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
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Abstract |
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Introduction |
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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
. Pur
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
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
(Chen et al., 1995
). Tat does not itself bind to JCV DNA, but Tat and Pur
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
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 TatPur
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.
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Methods |
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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 [-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
and GSTTat 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 phenolchloroform: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.
Binding of Tat or Tat mutant proteins to Pur
.
HIV-1 Tat proteins were bacterially produced as GST fusion proteins and coupled to glutathioneagarose beads. Beads coupled to equimolar amounts of Tat or each of the Tat mutants were reacted with a 20-fold excess of Pur (2x10-7 M), derived by thrombin cleavage of GSTPur
. GSTTat is not cleaved by thrombin in the binding buffer (50 mM TrisHCl, 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 SDSPAGE on a 10% gel, blotted to an Immobilon P membrane and probed with anti-Pur
monoclonal antibody 9C12. Detection was with the Pierce SuperSignal Enhancer system.
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Results |
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Maximal stimulation of JCV replication in vitro by HIV-1 Tat in the presence of cellular Pur
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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It is helpful to know the endogenous level of Pur in the HeLa cell extract since this would form a background for effects of added Pur
. In HeLa cells the levels of Pur
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
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
in the HeLa cell extract employed for in vitro replication is likely to be low relative to the levels of GSTPur
and GSTTat employed in the study.
Effects of mutant Tat proteins on JCV replication in vitro and on binding to Pur
A series of deletion and point mutations of Tat were used to examine the contributions of different Tat domains to both Pur binding and Tats 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
(Gallia et al., 1999a
; Wortman et al., 2000
). The bottom panel of Fig. 4
presents Pur
binding to different GSTTat mutants immobilized on glutathioneagarose beads, and the top panel presents effects of purified GSTTat and its mutants on replication initiated in vitro at the JCV origin. Since wild-type Tat with a deletion of aa 236, Tat86
(236), bound Pur
, as did Tat48, albeit weakly, Tat amino acids from 3748 were critical for binding to Pur
. There was no Tat mutation that restricted Pur
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
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
is the observation that when C22 was mutated in Tat48 it was more detrimental to both Pur
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
binding. This configuration could also be induced when the entire amino terminal region was deleted, as in Tat86
(236). This mutation still allowed Pur
binding although it diminished Tat replicative effects. This may indicate that amino acids in the region 236 promoted effects on replication complementary to effects requiring Pur
binding. Furthermore, C22 may have, through a conformational effect, influenced Pur
binding without actually contacting Pur
. While a global effect of C22 is important for both Pur
binding and replicative effects of Tat, it is clear that Tat domains other than those involved in Pur
binding are also critical for these effects. Tat72 bound Pur
very well whereas this deletion was detrimental to DNA replication. It is reasonable that certain domains of Tat could interact with Pur
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 3650 (Kashanchi et al., 1994
). The critical role of C22 in influencing Pur
binding may play a role not only in effects of Pur
on JCV DNA replication, but also in observed effects of Pur
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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Discussion |
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The interaction between Tat and Pur is remarkable in that the action of Pur
alone in JCV replication appeared inhibitory whereas the action of Tat and Pur
together was stimulatory. Overexpression of Pur
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
cDNA stimulated replication of the JCV-origin-containing plasmid, strongly suggesting that the effect of endogenous Pur
was also inhibitory. Any inhibitory effect of Pur
is likely to be at the level of initiation rather than elongation. In CV-1 cells microinjection of Pur
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
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
. The effect of Pur
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
to bind a variety of cell cycle regulatory proteins that it may not necessarily have access to in vivo. The inhibitory effect of Pur
at high concentrations may reflect the cellular function of Pur
, but it may also be due to an indiscriminate binding of Pur
to single-stranded DNA at replication bubbles or to competition with the essential single-stranded DNA-binding protein RPA. In contrast, Tat and Pur
acted synergistically to activate JCV replication. This suggests that the effect of Tat is not simply one of titrating away inhibitory Pur
. Rather, it is likely that Tat changes the configuration of Pur
to generate an altered activity of that protein. Such a change has previously been documented. In the presence of Tat the affinity of Pur
for its specific PUR element is strongly enhanced (Krachmarov et al., 1996
). Work is currently in progress to obtain genetically deficient Pur
cell lines, which will aid in dissecting the synergistic nature of interaction of Tat and Pur
.
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 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
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
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 with JCV regulatory sequences provide a very useful model system for dissecting molecular pathways of Tat pathogenicity. Pur
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
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
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
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
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 T1CDK9 complex (Mancebo et al., 1997
; Wei et al., 1998
). In the JCV replication system DNA unwinding by T-antigen may allow entry of Pur
, which preferentially binds to single-stranded DNA (Bergemann et al., 1992
). Tat is tethered to specific sequence elements at the origin through Pur
, whereupon Tat may then introduce protein kinases, or other cellular proteins, to influence the replication apparatus.
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Acknowledgments |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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, 621638.[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. 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 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 Kaposis 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.
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.
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 and the HIV-1 protein Tat. Proceedings of the National Academy of Sciences, USA 96, 11572-11577.
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.
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.
Itoh, H., Wortman, M. J., Kanovsky, M., Uson, R. R., Gordon, R. E., Alfano, N. & Johnson, E. M. (1998). Alterations in Pur 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 with the retinoblastoma protein, Rb, regulates binding to the Pur
single-stranded DNA recognition element. Journal of Biological Chemistry 270, 24352-24360.
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 transcriptionelongation complex. Proceedings of the National Academy of Sciences, USA 93, 2505-2510.
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, 1411214117; erratum 94, 9571.
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.
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.
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.
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, 944948.[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 proteinprotein 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.