Next Article 
Journal of Virology, May 2000, p. 3933-3940, Vol. 74, No. 9
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Chimeric Matrix Proteins Encoded by Defective Proviruses with
Large Internal Deletions in Human T-Cell Leukemia Virus Type
1-Infected Humans
Vladimir A.
Morozov,1,2
Sylvie
Lagaye,3
Graham P.
Taylor,4
Estella
Matutes,1 and
Robin A.
Weiss1,*
Institute of Cancer Research and Royal
Marsden Hospital, London SW3 6JB,1 and
Imperial College School of Medicine, Jefferiss Research
Trust Laboratories, London W2 1PG,4 United
Kingdom; Institute of Carcinogenesis, Cancer Research
Center of Russian Federation, 115478 Moscow,
Russia2; and Unité 342, INSERM,
St. Vincent de Paul Hôpital, 75014 Paris,
France3
Received 31 August 1999/Accepted 10 January 2000
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ABSTRACT |
Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic
agent of adult T-cell leukemia/lymphoma (ATLL), HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and other diseases. The mechanisms of virus pathogenesis are still obscure. The occurrence of defective proviruses in HTLV-1-infected cell lines and the peripheral blood mononuclear cells (PBMC) of infected individuals is a
frequent feature of virus infection. We detected defective proviruses
with large internal deletions in PBMC from ATLL and HAM/TSP
patients and in asymptomatic HTLV-1 carriers. Seventeen PCR-amplified
defective proviruses were sequenced, and three types of deletions were
found. Besides truncated MA and the 5' end of the genome, truncated
CA, truncated SU, and more frequently truncated TM linked to the
pX region were detected. Reverse transcription-PCR analysis of PBMC
from ATLL patients and asymptomatic carriers also revealed RNA
transcripts with large internal deletions. Analysis of two RT-PCR cDNA
clones confirmed a Gag-TM-pX structure of the transcripts. Most
defective proviruses contained numerous internal stop codons, but some
were capable of coding for the truncated MA linked to a variable
out-of-frame peptide. Cloned defective proviruses with long open
reading frames were subjected to in vitro transcription-translation
followed by radioimmunoprecipitation, which showed expression of
chimeric proteins between 8 and 12 kDa. Possible roles of defective
proviruses and chimeric proteins are discussed, although there is no
firm association with pathogenesis.
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INTRODUCTION |
The human T-cell leukemia virus type
1 (HTLV-1) is a retrovirus which causes two distinct pathologies:
adult T-cell leukemia/lymphoma (ATLL) (10, 27) and
chronic progressive myelopathy-tropical spastic paraparesis (TSP), also
called HTLV-1-associated myelopathy (HAM) (5, 26).
Recent studies have suggested that HTLV-1 is also associated with other
human diseases (23).
Previous comparative studies of different HTLV-1 isolates indicated the
following: the protein patterns of the viruses from cells of
individuals with ATL and HAM/TSP were identical (6), DNA
blotting (11, 33) and sequence comparisons of proviruses from ATLL and HAM/TSP revealed nearly 97% homology (4, 21), and restriction endonuclease analysis did not reveal any selected integration sites for the proviruses in the DNA of infected individuals (33). Occasional cases of ATLL associated with TSP-HAM
pathology have been reported (14, 24), and infection of a
patient with blood from a HAM/TSP donor resulted in HAM/TSP in the
recipient (8). Thus, different diseases in association with
one virus remains a paradox of HTLV-1. The mechanisms by which the
virus induces different diseases and the cofactors, either virus
associated or host related, that contribute to different forms of
disease manifestations or progression remain to be determined.
Defective proviruses have been observed in cells from ATLL patients and
could be important elements in pathogenesis (9, 17, 32). We
have therefore compared the presence of defective proviruses in HTLV-1
asymptomatic carriers and in patients with ATLL or HAM/TSP.
