overexpression upon replication initiated at the JCV origin in U-87MG human glial cells (Chang et al., 1996 ). Since Tat is known to bind to Pur{alpha} (Chepenik et al., 1998 ; Gallia et al., 1998 , 1999b ; Krachmarov et al., 1996 ; Wortman et al., 2000 ), and to alter its in vivo functions (Krachmarov et al., 1996 ), we sought to determine whether Tat modulates the replicative effect of Pur{alpha}. The effect of Tat upon replication initiated at the JCV origin in human cells was examined in vivo using human U-87MG glial cells transfected with plasmid pBLCAT3-Mad1L, bearing the JCV origin of replication (Fig. 1). The columns of the histogram were derived from densitometry of DpnI-resistant bands from a SacI digest of pBLCAT3-Mad1L, as revealed by hybridization. The first column, representing a relative density of zero, shows that replication did not occur in the absence of a plasmid, pJCT, expressing JCV large T-antigen. Overexpression of Pur{alpha} in these cells inhibited plasmid replication (columns labelled Pur{alpha}). Since high Pur{alpha} levels in G1 have also been observed to block the CV-1 cell cycle at the G1–S boundary (Stacey et al., 1999 ), it is conceivable that inhibition of the onset of replication is a cellular function of Pur{alpha}. However, U-87MG cells possess endogenous Pur{alpha}, and it is thus also conceivable that the inhibition does not represent a replicative function of the protein. Instead, overexpressed Pur{alpha} in the absence of partner proteins such as Tat, could exert a squelching effect by drawing associated proteins, such as cyclin A (Itoh et al., 1998 ), away from their site of action in replication. In any case, Tat expression reversed the inhibition by Pur{alpha} to levels seen with T-antigen alone (columns labelled Tat + Pur{alpha}). Expression of Tat in the glial cells yielded a dose-dependent activation of replication of two- to threefold under these transfection conditions. It may be notable that at the highest inhibitory dose of Pur{alpha}, stimulation by Tat was >20-fold. It is not known at precisely what levels Pur{alpha} is present in oligodendrocytes of the brain. However, it is clear that under all conditions used here Tat strongly enhanced replication initiated at the JCV origin.



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Fig. 1. Stimulation of DNA replication initiated at the JCV origin by the HIV-1 protein Tat in vivo. Plates of U-87MG human astrocytic glial cells were transfected with plasmid pBLCAT3-Mad1L as described in Methods. The following additional plasmids were cotransfected as indicated: pJCT, expressing JCV large T-antigen (10 µg); pCMV-Pur{alpha}, expressing the cellular protein Pur{alpha} (10 and 20 µg); and pTat, expressing the HIV Tat protein (5 and 10 µg). Intensities of fully DpnI-resistant SacI bands, representing replicated DNA, were determined as described in Methods. Relative band intensities were determined by normalizing to the lowest detectable band intensity, i.e. that obtained using 20 µg of pCMV-Pur{alpha} and no pTat, by assigning that intensity a value of 1. The relative intensities are presented in a histogram. T-antigen (pJCT) was omitted from the transfection shown in the first column of the histogram and was included in transfections for all other columns.

 
One caveat to be considered here is the possibility that Tat could influence replication through an ability to enhance expression of T-antigen from the pJCT vector thereby eliciting the appearance of enhancing replication. We have previously ruled out this possibility by assaying levels of T-antigen in transfected U-87MG cells via immunoblotting. In that experiment Tat exerted no such effect on the T-antigen expression vector (Chang et al., 1996 ). It is likely that Tat functions indirectly in that Tat does not directly bind to JCV DNA. To more precisely dissect the mechanism by which Tat exerts its effects on replication, we have employed an in vitro JCV DNA replication system.

Maximal stimulation of JCV replication in vitro by HIV-1 Tat in the presence of cellular Pur{alpha}
An in vitro system was employed to investigate whether Tat can directly influence JCV DNA replication. A system utilizing HeLa cell extracts has recently been shown to effectively replicate plasmids bearing a JCV origin (Nesper et al., 1997 ). Using a similar system it was affirmed in Fig. 2 that replication depends upon the presence of JCV T-antigen and upon the presence of a JCV origin of replication. The origin used for Fig. 2 was a 401 bp segment from the Mad-1 strain of JCV, a strain representative of rearranged strains frequently detected in brains of AIDS patients (Major et al., 1992 ; Newman & Frisque, 1997 ). In these experiments 32P-labelled plasmid DNA recovered from the replication reaction was linearized with HindIII and treated with DpnI. Resistance to restriction endonuclease DpnI is conferred upon plasmid DNA, propagated in dam+ strains of E. coli, upon replication in a mammalian system. Slight incorporation into DpnI-cleaved bands in the absence of T-antigen was due to repair activities and artefactual nick translation. Only the topmost band, 4·3 kb, representing full-length DpnI-resistant DNA was taken as a measure of replication. There was no incorporation into this band in the absence of T-antigen. In the presence of T-antigen incorporation was seen only when the plasmid template contained a JCV origin of replication.



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Fig. 2. Initiation of JCV DNA replication in vitro: requirement for T-antigen and an intact origin sequence. DNA replication reactions were carried out using an in vitro system employing HeLa cell extracts, and treatment with HindIII and DpnI was employed to assay for 32P-labelled, replicated DNA as described in Methods. A DpnI-resistant band at 4·3 kb represents fully replicated DNA. Plasmids subjected to the reaction were 400 ng of either pBLCAT3 or pBLCAT3 containing the Mad-1 strain origin (JCV-Mad1L). Reactions were performed using either 0 or 1·0 µg of T-antigen, purified as described.

