Journal of General Virology (1992), 73, 2669-2678. Printedin Great Britain

2669

Two major types of JC virus defined in progressive multifocal leukoencephalopathy brain by early and late coding region DNA sequences Grace S. Auit* and Gerald L. Stoner Laboratory of Experimental Neuropathology, National Institute o f Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, U.S.A.

A 610-bp region of the JC virus (JCV) genome sequenced from brains of 11 progressive multifocal leukoencephalopathy (PML) patients contains 20 sites of point mutations that allow reliable classification of JCV isolates into two types. These type-determining sites were located in the region extending from position 2131 in the VP 1 gene, through the intergenic region, to position 2740 in the T antigen gene. At these 20 sites the presence of different nucleotides creates two distinct patterns of substitution, with six isolates having the Type 1 pattern and five having the Type 2 pattern. Only four of the 1 ! isolates had 'crossovers' to the opposite type consensus DNA sequence at a small number of sites, indicating a very high type specificity. Additionally, three type-determining sites occur in the

non-coding region to the left of the origin of replication. Other mutations occurred at random sites, making each strain unique, although one strain, 105, is identical to the Type 1 consensus. The JCV prototype strain Mad-1 was found to be Type 1 and differs from the consensus sequence at five sites. The other previously sequenced JCV strain, GS/B, is Type 2. At three sites out of five in the T antigen C terminus there is a type-specific amino acid substitution; however, none of the type-determining mutations in the VP1 gene cause an amino acid substitution. Comparison of each type's consensus DNA sequence to that of BK virus suggests that Type 2 represents the ancestral JCV sequence from which Type 1 diverged during human evolution.

Introduction

1988; Coleman et al., 1983). Whether it establishes latency in the brain as well during primary infection is unknown. Alternatively, the virus may reach the brain from the kidney reservoir during activation to PML, or PML may result from reinfection. Each JCV isolate from the PML brain contains extensive sequence variation in the non-coding regulatory region (Grinnell et al., 1983b; Dfrries, 1984; Martin et al., 1985; Rentier-Delrue et al., 1981 ; Matsuda et al., 1987), whereas almost all isolates to date from non-PML kidney exhibit a well-defined promoter/enhancer sequence from which the PML brain isolates could be derived, termed archetype (Loeber & D6rries, 1988; Markowitz et al., 1991 ; Yogo et al., 1990). The method of viral transmission is also unclear. Most individuals become seropositive between the ages of 10 and 15 years, suggesting that infection occurs in childhood (Hogan et al., 1991). Transplacental infection has also been suggested, based on the viral reactivation in the kidney that occurs late in pregnancy (Daniel et al., 1981 ; Coleman et al., 1980). However the virus has not been isolated from foetal kidneys (Padgett & Walker, 1980) or from placenta, amniotic fluid, or neonatal urine (Coleman et al., 1980), although none of these has yet

The human polyomaviruses JC and BK (JCV and BKV) infect 70% to 90% of the population, as shown by the level of seropositivity in adults (Padgett & Walker, 1973; Walker & Padgett, 1983; Gardner, 1973). Following infection at unknown primary sites, the viruses persist in the kidney (Gardner et al., 1971 ; D6rries & ter Meulen, 1983; McCance, 1983). However, unlike BKV, under conditions of immune suppression JCV can productively infect glial cells of the brain, causing a fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML) (Astr6m et al., 1958; Padgett et al., 1971). This disease, once rare, is now a regular complication of human immunodeficiency virus (HIV) infection (Berger et al., 1987). HIV infection may actively promote JCV replication in the brain, as there is evidence that the tat protein of HIV strongly induces JCV late gene transcription (Chowdhury et al., 1990; Tada et al., 1990). The relationship between JCV infection in the kidney and the brain is unclear. The virus establishes itself in the kidney, and under certain conditions infectious virus is shed in the urine (Hogan et al., 1980; Arthur et al., 1985, 0001-1057 © 1992 SGM

2670

G. S. Ault and G. L. Stoner

been studied by more sensitive polymerase chain reaction (PCR) techniques. Identification of distinct viral genotypes that could be easily classified and reliably followed through the host would greatly aid the clarification of each of these questions. Although two distinct serotypes of BKV can be identified (Knowles et al., 1989; Tavis et al., 1989), no such serotypic subgroups have been found for JCV (Walker & Frisque, 1986). The many efforts to type JCV isolates by D N A sequence variation have focused on the non-coding regulatory region. The hypervariable regulatory region of virus isolated from PML brain has multiple sequence rearrangements, with extensive deletions and duplication, which vary with each isolate (GrinneU et al., 1983b; Martin et al., 1985; D6rries, 1984; Loeber & D6rries, 1988; Walker & Frisque, 1986; Major et al., 1992). Classification schemes based on the structure of the regulatory region have been described (Martin et al., 1985; Walker & Frisque, 1986), but it is not clear whether the patterns of sequence rearrangement reflect the genotype of the virus or result from host cell biology. Similarly, typing by restriction fragment length polymorphism (RFLP) (Yogo et al., 1990; Grinnell et al., 1983a, b; Martin & Foster, 1984) has not revealed the kind of clear-cut taxonomy which could lead to simple and unambiguous typing. In general, RFLP typing is a low resolution analysis which misses many informative sequence changes, and leaves doubt as to the actual sequence variations at the sites altered. Numerous point mutation variations between prototype JCV (Mad-l) and one other strain (designated GS) were observed in the protein-coding genes (Loeber & D6rries, 1988). It seemed likely that these regions, rather than the hypervariable regulatory region, would contain stable sequence variations that could reveal strain divergence and could serve as the basis for a clear-cut typing scheme. Accordingly, we have sequenced a 610 bp region of the VP1 and T antigen coding regions and have discovered 20 sites of point mutations that unequivocally define two separate types of JCV. Three additional typedetermining sites are found upstream of the early gene coding region.

