progressive multifocal leukoencephalopathy, has a highly restricted host range (for reviews, see references 17 and 28). It replicates efficiently only in primary ...
Vol. 54, No. 3
JOURNAL OF VIROLOGY, June 1985, p. 750-756
0022-538X/85/060750-07$02.00/0 Copyright © 1985, American Society for Microbiology
DNA Rearrangement in the Control Region for Early Transcription in a Human Polyomavirus JC Host Range Mutant Capable of Growing in Human Embryonic Kidney Cells TATSUO MIYAMURA,* AKEMI FURUNO, AND KUNITO YOSHIIKE Department of Enteroviruses, National Institute of Health, Shinagawa-ku, Tokyo 141, Japan Received 3 January 1985/Accepted 27 February 1985
A human polyomavirus JC virus (JCV) host range mutant (JC-HEK) can grow in human embryonic kidney cells, whereas the brain cell-tropic wild-type JCV strain (Mad-i) cannot; JC-HEK contains two complementing defective DNAs, JC-HEK-A and JC-HEK-B. We determined the nucleotide sequence of the putative transcriptional control region of JC-HEK-A DNA that can induce T-antigen synthesis in human embryonic kidney cells and compared it with the sequence of JCV Mad-i DNA. The JC-HEK-A control region was found to have a complex DNA rearrangement, namely, a partial local duplication of a noncoding region generating two extra replication origins and translocation of segments from the large-T-antigen gene (415 base pairs) and the VP-1 gene (78 base pairs). In the rearranged segment, JC-HEK-A had seven sets of the sequence 5'TGGA(T)A(T)A(T)3', which is found in the simian virus 40 enhancer core, whereas JCV Mad-1 had only one set in its control region. JC-HEK-A also had a 5'TGGAAGTGTAA3' sequence resembling the adenovirus early region 1A enhancer core sequence 5'AGGAAGTGAA3'. Because the viral enhancer is host discriminatory and because another human polyomavirus, BK virus, that grows well in human embryonic kidney cells has these signals in its control region, it is likely that some of the newly acquired signals in JC-HEK play an important role in the altered host range of JCV.
region for transcription is responsible for the host range mutation. In this study, the nucleotide sequence around the rearranged region (1,522 bp) in JC-HEK-A DNA was determined and compared with that of the corresponding region in wild-type JCV Mad-1 DNA (7, 8, 26). The changes in JC-HEK-A DNA were found to be complicated rearrangements of the sequences originating from the wild-type JCV DNA. It is interesting that, in the putative control region, JC-HEK-A has newly acquired sequences resembling the sequences of the enhancer cores of simian virus 40 (SV40) and adenovirus early region 1A (ElA).
Human polyomavirus JC virus (JCV) (30), isolated repeatedly from patients with the rare demyelinating disease progressive multifocal leukoencephalopathy, has a highly restricted host range (for reviews, see references 17 and 28). It replicates efficiently only in primary cultures of human fetal brain cells rich in spongioblasts. Although an extensive search for more readily available cell cultures to support the replication of this virus was unsuccessful (29), JCV was adapted to growth in originally nonpermissive human embryonic kidney (HEK) cells by serial high-multiplicity passages of JCV Mad-1 (27). Studies with the resulting host range mutant (JC-HEK) can provide insights into virus adaptation and the complex regulatory mechanism of viral replication. It has been shown previously that a JC-HEK plaque isolate contains two types of mutually complementing defective DNAs (43). Both have a large deletion, but one species of DNA (JC-HEK-A) has the intact early genome, and the other (JC-HEK-B) has the late genome. Furthermore, JCHEK-A has an insertion of ca. 800 base pairs (bp) in the noncoding region near the origin of DNA replication, whereas JC-HEK-B contains 650-bp tandem duplications mapping between 0.61 and 0.74 map units of the JCV Mad-1 genome. Upon transfection, JC-HEK-A DNA can direct T-antigen synthesis in HEK cells (43), whereas neither JC-HEK-B (43) nor JCV Mad-1 (9) DNA can. These studies have suggested that JC-HEK-A has a DNA rearrangement in its putative control region for transcription and the intact early genome. Because polyomavirus enhancers are host discriminatory (1, 2, 13, 22, 34) and because their structural changes sometimes affect the host range of viruses (10, 19, 20, 37-40), it is very likely that the DNA rearrangement in the JC-HEK-A control *