Detailed analysis of defective proviruses were previously carried out
on the MT-2 cell line (15, 16). In this cell line, besides a
complete provirus, seven defective proviruses were detected by Southern
hybridization, and four of them were found to be identical with a large
internal deletion resulting in the 5' portion of the gag
gene being linked to a pX region (15, 16). These proviruses contained an open reading frame (ORF) coding for a chimeric protein (p28) composed of Gag (MA [matrix] and truncated CA [capsid]) and a
short pX fragment (12). RNA transcripts for MT-2 cell defective provirus are packaged in virions and can be translated into a
p28 chimeric protein (25), and they establish new provirus in culture (1). HTLV-1 proviruses with internal deletions
have also been found in other cell lines (9, 33) and in
peripheral blood mononuclear cells (PBMC) of ATLL patients (18,
31), as well as defective proviruses in human immunodeficiency
virus infection (29).
Using the p28 provirus of MT-2 cells as a prototype, we designed a set
of PCR primers corresponding to the Gag and pX regions (25),
which allowed us to amplify proviruses with large internal deletions
provided they contained the corresponding gag and pX sequences but not the gag-pol-env-pX sequence of the
completed provirus. The aim of our research was to detect and analyze
possible defective proviruses with large internal deletions in
HTLV-1-infected individuals with different pathologies: HAM/TSP,
ATLL, and asymptomatic carriers. We report the identification and
nucleotide sequence analysis of different proviruses with large
internal deletions in DNA of patients with HTLV-1-associated
pathologies and show that some are transcribed and can be
translated into proteins.
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MATERIALS AND METHODS |
Patients.
Patients with ATLL or HAM/TSP and asymptomatic
HTLV-1 carriers were studied. HTLV-1 infection of each individual was
confirmed by Western blotting of serum and by PCR of PBMC DNA using
pol and tax pairs of primers. HAM/TSP patients
were also tested for the presence of complete provirus by Southern
hybridization with corresponding subgenomic probes. Blood was collected
by venipuncture, and PBMC were separated on a Ficoll-Hypaque gradient,
washed twice in phosphate-buffered saline (PBS), and stored at 
80°C
until use. Some PBMC samples and HTLV-1 immortalized cell lines from HAM/TSP patients were kindly provided by G. Peries (Hôpital St. Louis, Paris, France) and have been previously described (6, 7). Most PBMC samples from patients with ATLL, asymptomatic carriers, and some patients with HAM/TSP were from individuals studied
at the Royal Marsden Hospital and St. Mary's Hospital, London, United
Kingdom, with ethical approval.
Cells.
MT-2 and CEM cells were cultivated in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics as
previously described (3). Cells were passaged every 4 days.
Plasmids.
pMT-2-42 containing defective HTLV-1
provirus (12) was a gift from M. Hatanaka (Institute
of Virus Research, Kyoto University, Kyoto, Japan). The expression
vector pTargeT was purchased from Promega, Madison, Wis.
Sera and monoclonal antibodies.
Serum samples (verified by
different immunological tests) were obtained from two HTLV-1-infected
individuals with HAM/TSP. Monoclonal antibodies against p28, p19,
p24, and p21E were obtained from Chemicon Int. (Temacula, Calif.).
Anti-human immunoglobulin G-alkaline phosphatase (IgG-AP) and
anti-mouse IgG-AP were obtained from Boehringer-Mannheim, Mannheim, Germany.
Isolation of DNA and RNA.
Total genomic DNA was extracted
from 3 × 106 to 5 × 106 PBMC using
a DNA preparation kit (Promega) and was analyzed by agarose gel
electrophoresis. From each cell preparation, 107 cells were
used for DNA extraction by phenol-chloroform. Total RNA was isolated
from 3 × 106 to 5 × 106 PBMCs using
RNAzol B (Biogenetics, Poole, United Kingdom) according to the
manufacturer's protocol.
PCR and reverse transcription-PCR (RT-PCR).