(5" A A G A T C T A G A A G C A G A A G A C T C T G G A C A T G G 3'), altered to have an XbaI site; JRR-5, nt 4979 to 5009 (5' TCCATG-GATCCCTCCCTATTCAGCACTTTGT Y), altered to have a BamHI site; JRR-6, nt 315 to 285 (5" TTTCACTGCAGCCTTACGTG A C A G C T G G C G A Y), altered to have a PstI site.

Cloning and sequencing amplified DNA. PCR products were purified on ion-exchange push columns (PrimeErase Quik, Stratagene), ligated into pBluescript SK + vector (Stratagene), and transformed into Escherichia coli DH5c¢ cells (Bethesda Research Laboratories). Plasmid DNA isolated from positive clones was sequenced by the dideoxynucleotide chain termination method (United States Biochemical) and electrophoresis in 6 % polyacrylamide gels. Overlapping sequence from both strands was determined. Sequence data analysis. Sequence data were organized and analysed using the Genetics Computer Group sequence analysis software package (Devereux et al., 1984) on a VAX computer.

Results D N A sequence analysis identifies two J C V types

A 610 bp fragment of the JCV genome was cloned and sequenced following PCR amplification from brain tissue of 11 PML patients. All patients were from the United States, and six were also AIDS cases (Table 1). Fig. 1 indicates the amplified region, referred to as the V-T intergenic fragment, on a map of the JCV genome. The amplified fragment contains 12% of the entire JCV genome, and consists of the 3' 400 bp (nt 2131 to 2530) of the gene for the capsid protein VP1, the 75 bp intergenic

VPIJ

J

V-Tintergenic region

Large T antigen

f f ff

5130 bp

Small t antigen

Methods PCR amplification of viral DNA from brain tissue. Total DNA was extracted from frozen brain tissue using buffers and protocol from Stratagene. One-hundred Ixl PCR reactions containing 1 ~tg of this DNA were amplified for 35 cycles at 94 °C for 0.5 rain, 55 °(2 for I rain and 72 °C for 1 min, in a DNA Thermal Cycler (Perkin-Elmer Cetus). Taq polymerase and reagents were obtained from Perkin-Elmer Cetus. Primers were: VPV-3, nucleotide (nt) 2098 to 2127 in the JCV genome (5' C A C A A T C G A T T T T G G G A C A C T A A C A G G A G G y), altered to create a ClaI site near the 5" end (underlined); VPV-4, nt 2772 to 2742

Ori VP2

Agno Fig. 1. Map of JCV genom¢ indicating the cloned and sequenced region, termed the V-T intergenic region. Redrawn from Frisque et al. (1984).

Two types of JC virus in P M L brain

2671

T a b l e 1. PML patient data Coded identifier 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

(JCV strain)

Sex/age

Black/white

Place of death

Underlying disease

Duration of CNS symptoms

(106) (105) (104) (205) (103) (204) (203) (202) (102) (101) (201)

M/28 F/31 M/40 M/58 M/46 M/51 M/35 M/42 F/6 M/61 F/35

White Black White White White White White White White White Black

Morgantown, W.Va. Newark, N.J. Chicago Boston Kingston, Pa. Manitowoc, Wis. Newark, N.J. New York City Boston Boston Plainfield, N.J.

AIDS AIDS AIDS Thymoma AIDS CLL* AIDS AIDS CID]" CLL* SLE:I:

1 2 weeks 1 year 4-5 months Unknown 5 ~ months 1 month 2 months 3 months 7 months 2-3 months 1 month

AF CD DF GI KU LT PM RN SB SF WC

* Chronic lymphocytic leukaemia. I" Combined immune deficiency. :~ Systemic lupus erythematosus.

I

2131 I 2210 AAATGTTCCT CCAGTTCTTC ATATAACAAA CACTGCCACAACAGTGII-GC TTGATGAATT TGGTGTTGGGCCACTITGCA

101 102

.............................................. ............................................

102

....

G- ...........................

G- ...........................

A- .....

103

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

T- ...............................

103

---AA-

.......

G- ............................

C- ...........................

A- .....

104

..............................................

T- ...........

104

....

A- .......

G- ............................

C.- . . . . . . . . . . . . . . . . . . . . . . . . . . . .

105 106

............................................ .............................................

T- ................................ T- ................................

105

....

A- .......

G- ............................

C- ...........................

A- .....

106

....

A- .......

C-- . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C- ............................

A- .....

T- ............................... -1[ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C ....................

CCACTTTGCA

A .......

A- .....

