Acta Neuropathol (1992) 83:105 - 112
,kW
Heu patholosia (~ Springer-Verlag 1992
Regular papers Molecular characterization of a JC virus (Sap-l) clone derived from a cerebeilar form of progressive multifocai leukoencephalopathy*
H. Takahashi 1, Y. Yogo z, Y. Furuta 1, A. Takada 3, T. Irie 4, M. Kasai 4, K. Sano 1, Y. Fujioka l, and K. Nagashima 1 1 Department of Pathology, Hokkaido University School of Medicine, North 15 West 7, Kita-ku, Sapporo 060, Japan : Department of Viral Infection, The Institute of Medical Science, University of Tokyo, Tokyo, Japan 3 Department of Pathology, Sapporo Municipal Hospital, Sapporo, Japan 4 Department of Medicine, Sapporo Hokuyu Hospital, Sapporo, Japan Received June 21, 1991/Revised, accepted September 23, 1991
Summary. Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease caused by polyomavirus JC (JCV). In the majority of cases of P M L the cerebrum is mainly affected (cerebral PML) but on rare occasions lesions are restricted to the cerebellum and brain stem (cerebellar PML).We report a rare cerebellar P M L case which occurred in a Japanese patient undergoing prolonged hemodialysis treatment. To understand the molecular basis of the viral tissue tropism, we molecularly cloned JCV D N A and compared it with those of cerebral PML. O f ten clones analyzed nine showed identical fragment patterns after digestion with various restriction endonucleases, and we designated these clones Sap-1. It could be shown that the basic structures of the regulatory regions are similar between Sap-1 and isolates from cerebral PML. Restriction endonuclease mapping analysis was used to examine the genetic relationship between Sap-1 and urine-derived isolates containing the archetypal regulatory sequence. We found that Sap-1 was genetically related to an archetypal JCV isolate in Japan.
Key words: Polyomavirus JC - Progressive multifocal leukoencephalopathy (PML) - Cerebellar P M L - Molecular characterization
Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease caused by the human polyomavirus JC (JCV) [15, 17]. High-risk groups for P M L are patients with underlying diseases which decrease their immunological capacity and those who are immunosup* Supported by the Suhara Memorial Foundation (Sapporo), and a Special Grant-in-Aid for Promotion of Education and Science in Hokkaido University, provided by the Ministry of Education, Science and Culture. K. Nagashima is supported by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan, No. 02807036 Offprint requests to: K. Nagashima (address see above)
pressed therapeutically or after organ transplantation [22]. Although extremely rare, there have been a few documented cases in which P M L has occurred in patients with normal immunological function [19]. P M L cases can be classified into two types on the basis of involved structures in the central nervous system: cerebral P M L in which the cerebrum is mainly involved and cerebellar P M L in which the cerebellum and, to a lesser extent, the brain stem are involved [18]. In a few cerebral PML cases, the cerebellum and brain stem may also be involved but affected regions are generally small in these regions compared to the cerebrum. Cerebral PML occurs much more frequently than cerebellar PML. Although more than 100 documented cases of P M L have been reported to date, we are aware of only 12 cases of cerebellar P M L [8, 13, 18]. A t present it is not known what factors may influence the tropism of JC virus and determine the type of P M L that occurs. We recently encountered a case of cerebellar P M L that occurred in a Japanese patient who had received prolonged hemodialysis treatment. Since the JCV D N A has never been isolated from cerebellar P M L cases, we molecularly cloned the virus D N A , designated Sap-I, from the cerebellar lesions of this patient. The regulatory sequence of Sap-1 was compared to those previously cloned from cerebral P M L cases. Furthermore, we studied the genetic relationship between Sap-1 and archetypal isolates from the urine of healthy Japanese [231.
Case repor! The patient was a 64-year-old Japanese woman who had received prolonged hemodialysis treatment for chronic renal insufficiency believed to be secondary to chronic glomerulonephritis from 1982 until 1989. In March 1989 she developed bilateral ataxia and ophthahnoplegia. This was followed in June by sensory disturbances, pyramidal signs, a right hemiparalysis and a disturbance of consciousness. She died in July from respiratory failure. Computer tomography during her illness showed a low-density area in the right cerebeltar hemisphere and magnetic resonance imaging
106 revealed an area of high signal intensity in the white matter of the right cerebellar hemisphere. The patient was noted to have negative tuberculin skin reaction, and Tcell reactions to phytohemagglutinin (PHA), pokeweed mitogen (PWM) and concanavalin A (Con A) were decreased. Serum immunoglobulins were below normal. IgG was 944 mg/100 ml (normal 1,420_+330 rag/100 ml), IgA 156 rag/100 ml (245_+80 rag/100 ml), IgM 121 rag/100 ml (210 • 170 rag/100 ml), suggesting B cell dysfunction or abnormalities.