Sets of primers
for nested PCR corresponding to the long terminal repeat (LTR), Gag,
and pX regions of HTLV-1 were designed based on the relevant viral
sequences (30) and were synthesized by Oswel Labs
(Southampton, United Kingdom). HTLV-1 proviruses are available under
accession no. U19949 and D13784. Their sequences are as follows: 5'LTR
(forward), 5' GAC AAT GAC CAT GAG CCC CAA (positions 2 to 22); ATG gag
(forward), 5' TAG GCT ATG GGC CAA ATC TT (positions 798 to 817); A1 p28
(forward), 5' CAA ATC TTT TCC CGT AGC GCT AGC (positions 809 to 832);
A2 p28 (forward), 5' TCC AGT TAC GAT TTC CAC CAG TTG (positions 918 to 940); pX (reverse), 5' AGG AGG ATT TGA TGG GAG AGG TTA (ATK positions 6727 to 6704); B1 p28 (reverse), 5' GGA GGC GAT GTA GTT GCA ATA (ATK
positions 6683 to 6663); B2 p28 (reverse), 5' ATG TGC TTG GTT TAC AGG
GAT (ATK positions 6640 to 6659); Stop p28 (reverse), 5' GGT TAA TTA
TTG GCA GGG GAG (positions in ATK 6708 to 6687); HTLV-1 pol
and tax, as published by Kwok et al. (19); HTLV-1 pol (forward), 5' AGA TAC AGG AGC AGA CAT GAC (ATK positions
2242 to 2262); HTLV-1 pol (reverse), 5' GGA CTG GAA AAC ACT
ACA GTA (AKT positions 3547 to 3528); HTLV-1 tax forward, 5'
TTT CGG ATA CCC AGT CTA CG (ATK positions 7335 to 7354); and HTLV-1
tax reverse, 5' GAT AAC GCG TCC ATC GAT GG (ATK positions
7472 to 7491).
We used in each reaction 1 µg of DNA (equivalent to approximately
150,000 cells). Hot-start PCR amplification was performed as instructed
by the supplier of Taq polymerase (Perkin-Elmer/Roche, Nutley, N.J.). After initial denaturation at 95°C for 5 min, DNA was
subjected to 32 to 40 cycles of amplification (denaturation at 95°C
for 1 min, annealing at 48 to 55°C [dependent on primers] for
45 s, extension at 72°C for 1 min). After the last cycle, extension was carried out at 72°C for 10 min, and a 10-µl aliquot of each PCR mixture was electrophoresed in a 1.8% agarose gel containing ethidium bromide. Adequate conditions and amounts of DNAs
were confirmed by PCR with 
-globin primers, upstream (5'-ACA CAA CTG
TGT TCA CTA GC-3') and downstream (5'-CCA CTT CAT CCA CGT TCA CC-3').
Amplimers were analyzed by agarose (1.8%) electrophoresis with
ethidium bromide. High-fidelity PCR was performed with the 5'LTR-pX
pair of primers and amplification conditions suggested by the kit
supplier (Boehringer-Mannheim). Long PCR fragments were analyzed on 1%
agarose gel with ethidium bromide. RT-PCR was performed with a Titan
one-tube RT-PCR kit according to the protocol of the supplier
(Boehringer-Mannheim).
Southern blot hybridization.
After overnight digestion of 10 µg of each genomic DNA sample with SacI (Promega), gel
electrophoresis, and overnight transfer to a Hybond N membrane
(Amersham, Little Chalfont, United Kingdom), Southern hybridization was
done by using a digoxigenin (DIG) DNA labeling and detection kit
(Boehringer-Mannheim). For Southern blots of PCR products, 10 µl of
the PCR product was loaded per track. Conditions of prehybridization,
hybridization, washing, and color development were as proposed by the
manufacturer. The SacI-PstI fragment from
pMT-2-42 was used as a probe. DIG-labeled DNA molecular weight markers
III and VII (Boehringer-Mannheim) were used for gel calibration.
Conditions of DNA transfer and Southern hybridization of PCR-amplified
fragments were the same as for the genomic DNA.
Protein electrophoresis, Western blotting, and RIP.
Electrophoresis was performed in a precast sodium dodecyl
sulfate-Tricine 10 to 20% polyacrylamide gel (Novex, San Diego, Calif.). Rainbow markers (Amersham) were used for gel calibration. After electrophoresis, proteins were blotted for 2 h at 4°C on 0.22-µm-pore-size nitrocellulose membrane (BA83; Schleicher & Schuell, Dassell, Germany). Membranes were blocked with 8% dry milk in
PBS with 0.1% Tween 20 overnight at 4°C. After 2 h of incubation with a corresponding serum or monoclonal antibody, anti-human or anti-mouse IgG-AP (1:2,000 dilution; Boehringer-Mannheim) were used as the secondary antibody. After five washes (5 min each) in
PBS-Tween 20, color was developed with 4-chloro-1-naphthol (Bio-Rad,
Hercules, Calif.) in PBS with 0.01% H2O2 or BM
purple substrate (Boehringer-Mannheim). Virus-specific proteins
synthesized in the TNT in vitro transcription-translation system
(Promega) were precipitated with monoclonal antibodies or with
polyclonal sera from HTLV-1-infected individuals. One hour later,
protein G (Sigma, St. Louis, Mo.) was added to increase immune
complexes, and incubation was continued for 16 h. Precipitates
were washed five times in buffer with 0.5% NP-40, disrupted in
radioimmunoprecipitation (RIP) assay buffer at 100°C for 5 min, and
applied to the gel.