2

A A A T G T T C C T C C A G T T C F I C A T A T A A C A A A CACTGCCACA ACAGTGCTGC T i - C ~ T G A A T T

2

AGGGG~CAGAGGAACTTCCA GGGGACCCAGACATGATGAG ATAIGTTGAC AGATATGC~C AGTTGCA&~C ~ T G C T G

201

......

C- ...............................

201

....

G- . . . . . .

A- . . . . . . . . . . . . . . . . . . . . . . . . . . . .

T- ........................

202

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C- ................................

202

....

G- .......

A ............................

T - ...........................

G- .....

203

..............................................

C- ................................

203

....

G- .......

A .............................

T- ............................

G- .....

204

..............................................

T.................................

204

....

G- .......

A .............................

T- ............................

205

............................................

C- ...............................

205

....

G- .......

A .............................

T.............................

G......................................

TGGTGTTGGG

l0 II 12 13 2530 I AGGGAACAGAGGAGCTTCCA~ C C C A G ACATGATGAG ATACGTTGAC AGATATGGAC AGTTC~AGAC A A A A A T ~ G 101 . . . . A- . . . . . . G- . . . . . . . . . . . . . . . . . . . . . . . . C- . . . . . . . . . . . . . . . . . . . . . . . . . . . . A- . . . . .

C--A ......

C-- . . . . . G- .... VPl

2

3

4

1

AAGGTGACAA C T T A T A C T T G

I01

............

A--T .................

102

...........

A--C-

.................................

103

.............

A--C-

. . . . . . . . . . . . .

104

.............

A--C- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

105

.............

A--C- . . . . . . . . . . . . . . . . . . . .

106

.............

A--C-

2

AAC4~TGACAA

T C A G C T G l ~ G ATGTTTGTGG C A T G T T T A C T AACAGGTCTG GTTCCCAGCA GTGGAGAGGA C....................

TCAGCTGTTG ATGTTTGTGG C A T G T T T A C T

G- .......................

104

............................................................

A---T-

G- .......................

105

............................................................

A---T-

.............

G- .......................

106

.............................................................

A---T-

.............

2

TAATCAAAAG CCTTTATTGT

AACAGATCTG GTTCCCAGCA GTGGAGAGGA

.............

G--•

.............

G--T-

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

203

.............

G--T-

...............................

204

.............

G--T-

....................................

205

............

G--T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

AATATGCAGT A C A T T T T A A T

A- .......................

201

......

A- ......

202

.............................................................

A- .......................

203 204

........................................................... ..............................

A- ......

205

...........................................................

G----A

T ................

........................ T................

T A A C A ~ G C AGTTATTTTG A---T .............. A---T- ............. A---T- .............

5 6 7 2370 CTCTCCAGAT ATTTTAAGGT TCAGCTAAGGAAAAGGAC-GGTTAP@,AACCCCTACCCAA•T TCTTTCCTTC T[ACTGATTT

1

]01

~-C .......................

G

C- .............

101

102

--C-

103

. . . . . . C. . . . . . . . . . . . . . --C . . . . . . . . . . . . . . . . . . . .

G

104

--C ..................

G

105

--C-

........................ .......................

A .....

106

--C-

2

CTGTCC.AGAT

201

--C ........................

AT1TTAAGGT

G . . . . . . . . . . . . . . . . . . . . . . . . . . . .

T---G----

G G. . . . . . . . . . . .

G........

C....

............. ............

G---G- ....... T exon 2 eM

L~ . . . .

16 17 18 2690 GGGGAGGGGTCTTTGGTTTTTTGAAACATT GAAAGCCITT ACAGATGTGA AAAGTGCAGT ITTCCXGTGT GTCTGCACCA ..........

C......................................

A- .......

T- ..................

102

..........

CI

~ ........

TI . . . . . . . . . . . . . . . . . .

103

..........

C-- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A .........

T-- . . . . . .

C- .............

I 0 ~ . ..........

C- ......................................

A - G ......

T ...................

G .................................

C- .............

105

..........

C .......................................

A- ........

T ...................

~G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C- .............

106

..........

C .......................................

A- ........

T ...................

20

GGGGAGGGGTTTTTGGTTTTTTGAAACATT GAAAGCCN7 ACAGATGTGA TAAGTGCAGT GTTCCTGTGT GTCTGCACCA .......... T. . . . . . . . . . . A. . . . . . . . . . . . . . . . . . . . . . . . . . . T. . . . . . . . G Cr . . . . . . . . . . . . . . . . . . .......... T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • ........ G- . . . . . . T . . . . . . . . . . . .......... T. . . . . . . . . . . . . . G. . . . . . . . . . . . . . . . . . . . . . . . • ......... G- . . . . . . . . . . . . . . . . . . .......... • ....................................... T - G. . . . . . . G- . . . . . . . . . . . . . . . . . . .......... T- ...................................... T......... C-- . . . . . . . . . . . . . . . . . .

. . . . . . . .

G .......................

TCAGCTAAGA AAAAGGAGGG T T A A A A A C C C C T A C C C A A T T T C T T T T C T T C ...................................

TTACTGATTT

C .....

A ...................................

G ........

T- .............

203

--G- ........................

A ..................................

--G- ........................