Pathological findings Autopsy was carried out 3 h after death. The kidney showed marked contraction with multiple small cysts on the surface. The direct cause of death was pneumonia. Cerebellar sections revealed widespread tissue attenuation in the eerebetlar white matter. No abnormalities were noted in the cerebral white matter. On histological examination the attenuated cerebellar white matter was completely devoid of myelin sheaths, although axons were relatively preserved, showing primary demyelination (Fig. 1A). In the demyelinated lesions there were numerous enlarged nucleibearing oligodendroglia (Fig. 1B) and hypertrophic astrocytes, some of them showing nuclear atypism.
using polyclonal anti-JCV antibody raised by immunization with JCV (Tokyo-1 strain) [16].
In situ hybridization Localization of the JCVgenome at the cellular level was carried out using in situ hybridization methods. Briefly, 5-~xm-thick sections were cut from paraffin-embedded tissue. The sections were treated with 100 Bg/ml proteinase K for 15 rain at 37 ~ and refixed with 4 % paraformaldehyde. The JCV DNA probe was labeled with digoxigenin-dUTP (Boehringer Mannheim) by the random primer method [4]. Hybridization was carried out at 42~ for 16 h, followed by stringent washing, and then digoxigenin was reacted with alkaline phosphatase labeled anti-digoxigenin antibody. The final product was visualized by nitro blue tetrazolium for alkaline phosphatase [6].
Electron microscopy
Methods
A number of small pieces of cerebellar tissue were fixed in 2.5 % glutaraldehyde, 2 % paraformaldehyde and 6 % sucrose in 0.1 M sodium cacodylate buffer (pH 7.2). They were then fixed in 2 % osmium tetroxide, dehydrated, and embedded in a Epon-Araldyte mixture. Ultrathin sections prepared with a LKB-type 3 ultramicrotome were doubly stained with uranyl acetate and lead citrate and examined in a Hitachi H-800 electron microscope.
Immunochemical studies
Extraction and fractionation of tissue DNA
Immunohistochemical staining for JCV V antigen was carried out using the avidin-biotin-peroxidase complex (ABC) method [7],
Affected cerebellar tissue was digested with proteinase K in the presence of 1% sodium laurylsulfate in 10 mM Tris-HC1 (pH
Fig. 1A, B. Histological features of a cerebellar form of PML. A Diffuse and complete loss of myelin sheath in the cerebellar white matter. The peripheral areas indicated by arrows only contained myelin sheaths in the white matter. Kliiver-Barrera stain for myelin
sheath. B Numerous enlarged nuclei-bearing oligodendroglias were seen in the demyelinated lesions. Small nuclei at the right upper corner were those of cerebellar granule cells. Hematoxylineosin stain x 200
107 7.8)-10 mM EDTA at 56 ~ for 1 h. The digest was extracted once with phenol and once with chloroform-isoamyl alcohol (24:1). The aqueous phase was dialyzed extensively against 2 mM NaC1, 2 mM Tris-HCl (pH 7.8), 0.2 mM EDTA and DNA was concentrated by ethanol precipitation. DNA extracted from tissue was digested with EcoRI and electrophoresed on a 0.6 % agarose gel. HindIII-digested lambda DNA was co-electrophoresed and used as size markers. Portions of the gel containing DNA migrating in the 4000 to 6000 base pairs (bp) region was excised and electroeluted as described previously [20]. This size-fractionated DNA served as material for cloning of the JCV DNA.
DNA was linked to the vector at the EcoRI site. In addition, five cerebral PML-derived recombinant JCV DNA clones of which regulatory sequences had been determined previously were used to demonstrate structural features of regulatory regions common to cerebral PML-derived JC virus isolates. They included four clones from American patients (Madl-Br[5], Herl-Br[12], MadS-Br[12], Mad9-Br[12]) and one from a German patient (GS/B[ll]).
Cloning of J C V D N A
carried out as recommended by the suppliers. Digested DNAs were electrophoresed on 1.5 % or 1.8 % agarose gels or on a 3.5 % polyacrylamide gel in 45 mM Tris-borate, 1 mM EDTA [20], depending on the expected sizes of the DNA fragments. As size markers, the EcoT14I (StyI) fragments of lambda DNA or the Hinfl fragments of pUC19 were run in parallel. After electrophoresis, gels were stained with ethidium bromide and were photographed on a UV light transilluminator.
