Journal of General Virology (1993), 74, 2531-2537. Printed in Great Britain
2531
Complete sequence conservation of the human T cell leukaemia virus
type 1 t a x gene within a family cluster showing different pathologies
Marian E. Major,it * Simon Nightingale 2 and Ulrich Desselbergerit$
1 Regional Virus Laboratory, East Birmingham Hospital, Birmingham B9 5 S T and 2 Midland Centre f o r Neurosurgery
and Neurology, Birmingham B67 7JX, U.K.
We have amplified, through PCR, the full-length tax
gene of human T cell leukaemia virus type 1 (HTLV-1)
derived from proviral DNA of peripheral blood lymphocytes of five first degree relatives of Afro-Caribbean
origin. One patient (the father) had adult T cell
leukaemia (ATL), one (the mother) tropical spastic
paraparesis (TSP), and three (children) were healthy
asymptomatic carders. All five family members had
identical tax nucleotide sequences as determined by
direct sequencing of PCR products. This sequence was
compared with tax gene sequences of an unrelated TSP
patient of Afro-Caribbean origin, and of C8166 cells,
and found to have one and seven nucleotide differences,
respectively. At the amino acid level these three
sequences differed from the HTLV-1 prototype Japanese
strain (ATK-1). All sequence changes were clustered
towards the 3' end of the gene. These data demonstrate
the complete conservation of an HTLV-1 gene following,
presumably, horizontal and vertical transmission of the
virus. Clones of this gene showed more sequence
variation within the TSP patient than the ATL patient,
mostly consisting of point mutations; there was no
conservation of mutations between the two individuals.
These mutations occurred only in individual clones of
the ATL patient whereas those of the TSP patient were
found to be repeated in different clones. A tax-specific
cytotoxic T lymphocyte response was observed in two
asymptomatic carders with low antibody titres, whereas
none was detected in an individual with a high antibody
level. No tax-specific sequence was identified which may
have contributed to the apparently high degree of
transmission from mother to children (three of five
children tested) nor account for the differences between
disease symptoms in the parents.
Human T cell leukaemia virus type 1 (HTLV-1) is
strongly associated with adult T cell leukaemia (ATL)
(Yoshida et al., 1982) and the chronic neurological
disorder tropical spastic paraparesis (TSP) (Gessain et
al., 1985) also termed HTLV-l-associated myelopathy
(Osame et al., 1986), although the majority of infected
individuals are healthy asymptomatic carriers (Murphy
et aI., 1989). The HTLV-1 provirus is flanked by long
terminal repeats (LTRs) and contains the gag, pol and
env regions normally found in retrovirus genomes. In
addition, there is a region at the 3' end termed the pX
region (Seiki et al., 1983). This contains several open
reading frames (ORFs), for which only three proteins
have been clearly identified. These consist of the transactivator of transcription, Tax (40K), the regulator of
virion protein expression, Rex (27K), and a protein of
21K (p21 rex) of unknown function (Nagashima et al.,
1986). The products are expressed from the same
subgenomic, doubly spliced mRNA species (Seiki et al.,
1985), and the expression of tax and rex genes utilizes
different AUG codons both derived from the second
exon of the mRNA (Nagashima et al., 1986). Other
minor mRNA species encoded by the pX region have
been identified in cells infected with HTLV-1 (Berneman
et al., 1992a; Ciminale et al., 1992; Koralnik et al., 1992)
but there are conflicting data indicating the precise splice
junction sites and detection of transcripts in peripheral
blood mononuclear cells (PBMCs) from infected individuals is inconsistent (Koralnik et al., 1992). No
functions have been assigned to the predicted products of
the transcripts.
Tax is a nuclear protein, which stimulates transcription
of all viral genes from the 5' LTR (Cann et al., 1985;
Seiki et al., 1986) and has also been shown to activate
certain cellular genes, among them those encoding
interleukin 2 (IL-2), the alpha subunit of the IL-2
receptor (IL-2R~) and the granulocyte/macrophage
colony-stimulating factor (Wano et aL, 1988). Some of
these genes are involved in cell proliferation and control.