A

205

--G-

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I

8 9 2450 AATTAACAGA A ~ C T C C T A GAGTTGATGGGCAGCCTATGTATGC.-CATGGATC~]TCAAGT A G A ~ G G T T AGAGTTTTTG

101

...............

. . . . . . . . .

T ..............

G. . . . . . . . . . . . . . . . . . . . . . .

T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C. . . . . . . . . . . . . T- ............

/%- . . . . . . . . . . . . . . . . . . . . .

102

. . . . . . . . . . . . .

T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A- ....................

103

...............

T- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A ......................

104

---C

T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A- ....................

105

...............

T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A- .....................

%06

...............

1

A--

...........

.............

C- .............

................................

204

........................

-C ........

G---G~,~--GT---G-

C- . . . . . . . . . . . . .

G

202 --G- ........................

.................................

.............

A A A G T A T A A C CAGCTTTACT TGACAGTTGC A G T T A T T T T G

T.....................................................

Large

I

~J

2610

G- . . . . . . . . . . . . . . . . . . . . . . .

C....................

202

end

15

TAATCAAAAG CCTTTATrGT AATATGCAGT A C A T ~ A A T AAAGTATAAC C A ~ T A C T .............................................. AT. . . . . . . . . . . . . ........................................................... ................................ T. . . . . . . . . . . . . . . . . . . . . . C. . . . .

T...........

201

14

1 I01 102 103

G - ....................... G- .......................

C .................

. . . . . . . . . . . . . .

CTTGTAT[TG

2290

.....................................

2 AATTAACAGA AGC~CCCCTA GAGTTGATGGGCAC~CTATG T A T ~ T G G 201 - - - C . . . . . . . . . . T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G...................

ATGCTCAQST AGAGGAGGTTAGAGTTTTT6 A. . . . . . . . . . . . . . . . . . . . .

202

..............

C

203

...............

C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

204

..............

C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

205

..............

C ........................................

......................................

1 202 203 204 205

.....................................

A ...........

19 20 2740 GAC-C-CTTCTGAGAC.CTC-C-GAAAAGCATTGT GATTGTC, A T T CAGTGCITGA ..................... A- ............ ¥- ............. ..................... A- ............ T. . . . . . . . . . . . . . ..................... C--. . . . . . . . . . . . T. . . . . . . . . . . . . . 104 ..................... A- . . . . . . . . . . . . T .............. I I 01 102 103

105 106

..................... ..............

2

(~GGCTTCTG

T......

I~ ............ A .............

~". . . . . . . . . . . . . . T- .............

AGACCTGGGA AGAGCATTGT G A T T G A C ~ T T CAGTGCTTGA

C--. . . . . . . . . . . .

A- . . . . . . . . . . . . .

G- ....................

202

201 . . . . . . . . . . . . . . . . . . . . . .....................

G- ............

A- .............

G- ....................

203

.....................

6-

~-- . . . . . . . . . . . . .

G- . . . . . . . . . . . . . . . . . .

204

.....................

G- .....................

205 . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . .

A.............

A- .............

G- . . . . . . . . . . . .

A--C

...........

Fig. 2. Consensus sequences from 11 strains of JCV for Type 1 and Type 2 V - T intergenic regions. Line 1 is the Type I consensus, with sequences of individual Type 1 strains indicated on the lines below. Dashes indicate identity to consensus, except that at typedetermining sites (1 to 20) the sequences of all strains are indicated. Line 2 is the Type 2 consensus, with Type 2 strains below it. Numbering is based on the Mad-I strain (Frisqu¢ et al., 1984).

G. S. Ault and G. L. Stoner

2672

Table 2. Type-determining sites in non-coding region left of origin Nueleotide*

Table 3. Number of differences from consensus sequences of JCV JCV strain

JCV strain

5017

5026

5039

Type 1 Consensus

A

C

C

101

-t

-

-

102 103 104 105 106 Mad-l:~

-

-

-

Type 2 Consensus 201 202 203 204

T -

T -

G C -

205

-

-

-

GS/B§

-

-

* N u m b e r i n g a c c o r d i n g t o M a d - 1 s e q u e n c e ( F r i s q u e et aL, 1984). ~" D a s h e s i n d i c a t e i d e n t i t y to c o n s e n s u s . S e q u e n c e f r o m F r i s q u e et al. (1984). § S e q u e n c e f r o m L o e b e r & D 6 r r i e s (1988).

region (nt 2531 to 2605), and the 3" 135 bp (nt 2606 to 2740) of the T antigen-coding gene. Among the isolates sequenced, 53 sites of single base pair substitutions were found. There were no nucleotide additions or deletions. At 20 of these positions, the presence of different nucleotides divides the isolates into two groups, such that one group shares a nucleotide at the given site and the other shares another nucleotide. These 20 sites create two distinct patterns of nucleotide substitution, and allow two strain groups, termed Type 1 and Type 2, to be unequivocally identified. On the basis of these type-determining sites, six of the JCV isolates were classified as Type 1, and the other five were classified as Type 2. Fig. 2 shows a consensus sequence for each type, with the sequences of the individual isolates indicated beneath it. At 10 of the sites, one of the isolates is not true to type. In general, these changes involve a 'crossover' to the alternative nucleotide found in the other type, not a random substitution. The only exception to this crossover pattern is type-determining site 14 (nt 2592) in the 75 bp non-coding intergenic region, at which the sequence of Type 2 isolates is divided between G and T, whereas all Type 1 isolates have an A. At any given typedetermining site, only one isolate within a type has a crossover, with the exception of site 7 at which Type 2 isolates 201 and 204 both have a crossover. At position 19 one isolate of both types has crossed over. In all, seven isolates have no crossovers at type-determining sites, two isolates cross over at one o f the 20 sites, one at three sites, and one at six.