Two methods were used. In initial experiments, the EcoRIdigested and size-fractionated tissue DNA was ligated to lambda gtlO which had been digested with EcoRI and treated with bacterial alkaline phosphatase. The ligated DNA was packed in vitro using Gigapack (Strategene, Calif.) according to the protocol suggested by the manufacturer and was used to infect E. coti C600. Recombinant phages containing JCV DNA were obtained by two rounds of plaque hybridization [20] using 32P-labeled JCV DNA as the probe. Recombinant phages containing JCV DNA were purified as described previously [20]. Cloned JCV DNA was excised with EcoRl from recombinant phages and was subcloned into the plasmid vector pUC18. In subsequent experiments, the EcoRI-digested, size-fractionated DNA derived from a different cerebellar lesion was directly ligated with EcoRI-digested, bacterial-alkaline phosphatasetreated pUC19 and was used for E.coli DH5c~ transformation by high-voltage electroporation [3]. Recombinant plasmids were screened by colony hybridization [20] using 32p-labeled JCV DNA as the probe. Recombinant DNAs containing JCV (MY) DNA (pJC-MY), JCV (CY) DNA (pJC-CY, clone 1), and JCV (Tokyo-i) DNA (pJCT-Br) served reference DNAs. pJC-MY and pJC-CY were established previously from the urine of healthy Japanese [23]. pJCT-Br was previously cloned from the cerebral tissue of a Japanese PML patient [14]. Since, in pJC-MY and pJC-CY, JCV DNA was cloned at BamHI sites, the JCV DNA was exised from the vector and full-length JCV DNA was recloned into the EcoRI site and used for restriction enzyme analysis. In pJCT-Br, JCV
Restriction enzyme analysis Restriction endonucleases were obtained from Toyobo Co., (BglI, DdeI, HaeIII, HincII, Hinfl, NcoI, PvufI) and Bethesda Research Laboratories, (SstI [SacI]). Digestion with each enzyme was
D N A sequencing A NcoI-SstI fragment, containing a region from the start site of T antigens to the origin of DNA replication, was inserted into M13 mpl8 or rap19 between the Sinai and SstI sites. A HindIII-NcoI fragment, containing a region from the origin to the start site of late leader protein (agnoprotein), was inserted into M13 mpl8 or mpl9 between HindIII and Sinai sites. Single stranded DNAs purified from recombinant phages were sequenced using the chaintermination method [21]. Sequencing was carried out with overlapping clones representing both DNA strands.
Results
Demonstration of J C V infection in cerebellar lesions To c o n f i r m t h e diagnosis of P M L m a d e histologically, we a t t e m p t e d to d e t e c t J C V V a n t i g e n s b y i m m u n o h i s t o -
Fig. 2A,B. Demonstration of JC viral antigen and genome. A Enlarged nuclei labeled with anti-JCvirus (JCV) V antibody. Note the lack of antigen in the reactive astrocytes (arrows). Avidin-biotin-peroxidase complex method. B Enlarged nuclei were shown to contain JCV genome which is demonstrated as black signals using a non-radioactive in situ hybridization method (digoxigenin). A,B • 200
108
Restriction analysis of cloned JCV DNA
Fig. 3. Electron micrographrevealed a large number of papovavirus particles in an enlarged nucleus x 40,000
chemical method and JCV DNA by in situ hybridization. V antigens were detected in the enlarged nuclei of oligodendroglial cells in a demyelinating region of the cerebellum, but were not in atypical astrocytes (Fig. 2A). The nuclei in which V antigens were demonstrated were also positive for the viral DNA (Fig. 2B). Furthermore, using an electron microscope we could detect numerous polyomavirus virions in the abnormal nuclei of oligodendroglial cells (Fig. 3).
Molecular cloning of JCV DNA Following digestion of total DNA from cerebellar tissue with EcoRI, linear JCV DNA was detectable by agarose gel electrophoresis and by subsequent blot hybridization (data not shown). To clone the virus DNA whole tissue DNA was digested with EcoRI, separated by agarose gel electrophoresis and DNA migrating in the expected size region was eluted and ligated into either lambda or plasmid vectors. In initial experiments, JCV DNA was cloned using lambda gtlO and then subcloned with plasmid pUC18. Although many clones were obtained by this method, one clone, designated as Sap-1 clone 1 (c/l), was used for molecular characterization. In the subsequent experiments, we cloned JCV DNA directly into pUC19 plasmid using DNA derived from different areas of the cerebellum. Nine clones containing JCV DNA, Sap-1 cl2 to cI10, were obtained and all of were subjected to molecular characterization.