 
It has previously been reported that Tat effects on JCV late gene transcription are mediated through PUR elements in the promoter (Chowdhury et al., 1993 ; Krachmarov et al., 1996 ). Tat alters the ability of Pur{alpha} to bind these elements (Krachmarov et al., 1996 ), which are also part of the origin of replication. Footprinting has detailed the binding of Pur{alpha} and T-antigen to these elements in the Mad-1 promoter (Chen et al., 1995 ), certain of which are reported to be critical for JCV DNA replication (Chang et al., 1994 ; Lynch & Frisque, 1990 ). Therefore, we have examined the effects of modulating either of these proteins upon replication initiated at the Mad-1 origin in vitro. Pur{alpha} alone could not substitute for T-antigen in ability to initiate DNA replication. These data, constituting blank lanes for different doses of Pur{alpha}, are not shown. This control was necessary since PUR elements overlap T-antigen binding sites in the JCV origin (Chang et al., 1996 ). In Fig. 3 the effects of Tat and Pur{alpha}, either separately or together, upon replication initiated at the JCV origin are presented. Fig. 3(A) shows the effects of varying doses of GST–Tat upon replication with a constant level of GST–Pur{alpha}. An incremental stimulation of replication by Tat can be seen, beginning at 4x10-10 M, peaking at 4x10-8 M and persisting to 10-7 M. At that point molar levels of Pur{alpha} and Tat differ by less than an order of magnitude. Tat exerted maximal effects on replication at levels approximately five- to tenfold higher than that of plasmid DNA (10-8 M). At concentrations above 10-7 M effects of Tat in the presence of Pur{alpha} were markedly inhibitory (Fig. 3A, two rightmost lanes). The maximal stimulation by Tat together with Pur{alpha} vs T-antigen alone (lane 2) in this experiment was more than sixfold, as determined by densitometry of the 4·3 kb bands. Fig. 3(B) shows the effects of Tat and Pur{alpha} separately upon replication initiated by T-antigen. The autoradiograph for Fig. 3(B) was exposed longer than that for Fig. 3(A). This is evident by comparing the two leftmost lanes in Fig. 3(A) with the two lanes labelled 0 in Fig. 3(B), which represent the effects of T-antigen alone. In both (A) and (B) there is a stimulation of replication of more than twofold by 3x10-7 M Pur{alpha} in the absence of Tat. Stimulation persists to 7.5x10-7 M, but at levels higher than 10-6 M, Pur{alpha} is strikingly inhibitory. Given the enhanced exposure of the autoradiograph in Fig. 3(B), stimulation by Pur{alpha} alone is much less than that by Tat and Pur{alpha} together. This is further documented in Table 1, which provides quantitative data on effects of T-antigen, Tat and Pur{alpha} on replication initiated at the JCV origin at a variety of protein concentrations. Table 1 also provides an important control for the in vitro replication reactions: it documents the lack of effect of GST alone on the replication reaction. The right lanes of Fig. 3(B) show the effects of Tat on replication in the absence of added Pur{alpha}, and they reveal two important aspects of Tat activity. Firstly, there is only a slight effect of Tat on replication in the absence of added Pur{alpha}. This may not be clearly evident in the published photograph, but band densitometry indicates a 1·2-fold stimulation by 10-7 M Tat. This is vastly less than the approximately sixfold stimulation by that concentration of Tat in the presence of Pur{alpha}. The stimulation of replication by the two proteins is synergistic since the effects of 10-7 M Tat and 3x10-7 M Pur{alpha} together, nearly sixfold stimulation, are more than multiplicative of the effects of those concentrations of each protein alone, approximately 1·2-fold and twofold, respectively. Secondly, the effect of Tat alone at concentrations >10-7 M is not inhibitory to replication (Fig. 3B, two rightmost lanes), whereas those concentrations in the presence of Pur{alpha} are markedly inhibitory (Fig. 3A, two rightmost lanes). The effect of high concentrations of Tat in the presence of Pur{alpha} mimics the effect of higher concentrations of Pur{alpha} alone (Fig. 3B, second and third lanes). This is further evidence for a cooperative interaction of Tat and Pur{alpha}. In this and all other experiments purified bacterial GST was employed as a control. GST had only a slightly inhibitory effect upon labelling of the DNA in the presence of T-antigen (Table 1). The ability of Tat to affect replication at very low concentrations lends credence to the notion that Tat may have this effect in the brain. The effects of Tat and Pur{alpha} in the in vitro system may well reflect the effects of the two proteins in transfected cells (Fig. 1). The effects of high concentrations of Pur{alpha} are inhibitory to replication in vitro, as are the effects of overexpressed Pur{alpha} in the U-87MG cells. It must be cautioned, however, that the precise concentration of a protein at a location of activity in a cell cannot be determined due to compartmentalization.



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Fig. 3. Effects of Tat and Pur{alpha}, separately or coordinately, on JCV DNA replication in vitro. The in vitro JCV DNA replication system was employed as described in Methods and the legend to Fig. 2. Labelled DNA was treated with HindIII and DpnI as described. A DpnI-resistant band at 4·3 kb represents fully replicated plasmid DNA. (A) Effects of Tat on JCV DNA replication in the presence of added Pur{alpha}. Replication reactions were performed using either no T-antigen (left lane) or 1·0 µg of T-antigen (lanes labelled +). In the left three lanes Tat was absent, and GST–Pur{alpha} was either absent (lanes 1 and 2) or present at 3x10-7 M (lanes labelled +). In the seven rightmost lanes T-antigen was present, GST–Pur{alpha} was constant at 3x10-7 M, and GST–Tat was increased as follows: 4x10-10 M, 4x10-9 M, 2x10-8 M, 4x10-8 M, 10-7 M, 3x10-7 M and 6x10-7 M. (B) Effects of Tat and Pur{alpha}, added separately, on JCV DNA replication in vitro. Replication reactions were performed using either no T-antigen (left lane) or 1·0 µg of T-antigen (lanes labelled +). In the two lanes labelled 0, neither Tat nor Pur{alpha} were added. Note that DNA replication is evident in the centre 0 lane due to the presence of T-antigen. To the left of the centre 0 lane, Pur{alpha} concentrations are increased as follows: 3x10-7 M, 7·5x10-7 M, 1·5x10-6 M, 2·7x10-6 M. To the right of the centre 0 lane, Tat concentrations are increased as follows: 4x10-9 M, 2x10-8 M, 10-7  M, 3x10-7 M and 6x10-7 M.