Type 1 consensus

Type 2 consensus

Crossovers/ unique mutations

tot

4

22

1/3

102 103 104

1 6 5

21 24 25

0/1 1/5 0/5

105 106

0 3

20 23

0/0 0/3

5

23

1/4

22 22 22 23 22 22

14 2 2 9 2 5

6/8 0/2 0/2 3/6 0/2 2/3:~

M a d - 1* 201 202 203 204 205 GS/Bt

* J C V ( M a d - l ) s e q u e n c e f r o m F r i s q u e et aL (1984). $ G S / B s e q u e n c e f r o m L o e b e r & D 6 r r i e s (1988). M u t a t i o n a t p o s i t i o n 2 7 1 2 i n G S / B is listed a s a c r o s s o v e r , a l t h o u g h t h e b a s e is T, n o t t h e T y p e 1 c o n s e n s u s A .

Type-determining sites near the origin In addition to type-determining sites in the V - T intergenic region, the non-coding sequence to the left o f the origin contains three type-determining sites near the T antigen start site. These three sites are entirely consistent with the 20 sites of the coding region in designating virus type (Table 2). Sequences o f multiple clones of this region did not reveal any discrepancy of type determination with the coding region sequence. Eight clones of 102, four clones of 101,104, 105, 201,202 and 203, and two clones of 103, 106, 204 and 205 were identical at these three sites, and in all cases confirmed type classification based on the V - T intergenic fragment. One isolate, 201, had a crossover at the third of these sites.

Strain variation The mutations which are not type-determining are unique to one or two isolates. At seven of 33 'unique' sites where two isolates are mutated, the mutation can be in one or both types, and can be changed to the same or a different nucleotide. There is one unusual site, at position 2245, where three Type 1 isolates have a mutation to C whereas the other three and all Type 2 have T. Based on these unique sites, as well as differences at crossover sites, each of these 11 isolates can be distinguished from the others. The variation between the two types and between strains within a type can be quantified by the number of mutations of both kinds which distinguish each strain from the consensus sequence (Table 3). The degree o f divergence between Types 1 and 2 is illustrated by the

Two types of JC virus in P M L brain

2673

Table 4. Predicted amino acid differences in V - T intergenic region of JCV Amino acid*/nucleotide site VP1 JCV strain Type 1 Consensus 101 102 103 104 105 106 Mad-1t Type 2 Consensus 201 202 203 204 205 Mad-8:~ GStB§

T antigen

241/2190 265/2261 269/2274 329/2454 345/2502 Phe Ash . . . . . . . . . . . . . . . . . Ser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... --Asp . . . . . ......

Ser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phe . . . . . . . Phe . .

Gly Arg . . . . . . . . . . Gia --. . . . . . . . . . . . . . . . . . . Lys . . . . . . . . . . ...... . . . . . . . . . . . . ...... . .

. . . . . . . . . . . . . . .

653/2712 664/2679 667/2671 670/2661

Phe . . . . Ser . . . . . . . . . . . . . . Ser Ser Ser Ser . . . . . Ser Set Tyr . .

. .

Thr . . lie . . . . . . . . . ----Lys ---

. .

. .

. . . .

. -------

. .

. .

. . . . . . . .

Asn Phe . . . . . . ...... . . . . . . . . . . . . . His Tyr Pro Tyr His Tyr His Tyr His Tyr His Tyr Pro Tyr His Tyr

* Dashes indicate identity to Type 1 consensus sequence. t Sequence from Frisque et al. (1984). :~T antigen sequence is unpublished data of C. Myers and R. J. Frisque. No sequence available for VP1. § Sequence from l.x~ber & D6rries (1988).

number of mutations that distinguish Type 1 isolates from the consensus for Type 2, and vice versa. The variation within each type is determined from the number of mutations that distinguish each isolate from its own consensus. It is apparent that some strains have diverged from the consensus sequences more than others. However, typing was unambiguous in all cases; even the most divergent strain, 201, shares Type 2 consensus at 14 of 20 type-determining sites. In six cases, two or more clones of the V - T intergenic region from a single brain were sequenced. Two clones each from the Type 1 strains 103 and 104 were identical to each other. Three clones of the Type 2 strain 204 and five clones of strains 102 and 201 were also identical. A m o n g four clones of the Type 2 strain 203, one clone had a single base pair mutation at position 2261, changing a unique mutation site back to the consensus sequence. This low level of substitution may arise within the host during viral replication or from an error of the Taq D N A polymerase during P C R amplification. To date we have not observed sufficient variation between clones to suggest the presence of more than one viral strain in the same individual's brain. The sequence of the V - T intergenic region of Mad-1 (Frisque et al., 1984) was aligned with our sequences, and was classified as Type 1 (Table 3). There were four unique sites at which Mad-1 diverged from our Type 1 consensus sequence, as well as a crossover at the typedetermining site 15. One of the unique mutations, at

position 2502 in the VP1 protein, is an amino acid substitution also (Table 4). The GS/B strain (Loeber & D6rries, 1988) was classified as Type 2 (Table 3). This strain has two crossovers, at sites 16 and 19, and three unique mutations. GS/B is unusual in that it is the only strain which has a mutation at a type-determining site within a protein-coding sequence which does not 'cross over' to the Type 1 consensus, but has a different nucleotide (site 19). It results in a unique amino acid substitution (Table 4). Mad-1 and GS/B both are completely true to type in the three non-coding region type-determining sites to the left of the origin.

