JOURNAL OF VIROLOGY, Mar. 1979, p. 846-855 0022-538X/79/03-0846/10$02.00/0

Vol. 29, No. 3

Restriction Endonuclease Cleavage Map of the DNA of JC Virus JONATHAN D. MARTIN,* RICHARD J. FRISQUE, BILLIE L. PADGETT, AND DUARD L. WALKER Department of Medical Microbiology, University of Wisconsin Medical School, Madison, Wisconsin 53706

Received for publication 28 July 1978

A physical map of the sites cleaved by the following restriction endonucleases derived for the DNA of JC virus, a human polyomavirus: EcoRI, HpaI, and PstI (one site each); HindII (four sites); and HindIII (three sites). By agarose gel electrophoresis of fragmented DNA, the size of full-length DNA of JC virus was estimated to be 5,125 + 105 base pairs (98 ± 2% of the length of simian virus 40 DNA). was

JC virus (JCV) is the human polyomavirus associated with progressive multifocal leukoencephalopathy (16). It shares both T and V antigenic determinants with simian virus 40 (SV40) and with the other major polyomavirus of humans, BK virus (BKV; 15). JCV is highly neurooncogenic in hamsters (21) and has recently been shown to induce brain tumors in non-immunosuppressed adult owl monkeys (11). Comparisons of the genomes of JCV, BKV and SV40 have been limited and, to some extent, ambiguous. In DNA reassociation acceleration experiments (7), JCV was shown to be related more closely to BKV (with 25% DNA homology) than to SV40 (11% homology). The opposite relationship was obtained by competition hybridization on filters (12); JCV and SV40 DNAs were 40% homologous, whereas JCV and BKV DNAs showed 20% homology. (BKV and SV40 DNAs are 20 [8] to 40% [12] homologous, by the same techniques.) A set of sequences is common to all three viruses (7, 12) and, in SV40, is located primarily in the late region (7, 10, 12). To facilitate more detailed comparisons of the genomes of these viruses, we generated a restriction endonuclease cleavage map of JCV DNA. It is anticipated that such a map will be helpful in determining the regions of homology among the three viruses. (A preliminary presentation of these results was made at the 78th Annual Meeting of the American Society for Microbiology, Las Vegas, Nev., 14-19 May 1978.)

MATERIALS AND METHODS Media, cells, and viruses. Primary cultures of human fetal glial (PHFG) cells were grown either in Hams medium (F12) or in a medium composed of an equal-volume mixture of M199 with Hanks salts and McCoy medium (ISI Biologicals, Cary, Ill.). The mixed medium was considerably more effective than others

used in the past. Both media were buffered with 7 mM HEPES (N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid) and were supplemented with 10% heatinactivated fetal calf serum and antibiotics. CV-1 cells were grown in Eagle minimum essential medium (ISI Biologicals) supplemented as described above. Infected cultures of either cell line were maintained in minimum essential medium containing 3% heat-inactivated fetal calf serum. PHFG cells were prepared from abortuses as described previously (14). CV-1 cells were obtained originally from Flow Laboratories, Rockville, Md. Pools of JCV were obtained from PHFG cells infected with 100 to 200 hemagglutinating units of virus per 75-cm2 culture (13). One pool was obtained by infection at comparatively low multiplicity, i.e., with 10 hemagglutinating units per culture. Because exceptional cultures of spongioblasts are required for passage at low multiplicity, JCV could not be grown routinely in this manner. SV40 strain 776 was originally a gift of D. Nathans and was propagated in CV1 cells by infection at multiplicities of 0.1 to 1.0 PFU/ cell. Enzymes. Bacterial restriction endonucleases were purchased from Bethesda Research Laboratories, Inc., Rockville, Md., and from New England Biolabs, Inc., Beverly, Mass. Comparable enzymes from both sources were used interchangeably and were pooled when necessary. Bacterial alkaline phosphatase (Worthington BAPC; 37 U/mg) was treated as described by Smith and Birnstiel (21), except that it was dissolved to give 200 U/ml in a solution containing 10 mM Tris-hydrochloride, pH 7.4, 50 mM NaCl, 10 mM MgCl2, and 1 mM ,B-mercaptoethanol (TNMB). Polynucleotide kinase (20,000 U/mg) from T4-infected Escherichia coli was purchased from Miles Biochemicals, Inc., Elkhart, Ind. Preparation of viral DNA. Viral DNA was obtained from infected cells. Cultures of PHFG cells infected with JCV were frozen at -20°C after developing extensive cytopathic effects at 28 to 35 days after infection. CV-1 cultures infected with SV40 were frozen at 4 to 7 days after infection. Cells were collected from thawed cultures, and DNA was extracted by the method of Hirt (5). Covalently closed, super-