Owing to the lack of a specific integration site (Seiki et
t Presentaddress: Divisionof Virology,Departmentof Pathology,
Universityof Cambridge, CambridgeCB2 1QP, U.K.
$ Present address: ClinicalMicrobiologyand Public Health Laboratory, Addenbrooke'sHospital, CambridgeCB2 2QW, U.K.
0001-1827 © 1993 SGM
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2532
Short communication
al., 1984), or a classic oncogene in the viral genome (Seiki
et al., 1983), and the observation that extracellular Tax
can induce lymphocyte proliferation, possibly through
the activation of IL-2Re expression (Marriott et al.,
1992), the tax gene has been implicated in mechanisms of
initiation or maintenance of cellular transformation
which occur in infected individuals. Tax has been shown
to induce the expression of the nuclear factor NF-KB
which is involved in the activation of IL-2 and IL-2R~
expression (Leung & Nabel, 1988; Ruben et al., 1988).
The HTLV-1 enhancer region in the L T R contains a
cAMP-responsive element (CRE) which is crucial for
Tax-enhanced expression (Giam et al., 1986; Giam &
Xu, 1989). Recently, Tax has been shown to interact with
the CRE-binding protein and activating transcription
factor 1 (Zhao & Giam, 1992) suggesting an active role
for these DNA-binding proteins in Tax activation of the
HTLV-1 LTR.
Sequence variants of the HTLV-1 reverse transcriptase, pol and env genes and the L T R regions have been
described (Bangham et al., 1988; Malik et al., 1988;
Daenke et al., 1990), and it appears that no one variant
is associated with either ATL, TSP or the asymptomatic
carrier state. The tax gene of HTLV-1 is known to be
well conserved and intolerant of changes or mutations
(Smith & Greene, 1990). Semmes & Jeang (1992) made a
series of Tax mutations consisting mainly of single amino
acid changes, specifically targeting the serine residues
that may be involved in the phosphorylation of Tax.
Surprisingly, most of these single changes did not lead to
a significant loss in biological activity, with all the
mutants migrating to the nucleus. However, the potential
involvement of Tax in cellular proliferation and the
transformation process, the protein-protein interactions
necessary for its activating function and the association
of a tax-specific cytotoxic T lymphocyte (CTL) response
with both TSP (Jacobson et al., 1990) and asymptomatic
carriers (Parker et al., 1992) suggest that minor sequence
differences may contribute to the onset, or lack of
disease. Komurian et al. (1991) studied the extent of
sequence conservation across several regions of the
HTLV-1 genome, including the pX region. Sequences of
isolates from individuals of different geographical locations and exhibiting ATL, TSP and a B cell lymphoma
were compared but no specific mutations could be linked
to either A T L or TSP; healthy asymptomatic carriers
were not included.
We had the opportunity to examine the tax genes of
five HTLV-1-infected individuals in one family of AfroCaribbean origin, between them exhibiting ATL, TSP
and the healthy asymptomatic carrier state. The incidence of the virus and disease in this group is unusual.
Firstly, there was a high rate of transmission from
mother to children; three out of five children tested were
Table 1. H T L V - 1
virological states
infections in f a m i l y A : clinical and
Clinical
HTLV-1infection
state
Patient
Age
(age at
tax-specific
no.
Sex (years) onset) Serology*
PCR
A180
A132
A146
A147
A185
A141
A142
F
M
F
F
F
M
M
64
73t
32
23
30
24
26
TSP (52)
ATL (72)
Asym.
Asym.
Asym.
Asym.
Asym.
+
+
+
+
+
--~
+
+
+
+
+
-
-- ~
ND§
* Determined by PPAT, ELISA and immunofluorescence (Mowbray et al., 1989).
t Patient died in 1990.
:~ J. Mowbray, personal communication.