We performed a series of restriction enzyme analyses to compare the various Sap-1 clones to JCV clones from a cerebral PML case (Tokyo-1 [14] and to clones from the urine of healthy individuals (CY and MY) [23]. CY and MY represent the two JCV subtypes circulating in the Japanese population (unpublished results). JCV DNAs were recovered from Sap-1 clones and from reference clones by EcoRI digestion and were digested with BgllI, DdeI, HaeIII, HinclI, Hinfl, PvuII, or SstI (typical gel electrophoresis patterns are shown in Fig. 4.). The results can be summarized as follows: i. In analyses using enzymes other than SstI all the Sap-1 clones were identical to one another (Fig. 4A, B, D). However, SstI digestion showed that one Sap-1 clone, cl9, differed from the others (see below). We refer to those clones except cl9 as Sap-1 unless otherwise specified. ii. SstI digestion revealed that most Sap-1 clones with the exception of cl9 were similar to Tokyo-1 but differed from urine-derived clones (CY and MY). Sap-1 and Tokyo-1 did not generate one of the 260-bp fragments generated by CY and MY but instead gave rise to two smaller fragments, the sizes of which were slightly different between Sap-1 and Tokyo-1. It was previously reported that in Tokyo-1 these shorter fragments were generated by rearrangement of regulatory sequence, such as deletion and amplification, relative to the regulatory sequence of urine-derived isolates [23]. Therefore, it is likely that the regulatory region of most Sap-1 clones also underwent similar sequence rearrangement.The SstI cleavage pattern of cl9 is characterized by the fusion of a 260-bp fragment and the shortest fragment generated by the other Sap-1 clones (Fig. 4C). Probably, one of two SstI sites in duplicated segments was lost in c/9. iii. Two major urine-derived JCV strains (CYand MY) in Japan can be differentiated by digestion with several enzymes, including DdeI, HaeIII, and Hinfl (gel patterns after Hinfl and DdeI digestion are shown Fig. 4C and D, respectively). In analysis using these enzymes, gel patterns of Sap-1 and Tokyo-1 were identical to that of MY (Fig. 4C and D). iv. When HincII was used, all Sap-1 clones were distinguished from Tokyo-1 and MY (Fig. 4A). The difference in HincII cleavage profiles were ascribed to the presence in Sap-1 clones of an extra HincII cleavage site located in the T antigen-coding region (data not shown). This extra HinclI site has not been detected in the JCV isolates examined so far (unpublished results). However, no other changes were detected between Sap-1 and MY (and Tokyo-l) in analysis in which other restriction enzymes used including PvuII, except the difference due to rearrangement of regulatory sequences in Sap-1 and Tokyo-1 (see below).
Regulatory sequence of cloned JCV DNA For representative Sap-1 clones, cll, cl2, and c13, that appeared to be identical in restriction analysis, and cl9
109
Fig. 4A-D. Restriction enzyme analysis of JCV DNA clones. JCV DNA was recovered from ten JCV DNA clones from the current cerebellar progressive multifocal leukoencephalopathy (PML) case, a clone from a cerebral PML case (Tokyo-1), and two urine-derived clones (CY and MY), and digested with restriction enzymes HincII (A), Hinfl (B), SstI (C), and DdeI (D). Fragments generated were electrophoresed on a 1.5 % agarose gel (HincII and Hinfl), on a 1.8 % agarose gel (SstI), or on a 3.5 % polyacrylamide gel (DdeI). Fragments separated in gels were stained with ethidium
bromide and were photographed on a UV light transilluminator. Sizes of some EcoT14I fragments of lambda DNA (A-C) and those of Hinfl fragments of pUC19 (D) are indicated in bp on the right. Note that two fragments of 260 bp in size were generated after digestion of CYand MY with SstI (C). Faint bands are indicated by dots in C. Closed arrowheads on the left of B and D denote fragments which were produced from CY but not from MY, while open arrowheads indicate those which were produced from MY but not from CY
that differed from the other clones in an analysis with SstI, we sequenced the entire noncoding region spanning from the start site of T antigen to that of late leader protein (agnoprotein). The sequences cll, cl2, and cl3 were identical to one another but differed from that of cl9 (the sequences of cll and cl9 are shown in Fig. 5) This result, together with those of the restriction analysis described above, suggest that cll represents the major species present in the cerebellar lesion of the patient, while cl9 is probably a minor one, or the difference may be due to events during cloning procedures. In Fig. 6, the archetypal regulatory sequence (A), identified in JCVs excreted by nonimmunocompromised individuals [23], is shown at the top and those of cerebral PML-derived isolates (B) and those of Sap-1 clones (C) are given below, showing deletions relative to the archetype as gaps and repeats as parallel straight lines [23]. Comparison of the regulatory sequence of cll with the archetypal sequence (Fig. 6A, C) suggests that the regulatory sequence of cll may have arisen from three
independent events, duplication of 60-bp (nucleotides [nt] 43 to 101) and 3-bp sequences (nt 257 to 259) and deletion of a 82-bp sequence (nt 100 to 181). However, the sequence data can not indicate in which order these events would have occurred. From Fig. 6, it is evident that the regulatory sequences of cerebral PML-derived isolates share many of the structural features, with the single exception of Madll-Br: duplication of domain A, (47-bp sequence) and C (9-bp sequence) and deletion of domain B (55-bp sequence) (Fig. 6B). The major species of Sap-1 (c/l) had duplications of 59-bp (archetype nt 43 to 101) and 3-bp sequences (nt 257 to 259) and was lacking a 65-bp sequence (nt 117 to 181) (Fig. 6C). The 59-bp repeated segment contains most of domain A, except for a terminal 5-bp stretch. The 65-bp deleted sequence contained most of domain B except for one nucleotide. Although it was not duplicated, most of domain C, except for one nucleotide, was present in Sap-1 regulatory region. The duplication of domain C may not be important for the replication of JCV in brain