Identification of possible subtypes The individual strains within a type can be divided into two groups based on their alternative sequences at one or two sites and overall degree of divergence. Type 2 strains differ at site 14, at which three strains (205,203 and 202) have G whereas two (204 and 201) have T. The Type 1 sequence is A at this site. Strains 205, 203 and 202 also have very low divergence from the consensus, with no crossovers, whereas 204 and 201 are more divergent (Table 3) and share a crossover at site 7. Type 1 strains fall into two groups based on a single site, those having C at position 2245, and those having T. There are no other sites at which the Type 1 strains have any more than an individual point mutation or a single instance of crossover, and all the strains have a similar degree of

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G. S. Ault and G. L. Stoner s u b s t i t u t i o n s are p r e d i c t e d ( T a b l e 4). T h r e e o f these a r e c h a n g e s at t y p e - d e t e r m i n i n g sites 17, 18 a n d 19. A t 19, one strain o f e a c h t y p e has crossed over, a n d G S / B has a u n i q u e a m i n o acid. T w o o t h e r t y p e - d e t e r m i n i n g sites, at 16 a n d 20, d i d n o t result in an a m i n o a c i d change. T h e f o u r t h site o f a m i n o a c i d s u b s t i t u t i o n , a t p o s i t i o n 2679, is a site o f two u n i q u e m u t a t i o n s , one in a T y p e 1 s t r a i n a n d one in a T y p e 2 strain. A u n i q u e m u t a t i o n in s t r a i n 201 i m m e d i a t e l y before t y p e - d e t e r m i n i n g site 18 causes a different a m i n o a c i d s u b s t i t u t i o n f r o m the o t h e r T y p e 2 strains. O f the 29 m u t a t i o n s in the V P 1 c o d i n g regions o f o u r 11 isolates, only four are p r e d i c t e d to cause a n a m i n o a c i d s u b s t i t u t i o n ( T a b l e 4). T w o a m i n o a c i d c h a n g e s a r e at u n i q u e m u t a t i o n s o f a T y p e 1 strain, a n d t w o are at u n i q u e sites o f a T y p e 2 strain. Since n o type-specific a m i n o a c i d c h a n g e s h a v e b e e n identified in the r e g i o n o f the VP1 c a p s i d p r o t e i n w h i c h has b e e n studied, it is u n l i k e l y t h a t T y p e s 1 a n d 2 c a n be d i s t i n g u i s h e d serologically.

d i v e r g e n c e f r o m the consensus. T h e strains h a v e b e e n t e n t a t i v e l y g r o u p e d into w o r k i n g s u b t y p e s b a s e d o n these differences for p u r p o s e s o f f u r t h e r i n v e s t i g a t i o n .

Predicted amino acid changes T h e m a j o r i t y o f the m u t a t i o n s , w h e t h e r t y p e - d e t e r m i n ing or unique, are silent. H o w e v e r t h e r e are type-specific v a r i a n t s o f the T a n t i g e n protein. I n the 135 b p (6-5 Y/oo)o f the T a n t i g e n gene t h a t was s e q u e n c e d , four a m i n o a c i d Table 5. Type-specific restriction sites in the V - T intergenic region Restriction enzyme

Virus type

Site no.*

Recognition sequence

Hinf I SfcI AluI MaelI MseI Fnu4HI BgllI Sau3AI Sau96I, Avail DdeI

1 1 1 1 1 2 2 2 2 2

8 9 11 12 14 1 4 4 8 9

GACTC CTCAAG AGCT ACGT TTAA GCTGC AGATCT GATC GGACC CTCAG

Type-specific restriction enzyme sites S o m e o f the t y p e - d e t e r m i n i n g sites a l t e r a r e s t r i c t i o n e n z y m e r e c o g n i t i o n s e q u e n c e ( T a b l e 5), m a k i n g it possible to t y p e n e w isolates r e l i a b l y b y r e s t r i c t i o n

* See Fig. 2.