Cloning and sequencing.
PCR-amplified fragments were
purified on 1.5% low-melting-point agarose (Bethesda Research
Laboratories, Gaithersburg, Md.) and cloned in the pTargeT vector
(Promega). Recombinant clones were analyzed by restriction with
EcoRI (Promega) to confirm correct fragment insertion.
Inserts were sequenced using a ABI Prism dye terminator cycle
sequencing kit with AmpliTaq DNA polymerase (Applied Biosystems/Perkin-Elmer) and analyzed using an Applied Biosystems model
373 automatic DNA sequencer.
In vitro expression of cloned sequences.
Expression of
cloned amplimers was carried out in the TNT in vitro
transcription-translation system (Promega) with
L-[U-14C]leucine (Amersham) as specified by
the manufacturer. Synthesized proteins were analyzed by electrophoresis
or immunoprecipitated with HAM/TSP patient sera, followed by
electrophoresis of precipitated proteins on a 10% NuPAGE gel (Novex).
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RESULTS |
Defective proviruses revealed by genomic Southern blotting and
long-range PCR.
To estimate complete provirus in patient DNA,
Southern hybridization analyses of total genomic DNAs obtained from
ATLL- and HAM/TSP-derived cell lines were performed with
SacI, known to have cleavage sites in both LTRs of the
provirus (30, 33). Ten micrograms of DNA extracted from each
cell line was subjected to restriction endonuclease digestion followed
by electrophoresis and hybridization with the DIG-labeled subgenomic
probe. DNA from the human lymphoid cell line CEM was used as a negative
control. Southern blot analysis revealed that five of six
HAM/TSP-derived cell lines and one ATLL-derived cell line contained
both complete and defective proviruses; one ATLL-derived cell line was
found to contain only complete provirus (Fig.
1A). One to four defective proviruses
ranging from 7 to 2 kb were detected in these DNA samples. Similar
results were obtained following EcoRI digestion and Southern hybridization (data not shown).
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FIG. 1.
Complete and deleted HTLV-1 proviruses detected by
Southern hybridization in the DNA from TSP/HAM- and ATLL-derived cell
lines. (A) Total genomic DNA was cleaved with SacI and,
after electrophoresis and transfer, hybridized to DIG-labeled
subgenomic probe. Lanes: 1, DNA from MT-2 cells; 2 and 3, DNA from ATLL
cell lines; 4 to 9, DNA from TSP/HAM-derived cell lines; M, DIG-labeled
Boehringer III markers. Positions of full-size proviruses are indicated
by the arrow on the left. Most of deleted proviruses are localized
between 2 to 3.5 kb. (B) The same DNA samples tested by PCR and
Southern hybridization for the defective proviruses with large internal
deletions (A1-B1 pair of primers). M, DIG-labeled Boehringer VI
markers. Position of the p28 provirus is indicated by an arrow.
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PCR analysis performed with the A1-B1 primer pair on the same DNA
samples followed by Southern blot hybridization revealed
numerous
defective proviruses (Fig.
1B). Complete provirus was
analyzed in 20 asymptomatic carriers and 7 ATLL patients and was
detected in 8 and 6, respectively. However, because of low proviral
load especially in
asymptomatic carriers, we cannot exclude that
more samples contained
complete HTLV-1 genomes. Analysis of all
samples for the presence of
the HTLV-1 genome was confirmed by
PCR with the
pol-tax pair
of primers and by long-range PCR with
the 5'LTR-pX pair of primers
(data not
shown).
Defective proviruses with Gag-pX structure revealed by nested
PCR.
