Characterization of BK virus variants rescued from human ... - CiteSeerX

Journal of General Virology (1990), 71, 2731-2736. Printed in Great Britain

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Characterization of BK virus variants rescued from human tumours and tumour cell lines M . Negrini, 1 P. Rimessi, 1 C. Mantovani, 1 S. Sabbioni, 1 A. Corallini, 1 M . A. Gerosa 2 and G. Barbanti-Brodano 1. 1Institute o f Microbiology, School o f Medicine, University o f Ferrara, Via Luigi Borsari 46, 1-44100 Ferrara and 2Department o f Neurosurgery, University o f Verona, 1-37100 Verona, Italy

Episomal BK virus (BKV) DNA was detected in primary human brain tumours, in Kaposi's sarcoma and in cell lines from brain tumours, Ewing sarcoma and osteogenic sarcoma. Infectious BKV was rescued from several tumours and tumour cell lines by transfection of total cellular DNA into human embryonic fibroblasts. Restriction endonuclease and nucleotide sequence analysis showed that all the rescued viruses are similar to BKV-IR, a BK variant previously

isolated from a human tumour of pancreatic islets, indicating that a specific BKV strain may be associated with certain types of human tumours. All the variants contain a putative transposable element in the regulatory region of the viral genome. This region has mutagenic properties and enhancing activity in transformation, suggesting a possible role of these variants in tumour induction or progression.

BK virus (BKV) is a human papovavirus with a general distribution in the normal population world-wide (Gardner, 1973; Shah et al., 1973; Portotani et al., 1974; Brown et al., 1975). Primary infection occurs in childhood, after which BKV remains latent in the kidney and tonsils (Heritage et al., 1981; Chesters et al., 1983; Grinnell et al., 1983; Goudsmit et al., 1982). BKVinduced acute diseases in immunocompetent individuals affect mainly the upper respiratory apparatus (Goudsmit et al., 1981, 1982) and the urinary tract (Hashida et al., 1976; Mininberg et al., 1982). In immunodeficient or immunosuppressed hosts, BKV latent infection is reactivated (Andrews et al., 1988) and produces lesions in the urinary apparatus (Coleman et al., 1978), the most severe of which is haemorrhagic cystitis in bone marrow transplant recipients (Arthur et al., 1988). BKV transforms rodent, monkey and human cells (Portolani et al., 1975, 1978; Purchio & Fareed, 1979; Takemoto et al., 1979; Grossi et al., 1982) and is oncogenic in hamsters, mice and rats. Tumours induced in experimental animals include brain tumours, tumours of pancreatic islets, osteosarcomas and fibrosarcomas (Corallini et al., 1977, 1978, 1982; Costa et al., 1976; Uchida et al., 1976, 1979). In agreement with the indications of this experimental pathogenicity, BKV D N A was detected by Southern blot hybridization in human brain tumours and tumours of pancreatic islets (Corallini et al., 1987; D6rries et al., 1987).

Because of the relationship of Kaposi's sarcoma (KS) with immunosuppression, we have investigated tissue from classical and African KS for the presence of BKV DNA. In addition, we have analysed a number of cell lines derived from brain tumours and sarcomas. Agarose gel electrophoresis and nucleic acid hybridization were carried out according to Southern (1975). BKV D N A probes were labelled by primer extension (Feinberg & Vogelstein, 1984). Nucleotide sequence analysis was performed by the dideoxynucleotide chain-terminating method (Sanger et al., 1977). Mutagenesis of the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus was carried out according to the method of Brandt et al. (1987). To construct the recombinant D N A molecules, standard procedures were followed (Maniatis et al., 1982). Primary mouse embryo fibroblasts and Rat-1 cells were grown and propagated in Dulbecco's modified Eagle's minimum essential medium supplemented with 10~ foetal bovine serum. The results presented in Table I show that BKV DNA is associated with African KS, ependymoma, meningioma, glioblastoma, glioma, neuroblastoma, Ewing sarcoma and osteogenic sarcoma. Control human tumour cell lines from epithelioma (Hep-2), hepatoma (HepG2) and cervical carcinoma (KNC) were negative. BKV D N A was free in tumour cells (Fig. 1) and only in rare tumours were viral sequences integrated into cellular DNA. Infectious BKV was rescued from several positive