 

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Table 1. Effects of Tat and Pur{alpha} on JCV DNA replication in vitro

 
In the absence of Tat and Pur{alpha} the level of incorporation of dCMP into DpnI-resistant plasmid DNA, 3·2 pmol, translates into a level of 32 pmol dNMP per µg of input plasmid DNA. This is very comparable to levels of incorporation reported by Nesper et al. (1997) , who also employed a system using HeLa cell extracts and JCV T-antigen. Levels of incorporation reported here are also quite comparable to those reported for the original SV40 DNA in vitro replication system (Li & Kelly, 1984 ).

It is helpful to know the endogenous level of Pur{alpha} in the HeLa cell extract since this would form a background for effects of added Pur{alpha}. In HeLa cells the levels of Pur{alpha} fluctuate dramatically during the cell cycle (Itoh et al., 1998 ). In an asynchronous culture, in which most cells would be in late G1, a time when Pur{alpha} levels are relatively low, it can be estimated that the intracellular level of the protein is approximately 10-9 M. This previously published estimate is based on relative intensities of gel bands in immunoblots (Itoh et al., 1998 ), and it is undoubtedly crude. It is unlikely, however, to be in error by more than tenfold. Thus, the level of Pur{alpha} in the HeLa cell extract employed for in vitro replication is likely to be low relative to the levels of GST–Pur{alpha} and GST–Tat employed in the study.

Effects of mutant Tat proteins on JCV replication in vitro and on binding to Pur{alpha}
A series of deletion and point mutations of Tat were used to examine the contributions of different Tat domains to both Pur{alpha} binding and Tat’s replicative effects. The method of standardizing molar concentrations of the mutant Tat proteins has been described in two previous publications, which are in good agreement regarding effects of the mutations on Tat binding to Pur{alpha} (Gallia et al., 1999a ; Wortman et al., 2000 ). The bottom panel of Fig. 4 presents Pur{alpha} binding to different GST–Tat mutants immobilized on glutathione–agarose beads, and the top panel presents effects of purified GST–Tat and its mutants on replication initiated in vitro at the JCV origin. Since wild-type Tat with a deletion of aa 2–36, Tat86{Delta}(2–36), bound Pur{alpha}, as did Tat48, albeit weakly, Tat amino acids from 37–48 were critical for binding to Pur{alpha}. There was no Tat mutation that restricted Pur{alpha} binding while still allowing enhanced replication of the JCV origin-bearing plasmid. As seen in the top panel of Fig. 4, and consistent with Fig. 3(A), full-length, wild-type Tat86 produced a dramatic stimulation of replication of the JCV origin-bearing plasmid. Intriguingly, all of the examined Tat mutants inhibited overall replication, but to different degrees. The reason for such inhibition is not known, but it reflects the ability of different domains in the Tat protein to affect the replicative process. Both replicative effects of Tat and Tat ability to bind Pur{alpha} were influenced by a global, conformational effect of Tat aa C22. When C22 was mutated to G in either Tat72C22->G or Tat48C22->G, the resulting protein was especially inhibitory to replication. Consistent with mediation of Tat replicative effects through a Tat interaction with Pur{alpha} is the observation that when C22 was mutated in Tat48 it was more detrimental to both Pur{alpha} binding and JCV DNA replication than when C22 was mutated in Tat72. It is likely that presence of C22 induced a global change in Tat that helped configure it for Pur{alpha} binding. This configuration could also be induced when the entire amino terminal region was deleted, as in Tat86{Delta}(2–36). This mutation still allowed Pur{alpha} binding although it diminished Tat replicative effects. This may indicate that amino acids in the region 2–36 promoted effects on replication complementary to effects requiring Pur{alpha} binding. Furthermore, C22 may have, through a conformational effect, influenced Pur{alpha} binding without actually contacting Pur{alpha}. While a global effect of C22 is important for both Pur{alpha} binding and replicative effects of Tat, it is clear that Tat domains other than those involved in Pur{alpha} binding are also critical for these effects. Tat72 bound Pur{alpha} very well whereas this deletion was detrimental to DNA replication. It is reasonable that certain domains of Tat could interact with Pur{alpha} while other domains would remain accessible to interact with additional proteins or RNA that could influence the replication apparatus. It has been reported that Tat binding to transcription factor TFIID is dependent upon Tat residues 36–50 (Kashanchi et al., 1994 ). The critical role of C22 in influencing Pur{alpha} binding may play a role not only in effects of Pur{alpha} on JCV DNA replication, but also in observed effects of Pur{alpha} on HIV-1 transcription (Chepenik et al., 1998 ). It is notable that while Tat72 was capable of HIV-1 transcriptional activation, Tat72C22->G was not (Rhim et al., 1994 ). The specific importance of C22 is further emphasized by the observation that whereas Tat48 bound transcription factor TFIIH in vitro, Tat48C22->G did not (Parada & Roeder, 1996 ).



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Fig. 4. Effects of Tat mutations on the ability of Tat to bind to Pur{alpha} and to enhance replication initiated at the JCV origin. Top, mutational analysis of Tat protein domains involved in activation of replication initiated at the JCV origin. Replication reactions were performed as described for Fig. 2 in the presence of 1·0 µg of JCV T-antigen and 150 ng of Pur{alpha} (10-7 M). No Tat was added in lane 1. In the remaining lanes GST–Tat (Tat86) or GST–Tat mutants were present at the same molar concentration of 1·3x10-7 M, corresponding to 127 ng of wt Tat. Bottom, mutational analysis of Tat protein domains involved in binding to Pur{alpha}. Binding of Pur{alpha} to Tat or its mutant proteins fused to GST and immobilized on glutathione–agarose beads was performed as described in Methods. Shown is an immunoblot of Pur{alpha} eluted from the beads. For comparison, lanes showing mutant Tat protein effects on replication are positioned directly above respective lanes showing effects on binding to Pur{alpha}.