To detect defective proviruses with a Gag-pX0 structure,
further analysis of the defective proviruses was carried out by nested PCR with two sets of primers, followed by Southern hybridization. In
the first round of PCR, we used a pair of primers that corresponds to
the 5' LTR (2 to 22) and pX (6727 to 6704) nucleotides; nested PCR was
done with a second pair of primers, ATG (798 to 817) and stop p28 (6708 to 6687). Using these primers, we were able to detect proviruses with
internal deletions that contain both the 5' LTR and the beginning of
pX0 region. Another two primer pairs for nested PCR, representing the
N-terminal part of MA (A1; 809 to 832) and pX0 (B1; 6684 to 6664),
followed by internal MA (A2; 918 to 941) and pX0 (B2; 6639 to 6659),
allowed us to detect only the internal part of defective proviruses,
with or without a 5' LTR. Use of both pairs of primers allows efficient
amplification of deleted proviruses (Fig.
2).
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FIG. 2.
Detection of HTLV-1 proviruses with large internal
deletions by nested PCR-Southern blotting in the DNA from PBMCs of the
ATLL and TSP/HAM patients. PCR was performed with the A1-B1 and A2-B2
pairs of primers. Lanes: 1, DNA from MT-2 cells; 2, DNA from
noninfected CEM cells; 3 to 8, DNAs from ATLL patients; 9 to 12, DNAs
from TSP/HAM patients; 13, water control. Position of the p28 provirus
from MT-2 cells is marked by an arrow.
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To ensure that the defective proviruses are genuine and did not result
either from carryover contamination or from PCR artifacts,
the
specificity of the detection was further analyzed by testing
DNA from
MT-2 cells, known to contain seven defective proviruses,
and by spiking
DNA from HTLV-1-free CEM cells with plasmid DNA
containing a
full-length HTLV-1 genome. As shown in Fig.
3, deleted
proviral genomes were
amplified only from MT-2 cells, confirming
the specificity and validity
of our analysis of clinical specimens.
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FIG. 3.
Estimation of the specificity of defective provirus
amplification by PCR and nested PCR. (A) Amplification of defective
proviruses using the LTR-B1 pair of primers (35 cycles). Lanes: M,
markers; 1, DNA from MT-2 cells (200 ng); 2, DNA from noninfected CEM
cells (500 ng) mixed with 100 ng of plasmid expressing full-size
HTLV-1; 3, DNA from CEM cells; 4, no-DNA control. (B) Amplification of
defective proviruses using the A1-B1 pair of primers (35 cycles).
Lanes: M, markers; 1, DNA from CEM cells (500 ng); 2, DNA from CEM
cells mixed with 50 ng of pMT-2-42 (cloned p28 provirus); 3, DNA from
CEM cells (500 ng) mixed with 100 ng of pHTLV-1; 4, DNA from MT-2 cells
(200 ng). (C) Amplification of defective proviruses by nested PCR using
the A2-B2 pair of primers (30 cycles). One microliter from the first
reaction (B) was taken for nested PCR. Positions of amplimers are given
on the right. Five microliters of each PCR mixture was run on a 2%
agarose gel with ethidium bromide.
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Overall, 51 DNA samples, comprising 13 cell lines and 38 extracted
directly from PBMC of infected individuals, were examined
for the
defective proviruses (Table
1). After the
first round
of PCR especially with the 5'LTR-pX pair of primers, we
detected
amplimers only in a few samples, indicating a relatively low
load
of defective proviruses containing both the 5' LTR and
corresponding
pX region. However, after either Southern hybridization
or a second
PCR round with the 5'-Gag-pX pair of primers, numerous
defective
proviruses were detected in the majority of samples.
The sizes of the majority of Gag-pX0 amplimers varied from nearly 200 to 1,200 bp, signifying a spectrum of internal deletions.
Notably,
comparison of DNA from PBMC and from cultivated lymphocytes
of the same
patient with HAM/TSP indicated that the quantity of
the deleted
proviruses increased during in vitro culture, possibly
through mitosis,
although not all of the defective proviruses
proliferated equally (data
not shown). More efficient amplification
was observed with the MA-pX
pair of primers, indicating that many
of the analyzed proviruses either
lack a 5' LTR or have a mutated
5'
LTR.
Nucleotide sequence of the amplimers.
