Proc. Natl. Acad. Sci. USA Vol. 92, pp. 1087-1091, February 1995 Microbiology Cooperative action of cellular proteins YB-1 and Pura with the tumor antigen of the human JC polyomavirus determines their interaction with the viral lytic control element NANCIE N. CHEN*, CHUN-FAN CHANG*, GARY L. GALLIA*, DOUGLAS A. KERR*, EDWARD M. JOHNSONt, CHAVDAR P. KRACHMAROVt, SHARON M. BARRt, RICHARD J. FRISQUEi, BRIGITr BOLLAG*, AND KAMEL KHALILI*§ *Molecular Neurovirology Section, Jefferson Institute of Molecular Medicine, Department of Biochemistry and Molecular Biology and Jefferson Cancer Institute, Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA 19107; tDepartment of Pathology and Brookdale Center for Molecular Biology, Mt. Sinai School of Medicine, New York, NY 10029; and tDepartment of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802 Communicated by Duard Walker, University of Wisconsin, Madison, WI, October 14, 1994 (received for review July 22, 1994) ABSTRACT Human JC polyomavirus (JCV) is the etio- to cell-specific transcriptional factors, other regulatory pro- logic agent of the neurodegenerative disease progressive mul- teins found in various cells and tissues are believed to be critical tifocal leukoencephalopathy. By using JCV as a model, we in facilitating viral gene expression during the lytic cycle investigated the role of the viral early protein tumor antigen (13-18). Results from this (10-12, 17, 19) and other (9, 20) (TAg) in the binding of two cellular proteins, Pura and YB-1, laboratories have indicated that the 98-bp enhancer/promoter to JCV regulatory sequences. Results from band-shift assays sequence of JCV encompasses multiple cis-regulatory ele- with purified YB-1, Purca, and TAg indicated that efficient ments that interact specifically with nuclear proteins and binding ofPura, a strong activator ofearly gene transcription, modulate viral early and late gene transcription. A region to a single-stranded target sequence corresponding to the designated the lytic control element (LCE) in the 98-bp repeat viral lytic control element, is diminished in the presence ofthe proximal to the origin of DNA replication contains a pen- late gene activator YB-1, which recognizes the opposite strand tanucleotide repeat sequence, AGGGAAGGGA, juxtaposed of the Pura binding site. Of particular interest was the ability to a poly(dT-dA) tract that displays a single-stranded config- ofPura and TAg to enhance binding ofYB-1 to DNA molecules uration (21). This motif differentially affects viral gene ex- without being associated with this complex. Binding studies pression by positively and negatively modulating early and late using a mutant peptide encompassing the N terminus ofYB-1 promoter transcription (17, 19) and plays an important role in indicated that the C terminus ofYB-1 is important for its DNA viral DNA replication (22, 23). In this study we demonstrate binding activity. The ability of Purax and TAg to increase that two distinct cellular proteins, Pura and YB-1, activators binding ofYB-1 to DNA is independent ofthe YB-1 C terminus. for the early and late gene transcription of JCV, respectively, Similarly, results from band-shift assays using Pura variants modulate each other's binding to the LCE. Moreover, we show indicated that two distinct regions of this protein contribute that the viral early protein TAg is capable ofenhancing binding either to its ability to bind DNA or to its ability to enhance of YB-1 to DNA molecules. We propose a working model for YB-1 DNA binding activity. Based on the interaction of Pura, the potential role of LCE-binding proteins and the viral TAg YB-1, and TAg, and their binding to DNA, a model is proposed in programming the early-to-late shift of the viral gene tran- for the role ofthese proteins in transcription ofviral early and scription during the lytic cycle. late genes during the lytic cycle. Studies of the small DNA tumor viruses have provided insights MATERIALS AND METHODS into a number of regulatory pathways used in the synthesis and Expression and Purification of Pura and YB-1 from Bac- processing of eukaryotic mRNA. In particular, work on pa- teria. Maltose-binding protein (MBP)-Pura and MBP-YB-1 povaviruses including simian virus 40 (SV40) has led to the fusion proteins were produced in bacteria using the plasmids identification of several regulatory factors that modulate tran- pMAL-Pura and pMAL-YB-1, respectively. The expressor scription of eukaryotic genes (1, 2). This group of viruses plasmids were constructed by inserting cDNA sequences for utilizes the host cell machinery to promote viral transcription Pura (24) and YB-1 (25) in-frame into pMAL-cR1 vectors. bidirectionally during lytic infection, leading to the production Proteins were expressed in Escherichia coli and purified on of regulatory