0000-9635 © 1990SGM

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T a b l e 1. B K V D N A in Kaposi's sarcoma, brain tumours and tumour cell lines

DNA source Tumour Classical KS African KS Ependymoma Meningioma Tumour cell line Glioblastoma Glioma Neuroblastoma Astrocytoma Meningioma Ewing sarcoma Osteogenic sarcoma Giant cell sarcoma Haemangioendothelioma Chondrosarcoma Fibrous histiocytoma Sarcoma Control Hep-2, HepG2, KNC

Positive samples/ samples tested

Rescue of BKV DNA

0/7 4/13 2/3 2/2

2/4 2/2 2/2

1/ 10 1/1 2/6 0/1 0/1 1/5 2/10 0/7 0/1 0/1 0/1 0/2

1

2

FII

BKV-E BKV-KS85 BKV-KSI22

7

Fill

0/3

Fig. 1. Hybridization of five KS DNAs digested with the singlecutting restriction endonuclease BamHI (lanes 2 to 6), to a 32p-labelled BKV DNA probe. Lanes I and 7 contain uncut BKV DNA supercoiled circular form I (FI), relaxed circular form II (FII) and linear form III (FIII) (2-5 genome equivalents). The band comigrating with FII in lane 6 is an incomplete digestion product of BKV DNA.

Transformationt number of colonies/106 cells

Tumour inductionS/ number with tumours/number inoculated (~)

l07 106 105 107

55 19 3-5 17 2.5 0 ND§ 15.5 5"5 2-5

44/50 (88-0)

105 107 107 106 l0 s

6

0/l 1/2

Titre (p.f.u.)

106

5

FI m

Table 2. Transformation and tumour induction by BKV- W T and B K V strains rescued from human tumours

BKV-WT

4

1/1 0/1 2/2

t u m o u r s a n d t u m o u r cell lines b y t r a n s f e c t i o n o f h u m a n e m b r y o n i c f i b r o b l a s t s w i t h total cellular D N A ( T a b l e 1). The rescued viruses have the antigenic and biological properties of wild-type BKV. They transform primary m o u s e e m b r y o cells a n d i n d u c e e p e n d y m o m a s u p o n i n t r a c e r e b r a l i n o c u l a t i o n in n e w b o r n h a m s t e r s ( T a b l e 2). T r a n s f o r m i n g a c t i v i t y a n d o n c o g e n i c i t y a r e l o w e r i n the r e a c t i v a t e d v a r i a n t s , c o m p a r e d to w i l d - t y p e B K V ( B K V W T ) . H o w e v e r , histological e x a m i n a t i o n o f t u m o u r s

Inoculum*

3

9/16 (56.3) 8/11 (72-7) 10/18 (55-5)

* BKV-WT, wild-type BKV; BKV-E, BKV-KS85 and BKV-KS122, BKV strains rescued from an ependymoma and from two Kaposi's sarcomas, respectively. t Transformation experiments were carried out in primary mouse embryo fibroblasts. Values are the average counts from two Petri dishes. Tumours were induced by intracerebral inoculation of newborn hamsters. All tumours, without exception, were ependymomas. § ND, Not done.

Short Communication 1 2 3 4 5 6 7 8 9 I0 11 12 13 14 15 16 17

Fig. 2. Agarose gel electrophoresis of D N A s from BKV-WT (lanes 2, 6, 10, 14), BKV-KS85 (lanes 3, 7, 11, 15), BKV-KS122 (lanes 4, 8, 12,16) and BKV-IR (lanes 5, 9, 13, 17) digested with PvulI (lanes 2 to 5), BgllI (lanes 6 to 9), HindllI (lanes 10 to 13) and EcoRI (lanes 14 to 17). A, B, C and D indicate the four HindlII fragments of BKV-WT. The size marker (kb) in lane 1 is obtained by digestion of the Saccharomyces cerevisiae '2~t circle' plasmid D N A with several restriction endonucleases (BRL).