846

VOL. 29, 1979

coiled viral DNA (form I DNA) was isolated by two consecutive equilibrium centrifugations in CsCl-ethidium bromide solution (13); the DNA was banded by centrifugation at 124,000 x g (44,000 rpm; type 65 rotor) for 44 h at 150C. Ethidium bromide was removed from the isolated DNA by extraction four to six times with buffered isoamyl alcohol. The DNA was dialyzed against 10 mM Tris-hydrochloride, pH 7.4-1 mM EDTA (TE). The dialyzed preparations had ratios of absorbance at 260 nm to absorbance at 280 nm of 1.8 to 2.0. Cleavage of viral DNA with restriction endonucleases. All restriction enzymes were used in an amount (usually 2 to 3 commercial units) sufficient to digest 1 ,ug of SV40 form I DNA under our reaction conditions. All reactions were carried out in 50 ML of TNMB containing enzyme, 1 Mug of form I DNA, and 5 ug of autoclaved gelatin. The mixtures were incubated at 37°C for 1 to 3 h. The reactions were terminated by incubation at 60°C for 10 min or by storage at -70°C overnight, followed by heat inactivation. For digestion with two enzymes, the enzymes were added either simultaneously or consecutively. Restriction fragments of JCV DNA were analyzed by agarose gel electrophoresis with SV40 restriction fragments included as markers to calibrate the gel. Migration distances were measured from photographs of the gels. Independent measurements for the same or duplicate gels never differed by more than 0.5 mm, which was taken as the limit of our accuracy. Sizes of JCV fragments were obtained from calibration curves as a percentage of the SV40 genome. The sizes were normalized, to the nearest 0.5%, to fit 100% of the JCV genome. Agarose-ethidium bromide gel electrophoresis. Viral DNAs and their restriction endonuclease products were analyzed in horizontal slab gels (16 by 22 by 0.3 cm) containing 1% agarose (type 1; Sigma Chemical Co., St. Louis, Mo.) and 0.5 ,ug of ethidium bromide per ml (19). Samples of 50,ul received 25MLl of 1% agarose and were loaded. Electrophoresis was carried out at room temperature at 25 mA/slab for 6 to 8 h. The effective path length between wicks was 12 cm. Electrophoresis buffer was 40 mM Tris-acetate, pH 7.8-5 mM sodium acetate-1 mM EDTA (4, 7), containing 0.5 MLg of ethidium bromide per ml. DNA bands were visualized and were photographed (13) with UV light (model C-61 Chromato-vue transilluminator; U-V Products, Inc., San Gabriel, Calif.). 5'-Terminal labeling of JCV linear DNA. The HindIII fragments of JCV DNA were ordered by the method of Smith and Birnstiel (21), which necessitated the labeling of linear DNA at the 5' termini. Fulllength linear DNA was obtained by treatment of 2 Mug of form I DNA with EcoRI for 1 h in a reaction mixture (40 pd) as described above. The reaction was terminated by heat treatment and cooled. A 1-U (5pl) amount of alkaline phosphatase was added, and incubation was resumed at 37°C for 1 h. To inactivate the phosphatase, 4.5 pl of 0.1 M HCl was added to give -pH 5, and the mixture was held at room temperature for 10 min. It was neutralized by the addition of 1.5 pl of 1 M Tris-hydrochloride, pH 8.0, and was transferred to a tube in which 50 MCi (4 Ml) of [y-32P]ATP (2,700 Ci/mmol; a gift of J. Slightom and 0. Smithies)