Table 6. Alignment o f J C V Types 1 and 2 consensus sequences with three B K V strains at J C V type-determining sites* BVKt BKV position 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

2296 2343 2346 2385 2412 2439 2475 2505 2547 2574 2583 2613 2643 2711 2715 2740 2780 2802 2849 2863

JCV

Dun

MM

ASV

JCV position

T G T C T A T C G C A T A T G T A A G G

T G T C T A T C G C A T A T G T A A G G

T G T C T A T C G C G T A T G T A C G G

2177 2224 2227 2266 2293 2320 2356 2386 2428 2455 2464 2494 2524 2592 2596 2621 2661 2671 2712 2726

Mad-1 T~ A C G C G C T A A G C A~ A G~II C A~ T A T

JCV consesus Type 1

Type 2

T~ A C G C G C T A A G C A~ A T C As T A T

C G~ T~ A G A~ T~ C~ G~ G A~ T~ G G/T§ G~ T~ T G G~ A

* Alignments made with the GAP program of UWGCG sequence analysis package (Devereux et al., 1984). t BKV strains Dun and MM from GenBank. Sequence of ASV from Tavis et al. (1989). Sites at which the consensus sequence (or that of Mad-l) agrees with the BKV sequence. § T is represented twice at this site. IIMad-1 crosses over to the Type 2 consensus at this position.

Two types o f J C virus in P M L brain

digestion of this 610 bp fragment. Many of these restriction enzyme sites overlap sites which in our isolates have not shown any crossovers, although new isolates could potentially contain a mismatch at any one of the sites. Cleavage with M a e l I at site 12 yields fragments of 397 bp and 278 bp from the amplified region of Type 1 strains, whereas cleavage with B g l l I at site 4 yields fragments of 169 and 506 from the amplified region of Type 2 strains (data not shown). No crossovers have yet been found in the M a e l I or B g l l I sites. Evolution o f J C V types

To establish which JCV type best represents the ancestral sequence and which is the later derivative, we have compared the type-determining sites in the Type 1 and 2 V-T intergenic regions to the same region of the closely related human polyomavirus BKV. The BKV sequence matches the type 2 consensus at 11 of the 20 type-determining sites, whereas only three sites match the Type 1 consensus (Table 6). The remaining sites are ambiguous. This analysis suggests that the Type 2 sequence is closer to the original JCV sequence from which Type 1 has diverged.

Discussion With the detection of 20 type-determining point mutations in the protein-coding and intergenic region of the JCV genome, two virus genotypes can readily be identified. The pattern of base pair substitution at these sites is highly conserved within each type, so that reliable classification of a new JCV isolate as Type 1 or Type 2 requires determination of only a relatively small number of sites. Amplification of a smaller fragment for sequencing or for restriction analysis is a convenient and efficient approach. For example, a region of only 100 bp at the C terminus of the VP1 protein contains five typedetermining sites which are perfectly true to type between 10 of our 11 isolates, Mad-I and GS/B. We have used restriction enzyme M a e l I to cleave type-determining site 12 in Type 1 strains specifically within an amplified fragment of this region. A 100 bp fragment containing the B g l l I site (site 4) can be similarly amplified and cleaved to identify Type 2 strains. Restriction digestion of amplified fragments can produce accurate type determination; however it is important to determine sequence at several sites for a reliable classification. Since four of our 11 strains, as well as Mad-1 and GS/B, have at least one crossover to the consensus sequence of the opposite type, new isolates could potentially contain a mismatch at any one of the sites. Unique mutations also cannot be predicted, and can change restriction enzyme sites. Most isolates have

2675

no or few crossovers, but greater divergence is possible, given that one strain, 201, has crossovers at six typedetermining sites and eight unique sites. While this manuscript was in preparation, Yogo et al. (1991) reported the identification of two types of JCV cloned from urine and PML brain, which they termed Type A and Type B. Their grouping was based chiefly on RFLP analysis of the whole genome and additionally on sequence data from the non-coding region to the left of the origin of replication. Our non-coding region typedetermining sites correspond to the sites described by Yogo et al. (1991). Since we find these sites to be in full agreement with type-determining sites in the V-T intergenic region, it is clear that the RFLP-based Types A and B of Yogo et al. (1991) correspond to our Types 1 and 2, respectively. However, typing which is not based on known sequence variation tends to be ambiguous, making it difficult to include additional isolates (Grinnell et al., 1983b; Martin & Foster, 1984; Yogo et al., 1991; Takahashi et al., 1992; Tominaga et al., 1992). Using our simple system of type-determining sites, new sequences can be easily classified relative to known strains. It is not yet clear how our Types 1 and 2 relate to earlier classifications based on regulatory region organization (Walker & Frisque, 1986; Martin et al., 1985; Myers et al., 1989; Matsuda et al., 1987), but this is currently under investigation. The prototype strain Mad-1 was shown to be Type 1. The other strain for which a complete sequence has been determined, GS/B (Loeber & D6rries, 1988), is Type 2. Therefore Mad-1 can be considered to be the prototype for Type 1 whereas GS/B is the prototype Type 2 strain. In addition to type classification, finer distinction can be made between isolates of the same type by complete sequence comparison. Most of the 11 isolates obtained in this study are regarded as individual strains because they can be readily distinguished by differences of four or more unique mutations. In some cases the distinction is rather subtle. Type 1 strain 105 is identical to the consensus sequence, and strain 102 differs from it by only one unique mutation. Although this mutation is repeated in all clones of strain 102, in an isolate from which four clones were sequenced (203) a unique mutation was reversed to the consensus sequence in one clone, suggesting that mutation within the host or polymerase errors during PCR amplification can be a source of variation. The misincorporation frequency for Taq polymerase at the low concentration of nucleotides used here (200 ktMeach) has been calculated as 5 x 10-6 errors per nucleotide per cycle (Fucharoen et al., 1989; Goodenow et al., 1989), or 0.12 misincorporation in a 600 bp amplified fragment after 40 cycles. Thus it appears that the pattern of substituted sites is reproducible between different clones from the same brain sample