revealed that ependymomas induced by variants BKVE, BKV-KS85 and BKV-KS122 have a higher number of malignancy markers than BKV-WT-induced tumours since they show more atypic cells, more frequent mitoses, invasiveness and microscopic metastases in the subarachnoidal space (data not shown). The genomic DNA of all the rescued viruses, analysed by agarose gel electrophoresis after digestion with several restriction endonucleases, has the same restriction pattern. Fig. 2 shows a representative result obtained with the two variants rescued from KS. The large PvulI fragment migrates faster than in BKV-WT. The KS variants have lost one BgllI site and produce only three HindlII fragments instead of four since HindlII fragment D is missing. In addition HindlII fragments B and C are larger than the corresponding wild-type fragments and linear EcoRI-digested molecules migrate slightly faster than BKV-WT DNA. This restriction pattern is similar to that of BKV-IR (Fig. 2), a BKV variant rescued from a human tumour of pancreatic islets (Caputo et al., 1983; Pagnani et al., 1986) and is compatible with an insertion into the regulatory region of the viral genome, which is in HindlII fragment C, as well as with a deletion around map unit 0.54 where a BgllI site and the HindlII site between fragments B and D are located. Nucleotide sequence analysis established that the insertion and the deletion are similar in BKV-IR and in the rescued variants, differing in only a few nucleotides in the insertion (Fig. 3). The deletion, in different

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variants, is due to a loss of 253 bp between nucleotides 2792 and 3044. No rearrangements are observed in the regions flanking the deletion, where the nucleotide sequence is collinear with that of BKV-WT DNA (data not shown). The deleted nucleotides remove sequences coding for the middle and carboxy-terminal portions of the small t antigen and contain the donor splice site for the small t antigen 20S mRNA (nucleotides 2792 and 2793) (Yoshiike & Takemoto, 1986). The deletion falls within the sequence spliced out of the early region primary transcript (nucleotides 2725 to 3069) to yield the 19S mRNA for the large T antigen (Yoshiike & Takemoto, 1986) so that expression of the large T antigen is not affected. The insertion is made up of 80 nucleotides distributed in four clusters that produce rearrangements in the region of the enhancer repeats (Fig. 3). The inserted sequences are flanked by two 12 nucleotide direct repeats flanked by inverted repeats (Fig. 3) so that the whole insertion could fold over to form a putative transposable insertion sequence (IS) (Fig. 4). If this sequence is a mobile element able to transpose from the viral DNA to the cell genome, it could induce tumours by mutation or by insertional activation of cellular oncogenes. The latter hypothesis is supported by the observation that the IS sequence contains transcriptional enhancers in loop two of the early region (Fig. 4). To test these hypotheses, we constructed recombinant DNA molecules containing HindIII fragment C of BKVIR (pBode-IR) and of BKV-WT (pBode-WT) and compared their mutagenic effect and enhancer activity upon transformation. The plasmid pML-Tet, used to construct pBode-IR and pBode-WT, was a control. Induction of mutation was measured by transfection of recombinants into Rat-1 and CHO cells and selection in medium containing 6-thioguanine (0.1 mM) which allows detection of cells with mutations in the gene coding for HGPRT (Caskey & Kruh, 1979). The results presented in Table 3 show that the mutation frequency after transfection of pBode-IR is seven times greater than in cells transfected with pBode-WT. Comparable mutation frequencies have been obtained in six individual experiments. Similar IS-like structures, endowed with mutagenic activity, were detected in the transforming region of the genomes of herpes simplex virus type 2 and cytomegalovirus (Galloway et al., 1984; Brandt et al., 1987). To test the enhancer activity in transformation, BKV-IR or BKV-WT HindlII fragment C were placed upstream of the activated human c-ras oncogene and the recombinants were transfected into Rat-1 cells. As shown in Table 4, pBode-IR/c-rasA produces a significantly higher number of transformed foci than pBodeWT/c-rasA and pBR/c-rasA which does not contain BKV sequences (Pagnani et aL, 1988). However, it does not have the transforming activity of pBK/c-rasA, where

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3453 WT

3525

GC~ACAGGGAGGAGCTGCTTACCCATGGAATGCAGCCAAACCATGACCTCAGGAAGGAAAGTGCATGACTICACAGG

KS85 GCCA~AGGGAGGAGCTGCTTACCCAT~GAATGCAGCCAAACCATGACCTCAGGAAGGAAAGTGCATGACTGG~GCAG~A~CCAG~TGGCAG KS122 GCCACAGGGAGGAGCTGCTTACC~AT~GAATGCAGCCAAACCATGACCTCAGGAAGGAAAGTGCATGACTG(~3~AGC~AG~CAf~TGGcAG