§ ND, Not done.
positive for antibodies to HTLV-1 (Table 1). This
observation, coupled with the occurrence of different
diseases in the parents, suggested that these individuals
may harbour a particularly virulent strain of HTLV-1
exhibiting variation at the molecular level. Given the
high degree of tax gene conservation, analysis and
comparison of the nucleotide sequences would give an
indication of the route of transmission, conservation o f
the gene and dominance of virus populations during
infection from one individual to another over a period of
time and data indicating whether the same virus strain is
associated with both diseases.
We amplified and sequenced the tax genes from
proviral D N A obtained from peripheral blood samples
of these individuals and compared them with those from
an unrelated TSP patient (B301), also of Afro-Caribbean
origin, and of C8166 cells, a cell line carrying the HTLV1 proviral genome (Salahuddin et al., 1983).
Blood samples were obtained from seven members of
one family (family A) and the sera tested for antibodies
to HTLV-1. Both parents (A180 and A132) and three of
the children (A146, A147, A185) were found to be
positive for HTLV-1 whereas two children (A141 and
A142) were seronegative (Table 1). All the seropositive
individuals were found by PCR to possess copies of the
tax gene in their genomic DNA. Patient A180 (TSP) had
a characteristically high antibody titre whereas that of
patient A132 (ATL) was intermediate. A146 had an
elevated antibody titre, although the titres of asymptomatic carriers are normally significantly lower than those
of TSP patients; similar observations have been reported
for other individuals not exhibiting any defined clinical
state (Mowbray et al., 1989). A147 and A185 had low
HTLV-1 antibody titres. All the children in this study
were breast-fed from birth by their mother. D N A was
obtained from peripheral blood of an unrelated HTLV-1-
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2533
SA
7429
73A~TTATTATCA~c~TCccAGGGTTTc~c~CAGAGTcTTc~TTTcGc~ATACccAGTcTAcGTG'~TTGc~GAcTGTGTAcAAGGCc~.cTGGTGccCcATcTcTGGGGcÈACTATGTTc
7430
7549
GGCCCGCCTACATCGTCACGCCCTACTGGCCACCTGTCCAGAGCATCAGATCACCTGGGACCCCATCGATGGACGCGTTATCGGCTCAGCTCTACAGTTCCTTATCCCTCGACTCCCCTC
7670
7789
TGGATACATGGAACCCACCCTTGGGCAGCACCTCCCAACCCTGTCTTTTCCACAACCCCGGACTCCGGCCCCAAAACCTGTACAACCCTCTGGGGAGC-CTCCGTTGTCTGCATGTACCTCTA
7909
7910
~
t'~
[]
8029
AATTTCCCT¥CACCACAGGGGCCCTAATAATTCTACCCGAAGACTGTTTGCCCACCACCCTTTTCCAGCCTGCTA~GCACCCGTCACGCTAACAGCCTGGCAA~ACGGCCTCCTTCCGTT•
8030
8149
CCACTC~'CCCTCACCACTCCAGGC
CTTA'I~TGC~%'CATTTACC~%'T~CC-'-CCTATC-~TTT
CCGGC~2CCTGCCCT/~G~TGGCCAC~CATCTTTAGTACTACAGTCCTCCTCCTTTAT
8269
8150
•
ATTTCACAAATTTCAAACCAAGGCCTACCACCCCTCATTTCTACTCTCACACGGCCTCATACAGTACTCTTCCTTTCATAGTTTACATCTCCTGTTTGAAGAATACACCAACATCCCCAT
82;0CTCTACTTTTTAACGAAAAAGAGGCAGATGACAATGACCATGAGCCCCAAATATCCCCCGGGG~TTAGAGCCTCCCAGTGAkAAACAATTTCCGA~GAAACAGAAGT~23T8C 9A
Fig. 1. Nucleotide changes observed in HTLV-1 t a x gene. Symbols: IS], family A; V, patient B301 ; 0 , C8166 cell line. Refer to Table
2 for specific nucleotide and corresponding amino acid changes. SA, Splice acceptor site (Seiki et al., 1985). Boxed codons: CAC
represents codon 3 of mRNA; TGA, stop codon of t a x gene (Seiki et al., 1983).
infected TSP patient (B301) and of healthy uninfected
individuals.
