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Trans-Activation of the JC Virus Late Promoter by the Tat Protein

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Trans-Activation of the JC Virus Late Promoter by the Tat Protein Proc. Natl. Acad. Sci. USA Vol. 87, pp. 3479-3483, May 1990 Biochemistry Trans-activation of the JC virus late promoter by the tat protein of type 1 human immunodeficiency virus in glial cells HIROOMI TADA*, JAY RAPPAPORTt, MONIR LASHGARI*, SHOHREH AMINI*, FLOSSIE WONG-STAALt, AND KAMEL KHALILI*t *Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107; and TLaboratory of Tumor Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 Communicated by Robert C. Gallo, February 9, 1990 ABSTRACT Progressive multifocal leukoencephalopathy culture (18). It has been shown that the highly restricted host (PML) is a demyelinating disease of the central nervous system range/tissue specificity of JCV to glial cells rests in the caused by the JC virus (JCV), a human papovavirus. PML is expression of its viral genome (20-22). It would appear that a relatively rare disease seen predominantly in immunocom- the JCV control region contains a regulatory element(s) that promised individuals and is a frequent complication observed is recognized by trans-acting factors present predominantly in AIDS patients. The significantly higher incidence ofPML in in glial cells. AIDS patients than in other immunosuppressive disorders has Although PML is a relatively infrequent disorder, latent suggested that the presence of human immunodeficiency virus infection with JCV appears to be fairly common (15). Virus type 1 (HIV-1) in the brain may directly or indirectly contribute reactivation and resulting neuropathology appears to be a to the pathogenesis ofthis disease. In the present study we have consequence of the suppression of cell-mediated immunity. examined the expression of the JCV genome in both glial and The striking similarity between PML and AIDS leukoenceph- non-glial cells in the presence ofHIV-1 regulatory proteins. We alopathy suggests that JCV reactivation is a consequence of rind that the HIV-1-encoded trans-regulatory protein tat in- HIV infection, either directly by HIV-encoded trans-acting creases the basal activity of the JCV late promoter, JCVL, in factors or secondarily through T4 cell depletion. Since glial glial cells. In a reciprocal experiment, the JCV early protein, cells are productively infected by both JCV and HIV-1, JCV the large tumor antigen, stimulates expression from JCVL and reactivation through superinfection could be an in vivo mech- HIV-1 long terminal repeat promoter in both glial and non-glial anism of pathogenesis. cells. This trans-activation occurs at the level ofRNA synthesis, In addition to the standard retroviral genes gag, pol, and as measured by the rate of transcription, stability of the env, the HIV-1 genome encodes several accessory proteins message, and translation. We conclude that the presence of the (vif, vpr, tat, rev, and nef) (23). The best studied of these HIV-1-encoded tat protein may positively affect the JCV lytic proteins, tat, rev, and nef, have regulatory roles in viral gene cycle in glial cells by stimulating JCV gene expression. Our expression. We examined the relationship between JCV and results suggest a mechanism for the relatively high incidence of HIV-1 gene expression by testing the ability of HIV-1- PML in AIDS patients than in other immunosuppressive encoded trans-regulatory proteins to activate the JCV pro- disorders. Furthermore, our rindings indicate that the HIV-1 moter. Our results indicate that the HIV-1-encoded protein regulatory protein tat may stimulate other viral and perhaps tat stimulates expression of the JCV late (JCVL) promoter cellular promoters, in addition to its own. predominantly in cells of human glial origin. This stimulatory effect occurs primarily at the level of RNA synthesis. The AIDS is associated with a variety of neurologic disorders potential ofthe JCV-encoded trans-activator, the large tumor (1-5). Opportunistic infection of the central nervous system antigen (T antigen), to affect HIV long terminal repeat (CNS) and primary CNS lymphoma are often found in the (LTR)-directed gene expression was also examined. later stages of this disease (3, 5, 6). In addition to inducing these secondary manifestations of immune suppression, hu- MATERIALS AND METHODS man immunodeficiency virus (HIV) is thought to play a direct role in neuropathogenesis (7). HIV has been detected in brain Cells and Transfection Procedure. U-87MG (HTB-14) is an tissue and cerebrospinal fluid by a variety of procedures established human glioblastoma cell line, which was obtained (8-12). Furthermore, recent studies have clearly demon- from American Type Culture Collection. H9 is a continuous strated the presence of HIV-1 virus in oligodendroglial and line of human T4 lymphocyte cells. The HeLa cell line was astroglial