MATERIALS AND METHODS DNA. JC-HEK-A DNA (43) inserted into bacterial plasmid pBR322 at the EcoRI cleavage site (pJC522) or at the BamHI cleavage site (pJC010) was used. The cleavage map of JCHEK-A(pJC522) and the area sequenced in this study are shown in Fig. 1. The recombinant plasmids were grown in Escherichia coli K-12 (strain HB101) and were purified as described previously (43). Enzymes. Restriction endonucleases were purchased from Takara Shuzo Co., Ltd., Kyoto, Japan (BamHI, HaeIII, HindIII, and Hinfl) and from Bethesda Research Laboratories, Inc., Gaithersburg, Md. (BamHI, Hindlll, and Sau3A). The reaction conditions were in accordance with the recommendations of the suppliers. Bacterial alkaline phosphatase and T4 polynucleotide kinase were from Boehringer Mannheim Biochemicals, Indianapolis, Ind. DNA sequencing. pJC522 DNA was digested with HindIll. Of six fragments generated, five (140 to 800 bp) were separated by electrophoresis in 5.5% preparative polyacrylamide gels and selectively recovered by electroelution. The largest fragment (ca. 6,000 bp), mostly from pBR322 and
Corresponding author. 750
JCV DNA REARRANGEMENT
VOL. 54, 1985
EcoRI -a MAP UNIT
EgoRI
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FIG. 1. Restriction endonuclease map of JC-HEK-A(pJC522). The physical and functional maps of JCV Mad-1 DNA are based on its total (8). Circular JCV Mad-1 DNA 5,130 bp long was cut open at its EcoRI site. JC-HEK-A DNA was aligned to JCV Mad-1 DNA to show that it retains the intact early genome. The shaded area in JC-HEK-A is the rearranged region. A, B, and C in the boxes refer to the HindIII fragments of JCV DNA. The multiple horizontal lines represent the pBR322 portion of pJC522. The thick horizontal line with two arrowheads indicates the area sequenced in the present study. Abbreviations: E, EcoRI; H, HindIII; B, BamHI; Ha, HaeIII; S, Sau3A; and Hf, Hinfl. sequence
the T-antigen-coding region, was not recovered. The nucleotide sequences of the HindIII fragments less than 200 bp long were determined by the method of Maxam and Gilbert (24) with [-y-32P]ATP (specific activity, 3,000 Ci/mmol; Amersham International plc, Buckinghamshire, England). The remaining 800-bp HindlIl fragment was further digested with Hinfl and HaeIII. The nucleotide sequences of the 130-bp HindIII-Hinfl fragment, the 190-bp Hinfl-Hinfl fragment, the 170-bp Hinfl-HaeIII fragment, the 30-bp HaeIII-HaeIII fragment, and the greater part of the 270-bp HaeIII-HaeIII fragment were determined by the method of Maxam and Gilbert (24). The 800-bp HindIlI fragment from pJC010 was further digested with Sau3A, and the resulting fragments were inserted between the HindlIl and BamHI sites or in the BamHI site of M13mplO and M13mpll vectors (25). The sequences were determined by the dideoxy method (35) with [a-32P]dATP (specific activity, 800 Ci/mmol; Amersham International). The M13 cloning and sequencing kits were obtained from Amersham International. Labeled DNAs were
electrophoresed on either 6 or 8% polyacrylamide gels with 8.3 M urea. Gels were subjected to autoradiography at -700C. RESULTS Defective JC-HEK-A DNA is believed to have the rearranged segment near the DNA replication origin and the intact early genome (Fig. 1), based on the alignment of the JC-HEK-A physical map to the JCV Mad-1 physical and functional maps (8, 17, 43) and on the ability of JC-HEK-A DNA to direct T-antigen synthesis in HEK cells (43). The nucleotide sequence of the rearranged region (the putative transcriptional control region [Fig. 1]) was determined (Fig. 2). From a comparison of the sequence with the total sequence of wild-type JCV Mad-1 (8), the origins of the rearranged DNA segments were identified and are schematically depicted as segments A, B, C, D, E, F, G, and H in Fig. 3. DNA rearrangement. The DNA replication origin charac-