PHYSICAL MAP OF JCV DNA

847

had been dried. After dissolution of the ATP, 2.2 U (4

pl) of polynucleotide kinase was added. Labeling was

carried out at 37°C for 30 min and was stopped by the addition of 2Ml of 5 mM ATP and incubation at 60°C for 10 min. A portion was precipitated with trichloroacetic acid in the presence of pyrophosphate (9). Specific activities of 135,000 cpm/Mug were obtained. SV40 DNA was cut with EcoRI and was labeled similarly in a control reaction. The labeling of this DNA was twofold less efficient. Most of the unincorporated [y-32P]ATP was removed from the final reaction mixtures by chromatography through a 0.9-ml column of Sephadex G-25 (Pharmacia Fine Chemicals, Piscataway, N.J.) in TE in a 1-ml syringe. The labeled DNA was recovered in 100 to 150 Ml. Partial Hindl digestion of end-labeled JCV linear DNA. The HindIII fragments of JCV DNA were ordered with respect to one end of the EcoRI linear DNA (21). The terminally labeled DNA (-2 Mg in 100Ml) was adjusted to conditions for digestion with restriction endonucleases and was treated for 1 h at 37°C with HpaI in slight excess. After heat inactivation, aliquots (-10,000 cpm) each received 1 Mg of salmon DNA (Sigma Chemical Co.) and from 0.1 to 1.5 times the amount of HindIll required to give complete digestion of 1 Mig of SV40 DNA under standard conditions. All reactions were carried out at 37°C for 1 h and were stopped by incubation at 60°C for 10 min.

The products were analyzed by agarose gel electrophoresis as described above. Unlabeled SV40 restriction fragments and end-labeled SV40 linear DNA were included to size the JCV DNA fragments. Autoradiography. Slabs containing "P-labeled JCV fragments were photographed with UV light and then were dried as described by Smith and Birnstiel (21). After preliminary autoradiography, the portion (-2 cm) of the slab containing residual, unincorporated [y-32P]ATP was removed. The dried slab then was exposed to Kodak NS-5-T X-ray film for 12 h at room temperature.

RESULTS Susceptibility of JCV DNA to restriction enzymes. Several bacterial restriction endonucleases were examined for their utility in producing a physical map of JCV DNA. The enzymes EcoRI, HpaI, and PstI each cleaved form I DNA of JCV at only one site (Fig. 1, slots 4, 10, and 12, respectively). All of the molecules were cleaved completely into linear (form III) DNA. However, under our reaction conditions, which resulted in complete digestion of BKV and SV40 DNAs, JCV DNA was partially resistant to BamHI. A fraction of the DNA was resistant even at concentrations of enzyme that were 2- to 10-fold in excess (data not shown). This resistance is a consequence of the heterogeneity of the DNA (7, 12, 13; see below). The DNA appeared to be completely resistant to HhaI, which cleaved both BKV and SV40 DNAs (data not shown).

848

MARTIN ET AL.

J. VIROL.

FIG. 1. Restriction endonuclease cleavage patterns of JCV DNA. Samples (1 pg) of prototype JCV DNA were treated at 37°C for 3 h with buffer alone or with two commercial units of the enzymes indicated on the figure. Equivalent reactions containing SV40 DNA were included to serve as controls and to provide calibration markers. The reactions were stopped by incubation at 60°C for 10 min. Agarose was added to the samples, and electrophoresis was carried out in a horizontal agarose-ethidium bromide slab gel for 8 h at 25 mA. The photograph (left) was taken with UV light. The drawing to the right indicates the positions of all bands seen in the original photograph. The dashed lines denote presumably defective molecules or fragments of JCV DNA.