To analyze the
internal structure of the PCR-amplified proviral sequences, 17 PCR
amplimers from different patients (11 HAM/TSP, 3 ATLL, and 3 carriers) were purified on low-melting-point agarose and sequenced
directly in both directions using internal primers. Most but not all
deleted proviruses contained stop codons within the sequenced fragments.
As expected from the primers used, the internal parts of all defective
proviruses were flanked at the 5' by the Gag (MA) fragment
and at the
3' by pX sequences, whereas the internal parts (between
MA and pX) of
these proviruses were quite different. Two proviruses
contained the
3'-terminal part of SU (surface) linked to the 5'-terminal
part of TM
(transmembrane), and 12 of 14 deleted proviruses contained
3'-terminal
fragments of TM. Two proviruses contained only 5'-terminal
fragments of
CA. However,
pol-related sequences were not revealed
in any
of these proviruses. Based on genomic structure, we grouped
defective
proviruses into three principal classes (Table
2).
The MA stretch in all three classes
of proviruses was relatively
long, from 70 to 360 bp. Differences in
size were observed between
TM stretches in proviruses from classes 1 and 2, from 6 to nearly
530 bp. CA fragments in both CA-containing
proviruses (class 3)
were nearly 120 bp. Class 3 strongly resembles the
p28 provirus
present and expressed in MT-2 cells (
15,
25).
No clear association
with pathology was observed for any of the
identified groups of
deleted proviruses, although class 2 was more
frequent in HAM/TSP
patients.
Four proviruses with a 5' LTR and long ORFs and the
MA-
TM-pX
structure from HAM/TSP patients were sequenced from the first
ATG
(position 804) to the pX region (position 6670). Sequence
alignments
are shown in Fig.
4. The size of MA in
these proviruses
varied from 126 bp (case 2) to 330 bp (case 4). TM
varied from
6 bp (case 4) to 363 bp (case 3). There was a high degree
of similarity
to the prototype HTLV-1 clone pATK, with identical
mutations in
all MA, TM, and pX fragments between sequenced proviruses.
For
example in MA of all four proviruses, G was substituted for A
(position 889), in TM of all three (containing the corresponding
sequence), T was substituted for C (position 6532) and A
was substituted
for C (position 6631), and in pX of all four
proviruses, G was
substituted for A (position 6659) and T was
substituted for C
(position 6664). It is important to point out that
these sequences
do not represent cross-contamination in PCRs and that
all five
base substitutions were exactly the same as in a sequenced
HTLV-1
provirus from a HAM/TSP patient (
4).
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FIG. 4.
Nucleotide alignments of the four deleted
HTLV-1 proviruses with MA-TM-pX structure and complete HTLV-1
(ATK). All proviruses were detected in TSP/HAM patients. The upper line
is the nucleotide sequence of ATK; nucleotide positions are given in
parentheses. Only differences in the defective proviruses are
indicated. Aligned identical bases are indicated (---). Termination of
codon of the env gene is underlined. Numbers 1 to 4 correspond to the proviruses detected in different individuals.
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Analysis of proviruses 1 to 3 indicated that they are capable of
coding for 8 to 12-kDa proteins. They were initiated at the
first ATG
(position 804) followed by an MA fragment at the N-terminus
and a
chimeric sequence at the C-terminal part of the molecule.
The chimeric
C-terminal part was derived as a consequence of single
nucleotide
deletions or insertions resulting in a change of the
reading frame as
follows: in the first provirus, insertion of
C (position 133); in the
second provirus, deletion of G (position
6503); and in the third
provirus, insertion of G (between 1133
and 1134). These mutations
occurred in the regions of MA-TM junctions,
and provirus 3 was detected
in PBMC of a patient who rapidly developed
HAM/TSP after blood
transfusion (
8). Provirus 4 contained deletion
of G
(position 807) in the second codon, so that the putative
synthesized
protein is totally out of frame, yet the coding capacity
of this
unusual ORF is nearly 4.2 kDa. Structures of the ORFs
of these
proviruses are shown in Fig.
5.
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FIG. 5.
Deduced protein sequences coded by the ORFs of defective
HTLV-1 proviruses. Virus-specific sequences are marked in bold; mutated
in-frame amino acids (aa) are given in small letters; out-of-frame
amino acids are underlined.
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Detection of chimeric transcripts in PBMC by RT-PCR.