Genomic DNA was extracted from l0 G to 107 fresh
PBMCs, isolated on a Ficoll-Hypaque gradient, or
frozen whole blood, using the proteinase K-SDS method
(Sambrook et al., 1989). The tax gene was amplified from
1 lag DNA using a nested PCR (Simmonds et al., 1990)
in buffer containing 10 mM-Tris-HC1 pH 8.3, 50mMKC1, 1"5 mM-MgC12, 0"001% gelatine and 200 laM each of
dATP, dGTP, dCTP and dTTP (Pharmacia). The
amplification primers, synthesized on an Applied Biosystems 381A DNA synthesizer, had the following
sequences: (i) HT0, 5' TCGCTGCCGATCACGATGCGTT 3' (sense) corresponding to nucleotides (nt) 7067 to
7088 of the HTLV-1 (ATK-1) prototype sequence (Seiki
et al., 1983); (ii) HT2B, 5' TTGAGCCATATGCGTGGCATGA 3' (antisense, nt 8608 to 8587); (iii) pX52,
5' TTCCTCCACCAGCAGGTCCT 3' (sense, nt 7238
to 7257); (iv) pXLTR2, 5' GGAGGTCTGAGCTTATGATT 3' (antisense, nt 8497 to 8478). All primers were
used at a final concentration of 0' 1 laM in a 50 lal reaction
volume with 0"5 to 1 unit of Taq DNA polymerase
(Cetus Corporation). Primer pX52 was biotinylated at
the 5' end using a biotinylated phosphoramidite derivative (Cambio), to facilitate direct sequencing of the
amplified product (see below). PCR reaction mixtures
were initially denatured at 99 °C for 5 rain before the
addition of the Taq polymerase, followed by 35 cycles of
93 °C for 1 min, 55 °C for 1 min and 72 °C for 1.5 min,
with a final extension period of 10 min. One lal from the
first reaction was used in a final volume of 50 lal in the
second PCR reaction incorporating the second primer
pair (pX52 and pXLTR2). Five lal aliquots of each
reaction were analysed on ethidium bromide-stained 1%
agarose gels before sequencing; a single band of the
expected size (1256 bp) was obtained from all antibodypositive samples after the second reaction (data not
shown). Preparations and amplifications of genomic
DNA were carried out together with samples from
uninfected negative controls. These were included in
subsequent PCR and no products above 200 bp were
observed in any of these controls using tax-specific
primers. PCR were normalized for total amounts of
DNA and for efficiency as template by using primers
specific for the human/%globin gene (Cann et al., 1990).
An amplification product of the expected size (109 bp)
was observed in all samples containing human DNA
(data not shown).
The biotinylated amplified product was rendered
single-stranded following immobilization on streptavidin-coated magnetic beads (Dynal) and treatment with
0.15 M-NaOH for 10 min at room temperature. The nonbiotinylated strand was removed and the beads were
washed before resuspension in distilled water. The DNA
sequence of the positive strand was obtained by the
dideoxynucleotide chain termination method (Sanger et
al., 1977) using [35S]dATP (> 1000 Ci/mmol) (Amersham) and Sequenase T7 DNA polymerase (United
States Biochemicals). The sequence of the negative strand
was obtained from the double-stranded amplified product following the method of Winship (1989). Oligonucleotide primers for sequencing reactions were synthesized
according to the published sequence (Seiki et al., 1983)
and located at approximately 250 bp intervals. For
analysis of variation within individuals, PCR products
were cloned into the Invitrogen TA cloning vector
(Invitrogen Corporation, British Biotechnology) following the manufacturer's instructions.