It was reported previously that digestion of JCV DNA with unfractionated R-Hind yields five fragments (7, 12). Undoubtedly, two small Hind fragments were not retained in the gels of Howley et al. (7) or Osborn et al. (12), because we have observed repeatedly that HindII produces four fragments (Fig. 1, slot 6), HindIII produces three fragments (Fig. 1, slot 8), and HindII + III produces seven fragments (data not shown). The order of these fragments was determined with respect to each other and with respect to the sites cleaved by EcoRI, HpaI, and PstI (see below). Because we wanted to map the genome of the prototype virus, throughout most of this work we used DNA of the Mad-I isolate of JCV (12, 16). This DNA is typically heterogeneous (Fig. 1, slots 2 and 4). Therefore, it is important to know that a map derived from experiments with heterogeneous DNA would be representative of homogeneous DNA. Usually, homogeneous DNA is obtained from cloned virus propagated by passage at low multiplicity of infection. Because of the stringent cell system required for production of JCV in vitro (i.e., the spongioblasts in PHFG cultures) and because of the limited cytopathic effect produced in that system, JCV at present cannot be cloned. However, we have had limited success in producing Mad1 in exceptional glial cultures infected at a com-

paratively low multiplicity (10 rather than 100 hemagglutinating units per culture). The DNA obtained from cells infected with virus from one such passage (JCI,Mp) is considerably more homogeneous (Fig. 2, slots 3 and 6) than typical DNA (Fig. 2, slots 2 and 5). (Unfortunately, the amount of such homogeneous DNA was insufficient to be used in the mapping experiments described below.) More to the point here is the fact that the products of HindII cleavage of homogeneous DNA (Fig. 2, slot 9) are identical in size to the major products of HindII cleavage of heterogeneous DNA (Fig. 2, slot 8). (The minor fragments are a consequence of the heterogeneity of the DNA and are presumably defective fragments.) Furthermore, the DNA fragments produced by cleavage of homogeneous DNA with HindII + III gave an electrophoretic pattern identical to that of the major fragments in a HindII + III digest of heterogeneous DNA (data not shown). Thus, a physical map derived with typical heterogeneous DNA of prototype virus is very likely representative of the DNA which may eventually be obtained from cloned virus. Supportive of this conclusion is the fact that homogeneous DNA of Mad-i and homogeneous DNA obtained from another JCV isolate (Mad-4) give identical HindII and HindII + III cleavage patterns (data not shown). Sizes of JCV DNA fragments. Restriction

VOL. 29, 1979

PHYSICAL MAP OF JCV DNA

849

The sizes of the SV40 fragments were recorded as a percentage of the genome by the method of 0

L)

c

rr°

I

bi

-- s I E qt Ji t > u u > 0> cn u , 0 en -, n o

_

2 3 4 5 6 7 8 9 FIG. 2. Comparison of heterogene ous and homoJCV DNAs. Typical heter( ogeneous DNA (JC) was obtained from PHFG cultures infected in a routine manner, as described in the t4 ext. Homogeneous DNA (JCLMp) was prepared from .PHFG cultures infected at a lower multiplicity, as iridicated in the text. Samples (I pg) of both DNAs uxere treated as described in the legend to Fig. 1 e that the incubation time for endonuclease dig digests O The products, along with those in coi SV40 DNA, were analyzed as describe din the legend to Fig. 1. geneous

rxcept

netrol

endonuclease fragments of JCV D NA were separated for determination of size in horizontal slab gels of 1% agarose containing ethidium bromide (19). Included in each slab as calibration markers were SV40 fragments prc)duced by the enzymes used to map JCV DNA. Figure 1 is a typical display of JCV fragmenIts and SV40 markers; Table 1 lists the sizes 4of these JCV fragments. It should be noted that in our gels we did not detect the two smallest Hin.dIl fragments of SV40 DNA (23; Fig. 1, slot 5) o. r the smallest HpaI fragment (18; Fig. 1, slot 9). Furthermore, we did not resolve the SV40 Hind[III fragments B and C (23; Fig. 1, slot 7) or the HindII + III fragments C and D, F and G, and H through K (1; data not shown). We have assuimed that the leading edges of these unresolved bands are indicative of the rate of migration--and therefore of the size-of the smaller fragnients in each group.