752
J. VIROL.
MIYAMURA, FURUNO, AND YOSHIIKE EcoRI
1150 A X 1110 5'-AA TTCTGGCCAC ACTGTAACAA GGAAGCATGT CCCTATTTGG TAAGAGCTCC CTAGGTATGA GCTCATGCTT
1010 1050 F 1100 GGCTGGCAGC CATCCCTTCL CTTGTGCTTT GTTTACTGGC TGTTAGCTGG TTGGCTCCCT AGGTATGAGC TCATGCTTGG CTGGCAGCCA TCCCTTCCCT * 1000 910 950 TTTTTTTATA TATACA AG GCCGAGGCCG CCTCCGCCTC CAAGCTTACT CAGAAGTAGT AAGGGCGTGG AGGCTTTTTA GGAGGCCAGG GAAATTCCCG
900 * 850 TTGGCTCCCT AGGTATGAGC TCATGCTTGG CTGGCAGCCA TCCCTTCCCT TTTTTTTATA TATACAGGAG GCCGAGGCCG
810
CCTCC5CCTC CAAGCTTACT
800 710 750 CAGMGTAGT MGGGCGTGG AGGCTTTTTA GGAGGCCAGG GAAATTCCCT TGTTTTTCCC TTTTTTGCAG TAATTTTTTG CTGCAAAAAG CTAAAATGGA 700 610 650 .40 CAAAGTGCTG AATAGGGAGG AATCCATGGA GCTTATGGCT ATTCAAGGGG CCAATAGACA GTGGCAAAAC TACTTTAGCT GCAGCTtTAC TTGATCTCTG 600 510 550 TGGGGGAAAG TCATTAAATG TTAATATGCC ATTAGAAAGA TTAAACTTTG AATTAGGAGT GGGTATAGAT CAGTTTATGG TTGTATTTGA GGATGTAAAA 410 500 450 GGCACTGGTG CAGAGTCAAG GGATTTACCT TCAGGGCATG GCATAAGCAA CCTTGATTGC TTAAGAGATT ACTTAGATGG AAGTGTAAAA GTTAATTTAG
_3 1 0 400 AGAGAAAACA CCAAAACAAA AGAACACAGG TGTTTCCACC TGGAATTGTA ACCATGAATG AATATTCAGT GCCTAGAACT TTACAGGCCA GATTTGTAAG 300 210 250 GCAGATAGAT TTTAGACCAA AGGCCTACCT GAGAAAATCA CTAAGCTGCT GTGGCATTCT TTGGAAAAAT TGTTCCATCT GGGTACTTTG TTCTGTAATT
-p. AAAAAGCACC CCCTGTAATT CTAAAGCCTG TCCCTATTTG GTAAGAGCTC CCTAGGTATG AGCTCATGCT TGGCTGGCAG CCATCCCTTC qCTTGTGCT 150
200
4
50 100 _ 10 TTGTTTACTG GCTGTTAGCT GGTTGGCTCC CTAGGTATGA GCTCATGCTT GGCTGGCAGC CATCCCTTCC CTTTTTTTTA TATATACAG AGGCCGAGGC *
- 50 -90 CGCCTCCGCC TCCAAGCTTA CTCAGAAGTA GTAAGGGCGT GGAGGCTTTT TAGGAGGCCA GGGAAATTCC CTTGTTTTTC CCTTTTTTGC AGTAATTTTT t & T
-100
TGCTGCAAAA AGCTAAAATG FIG. 2. Nucleotide sequence of the rearranged region in JC-HEK-A. The sequence of the L strand from the EcoRI site to the initiation site for the T-antigen-coding region is given. The cytosine at the center of one of the three DNA replication origins was arbitrarily designated as nt 0; the numbering increases toward the left (the late side of the genome). That cytosine and two others at the centers of the extra DNA replication origins are marked with stars. The vertical arrows indicate the junction sites. The horizontal arrows indicate the SV40 enhancer core sequence 5'TGGA(T)A(T)A(T)3' (41). The arrow with an open head shows the sequence 5'TGGAAGTGTAA3' resembling the adenovirus ElA enhancer core sequence 5'AGGAAGTGAA3' (16). The complete 98-bp elements (7) are enclosed in brackets.
teristic of JCV and markedly similar to those of SV40 and human polyomavirus BK (BKV) (26) was located at three sites in the 1,520-bp segment of JC-HEK-A DNA sequenced in this study. The DNA replication origin contains four T-antigen recognition sequences, 5'GAGGC3', arranged in twofold symmetry (4). The cytosine at the center of symmetry in one of the three replication origins has been arbitrarily designated nucleotide zero (nt 0) (Fig. 2). Numbering increases toward the left (to the late genome), like the Frisque numbering for JCV Mad-1 (8), and decreases toward the right (to the early genome) with minus signals. The cytosines at the centers of the other two replication origins are at nt
822 and 972 (Fig. 2). Based on a comparison with the JCV Mad-1 sequence (8), the section from nt 110 to 1125 (Fig. 2) was found to be the rearranged segment of JC-HEK which substituted for the deletion of Mad-1 nucleotides (Mnt) 110 to 1,629 from wild-type JCV DNA. Although the rearrangement in JC-HEK-A was highly complex, all the sequences found in the rearranged section originated from the wild-type JCV sequence (Fig. 3). Segment A had the sequence from the VP-1 gene with a deletion between nt 1130 and 1131 (Mnt 1635 to 1684). Segment H was composed of one 98-bp element, which tandemly repeated in the Mad-1 control region, the DNA replication
VOL. 54, 1985
JCV DNA REARRANGEMENT 1000
li
500 0
i
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i
l
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BKV
