Download Two major types of JC virus defined in progressive multifocal

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
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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).
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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).
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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
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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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2674
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.
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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
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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.
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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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