E

GCCA~AGGGAGGAGCTGCTT-~CAT~GAATGCAG~CAAACCATGACCTCAGGAAGGAAAGTGCATGA~TG(7~AG~CA~CA~2GG~AG

CC IR

GCCACAGGGAGGAGCTGCTTACCCAT~GAATGCAGCCAAACCATGACCTCAGGAAGGAAAGTGCATGACTG(]GCAG~CAGCCAG~rGG~AG GCCACAGGGAGGAGCTGCTTACCCAT~GAATGCAGCCAAACCATGACCTCAGGAAGGAAAGTGCATGACTGG~G~AGC~AGCCA(~GG~AG 3200 ~ 3286 3529 GGAATGCAGCCAAACCATGACCTCAG

GAAGGAAAGTGCATGAC~

3585 ~ACAGGGAGGAGCTGC

TTAATACCCATIGGAATGCAGCCAAACCATGACCTCAGCCATGACCTCAGGAAGGAAAGTGCATGACTGGGCAGCCAGCCAGTGGCAGTTA TTAATACCCATIGGAAT-CAGCCAAACCATGACCTCAGCCATGACCTCAGGAAGGAAAGTGCATGACTGGGCAGCCAGCCAGTGGCAGTTA TTAATACCCAT~GAATGCAGCCAAACCATGACCTCAGCCATGACCTCAGGA-GGAAAGTGCATGACTGGGCAGCCAGCCAGTGGCAGTTA TTAATACCCAT~GAATGCAGCCAAACCATGACCTCAGCCATGACCTCAGGAAGGAAAGTGCATGACTGGGCAGCCAGCCAGTGGCAGTTA TTAATACCCA%~GAATGCAGCCAAACCATGACCTCAGCCATGACCTCAGGAAGGAAAGTGCATGACTGGGCAGCCAGCCAGTGGCAGTTA 3290 1 l 3376 3589 3663 TTACCCATGGAATGCAGCCAAACCATGACCTCAGGAAGGAAAGTGCATGAC"•]GGGCAGCCAGCCAGTGGCAGTTAATA ATACCCATIGGAATGCAGCCAAACCATGACCTCAGGAAGGAAAGTGCATGACTGGGCAGCCAGCCAGTGGCAGTTAAT/~TAAACACAAGA ATACCCATIGGAATGCAGCCAAACCATGACCTCAGGAAGGAAAGTGCATGACTGGGCAGCCAGCCAGTGGCAGTTAAT6]ATAAACACAAGA ATACCCATIGGAATGCAGCCAAACCATGACCTCAGGAAGGAAAGTGCATGACTGGGCAGCCAGCCAGTGGCAGTTAAT/~%TAAACACAAGA ATACCCATIGGAATGCAGCCAAACCATGACCTCAGGAAGGAAAGTGCATGACTGGGCAGCCAGCCAGTGGCAGTTAAT/~TAAACACAAGA ATACCCAT~GAATGCAGCCAAACCATGACCTCAGGAAGGAAAGTGCATGACTGGGCAGCCAGCCAGTGGCAGTTAATA~TAAACACAAGA 3380 3466 3667 3728 GTGAAACCCCGCCGACAGAC -ATGTTTTGCGAGCCTAGGAATCTTGGCCTTGTCCCCAGTTAAACT GGAAGTGGA/M~CTGGCCA/M~.GGA-GTGGAA/X~CAGCCASACACIA-CTGTTTTGCGAGCCTAGG~TCTTGGCCTTGTCCCCAGTT~CT GGAAGTGGAAACTGGCC~u~AGGA- GTGGAAA,#CICAGCCAGACAGIACATGTTTTGCGAGCCTAGGAATCTTGGCCTTGTCCCCAGTT~CT GGAAGTGG~CTGGCC~GGAGTGGAJ~u~SCAGCCAGACA(IACATGTTTTGCGAGCCTAGGAATCTTGGCCTTGTCCCCAGTTA~CT GG~GTGG~u~.ACTGG C C AAAGGAAG TGGAA~CAGCCAGACAGIA- ATGTTTTGCGAG CCTAGG~TCTTGGCCTTGTC C C C A G T T ~ C T GGAAGTGG.~u~a~CTGGCC~GG~GTGGA~CAGCCAGACAqA- ATGTTTTGCGAGCCTAGG~TCTTGGCCTTGTCCCCAGTT~CT 3470 l l{ 3556 Fig. 3. Alignment of nucleotide sequences of the insertion in HindlIl fragment C of the genomes from four BKV variants rescued from two KS (KS85 and KS122); from an ependymoma (E) and from a glioblastoma (CC). The sequences of BKV-WT (WT) and BKV-IR (IR), previously rescued from a tumour of pancreatic islets, are shown for comparison. Sequences bounded by square brackets are the transcriptional enhancer repeats; sequences enclosed in boxes are the 12 nucleotide direct repeats located at the 5' and 3' ends of the insertion. Arrows indicate differences in sequence affecting single nucleotides. Nucleotides were numbered clockwise taking the unique EcoRI restriction site as map unit 0. Upper numbers refer to BKV-WT sequence, lower numbers refer to the nucleotide sequences of the four variants and of BKV-IR. Homologous nucleotides are indicated by dots.