Danna et al. (1). Therefore, the sizes of JCV fragments were measured as a percentage (to

the nearest 0.5%) of the genome of SV40, as is shown in Table 1. Because JCV DNA appears to be smaller than that of SV40 (7, 13; see below) and because the sum of each set ofJCV fragment sizes was not 100%, the measured values were normalized to give the appropriate percentage of the genome of JCV (Table 1). The lengths in base pairs of JCV DNA fragments were estimated from the percentage of the SV40 genome that each JCV fragment measured and from an assumed length of 5,230 base pairs for full-length SV40 DNA (17). We estimated that linear JCV DNA is 94 to 96% of the length of SV40 linear DNA (Table 1, 4,900 to 5,000 base pairs). This should be an underestimate of the size of nondefective, fulllength JCV DNA because the measurement is biased toward the smaller, presumably defective molecules (13). A second estimate was derived from the sums of the fragments produced in digestions with HindII (those indicated in Tables 1 and 2). The sizes of the products from those particular digestions were derived from the most accurate region of the calibration curve (i.e. c50% of the length of SV40 DNA). From 11 such estimates an average length of 5,125 ± 105 base pairs (98 ± 2% of SV40 DNA) was determined for full-length JCV DNA. Heterogeneous JCV DNA is cleaved by

HindII into four major and three to four minor fragments (Fig. 1, slot 6). The four major fragments appear to comprise 100% of the genome (Table 1). This conclusion was verified by the observation that only these fragments were produced by HindII cleavage of homogeneous DNA obtained from another JCV isolate (Mad-4) and from Mad-i passed at comparatively low multiplicity, as mentioned above. The minor bands appear to be products of defective molecules. Minor bands also are seen after treatment with HindIII (Fig. 1, slot 8). The enzyme produces three major fragments which apparently comprise full-length molecules (Table 1). Localization of single restriction sites in JCV DNA. The first step in mapping the genome of JCV was the orientation of single-cut enzyme sites with respect to each other. This was accomplished by digesting the DNA with the three possible pairs of enzymes which cleave the DNA at one site, i.e., EcoRI, HpaI, and PstI. The products of these double digests and their respective sizes are given in Table 2. The resultant three pairs of fragments defined an unambiguous order for the sites cleaved by these

850

MARTIN ET AL.

J. VIROL.

TABLE 1. Sizes of JCV DNA fragments from single-enzyme digests JCV fragment size expressed as % of genome of: No. of base pairn'

No. of expta

JCV fragment

94.5 ± 2.5 96.0 ± 0.5 96.5

JCVb 100.0 100.0 100.0

4,942 ± 131 5,021 ±26 5,047

50.0 ± 1.5 21.0 ± 0.5 17.0±0.5 11.5 ± 0.5 99.5 ± 2.0

50.0 ± 0.5 21.0 ± 0.5 17.0±0.5 12.0 ± 0.5 100.0± 0.5

2,615 ± 78 1,098 ± 26 889±26 601 ± 26 5,204 ± 105

SV40

EcoRI linear HpaI linear PstI linear

3 2 1

HindII fragment: A B C D Total

4

4

2 HindIII fragment: 91.0 ± 1.0 4,759 ± 52 A 88.5 ± 0 8.0 ± 0.5 418 ± 26 B 8.0 ± 0.5 4.0±0 C 4.0±0 209±0 2 5,361 ± 78 100.0±0 Total 102.5 ± 1.5 a Number of experiments from which a mean was derived. b In this and in subsequent tables the percentage of the JCV genome is a normalized value obtained as described in the text. 'The number of base pairs was determined by multiplying the percentage of the genome of SV40 by 5,230, an assumed total number of base pairs for SV40 DNA (17).