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Short communication
Table 2. H T L V - 1 tax variation in different isolates
Table 3(a). H T L V - 1 tax variation within individual
A TL patient
Origin of sequence
Nueleotide
Amino acid
Family
Patient
No.*
Change
A
B301
7644
7833
7919
7981
7984
8013
8190
8230
8335
8336
8366
A~G
A~G
C ~ T
C~T
G~A
A~C
C--*T
G~A
G~A
C~G
A ~C
+;~
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
C8166
cells
+
+
+
+
Nucleotide
No.t
Change
No.*
Change
109
172
M~V
I--+V
221
222
232
A~V
R~K
N~H
Deletion
A ~ G
T ~ C
C~T
T ~ C
T ~ A
Deletion
294
S~N
329
G~E
7331
7343
7475
7491
7515
7633
7661
7840
8012
8120
8249
8308
8353
* Nucleotide positions are according to H T L V - 1 ( A T K - 1 ) sequence
(Seiki et al., 1983).
t Amino acid numbering is according to published sequence (Seiki
e t al., 1985).
+ , Change in isolate when compared to published sequence (Seiki
e t al., 1983).
Seven full-length HTLV-1 tax sequences were obtained
from direct analysis of amplified products, five from
family A, one from an unrelated TSP patient (B301) and
one from C8166 cells. Specific sites of variation of these
sequences are shown in Fig. 1, the specific nucleotide
changes and the predicted amino acid changes in Table
2. The sequence was very well conserved for all samples
( > 99% identity) and complete conservation was observed within the five family members. Some variation
was found in the unrelated TSP patient (B301) and the
C8166 cells. The negative controls employed at each
stage and the inclusion of unrelated samples were found
to differ at the nucleotide level from those of the study
group which confirmed that the results were not due to
carry-over of the PCR product.
A similar observation of gene conservation has been
reported by Gessain et al. (1992) in a case of HTLV-1
transmission involving three individuals. In this instance
part of the env gene sequence was compared but was
found to be identical for all three subjects. Komurian et
al. (1991) studied ORFs II, III and IV of the pX region
in patients from Japan, the Caribbean and the Ivory
Coast. These were also compared to the prototype
sequence ATK-1 (Seiki et al., 1983). Of the nine
sequences analysed, that with the largest number of
amino acid changes came from the patient with B cell
lymphoma but no signs of ATL or TSP (Gro isolate).
This sequence was compared with that of family A,
shown in Table 4. Interestingly, all the nucleotide changes
observed in family A are present in the Gro isolate, who
was also of Caribbean origin, with one additional point
mutation in Gro at position 8145, predicting an amino
acid change at position 276. This degree of change is
comparable with that seen between family A and B301 in
Deletion
A~G
A~G
A --* G
A --* G
A ~ G
Amino acid
Clone
no.
9
12
2
11
8
7
1
7
1
9
12
10
4
No.t
Change
58
66
105
P~S
S~P
F --* Y
330
345
D ~ G
E ~ G
* Nucleotide positions are according to H T L V - 1 ( A T K - 1 ) sequence
(Seiki et al., 1983).
t Amino acid numbering is according to published sequence (Seiki
et al., 1985).
Table 3(b). H T L V - 1 tax variation within individual
T S P patient
Nucleotide
No.*
Change
7359
7380
7401
7406
7495
7511
7598
7606
7632
7635
7671
7673
7721
7857
7907
7975
7984
8076
8077
8132
8200
8230
8314
8368
8370
G ~ A
G~A
T ~ C
C ~ T
T ~ C
C~T
A ~ T
A --+ G
T ~ C
C ~ T
G--+A
A --* G
A ~ G
G ~ A
T ~ C
A ~ T
G ~ A
G ~ A
G ~ A
A~G
A ~ G
G ~ A
A~C
A ~ G
A~G
Amino acid
Clone
no.
7
4
7
8
7
7
4
6
4 and
1
4 and
11
1
6
4
6
4 and
4
4
7
2 and
1
2
10
4
1
11
11
9
No.t
Change
14
21
28
G ~ R
G~R
W ~ R
64
I~ T
96
105
106
118
H --* R
F ~ L
L ~ F
G~R
180
G ~ R
219
222
Q ~ L
R ~ K
253
G ~ N
294
304
332
350
351
H ~ R
S~ N
E~A
E ~ G
T~A
*, ~', See footnotes to Table 3 (a).
this study (Table 2). No clonal variation was studied by
Komurian et al. (1991) and it is unknown whether
HTLV-1 contributed to the patient's B cell lymphoma.