110
A
LPI
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,kW
Heu patholosia (~ Springer-Verlag 1992
Regular papers Molecular characterization of a JC virus (Sap-l) clone derived from a cerebeilar form of progressive multifocai leukoencephalopathy*
H. Takahashi 1, Y. Yogo z, Y. Furuta 1, A. Takada 3, T. Irie 4, M. Kasai 4, K. Sano 1, Y. Fujioka l, and K. Nagashima 1 1 Department of Pathology, Hokkaido University School of Medicine, North 15 West 7, Kita-ku, Sapporo 060, Japan : Department of Viral Infection, The Institute of Medical Science, University of Tokyo, Tokyo, Japan 3 Department of Pathology, Sapporo Municipal Hospital, Sapporo, Japan 4 Department of Medicine, Sapporo Hokuyu Hospital, Sapporo, Japan Received June 21, 1991/Revised, accepted September 23, 1991
Summary. Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease caused by polyomavirus JC (JCV). In the majority of cases of P M L the cerebrum is mainly affected (cerebral PML) but on rare occasions lesions are restricted to the cerebellum and brain stem (cerebellar PML).We report a rare cerebellar P M L case which occurred in a Japanese patient undergoing prolonged hemodialysis treatment. To understand the molecular basis of the viral tissue tropism, we molecularly cloned JCV D N A and compared it with those of cerebral PML. O f ten clones analyzed nine showed identical fragment patterns after digestion with various restriction endonucleases, and we designated these clones Sap-1. It could be shown that the basic structures of the regulatory regions are similar between Sap-1 and isolates from cerebral PML. Restriction endonuclease mapping analysis was used to examine the genetic relationship between Sap-1 and urine-derived isolates containing the archetypal regulatory sequence. We found that Sap-1 was genetically related to an archetypal JCV isolate in Japan.
Key words: Polyomavirus JC - Progressive multifocal leukoencephalopathy (PML) - Cerebellar P M L - Molecular characterization
Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease caused by the human polyomavirus JC (JCV) [15, 17]. High-risk groups for P M L are patients with underlying diseases which decrease their immunological capacity and those who are immunosup* Supported by the Suhara Memorial Foundation (Sapporo), and a Special Grant-in-Aid for Promotion of Education and Science in Hokkaido University, provided by the Ministry of Education, Science and Culture. K. Nagashima is supported by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan, No. 02807036 Offprint requests to: K. Nagashima (address see above)
pressed therapeutically or after organ transplantation [22]. Although extremely rare, there have been a few documented cases in which P M L has occurred in patients with normal immunological function [19]. P M L cases can be classified into two types on the basis of involved structures in the central nervous system: cerebral P M L in which the cerebrum is mainly involved and cerebellar P M L in which the cerebellum and, to a lesser extent, the brain stem are involved [18]. In a few cerebral PML cases, the cerebellum and brain stem may also be involved but affected regions are generally small in these regions compared to the cerebrum. Cerebral PML occurs much more frequently than cerebellar PML. Although more than 100 documented cases of P M L have been reported to date, we are aware of only 12 cases of cerebellar P M L [8, 13, 18]. A t present it is not known what factors may influence the tropism of JC virus and determine the type of P M L that occurs. We recently encountered a case of cerebellar P M L that occurred in a Japanese patient who had received prolonged hemodialysis treatment. Since the JCV D N A has never been isolated from cerebellar P M L cases, we molecularly cloned the virus D N A , designated Sap-I, from the cerebellar lesions of this patient. The regulatory sequence of Sap-1 was compared to those previously cloned from cerebral P M L cases. Furthermore, we studied the genetic relationship between Sap-1 and archetypal isolates from the urine of healthy Japanese [231.
Case repor! The patient was a 64-year-old Japanese woman who had received prolonged hemodialysis treatment for chronic renal insufficiency believed to be secondary to chronic glomerulonephritis from 1982 until 1989. In March 1989 she developed bilateral ataxia and ophthahnoplegia. This was followed in June by sensory disturbances, pyramidal signs, a right hemiparalysis and a disturbance of consciousness. She died in July from respiratory failure. Computer tomography during her illness showed a low-density area in the right cerebeltar hemisphere and magnetic resonance imaging
106 revealed an area of high signal intensity in the white matter of the right cerebellar hemisphere. The patient was noted to have negative tuberculin skin reaction, and Tcell reactions to phytohemagglutinin (PHA), pokeweed mitogen (PWM) and concanavalin A (Con A) were decreased. Serum immunoglobulins were below normal. IgG was 944 mg/100 ml (normal 1,420_+330 rag/100 ml), IgA 156 rag/100 ml (245_+80 rag/100 ml), IgM 121 rag/100 ml (210 • 170 rag/100 ml), suggesting B cell dysfunction or abnormalities.