2676

G. S. Ault and G. L. Stoner

with a possible uncertainty of one or two nucleotide changes among the 20 to 26 mutations which distinguish any strain from the other type. Very closely related strains which differ at only one or two unique sites could represent isolation of the same strain from different individuals. Since we have isolated both JCV types from PML brains, it is clear that both types have characteristic JCV neurovirulence. Also, since PML in AIDS cases included both Type 1 and Type 2, induction by HIV is not unique to one or the other type. To date we have not isolated two types, or two different strains, from a single individual's brain. The possibility of multiple infection still exists but appears to be an uncommon event based on the observation that multiple clones from each of the brains in no case revealed more than one strain. Mutations at type-determining sites are referred to as 'crossovers', since they usually represent a switch to the opposite type sequence, rather than a random mutation. The reason for this pattern is unknown, but these crossovers may represent true genetic recombination occurring in potential (rare) cases of individuals infected with both virus types. If so, our strain 201, in which five of the six crossovers are clustered, could represent such an event. Alternatively, if Type 2 represents the ancestral JCV sequence from which Type 1 diverged, Type 1 crossovers could be the result of back mutations to the ancestral sequence, whereas crossovers in a Type 2 sequence would then be 'forward' mutations occurring after sequence divergence. Another factor may be constraints on protein sequence; at several sites mutation to either of the other two nucleotides would result in an amino acid substitution. The only mutations at typedetermining sites that are not crossovers in our data are site 14 in the intergenic region. The one instance of a non-crossover mutation at a type-determining site in a protein-coding sequence was in the strain GS/B of Loeber & D6rries (1988). A type-specific ,amino acid substitution of serine for phenylalanine in our isolates results from the mutation at site 19 in the T antigen gene, with a crossover in one strain of each type, and a substitution of tyrosine in strain GS/B. The amino acid sequence of the VP1 protein is conserved between the types. In the C-terminal 132 amino acids that could be predicted from our DNA sequence, only four amino acid substitutions occur, all of them at strain-specific rather than type-determining sites. Unless the N-terminal half of VP1 is found to be different, Type 1 and Type 2 viruses probably cannot be distinguished with serological assays, consistent with previous observations (Walker & Frisque, 1986). The situation may be different for the T antigen. In the short section of this 688 amino acid protein for which we can predict the sequence, there are three type-determin-

ing sites that result in an amino acid difference between Type 1 and Type 2. Therefore it may be possible to produce monoclonal antibodies specific to each type, to probe for early gene expression in tissues (Stoner et al., 1986). However our results suggest that it is unlikely that the two types differ in biological function of the T antigen. By analogy with the simian virus 40 (SV40) T antigen, the C-terminal region may have a host range function and be involved in capsid assembly (Zhu et al., 1991 ; Livingston & Bradley, 1987), and is less conserved than the N-terminal portion of the protein (Loeber & D6rries, 1988), which contains the domains involved in DNA binding, stimulation of replication and transactivation of late genes (Livingston & Bradley, 1987). Both major JCV strains are prevalent in the United States. Previously, Asian isolates were found to be only Type 2, whereas European isolates were mostly Type 1 (Yogo et al., 1991). To date, no African strains of JCV have been sequenced. PML is relatively rare in African AIDS patients (Williams, 1991) and the possibility of a third major strain of JCV with altered neurovirulence should be considered. We have asked which type is more representative of the ancestral JCV sequence, by assuming that the sequence JCV and BKV have in common can be considered 'ancestral'. Interestingly, this analysis identifies the Type 2 consensus sequence as much closer to the ancestral sequence, and thus closer to the original JCV, whereas Type 1 apparently is the later variant. Since related polyomaviruses such as mouse polyomavirus, SV40 (macaque) and BK virus (human) appear to have evolved from a common ancestor and diverged with their host species (Soeda et al., 1980), the existence of JCV Types 1 and 2 may reflect a major early divergence in the human family. A molecular clock based on the degree of nucleotide substitution between JCV Types 1 and 2 may permit speculation on the time during virus evolution at which divergence of these two strains began. The ability to distinguish JCV types easily and reliably can give insight into many questions about the epidemiology and biology of the virus. Simple typing procedures will facilitate transmission studies. For example, it should be possible to explore the question of perinatal transmission based on urine isolates, by asking whether offspring have the maternal virus type more frequently than that type occurs in the general population. Also, the question of whether the brain and the kidney isolates are the result of a single infection can be addressed. Previously it was reported that JCV isolates from brain and kidney of a single PML patient had identical sequences in all but the rearranged regulatory region (Loeber & D6rries, 1988). We are currently examining kidney tissue from patients in the present study to determine the relationship of the two forms of the virus.

Two types o f J C virus in P M L brain

We thank Richard J. Frisque for the use of sequence data prior to publication and Duard L. Walker for providing the PML patient tissues. This work was done in the Section on Neurotoxicology, Laboratory of Experimental Neuropathology, NINDS. The support and encouragement of Henry deF. Webster are gratefully acknowledged.

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(Received 23 April 1992; Accepted 22 June 1992)