The sequences described above were determined
directly on the uncloned PCR product to obtain the
majority sequence present in each sample. To analyse the
variation within single individuals, 12 clones of PCR
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Table 4. Comparison of H T L V-1 sequences from family'
A and isolate Gro
Nucleotide
Table 5. H T L V - 1 antibody titres in three asymptomatic
carriers of family A : comparison of C T L responses
PPAT titre in
samples from
Isolate
sequence
Amino acid
No.*
Original
base*
Family
A
Grot
7644
7833
7919
7981
8013
8145
8230
8335
8336
8366
A
A
C
C
A
T
G
G
C
A
G
G
T
T
C
T
A
A
G
C
G
G
T
T
C
C
A
A
G
C
2535
No.$ Change
109
172
M---,V
I--*V
221
232
276
294
A~V
N~H
F~L
S~N
329
G~E
* Nucleotide positions and bases are according to HTLV-1 (ATK1) sequence (Seiki et al., 1983).
? Sequence of isolate Gro, from Komurian et al. (1991).
:~ Amino acid numbering is according to published sequence (Seiki
et at., 1985).
products obtained from the A T L (A132) and TSP (A180)
patients were sequenced. The changes observed are
shown in Table 3(a, b). Nucleotide changes have
occurred at r a n d o m in different clones of the A T L patient
and nowhere twice, with a number of deletions leading to
truncated proteins. Given the clonal proliferation which
occurs in the lymphocytes of A T L patients during
disease progression such similarity is expected. There is
the tendency of A to G mutations (six of 13 positions)
which m a y be specifically due to Taq polymerase errors.
Therefore, it is considered that the changes observed in
this patient are most likely due to the enzyme used for
amplification. More variation is observed for the cloned
P C R product of A180 (the TSP patient), with some
changes occurring in more than one clone and particular
clones (1, 4, 7 and 11) exhibiting relatively extensive
variation. This may reflect the presence of mixed
populations in TSP patients due to the absence o f clonal
proliferation. It should be noted that there are no
nucleotide changes c o m m o n to the clones of both A132
and A180.
N o n e of the base changes and subsequent amino acid
variation observed here correlate to those studied by
Smith & Greene (1990). However, tax variations within
the individual patients coincided with two sites mutated
by Semmes & Jeang (1992), both of which altered serine
residues. The first occurred in the A T L patient at
nt 7515, affecting amino acid 66 (Table 3 a), resulting in
the substitution of proline, as opposed to alanine. The
second mutation occurred in the TSP patient at nt 8230,
amino acid 304 (Table 3b), yielding an asparagine
substitution instead of alanine. Semmes & Jeang (1992)
observed slight reductions in trans-activation from the
substitution at amino acid 66 only. Both studies on Tax
Patient
no.
Age
CTL
response
1989
1993
A146
A147
A185
32
23
30
-*
+*
+t
32768
128
64
16384
512
256
* Data from Parker et al. (1992).
t Data from C. Parker, personal communication.
mutations identified mutants which rendered the protein
non-functional in one or other of the pathways previously mentioned. Therefore it is possible that several of
the changes observed in the TSP patient (A180) are
within as yet unidentified domains which m a y render the
product non-functional in one or both of these potential
activation pathways.
Tax is expressed from a spliced m R N A (Seiki et al.,
1985). Amplification from genomic D N A as carried out
in this study did not allow the analysis of 4 bp of the env
region which contributes to the coding sequence of tax.
This study did, however, include the splice acceptor site
of the third exon of the tax~rex message; no mutations
were observed.
In contrast to the conservation of the tax gene between
the three daughters, their C T L responses have been
shown to differ. Observations of A146 and A147 have
been reported previously (Parker et al., 1992), there
called H C and HA, respectively. Fresh CD8 + cells from
A147 only were found to produce a specific response to
target cells infected with a vaccinia virus tax recombinant
whereas no such response could be detected for cells
from A146. Subsequently, cells from A185, cultured for
48 h, have also been shown to m o u n t a tax-specific
response (C. Parker, personal communication). Table 5
shows the anti-HTLV-1 antibody titres of the three
subjects, determined by the Serodia passive particle
agglutination (PPAT) assay (Mast Diagnostics) compared with titres from previous samples. Tax-specific
C T L responses were observed in the two asymptomatic
carriers with low humoral antibody titres, whereas none
was detected in the individual with a high antibody level.