 
Effects of rearrangements in JCV origin sequences upon enhancement of replication by Tat
Tat exerted a differential effect upon replication of Mad-1 vs archetype strains of JCV. Due to the nature of sequence differences between strains, it is generally believed that Mad-1 and other strains found in PML have arisen from the archetype through rearrangement. Detection of both archetype and several rearranged variants of JCV in multiple tissues from a paediatric PML patient have raised the question of whether archetype and rearranged strains of JCV have different replication capabilities in different tissues (Newman & Frisque, 1997 ). In Fig. 5(A) a comparison is presented of the abilities of Tat and Pur{alpha} to enhance replication initiated at either the Mad-1 or archetypal origin in the in vitro system. For this figure a low concentration of T-antigen was used to insure that both plasmids would be in the linear response range. The two origins have previously been shown to sustain T-antigen-dependent replication in glial cells (Ault, 1997 ). It can be seen in Fig. 5(A) that both origins initiated full-length plasmid replication in the presence of T-antigen. Replication initiated at both origins was enhanced by Tat and Pur{alpha}. However, in the presence of Pur{alpha} the Mad-1 origin responded dramatically to the presence of Tat (7·2-fold stimulation vs Pur{alpha} alone), while the archetype origin responded weakly (1·6-fold stimulation vs 450 ng of Pur{alpha} alone) as revealed by densitometry of the bands at 4·3 kb in Fig. 5(A). This experiment indicates that Tat and Pur{alpha} affect initiation, rather than some aspect of elongation, since the two plasmids are of nearly the same size and since vector sequences outside the origins are identical. Nonetheless, further experiments will be necessary to determine whether effects are on initial unwinding or on entry of proteins comprising the replication apparatus. This result highlights the importance of JCV origin auxiliary sequences to overall replication.



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Fig. 5. Differential effects of Tat and Pur{alpha} on DNA replication initiated at origins of Mad-1 vs archetype JCV strains. The in vitro DNA replication system was employed as described in Methods. Labelled plasmids were linearized with HindIII and treated with DpnI as described for Fig. 2 to assess replication. (A) Comparison of plasmid replication initiated at Mad-1 or archetype origins. The plasmids subjected to replication were pBLCAT3-Mad1L and pJCV archetype, containing the Mad-1 and archetype replication origins, respectively. For these plasmids each origin insert was cloned into the BamHI site of pBLCAT3. The origin inserts were generated by PCR using the same primer set: forward, 5'-CATTTTTGCTTTTTGTAGC and reverse, 5'-CCAAAACAGCTCTGGCTCGC, both coupled to BamHI linkers. The BamHI Mad-1 insert was thus 401 bp, and the archetype insert was 391 bp. In the replication system SV40 T-antigen was supplied at 0·25 µg; GST–Pur{alpha} at 150 or 450 ng; GST–Tat at 75 or 150 ng. A DpnI-resistant band at 4·3 kb represents full-length replication for each plasmid. (B) Comparison of sequences in the late-promoter sides of Mad-1 and archetypal origins. Numbering is that of Frisque et al. (1984) . While there is considerable sequence variation among both strain types, the Mad-1 insert used here can be considered to be derived from the archetype strain by deletion of 23 and 66 bp segments (shaded boxes) and duplication of a resulting 98 bp segment, as indicated by dashed lines. The original sequences at break points are shown above the archetype map. The asterisk denotes common sequence variants at one break point. New sequences created by the deletions and repetition are shown below the Mad-1 map. The L and arrow indicate the direction of late gene transcription.

 
The sequences of the two origins are schematized in Fig. 5(B), which specifies new sequences created in the Mad-1 variant by deletion of 23 and 66 bp regions from the archetype and by repetition of a resulting 98 bp segment. The greater response of the Mad-1 origin to Tat and Pur{alpha} may reflect the fact that the A–T tract of the first 98 bp Mad-1 repeat is between two sets of Pur{alpha}-binding elements: the GAGGC T-antigen binding repeat, located at -13 to +12 on the Mad-1 map at bottom of Fig. 5, and an AGGGA repeat created by the rearrangement, symbolized by the line at +37. It has previously been shown that Pur{alpha} binds to both of these elements (Chang et al., 1996 ). The repeated AGGGA pentamer is critical for JCV DNA replication (Chang et al., 1994 ; Lynch & Frisque, 1990 ). Previous footprinting studies have documented a cooperative effect of Pur{alpha} and T-antigen on binding to DNA between the AGGGA and GAGGC repeats (Chang et al., 1996 ). New studies, to be presented elsewhere, reveal effects of Pur{alpha} and Tat on local DNA unwinding at the A–T tract in the different JCV origins. While there may be conditions, other than those used here, under which Tat can more strongly alter archetype DNA replication, the present results highlight the different replicative capacities of the two origins in a given system.


   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
The present results indicate that, in addition to the demonstrated effects of Tat on JCV gene transcription (Chowdhury et al., 1993 ; Krachmarov et al., 1996 ), Tat may act to directly stimulate JCV DNA replication. Aside from the possibility of cell coinfection with HIV-1 and JCV, which has not been demonstrated, it is conceivable that Tat, secreted from an HIV-1-infected cell, could enter a neighbouring cell harbouring JCV. In fact, fusion with a Tat domain has been used as a method of transducing cells directly with protein (Schwarze et al., 1999 ). Since the mechanism by which Tat is secreted and taken up by cells is essentially that of diffusion, concentrations of the protein in cells adjacent to those producing Tat could be effectively high, although that remains to be experimentally demonstrated for cells in the brain. Tat, in conjunction with Pur{alpha}, can activate the late promoter of JCV (Chen et al., 1995 ; Chowdhury et al., 1993 ; Krachmarov et al., 1996 ). Finally, Tat can enhance JCV DNA replication, activated by T-antigen. While such a mechanism is demonstrably applicable to brain, it may also be applicable to other tissues infected by HIV-1 since both Pur{alpha} and DNA or RNA PUR elements are ubiquitously present in human tissues. It should be noted that potential exposure to Tat would not fully explain the activation of JCV infection in humans. Many rearrangements of JCV regulatory sequences have been observed in PML, and we have here examined only one of them. It remains to be determined whether any pattern of rearrangements is specifically associated with AIDS. Clearly, rearrangement of JCV from the archetypal strain is an important aspect of activation in AIDS, and presently little is known about where this rearrangement occurs or what induces it (Major et al., 1992 ). To help assess relevance of the present results to PML, future experiments should involve transfection of human oligodendrocyte cultures with origin-containing clones from different JCV strains followed by a detailed analysis of response to Tat.