T a b l e 3. Mutagenic activity o f the putative 1S sequence for the H G P R T locus o f Rat-1 cells Transfected D N A (30 ~tg) pBode-IR pBode-Wt pML-Tet None

Mutants/ 10v cells

Plating efficiency (~)*

84 11 8 6

37.5 36.5 38.1 41-2

Mutation frequencyt 2.3 × 3.1 × 2.1 x 1-5 x

10-5 10-6 10-6 10 -6

Enhancement factor 15.3 2.1 1.4 0

* Plating efficiency was calculated as the ratio between the number of colonies and the number of cells plated (2 x 102/10 cm Petri dish) under non-selective conditions. t The mutation frequency (Mr) was calculated by the formula Mf = M/CE, where M is the number o f mutants obtained, C is the total number of cells plated (1.5 x 107) and E is the plating efficiency. For each transfection, 30 Petri dishes of 10 cm diameter, each containing 5 x 10s cells, were prepared as described previously (Brandt et al., 1987).

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growth factors (Nakamura et al., 1988; Salahuddin et al., 1988; Ensoli et al., 1989a, b). Nevertheless, the physical structure of the putative IS-like sequence present in the regulatory region of BKV variants as well as its mutagenic effect and enhancing activity in transformation are worthy of further investigation in order to determine its possible role in tumour induction or progression. I-A I-A G-G A-T

We thank A. Bevilacqua, P. Zucchini and M. Bonazzi for excellent technical assistance and typing the manuscript. This work was supported by funds from Consiglio Nazionale delle Ricerche, Progetto Finalizzato 'Oncologia'; from Ministero della Sanit~t, Istituto Superiore di SanitY., 'Progetto A.I.D.S. 1989', Rome, Italy; and from Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.).

C-G2G

G-A G.A .

T-A

.

GCAGCCAGCCAG GCAGCCAGACAG 3272

3509

Fig. 4. The IS-like structure in the insertion of BKV variants rescued from human tumours and tumour cell lines. The two 12 nucleotide direct repeats are shown at the base of the stem-loop structure. Asterisks mark two nucleotides that differ in the direct repeats. Dots indicate mismatched pairs in the stem. The loop of 200 nucleotides contains two of the three transcriptional enhancers of the early region. Nucleotides were numbered taking the unique EcoRI restriction site as map unit 0.

Table 4. Transformation of Rat-1 cells Transfected DNA (2 ~g/106 cells)

A B C D

pBode-IR/c-rasA pBode-WT/c-rasA pBR/c-rasA pBK/c-rasA

Number of colonies/106 cells* 137.6 + 82.0 + 65.0 + 179.6+

13.5 8.7 A/B P < 0.001~ 5.6 A/C P < 0.001 19.1 A/D P < 0.01

* Values are the average of three Petri dishes. t Significance of difference between A and B.

the entire BKV early region cooperates with activated c-ras in the induction of the transformed phenotype (Pagnani et al., 1988). These experiments show that a specific BKV variant is associated with human brain tumours, osteosarcomas and African KS. This is a more aggressive, systemic form of the tumour, resembling KS in AIDS patients. Because of its ubiquity in the human population, it is possible that BKV is a passenger in these tumours. In addition, the pathogenesis of KS may be related to other viruses, such as cytomegalovirus (Giraldo & Beth, 1986), to activation of oncogenes (Delli Bovi & Basilico, 1987; Delli Bovi et al., 1987) or to abnormal production of cytokines and

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(Received 20 April 1990; Accepted 12 July 1990)