This is particularly interesting given the identical gene
sequence shown to be present in these family members.
Comparisons of recent antibody titres with those from
1989 do show a slight elevation in A146 and A185 but
not to the level of that observed in A147. The increased
antibody titre in A147 m a y or may not be due to a high
initial load of infectious virus; it is possible that the lack
of a C T L response in this individual is linked to her
initial reaction to infection which resulted in this high
level of humoral antibody.
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The conservation o f the t a x gene observed in this study
between individuals suggests that the passage o f infection
was horizontally between h u s b a n d and wife and vertically f r o m m o t h e r to children, presumably via breast
milk which has been shown to be a m a j o r route o f
vertical transmission ( A n d o et al., 1990). The lack o f
change in the t a x gene a m o n g these family members
demonstrates considerable stability over extensive periods o f time assuming the children (aged between 27 and
30 years) were infected at, or shortly after, birth.
A l t h o u g h infection by sexual transmission later in life
c a n n o t be excluded, the complete identity o f sequences in
the family members is more suggestive o f transmission
within the family cluster. Single nucleotide changes were
observed in comparisons o f the sequence f r o m this
family with that o f an unrelated T S P patient (B301),
which did not lead to variation between the two at the
amino acid level, and with a sequence f r o m a B cell
l y m p h o m a patient (Gro) in an entirely separate study
( K o m u r i a n et al., 1991). These changes were at positions
8191 and 8145 for patients B301 and Gro, respectively.
This is not unexpected given the evidence that isolates
f r o m similar geographical areas have higher sequence
conservation than those f r o m different locations (Malik
et al., 1988; Schulz et al., 1991 ; Berneman et al., 1992b).
Patient B301 emigrated to the U.K. f r o m the same
geographical areas as the parents (A132 and A180), and
the G r o isolate was also o f A f r o - C a r i b b e a n origin.
F r o m the result o f this study there appears to be no
d o m i n a n t t a x sequence associated with any particular
clinical state. M o r e specifically, identical consensus t a x
sequences were observed in all three o u t c o m e s o f
infection. There was no unusual sequence variant which
m a y be responsible for a high rate o f infectivity f r o m
m o t h e r to children, a l t h o u g h this characteristic m a y be
as m u c h a function o f the env gene as the t a x gene. There
is also no particular sequence which m a y be associated
with a C T L response.
The population within the T S P patient is m o r e variable
than that within the A T L patient, suggesting the
possibility that subpopulations m a y be relevant in disease
outcome. F r o m previous studies on Tax mutations
(Smith & Greene, 1990; Semmes & Jeang, 1992) it
appears that single amino acid substitutions are less
disruptive o f Tax function than consecutive changes in
adjacent residues. These latter changes were observed in
the consensus sequence o f the C8166 control cells and the
cloned p o p u l a t i o n o f the T S P patient only. They m a y
affect function t h r o u g h destabilization o f the protein or
lack o f nuclear localization, rather than increasing
activity. C o m p a r i s o n s between regions o f the gene cloned
f r o m different TSP patients and functional studies o f
these clones would be required to determine the true
significance o f this variation. Identical tax genes m a y be
activated or expressed at different levels in these patients
or may not lead to extensive trans-activation of cellular
genes owing to differences in host cell factors. Analysis
and comparisons of the mRNA sequences present in
these individuals, particularly between the three asymptomatic carriers given their CTL data, and the response of
cellular genes to trans-activation will be useful in
determining possible factors relevant in the transition
from the asymptomatic to the diseased state.
We would like to thank Claire Parker for communicating the CTL
data on the asymptomatic carriers and Charles Bangham for fruitful
discussions. This work was supported by a grant from the Medical
Research Council.
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