The interaction between Tat and Pur{alpha} is remarkable in that the action of Pur{alpha} alone in JCV replication appeared inhibitory whereas the action of Tat and Pur{alpha} together was stimulatory. Overexpression of Pur{alpha} upon transfection inhibited replication initiated at the JCV origin in Fig. 1, confirming earlier observation of that effect (Chang et al., 1996 ). In the earlier study anti-sense expression of Pur{alpha} cDNA stimulated replication of the JCV-origin-containing plasmid, strongly suggesting that the effect of endogenous Pur{alpha} was also inhibitory. Any inhibitory effect of Pur{alpha} is likely to be at the level of initiation rather than elongation. In CV-1 cells microinjection of Pur{alpha} in S phase had no effect on ongoing cellular DNA synthesis although injected cells completing replication were blocked from entering mitosis (Stacey et al., 1999 ). Tat clearly reversed the inhibitory effect of overexpressed Pur{alpha} in vivo (Fig. 1). The stimulation observed with Tat alone on JCV-initiated replication in the U-87MG cells is likely to be due to the same type of interaction of Tat with endogenous Pur{alpha}. The effect of Pur{alpha} alone in the in vitro JCV replication system was stimulatory at low concentrations (Fig. 3B, Table 1). This may reflect the known ability of Pur{alpha} to bind a variety of cell cycle regulatory proteins that it may not necessarily have access to in vivo. The inhibitory effect of Pur{alpha} at high concentrations may reflect the cellular function of Pur{alpha}, but it may also be due to an indiscriminate binding of Pur{alpha} to single-stranded DNA at replication bubbles or to competition with the essential single-stranded DNA-binding protein RPA. In contrast, Tat and Pur{alpha} acted synergistically to activate JCV replication. This suggests that the effect of Tat is not simply one of titrating away inhibitory Pur{alpha}. Rather, it is likely that Tat changes the configuration of Pur{alpha} to generate an altered activity of that protein. Such a change has previously been documented. In the presence of Tat the affinity of Pur{alpha} for its specific PUR element is strongly enhanced (Krachmarov et al., 1996 ). Work is currently in progress to obtain genetically deficient Pur{alpha} cell lines, which will aid in dissecting the synergistic nature of interaction of Tat and Pur{alpha}.

Results from the in vivo and in vitro JCV DNA replication systems are in reasonably good agreement. In both cases Tat stimulated replication initiated at the JCV origin. In both cases Pur{alpha} exhibited an inhibitory effect, either when overexpressed in vivo or at higher concentrations in vitro. Future experiments using cells with genetically inactivated PURA genes may provide insight into the stimulatory effect of Pur{alpha} seen at low concentrations in vitro. Note that one might not necessarily expect any in vivo or in vitro replication systems to be in complete agreement due primarily to issues of compartmentalization. The access of many known regulatory proteins to the replication apparatus is controlled in vivo by nuclear import or exclusion and by post-synthetic modifications, processes difficult to duplicate in vitro. Recent studies indicate that Pur{alpha} nuclear localization is highly regulated by cell cycle-dependent signals (Barr & Johnson, 2001 ).

Aside from any potential relevance to PML, the interactions of Tat and Pur{alpha} with JCV regulatory sequences provide a very useful model system for dissecting molecular pathways of Tat pathogenicity. Pur{alpha} is expressed in every human cell type thus far examined. PUR elements, such as the Tat-responsive element in the JCV origin/promoter region, are present in many cellular gene promoters, in origins of replication and in human telomeric repeats. In addition, both Tat and Pur{alpha} are known to interact with specific RNA sequences (Chepenik et al., 1998 ; Herault et al., 1995 ; Kobayashi et al., 2000 ; Tretiakova et al., 1998 ). Characterization of the Tat and Pur{alpha} interaction with PUR elements may help elucidate mechanisms of HIV-1 pathogenicity in AIDS as well as principles of normal cellular regulation.

Results from the in vitro replication system illuminate aspects of initiation of JCV DNA replication. The importance of auxiliary sequences adjacent to virus origins has previously been noted (Gutierrez et al., 1990 ; He et al., 1993 ; Li & Botchan, 1993 ). Auxiliary sequences near the SV40 origin strongly facilitate DNA unwinding by T-antigen while only weakly influencing the binding of that protein to the origin (Gutierrez et al., 1990 ). While effects of Tat and Pur{alpha} on JCV replication are clearly dependent on T-antigen, it remains to be determined whether they act at the step of initial DNA unwinding or on the DNA synthetic apparatus. In contemplating how Tat and Pur{alpha} stimulate JCV replication, parallels may be found in Tat effects on transcription. Tat stimulates transcription of HIV-1 through interaction with an RNA element, TAR, present within the 5' untranslated leader of HIV-1 transcripts (Berkhout et al., 1989 ; Churcher et al., 1993 ; Dingwall et al., 1990 ; Hamy et al., 1993 ; Kamine et al., 1991 ; Luo et al., 1993 ). Tat enhances HIV-1 transcription, at least in part, by binding to TAR and introducing protein kinases that phosphorylate and enhance processivity of RNA polymerase II (Parada & Roeder, 1996 ; Inamoto et al., 1997 ; Keen et al., 1996 ; Wei et al., 1998 ). These kinases reportedly include the p-TEFb kinase, a cyclin T1–CDK9 complex (Mancebo et al., 1997 ; Wei et al., 1998 ). In the JCV replication system DNA unwinding by T-antigen may allow entry of Pur{alpha}, which preferentially binds to single-stranded DNA (Bergemann et al., 1992 ). Tat is tethered to specific sequence elements at the origin through Pur{alpha}, whereupon Tat may then introduce protein kinases, or other cellular proteins, to influence the replication apparatus.