tumor antigen (TAg) and structural capsid amylose affinity columns (New England Biolabs). Pura dele- proteins at early and late phases of infection, respectively (3, tion mutants were expressed as glutathione S-transferase 4). Accumulation of TAg at the early phase of infection is fusion proteins in E. coli BL21 Lys.S and purified with critical for the transcriptional switch from early to late pro- glutathione-agarose (Sigma) (26). Protein concentration was moters and the progression of subsequent events during the measured by the Bradford assay (Bio-Rad), and the purity of lytic cycle, including DNA replication in permissive cells (3, 4). proteins was examined by SDS/PAGE. The human JC polyomavirus (JCV) replicates efficiently in Expression and Purification of JCV and SV40 TAg from glial cells and has been repeatedly isolated from demyelinated Insect Cells. A JCV DNA fragment (nt 5130-4771/4426- plaques within the brain ofpatients with progressive multifocal 2473) containing exons 1 and 2 of the TAg gene was inserted leukoencephalopathy, a neurodegenerative disease (for re- into the baculovirus cloning vector pVL1392 (Invitrogen) views, see refs. 5-7). Several studies have established that the downstream from the polyhedrin promoter. This construct, restricted host range of JCV to glial cells is determined at the pVLl392-JCT (Int-), was used to generate the virus B-JCT by level of viral gene transcription that is mediated by glial- recombination with wild-type baculovirus Autographa califor- enriched DNA-binding regulatory proteins (8-12). In addition Abbreviations: JCV, JC polyomavirus; TAg, tumor antigen; SV40, simian The publication costs of this article were defrayed in part by page charge virus 40; LCE, lytic control element; MBP, maltose-binding protein; payment. This article must therefore be hereby marked "advertisement" in CSD, cold-shock domain. accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 1087 Downloaded by guest on September 27, 2021 1088 Microbiology: Chen et al. Proc. NatL Acad Sci USA 92 (1995) nica nuclear polyhedrosis virus, AcMNPV. The recombinant A baculovirus 941T that expresses the SV40 TAg was a gift from Late Early M. Summers (Texas A&M University) and R. Lanford (South- west Foundation for Biomedical Research). To prepare puri- /T1 fied JCV and SV40 TAgs, Sf9 insect cells were infected with :ML3~~ B-JCT or 941T virus and lysed 48 h later. Viral proteins were B LCE TATA purified by binding to immunoaffinity columns containing monoclonal antibody PAb2000 (anti-JCV TAg) or PAb9O1 (anti-SV40 TAg) and eluting with triethylamine buffer con- 5' AGCCATCCCTTCCCT 3'(E) taining 20 mM triethylamine (pH 11.3), 10% glycerol, and 3 TCGGTAGGGAAGGGA 5' (L) leupeptin (10 ,g/ml). Band-Shift Assay. Binding reactions with purified MBP- B YB-1 and MBP-Pura were performed in 12 mM Hepes, pH Proteins: y Q A' 7.9/4 mM Tris HCl, pH 7.5/60 mM KCl/5 mM MgCl2/0.8 mM dithiothreitol/0.5 jig of poly(dI-dC). DNA-protein complexes .. ,.:.. ... were allowed to form during a 20- to 30-min incubation of the yP- protein sample with 50,000 cpm of probe DNA on ice. p . Reaction products were analyzed on a 9% polyacrylamide/ 0.5 x TBE gel (27) and detected by autoradiography. Binding activities were quantified by laser densitometry of autoradio- grams. RESULTS Interaction of YB-1 and Purax with LCE. The pentanucle- otide repeat sequence of LCE (Fig. 1A) is a potential target for the binding of Pura, a single-stranded DNA-binding protein that exhibits specific affinity for a purine-rich sequence ele- ment present in several gene flanking regions and origins of DNA replication (24, 28). Pura preferentially binds to closely spaced repeats of the triplet GGN. The cDNA encoding Pura 1 2 3 1 2 3 1 2 3 was cloned into a prokaryotic expression system (pMAL-cR), Probe: E and the bacterially expressed Pura protein exhibits binding L EL affinity to the pentanucleotide repeat located on the late FIG. 1. (A) Structural organization of the JCV Madl control strand of the LCE (PL), but not to that on the early strand (PE)- region that encompasses two complete 98-bp repeats, each containing Pura also binds, although weakly, to the duplex LCE (PEL) a stretch of A/T sequence and the LCE and B domains on the early side of the 98-bp repeat. Ori and core designate the positions of the (Fig. 1B). origin of DNA replication and a domain homologous to an octamer By using the DNA fragment derived from the central region enhancer sequence in SV40, respectively. The nucleotide composition of the JCV 98-bp repeat as a probe, we isolated a recombinant of LCE and its localization with respect to the B domain and TATA YB-1 clone with the ability to bind to a C + T-rich sequence region are shown. Arrows indicate the direction of transcription of of LCE (D.A.K., C.F.C., N.N.C., G.L.G., G.