TABLE 2. Restriction fragments obtained from double-enzyme digests of JCV DNA of ~No. expt"

Fragment Fragment + EcoRI ment:

HpaI

frag-

ofgt eof ggenome)

o o

bs

No. Of expt'It

Fragment

pairs

PstI + Hindll frag-

2

(% Length No. of base of ge-

nome)

pairs

1

ment:

85.0 ± 0 15.0±0 100.0 ± 0

A B

Total PstI + EcoRI frag-

4,446 ± 52 784 ±0 5,230 + 52

3

51.5 15.5 4.5 17.0 11.5 100.0

A

B, B., C D Total

2,615 784 235 863 575 5,073

ment:

68.5 ± 0 5 3,713 + 78 31.5 ± 1.0 1,700 ± 78 100.0 ± 0 5,413 + 157

A B Total

HindIII + Hindll frag-

Al

2

ment:

19.0 ± 0 941 ± 0 17.0±0 837±0 8.0 ± 0 418 ± 0 4.0 ± 0 209 ± 0 22.0 + 0.5 1,098 + 26 18.0 ± 0 889 ± 0 12.0±0 575±0 100.0 ± 0.5 4,942 ± 26

A, PstI + HpaI fragment: A B Total

2

EcoRI + HindII frag-

2

ment:

A B

53.0 ± 0 47.0 ± 0 100.0 ± 0

21.0 ± 0.5 1,072 ± 26 15.0 ± 0.5

C2 D Total

HpaI

+ Hindll ment:

A B C D Total

A.1 A4 B C D Total

HindIII EcoRI + 50.0 ± 1.0 2,563 + 52

C,

"

2,824 + 0 2,510 ± 0 5,361 ± 26

758 ± 26 131±26

2.5±0.5 12.0±0 575±0 100.0 ± 0 5,125 ± 0

frag-

frag-

3,007±26

4.0±0

3,00 ± 0 471±0 209±0

66.0 ± 0 22.0 ± 0 8.5±0 3.5±0 100.0 + 0

3,504 ± 52 1,151 ± 0 471±26 183±0 5,308 ± 78

33.0 ± 0 8.5±0

Al B C Total

PstI + HindIII frag-

2

2

en 55t00

100.5 ± 0

5,518 ± 26

2

ment: 50.5 ± 0.5 2,589 ± 26 21.0 ± 0.5 1,072 26 17.0±0 889±0 12.0±0 601±0 100.5 ± 0.5 5,125 ± 0

Number of experiments from which a mean was derived.

Al A, B C Total

VOL. 29, 1979

PHYSICAL MAP OF JCV DNA

851

enzymes. By assigning map position 0 to the a b c d e f EcoRI site and arbitrarily proceeding clockwise nOr ig *ain-_. through the PstI site to a unit length of 1.0, one can place the PstI site at map position 0.315 and the HpaI site at map position 0.850. Order of the HindH sites in JCV DNA. 8 5% ) /o a "mm The sites cleaved by HindII were ordered with X67% 64O%respect to the EcoRI, HpaI, and PstI sites. In 856%°-'_Mm addition, the HindII fragments cleaved by HindIII were identified. JCV DNA was digested with HindII together with each of the other four enzymes as indicated in Table 2. The EcoRI site is located in HindII fragment C, and the PstI 5site is in HindII fragment B. As expected (23), the HpaI site is one of the four sites cleaved by HindII. Seven fragments were observed in a FIG. 4. Autoradiograph of the products of end-ladouble digestion with HindII and HindIII. All beled linear DNA partially digested with three HindIII sites could be localized to HindII HindIII.JCV JCV DNA was digested completely with fragment A (Table 2). EcoRI, the linear DNA was labeled at the 5' termini, The results of the double digestions indicated and the labeled DNA was digested completely with in Table 2 established the placement of the HpaI, as described in the text. The DNA and added HindII fragments A and B as shown in Fig. 5. salmon sperm DNA were subjected to partial or comHowever, the order of fragments C and D re- plete digestion by HindIII at 37°C for I h, and the mained ambiguous because the EcoRI cleavage products were displayed in an agarose slab. (a site was at or near the center of this region; through d) Products of digestion of HpaI + EcoRI (100,000 cpm + 1 pg of salmon sperm DNA) identical sizes of the products of HindII + EcoRI fragments with 0.2 (a), 0.3 (b), 0.6 (c), or 1.6 U (d) of HindIII. (e) -,

,.-

'- '