   Acknowledgments
 
We thank John N. Brady for the gift of plasmids for the bacterial expression of GST–Tat mutants. Work was supported by NIH grants NS35000 (E.M.J. and K.K.) and CA55219 (E.M.J.).


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Amirhaeri, S., Wohlrab, F., Major, E. O. & Wells, R. D. (1988). Unusual DNA structure in the regulatory region of the human papovavirus JC virus. Journal of Virology 62, 922-931.[Medline]

Ault, G. S. (1997). Activity of JC virus archetype and PML-type regulatory regions in glial cells. Journal of General Virology 78, 163-169.[Abstract]

Bagasra, O., Lavi, E., Bobroski, L., Khalili, K., Pestaner, J. P., Tawadros, R. & Pomerantz, R. J. (1996). Cellular reservoirs of HIV-1 in the central nervous system of infected individuals: identification by the combination of in situ polymerase chain reaction and immunohistochemistry. AIDS 10, 573-585.[Medline]

Barr, S. M. & Johnson, E. M. (2001). Ras-induced colony formation and anchorage-independent growth inhibited by elevated expression of Pur-alpha in NIH3T3 cells. Journal of Cellular Biochemistry 81, 621–638.[Medline]

Bergemann, A. D., Ma, Z.-W. & Johnson, E. M. (1992). Sequence of cDNA comprising the human pur gene and sequence-specific single-stranded-DNA-binding properties of the encoded protein. Molecular and Cellular Biology 12, 5673-5682.[Abstract]

Berger, J. R. & Major, E. O. (1999). Progressive multifocal leukoencephalopathy. Seminars in Neurology 19, 193-200.[Medline]

Berger, J. R., Kaszovitz, B., Post, M. J. & Dickinson, G. (1987). Progressive multifocal leukoencephalopathy associated with human immunodeficiency virus infection. A review of the literature with a report of sixteen cases. Annals of Internal Medicine 107, 78-87.[Medline]

Berkhout, B., Silverman, R. H. & Jeang, K.-T. (1989). Tat trans-activates the human immunodeficiency virus through a nascent RNA target. Cell 59, 273-282.[Medline]

Chang, C.-F., Tada, H. & Khalili, K. (1994). The role of a pentanucleotide repeat sequence, AGGGAAGGGA, in the regulation of JC virus DNA replication. Gene 148, 309-314.[Medline]

Chang, C. F., Gallia, G., Muralidharan, V., Chen, N. N., Zoltick, P., Johnson, E. M. & Khalili, K. (1996). Evidence that replication of human neurotropic JC virus DNA in glial cells is regulated by a sequence-specific single-stranded DNA-binding protein Pur{alpha}. Journal of Virology 70, 4150-4156.[Abstract]

Chen, N. N., Chang, C.-F., Gallia, G. L., Kerr, D. A., Johnson, E. M., Krachmarov, C. P., Barr, S. M., Frisque, R. J., Bollag, B. & Khalili, K. (1995). Cooperative action of cellular proteins YB-1 and Pur{alpha} with the tumor antigen of the human JC polymovirus determines their interaction with the viral lytic control element. Proceedings of the National Academy of Sciences, USA 92, 1087-1091.[Abstract]

Chepenik, L. G., Tretiakova, A. P., Krachmarov, C. P., Johnson, E. M. & Khalili, K. (1998). The single-stranded DNA binding protein, Pur-alpha, binds HIV-1 TAR RNA and activates HIV-1 transcription. Gene 210, 37-44.[Medline]

Chowdhury, M., Kundu, M. & Khalili, K. (1993). GA/GC-rich sequence confers Tat responsiveness to human neurotropic virus promoter, JCVL, in cells derived from central nervous system. Oncogene 8, 887-892.[Medline]

Churcher, M. J., Lamont, C., Hamy, F., Dingwall, C., Green, S. M., Lowe, A. D., Butler, P.-J. G., Gait, M. J. & Karn, J. (1993). High affinity binding of TAR RNA by the human immunodeficiency virus type-1 tat protein requires base-pairs in the RNA stem and amino acid residues flanking the basic region. Journal of Molecular Biology 230, 90-110.[Medline]

Daniel, D. C. & Johnson, E. M. (1989). Selective initiation of replication at origin sequences of the rDNA molecule of Physarum polycephalum using synchronous plasmodial extracts. Nucleic Acids Research 17, 8343-8362.[Abstract]

Desai, K., Loewenstein, P. M. & Green, M. (1991). Isolation of a cellular protein that binds to the human immunodeficiency virus Tat protein and can potentiate transactivation of the viral promoter. Proceedings of the National Academy of Sciences, USA 88, 8875-8879.[Abstract]

Dingwall, C., Ernberg, I., Gait, M. J., Green, S. M., Heaphy, S., Karn, J., Lowe, A. D., Singh, M. & Skinner, M. A. (1990). HIV-1 tat protein stimulates transcription by binding to a U-rich bulge in the stem of the TAR RNA structure. EMBO Journal 9, 4145-4153.[Abstract]

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]

Ensoli, B., Buonaguro, L., Barillari, G., Fiorelli, V., Gendelman, R., Morgan, R. A., Wingfield, P. & Gallo, R. C. (1993). Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein in cell growth and viral transactivation. Journal of Virology 67, 277-287.[Abstract]

Ezhevsky, S. A., Nagahara, H., Vocero-Akbani, A. M., Gius, D. R., Wei, M. C. & Dowdy, S. F. (1997). Hypo-phosphorylation of the retinoblastoma protein (pRb) by cyclin D:Cdk4/6 complexes results in active pRb. Proceedings of the National Academy of Sciences, USA 94, 10699-10704.[Abstract/Free Full Text]