cells of patients with AIDS (13). The presence of derived from a cervical carcinoma as described (24). All cell HIV in brain appears to be associated with white matter types except H9 were maintained in Dulbecco's modified changes, which include vacuolar degeneration, enlarged as- Eagle's medium supplemented with 10% (vol/vol) fetal bo- trocytes, and demyelination. Similar histopathology is also vine serum and plated in 60-mm dishes for 24 hr prior to observed in patients with the demyelinating disease progres- transfection. Cells were transfected by the calcium phos- sive multifocal leukoencephalopathy (PML) (14-16). phate/DNA coprecipitation method (25). Transfected plas- Demyelination in brains ofpatients with PML is caused by mid DNA was kept constant at 15 ,ug per dish by adding the destruction of oligodendrocytes, the myelin-producing pUC19 plasmid DNA with the test plasmids and was copre- cells of the CNS. The JC virus (JCV), a human papovavirus, cipitated with calcium phosphate in a final volume of 1.5 ml. has been repeatedly isolated from brain plaques of PML H9 cells were transfected by using the DEAE-dextran patients and is thought to be the etiologic agent ofthis disease method (26). (14-19). This virus preferentially infects oligodendroglial cells of the CNS and propagates only in glial cells in tissue Abbreviations: CNS, central nervous system; HIV-1, human immu- nodeficiency virus type 1; PML, progressive multifocal leukoen- cephalopathy; JCV, JC virus; LTR, long terminal repeat; CAT, The publication costs of this article were defrayed in part by page charge chloramphenicol acetyltransferase; T antigen, large tumor antigen; payment. This article must therefore be hereby marked "advertisement" JCVL promoter, JCV late promoter. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 3479 Downloaded by guest on October 1, 2021 3480 Biochemistry: Tada et al. Proc. Natl. Acad. Sci. USA 87 (1990) Plasmids. pJCL-CAT (22) was previously constructed by DNA spotted on the nitrocellulose filter was performed by a cloning the 286-base-pair (bp) Pvu II-HindIII fragment (map standard procedure using dot-blot techniques (34). units 0.67-0.72) of JCV, containing the late gene control region, into the Bgl II site of pCAT3M (27). pCV-1 contains the tat, rev, and nef(28) open reading frames of HIV-1. ptat RESULTS is a recombinant plasmid expressing only the tat protein (29). Transcriptional Activities ofthe JCVL and HIV-i Promoters pBJC-T plasmid was constructed by placing the JCV DNA in Glial and Non-Glial-Origin Cell Lines. The activity of the fragment that codes for viral early region under the control of JCVL promoter and the HIV-1 LTR was first compared in the the herpes simplex virus ICP4 promoter (kindly provided by presence and absence of the JCV T antigen and HIV-1 J. Remenick, National Cancer Institute). trans-regulatory proteins. Indicator plasmids containing the Chloramphenicol Acetyltransferase (CAT) Assay. All ex- bacterial CAT gene, under the control of the HIV-1 LTR tracts were made 48 hr posttransfection, and CAT enzyme (pHIV-CAT) or the JCVL enhancer/promoter (pJCL-CAT), assays were performed as described (30). were transfected into glial (HTB-14) and non-glial (HeLa and S1 Nuclease Analysis. Total cellular RNA was prepared by H9) cells alone or with plasmids encoding HIV-1 tat (pCV-1) the hot acid phenol procedure 48 hr posttransfection (31). or JCV T antigen (pBJC-T). Fig. 1 illustrates the organization Input DNA was removed by treatment with DNase I (10 of the JCV and HIV-1 genomes and the plasmids that were tug/ml) in the presence ofRNasin (Promega Biotec). RNA (50 used in this study. As shown in Fig. 1B, pCV-1 contains a pig) was probed for CAT mRNA with a single-stranded DNA cDNA fragment from the HIV-1 genome harboring three probe uniformly labeled with [32P]dCTP (400 Ci/mmol; 1 Ci overlapping open reading frames, those for tat, rev, and nef, = 37 GBq) during primer extension synthesis from an M13 which are expressed from the adenovirus major late promoter phage vector. RNA was analyzed by S1 nuclease protection (28). In the pBJC-T construct, the JCV T antigen is consti- as described (32). tutively expressed by the herpes simplex virus immediate .Nuclear Run-On Assay. Nuclei were prepared 48 hr post- early promoter, ICP4 (Fig. 1). In extracts examined 48 hr transfection from HTB-14 cells in two 100-mm plates con- after transfection with pJCL-CAT, virtually no CAT activity taining 30 ttg of plasmid DNA and resuspended in hypotonic was detected in glial and non-glial (H9 and Hela) cells after buffer [10 mM KCl, 1.5 mM MgCI2, 10 mM Hepes (pH 7.9), a 120-min enzyme reaction (Fig. 2 A-C, lane 4). Except in and 5 mM dithiothreitol) for 10 min at 0°C. Cells were glial cells (Fig. 2A, lane 1), transfection of pHIV-CAT alone disrupted with a Dounce homogenizer. Nuclei were isolated indicated no detectable CAT activity (Fig.
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