Pathological findings Autopsy was carried out 3 h after death. The kidney showed marked contraction with multiple small cysts on the surface. The direct cause of death was pneumonia. Cerebellar sections revealed widespread tissue attenuation in the eerebetlar white matter. No abnormalities were noted in the cerebral white matter. On histological examination the attenuated cerebellar white matter was completely devoid of myelin sheaths, although axons were relatively preserved, showing primary demyelination (Fig. 1A). In the demyelinated lesions there were numerous enlarged nucleibearing oligodendroglia (Fig. 1B) and hypertrophic astrocytes, some of them showing nuclear atypism.
using polyclonal anti-JCV antibody raised by immunization with JCV (Tokyo-1 strain) [16].
In situ hybridization Localization of the JCVgenome at the cellular level was carried out using in situ hybridization methods. Briefly, 5-~xm-thick sections were cut from paraffin-embedded tissue. The sections were treated with 100 Bg/ml proteinase K for 15 rain at 37 ~ and refixed with 4 % paraformaldehyde. The JCV DNA probe was labeled with digoxigenin-dUTP (Boehringer Mannheim) by the random primer method [4]. Hybridization was carried out at 42~ for 16 h, followed by stringent washing, and then digoxigenin was reacted with alkaline phosphatase labeled anti-digoxigenin antibody. The final product was visualized by nitro blue tetrazolium for alkaline phosphatase [6].
Electron microscopy
Methods
A number of small pieces of cerebellar tissue were fixed in 2.5 % glutaraldehyde, 2 % paraformaldehyde and 6 % sucrose in 0.1 M sodium cacodylate buffer (pH 7.2). They were then fixed in 2 % osmium tetroxide, dehydrated, and embedded in a Epon-Araldyte mixture. Ultrathin sections prepared with a LKB-type 3 ultramicrotome were doubly stained with uranyl acetate and lead citrate and examined in a Hitachi H-800 electron microscope.
Immunochemical studies
Extraction and fractionation of tissue DNA
Immunohistochemical staining for JCV V antigen was carried out using the avidin-biotin-peroxidase complex (ABC) method [7],
Affected cerebellar tissue was digested with proteinase K in the presence of 1% sodium laurylsulfate in 10 mM Tris-HC1 (pH
Fig. 1A, B. Histological features of a cerebellar form of PML. A Diffuse and complete loss of myelin sheath in the cerebellar white matter. The peripheral areas indicated by arrows only contained myelin sheaths in the white matter. Kliiver-Barrera stain for myelin
sheath. B Numerous enlarged nuclei-bearing oligodendroglias were seen in the demyelinated lesions. Small nuclei at the right upper corner were those of cerebellar granule cells. Hematoxylineosin stain x 200
107 7.8)-10 mM EDTA at 56 ~ for 1 h. The digest was extracted once with phenol and once with chloroform-isoamyl alcohol (24:1). The aqueous phase was dialyzed extensively against 2 mM NaC1, 2 mM Tris-HCl (pH 7.8), 0.2 mM EDTA and DNA was concentrated by ethanol precipitation. DNA extracted from tissue was digested with EcoRI and electrophoresed on a 0.6 % agarose gel. HindIII-digested lambda DNA was co-electrophoresed and used as size markers. Portions of the gel containing DNA migrating in the 4000 to 6000 base pairs (bp) region was excised and electroeluted as described previously [20]. This size-fractionated DNA served as material for cloning of the JCV DNA.
DNA was linked to the vector at the EcoRI site. In addition, five cerebral PML-derived recombinant JCV DNA clones of which regulatory sequences had been determined previously were used to demonstrate structural features of regulatory regions common to cerebral PML-derived JC virus isolates. They included four clones from American patients (Madl-Br[5], Herl-Br[12], MadS-Br[12], Mad9-Br[12]) and one from a German patient (GS/B[ll]).
Cloning of J C V D N A
carried out as recommended by the suppliers. Digested DNAs were electrophoresed on 1.5 % or 1.8 % agarose gels or on a 3.5 % polyacrylamide gel in 45 mM Tris-borate, 1 mM EDTA [20], depending on the expected sizes of the DNA fragments. As size markers, the EcoT14I (StyI) fragments of lambda DNA or the Hinfl fragments of pUC19 were run in parallel. After electrophoresis, gels were stained with ethidium bromide and were photographed on a UV light transilluminator.