Frankel, A. D. & Pabo, C. O. (1988). Cellular uptake of the Tat protein from human immunodeficiency virus. Cell 55, 1189-1193.[Medline]

Frisque, R. J., Bream, G. L. & Cannella, M. T. (1984). Human polyomavirus JC virus genome. Journal of Virology 51, 458-469.[Medline]

Gallia, G. L., Safak, M. & Khalili, K. (1998). Interaction of the single-stranded DNA-binding protein Puralpha with the human polyomavirus JC virus early protein T-antigen. Journal of Biological Chemistry 273, 32662-32669.[Abstract/Free Full Text]

Gallia, G. L., Darbinian, N., Tretiakova, A., Ansari, S., Ansari, S. A., Rappaport, J., Brady, J., Wortman, M. J., Johnson, E. M. & Khalili, K. (1999a). RNA-dependent interaction between the cellular protein Pur{alpha} and the HIV-1 protein Tat. Proceedings of the National Academy of Sciences, USA 96, 11572-11577.[Abstract/Free Full Text]

Gallia, G. L., Darbinian, N., Tretiakova, A., Ansari, S., Rappaport, J., Wortman, M. J., Johnson, E. M., Brady, J. N. & Khalili, K. (1999b). Association of HIV-1 Tar with the cellular protein, Pur-alpha, is mediated by RNA. Proceedings of the National Academy of Sciences, USA 96, 11572-11577.[Abstract/Free Full Text]

Gutierrez, C., Guo, Z. S., Roberts, J. & DePamphilis, M. L. (1990). Simian virus 40 origin auxiliary sequences weakly facilitate T-antigen binding but strongly facilitate DNA unwinding. Molecular and Cellular Biology 10, 1719-1728.[Medline]

Hamy, F., Asseline, U., Grasby, J., Iwai, S., Pritchard, C., Slim, G., Butler, P.-J. G., Karn, J. & Gait, M. J. (1993). Hydrogen-bonding contacts in the major groove are required for human immunodeficiency virus type-1 tat protein recognition of TAR RNA. Journal of Molecular Biology 230, 111-123.[Medline]

He, Z., Brinton, B. T., Greenblatt, J., Hassell, J. A. & Ingles, C. J. (1993). The transactivator proteins VP16 and GAL4 bind replication factor A. Cell 73, 1223-1232.[Medline]

Herault, Y., Chatelain, G., Brun, G. & Michel, D. (1995). RNA-dependent DNA binding activity of the Pur factor, potentially involved in DNA replication and gene transcription. Gene Expression 4, 85-93.[Medline]

Hirt, B. (1967). Selective extraction of polyoma DNA from infected mouse cell cultures. Journal of Molecular Biology 26, 365-369.[Medline]

Hofman, F. M., Wright, A. D., Dohadwala, D. F., Wong-Staal, F. & Walker, S. M. (1993). Exogenous tat protein activates human endothelial cells. Blood 82, 2774-2780.[Abstract]

Inamoto, S., Segil, N., Pan, Z. Q., Kimura, M. & Roeder, R. G. (1997). The cyclin-dependent kinase-activating kinase (CAK) assembly factor, MAT1, targets and enhances CAK activity on the POU domains of octamer transcription factors. Journal of Biological Chemistry 272, 29852-29858.[Abstract/Free Full Text]

Itoh, H., Wortman, M. J., Kanovsky, M., Uson, R. R., Gordon, R. E., Alfano, N. & Johnson, E. M. (1998). Alterations in Pur{alpha} levels and intracellular localization in the CV-1 cell cycle. Cell Growth & Differentiation 9, 651-665.[Abstract]

Jeang, K.-T., Chun, R., Lin, N. H., Gatignol, A., Glabe, C. G. & Fan, H. (1993). In vitro and in vivo binding of human immunodeficiency virus type 1 Tat protein and Sp1 transcription factor. Journal of Virology 67, 6224-6233.[Abstract]

Johnson, E. M. & Jelinek, W. R. (1986). Replication of a plasmid bearing a human Alu-family repeat in monkey COS7 cells. Proceedings of the National Academy of Sciences, USA 83, 4660-4664.[Abstract]

Johnson, E. M., Chen, P.-L., Krachmarov, C. P., Barr, S., Ma, Z.-W. & Lee, W.-H. (1995). Association of human Pur{alpha} with the retinoblastoma protein, Rb, regulates binding to the Pur{alpha} single-stranded DNA recognition element. Journal of Biological Chemistry 270, 24352-24360.[Abstract/Free Full Text]

Kamine, J., Loewenstein, P. & Green, M. (1991). Mapping of HIV-1 Tat protein sequences required for binding to Tar RNA. Virology 182, 570-577.[Medline]

Kashanchi, F., Piras, G., Radonovich, M. F., Duvall, J. F., Fattaey, A., Chiang, C.-M., Roeder, R. G. & Brady, J. N. (1994). Direct interaction of human TFIID with the HIV-1 transactivator Tat. Nature 367, 295-299.[Medline]

Keen, N. J., Gait, M. J. & Karn, J. (1996). Human immunodeficiency virus type-1 Tat is an integral component of the activated transcription–elongation complex. Proceedings of the National Academy of Sciences, USA 93, 2505-2510.[Abstract/Free Full Text]

Kobayashi, S., Agui, K., Kamo, S., Li, Y. & Anzai, K. (2000). Neural BC1 RNA associates with pur alpha, a single-stranded DNA and RNA binding protein, which is involved in the transcription of the BC1 RNA gene. Biochemical and Biophysical Research Communications 277, 341-347.[Medline]

Krachmarov, C. P., Chepenik, L. G., Barr-Vagell, S., Khalili, K. & Johnson, E. M. (1996). Activation of the JC virus Tat-responsive transcriptional control element by association of the Tat protein of human immunodeficiency virus 1 with cellular protein Pur alpha. Proceedings of the National Academy of Sciences, USA 93, 14112–14117; erratum 94, 9571.[Abstract/Free Full Text]

Li, R. & Botchan, M. R. (1993). The acidic transcriptional activation domains of VP16 and p53 bind the cellular replication protein A and stimulate in vitro BPV-1 DNA replication. Cell 73, 1207-1221.[Medline]

Li, J. J. & Kelly, T. J. (1984). Simian virus 40 DNA replication in vitro. Proceedings of the National Academy of Sciences, USA 81, 6973-6977.[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]

Lynch, K. J. & Frisque, R. J. (1990). Identification of critical elements within the JC virus DNA replication origin. Journal of Virology 64, 5812-5822.[Medline]

Lynch, K. J. & Frisque, R. J. (1991). Factors contributing to the restricted DNA replicating activity of JC virus. Virology 180, 306-317.[Medline]

Major, E. O., Amemiya, K., Tornatore, C. S., Houff, S. A. & Berger, J. R. (1992). Pathogenesis and molecular biology of progressive multifocal leukoencephalopathy, the JC virus-induced demyelinating disease of the human brain. Clinical Microbiology Reviews 5, 49-73.[Abstract]

Mancebo, H. S., Lee, G., Flygare, J., Tomassini, J., Luu, P., Zhu, Y., Peng, J., Blau, C., Hazuda, D., Price, D. & Flores, O. (1997). P-TEFb kinase is required for HIV Tat transcriptional activation in vivo and in vitro. Genes & Development 11, 2633-2644.[Abstract/Free Full Text]

Nesper, J., Smith, R. W., Kautz, A. R., Sock, E., Wegner, M., Grummt, F. & Nasheuer, H. P. (1997). A cell-free replication system for human polyomavirus JC DNA. Journal of Virology 71, 7421-7428.[Abstract]

Newman, J. T. & Frisque, R. J. (1997). Detection of archetype and rearranged variants of JC virus in multiple tissues from a pediatric PML patient. Journal of Medical Virology 52, 243-252.[Medline]

Ohana, B., Moore, P. A., Ruben, S. M., Southgate, C. D., Green, M. R. & Rosen, C. A. (1993). The type 1 human immunodeficiency virus Tat binding protein is a transcriptional activator belonging to an additional family of evolutionarily conserved genes. Biochemistry 90, 138-142.

Parada, C. A. & Roeder, R. G. (1996). Enhanced processivity of RNA polymerase II triggered by Tat-induced phosphorylation of its carboxy-terminal domain. Nature 384, 375-378.[Medline]

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]

Schwarze, S. R., Ho, A., Vocero-Akbani, A. & Dowdy, S. F. (1999). In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 285, 1569-1572.[Abstract/Free Full Text]

Shahabuddin, M., Bentsman, G., Volsky, B., Rodriguez, I. & Volsky, D. J. (1996). A mechanism of restricted human immunodeficiency virus type 1 expression in human glial cells. Journal of Virology 70, 7992-8002.[Abstract]

Stacey, D. W., Hitomi, M., Kanovsky, M., Gan, L. & Johnson, E. M. (1999). Cell cycle arrest and morphological alterations following microinjection of NIH3T3 cells with Pur alpha. Oncogene 18, 4254-4261.[Medline]

Stoner, G. L., Ryschkewitsch, C. F., Walker, D. L. & Webster, H. D. (1986). JC papovavirus large tumor (T)-antigen expression in brain tissue of acquired immune deficiency syndrome (AIDS) and non-AIDS patients with progressive multifocal leukoencephalopathy. Proceedings of the National Academy of Sciences, USA 83, 2271-2275.[Abstract]

Tada, H., Rappaport, J., Lashgari, M., Amini, S., Wong-Staal, F. & Khalili, K. (1990). Trans-activation of the JC-virus late promoter by the tat protein of type 1 human immunodeficiency virus in glial cells. Proceedings of the National Academy of Sciences, USA 87, 3479-3483.[Abstract]

Taylor, J. P., Pomerantz, R. J., Raj, G. V., Kashanchi, F., Brady, J. N., Amini, S. & Khalili, K. (1994). Central nervous system-derived cells express a kappa B-binding activity that enhances human immunodeficiency virus type 1 transcription in vitro and facilitates TAR-independent transactivation by Tat. Journal of Virology 68, 3971-3981.[Abstract]

Tornatore, C. S., Chandra, R., Berger, J. R. & Major, E. O. (1994). HIV-1 infection of subcortical astrocytes in the pediatric central nervous system. Neurology 44, 481-487.[Abstract]

Tretiakova, A., Gallia, G. L., Shcherbik, N., Jameson, B., Johnson, E. M., Amini, S. & Khalili, K. (1998). Association of Puralpha with RNAs homologous to 7 SL determines its binding ability to the myelin basic protein promoter DNA sequence. Journal of Biological Chemistry 273, 22241-22247.[Abstract/Free Full Text]

Vazeux, R., Cumont, M., Girard, P. M., Nassif, X., Trotot, P., Marche, C., Matthiessen, L., Vedrenne, C., Mikol, J., Henin, D. and others (1990). Severe encephalitis resulting from coinfections with HIV and JC virus. Neurology 40, 944–948.[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]

Wortman, M. J., Krachmarov, C. P., Kim, J. H., Gordon, R. G., Chepenik, L. G., Brady, N. N., Gallia, G. L., Khalili, K. & Johnson, E. M. (2000). Interaction of HIV Tat with Pur-alpha in nuclei of human glial cells: characterization of RNA-mediated protein–protein binding. Journal of Cellular Biochemistry 77, 65-74.[Medline]

Yu, L., Zhang, Z., Loewenstein, P. M., Desai, K., Tang, Q., Mao, D., Symington, J. S. & Green, M. (1995). Molecular cloning and characterization of a cellular protein that interacts with the human immunodeficiency virus type 1 Tat transactivator and encodes a strong transcriptional activation domain. Journal of Virology 69, 3007-3016.[Abstract]

ZuRhein, G. M. & Chou, S. M. (1965). Particles resembling papovavirions in human cerebral demyelinating disease. Science 148, 1477-1479.

Received 5 February 2001; accepted 7 March 2001.