Two methods were used. In initial experiments, the EcoRIdigested and size-fractionated tissue DNA was ligated to lambda gtlO which had been digested with EcoRI and treated with bacterial alkaline phosphatase. The ligated DNA was packed in vitro using Gigapack (Strategene, Calif.) according to the protocol suggested by the manufacturer and was used to infect E. coti C600. Recombinant phages containing JCV DNA were obtained by two rounds of plaque hybridization [20] using 32P-labeled JCV DNA as the probe. Recombinant phages containing JCV DNA were purified as described previously [20]. Cloned JCV DNA was excised with EcoRl from recombinant phages and was subcloned into the plasmid vector pUC18. In subsequent experiments, the EcoRI-digested, size-fractionated DNA derived from a different cerebellar lesion was directly ligated with EcoRI-digested, bacterial-alkaline phosphatasetreated pUC19 and was used for E.coli DH5c~ transformation by high-voltage electroporation [3]. Recombinant plasmids were screened by colony hybridization [20] using 32p-labeled JCV DNA as the probe. Recombinant DNAs containing JCV (MY) DNA (pJC-MY), JCV (CY) DNA (pJC-CY, clone 1), and JCV (Tokyo-i) DNA (pJCT-Br) served reference DNAs. pJC-MY and pJC-CY were established previously from the urine of healthy Japanese [23]. pJCT-Br was previously cloned from the cerebral tissue of a Japanese PML patient [14]. Since, in pJC-MY and pJC-CY, JCV DNA was cloned at BamHI sites, the JCV DNA was exised from the vector and full-length JCV DNA was recloned into the EcoRI site and used for restriction enzyme analysis. In pJCT-Br, JCV
Restriction enzyme analysis Restriction endonucleases were obtained from Toyobo Co., (BglI, DdeI, HaeIII, HincII, Hinfl, NcoI, PvufI) and Bethesda Research Laboratories, (SstI [SacI]). Digestion with each enzyme was
D N A sequencing A NcoI-SstI fragment, containing a region from the start site of T antigens to the origin of DNA replication, was inserted into M13 mpl8 or rap19 between the Sinai and SstI sites. A HindIII-NcoI fragment, containing a region from the origin to the start site of late leader protein (agnoprotein), was inserted into M13 mpl8 or mpl9 between HindIII and Sinai sites. Single stranded DNAs purified from recombinant phages were sequenced using the chaintermination method [21]. Sequencing was carried out with overlapping clones representing both DNA strands.
Results
Demonstration of J C V infection in cerebellar lesions To c o n f i r m t h e diagnosis of P M L m a d e histologically, we a t t e m p t e d to d e t e c t J C V V a n t i g e n s b y i m m u n o h i s t o -
Fig. 2A,B. Demonstration of JC viral antigen and genome. A Enlarged nuclei labeled with anti-JCvirus (JCV) V antibody. Note the lack of antigen in the reactive astrocytes (arrows). Avidin-biotin-peroxidase complex method. B Enlarged nuclei were shown to contain JCV genome which is demonstrated as black signals using a non-radioactive in situ hybridization method (digoxigenin). A,B • 200
108
Restriction analysis of cloned JCV DNA
Fig. 3. Electron micrographrevealed a large number of papovavirus particles in an enlarged nucleus x 40,000
chemical method and JCV DNA by in situ hybridization. V antigens were detected in the enlarged nuclei of oligodendroglial cells in a demyelinating region of the cerebellum, but were not in atypical astrocytes (Fig. 2A). The nuclei in which V antigens were demonstrated were also positive for the viral DNA (Fig. 2B). Furthermore, using an electron microscope we could detect numerous polyomavirus virions in the abnormal nuclei of oligodendroglial cells (Fig. 3).
Molecular cloning of JCV DNA Following digestion of total DNA from cerebellar tissue with EcoRI, linear JCV DNA was detectable by agarose gel electrophoresis and by subsequent blot hybridization (data not shown). To clone the virus DNA whole tissue DNA was digested with EcoRI, separated by agarose gel electrophoresis and DNA migrating in the expected size region was eluted and ligated into either lambda or plasmid vectors. In initial experiments, JCV DNA was cloned using lambda gtlO and then subcloned with plasmid pUC18. Although many clones were obtained by this method, one clone, designated as Sap-1 clone 1 (c/l), was used for molecular characterization. In the subsequent experiments, we cloned JCV DNA directly into pUC19 plasmid using DNA derived from different areas of the cerebellum. Nine clones containing JCV DNA, Sap-1 cl2 to cI10, were obtained and all of were subjected to molecular characterization.
We performed a series of restriction enzyme analyses to compare the various Sap-1 clones to JCV clones from a cerebral PML case (Tokyo-1 [14] and to clones from the urine of healthy individuals (CY and MY) [23]. CY and MY represent the two JCV subtypes circulating in the Japanese population (unpublished results). JCV DNAs were recovered from Sap-1 clones and from reference clones by EcoRI digestion and were digested with BgllI, DdeI, HaeIII, HinclI, Hinfl, PvuII, or SstI (typical gel electrophoresis patterns are shown in Fig. 4.). The results can be summarized as follows: i. In analyses using enzymes other than SstI all the Sap-1 clones were identical to one another (Fig. 4A, B, D). However, SstI digestion showed that one Sap-1 clone, cl9, differed from the others (see below). We refer to those clones except cl9 as Sap-1 unless otherwise specified. ii. SstI digestion revealed that most Sap-1 clones with the exception of cl9 were similar to Tokyo-1 but differed from urine-derived clones (CY and MY). Sap-1 and Tokyo-1 did not generate one of the 260-bp fragments generated by CY and MY but instead gave rise to two smaller fragments, the sizes of which were slightly different between Sap-1 and Tokyo-1. It was previously reported that in Tokyo-1 these shorter fragments were generated by rearrangement of regulatory sequence, such as deletion and amplification, relative to the regulatory sequence of urine-derived isolates [23]. Therefore, it is likely that the regulatory region of most Sap-1 clones also underwent similar sequence rearrangement.The SstI cleavage pattern of cl9 is characterized by the fusion of a 260-bp fragment and the shortest fragment generated by the other Sap-1 clones (Fig. 4C). Probably, one of two SstI sites in duplicated segments was lost in c/9. iii. Two major urine-derived JCV strains (CYand MY) in Japan can be differentiated by digestion with several enzymes, including DdeI, HaeIII, and Hinfl (gel patterns after Hinfl and DdeI digestion are shown Fig. 4C and D, respectively). In analysis using these enzymes, gel patterns of Sap-1 and Tokyo-1 were identical to that of MY (Fig. 4C and D). iv. When HincII was used, all Sap-1 clones were distinguished from Tokyo-1 and MY (Fig. 4A). The difference in HincII cleavage profiles were ascribed to the presence in Sap-1 clones of an extra HincII cleavage site located in the T antigen-coding region (data not shown). This extra HinclI site has not been detected in the JCV isolates examined so far (unpublished results). However, no other changes were detected between Sap-1 and MY (and Tokyo-l) in analysis in which other restriction enzymes used including PvuII, except the difference due to rearrangement of regulatory sequences in Sap-1 and Tokyo-1 (see below).
Regulatory sequence of cloned JCV DNA For representative Sap-1 clones, cll, cl2, and c13, that appeared to be identical in restriction analysis, and cl9
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Fig. 4A-D. Restriction enzyme analysis of JCV DNA clones. JCV DNA was recovered from ten JCV DNA clones from the current cerebellar progressive multifocal leukoencephalopathy (PML) case, a clone from a cerebral PML case (Tokyo-1), and two urine-derived clones (CY and MY), and digested with restriction enzymes HincII (A), Hinfl (B), SstI (C), and DdeI (D). Fragments generated were electrophoresed on a 1.5 % agarose gel (HincII and Hinfl), on a 1.8 % agarose gel (SstI), or on a 3.5 % polyacrylamide gel (DdeI). Fragments separated in gels were stained with ethidium
bromide and were photographed on a UV light transilluminator. Sizes of some EcoT14I fragments of lambda DNA (A-C) and those of Hinfl fragments of pUC19 (D) are indicated in bp on the right. Note that two fragments of 260 bp in size were generated after digestion of CYand MY with SstI (C). Faint bands are indicated by dots in C. Closed arrowheads on the left of B and D denote fragments which were produced from CY but not from MY, while open arrowheads indicate those which were produced from MY but not from CY
that differed from the other clones in an analysis with SstI, we sequenced the entire noncoding region spanning from the start site of T antigen to that of late leader protein (agnoprotein). The sequences cll, cl2, and cl3 were identical to one another but differed from that of cl9 (the sequences of cll and cl9 are shown in Fig. 5) This result, together with those of the restriction analysis described above, suggest that cll represents the major species present in the cerebellar lesion of the patient, while cl9 is probably a minor one, or the difference may be due to events during cloning procedures. In Fig. 6, the archetypal regulatory sequence (A), identified in JCVs excreted by nonimmunocompromised individuals [23], is shown at the top and those of cerebral PML-derived isolates (B) and those of Sap-1 clones (C) are given below, showing deletions relative to the archetype as gaps and repeats as parallel straight lines [23]. Comparison of the regulatory sequence of cll with the archetypal sequence (Fig. 6A, C) suggests that the regulatory sequence of cll may have arisen from three
independent events, duplication of 60-bp (nucleotides [nt] 43 to 101) and 3-bp sequences (nt 257 to 259) and deletion of a 82-bp sequence (nt 100 to 181). However, the sequence data can not indicate in which order these events would have occurred. From Fig. 6, it is evident that the regulatory sequences of cerebral PML-derived isolates share many of the structural features, with the single exception of Madll-Br: duplication of domain A, (47-bp sequence) and C (9-bp sequence) and deletion of domain B (55-bp sequence) (Fig. 6B). The major species of Sap-1 (c/l) had duplications of 59-bp (archetype nt 43 to 101) and 3-bp sequences (nt 257 to 259) and was lacking a 65-bp sequence (nt 117 to 181) (Fig. 6C). The 59-bp repeated segment contains most of domain A, except for a terminal 5-bp stretch. The 65-bp deleted sequence contained most of domain B except for one nucleotide. Although it was not duplicated, most of domain C, except for one nucleotide, was present in Sap-1 regulatory region. The duplication of domain C may not be important for the replication of JCV in brain
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