To
confirm that expression of defective proviruses with large internal
deletions could take place in vivo, we isolated total RNA from fresh
PBMC of 6 ATLL and 14 asymptomatic carriers for RT-PCR or RT-PCR plus
Southern hybridization with the A1-B1 pair of primers and corresponding
probes (Fig. 6). The cDNAs ranging from
nearly 280 to 950 bp were revealed in 11 of 14 asymptomatic carriers
and in 5 of 6 ATLL patients. Short transcripts (nearly 280 bp) were
more frequently detected. No DNA contamination was found in the same
samples examined by direct PCR with the same pair of primers. Some of
these amplimers were sequenced using the A2-B2 pair of primers;
alignment of the two cDNAs and complete HTLV-1 provirus (Fig.
7) shows high homology between the two
cDNAs. Seven substitutions were detected in a 192-bp MA fragment of
both amplimers, resulting in five amino acid changes. Replacements found were Ala for Val (positions 976 and 977), Arg for Trp (position 978), Ser for Leu (position 1114), Asp for Ala (position 1129), and Pro
for Ala (positions 1134 and 1136). In both cDNAs, the first putative
(since the 5' end of the proviruses was not sequenced) stop codon (TAA)
was detected between MA and TM as a result of a G substitution for T
(position 1137). Partial sequence analysis of two other transcripts
also demonstrated a MA-TM-pX structure.
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FIG. 6.
Transcripts of deleted HTLV-1 proviruses detected in
PBMCs of ATLL and asymptomatic carriers by RT-PCR and Southern
hybridization. Lanes: M, size markers; , no RNA; +, RNA from MT-2
cells; 1 to 3, RNA from asymptomatic HTLV-1 carriers; 4 to 8, RNA from
ATLL patients.
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FIG. 7.
Sequence alignments of the RT-PCR-amplified transcripts
from PBMC of the asymptomatic carries (A and B) and ATK. Only the
sequence differences between ATK and amplimers are indicated.
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Expression of defective proviruses in vitro.
To confirm
the possible expression and immunoreactivity of the chimeric proteins,
we cloned defective proviruses 1 to 3 into the pTargeT expression
vector and analyzed protein expression in the TNT in vitro
transcription-translation system. After incubation of plasmids in TNT
reaction mixture, the expressed proteins were immunoprecipitated with
HAM/TSP patient serum or monoclonal antibodies to p19/p28,
electrophoresed, and analyzed by fluorography (Fig. 8). Protein expression was detected in
all three reaction tubes, and all three synthesized proteins were
recognized by patient sera and by monoclonal antibody against p19 and
p28. The sizes of the synthesized proteins varied from nearly 8 to 12 kDa and corresponded well to the coding capacities of the ORFs deduced from the nucleotide sequences of cloned proviruses. These results were
confirmed by Western blotting (data not shown). Reactivity with patient
serum as well as with anti-MA monoclonal antibody clearly indicated
that the N-terminal parts of these proteins are intact and immunogenic;
if expressed in vivo, these proteins might thus be myristylated and
associated with the plasma membrane.
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FIG. 8.
RIP analysis of the proteins coded by three HTLV-1
defective proviruses with long ORFs that were expressed in vitro.
Lanes: M, 14C-rainbow markers (Amersham); 1, positive
control of in vitro transcription-translation reaction; 2 to 4, RIP
with TSP/HAM patient serum; 5 to 7, RIP with monoclonal antibodies to
p17/p28. Sizes of the proteins are given in kilodaltons on the left.
|
|
| |
DISCUSSION |
HTLV-1 is associated with several independent pathologies in
humans, although over 90% of infected individuals remain
asymptomatic throughout life. The two main diseases are ATLL and
HAM/TSP. Analysis of virus variants associated with these
pathologies did not reveal principal differences, indicating that
there are no pathology-specific sequences or mutations. Thus, other
elements or factors might be linked to pathology progression.
The formation of defective proviruses is known to take place in cells
infected with replication-competent retroviruses. These deletions could
occur at different steps of the virus replication cycle: reverse
transcription, proviral integration, or DNA replication. In
evolutionary terms, one might consider exogenous deleted proviruses as
intermediate forms between complete exogenous and silent
endogenous viruses. Thus, analysis of these proviruses could shed
light not only on virus-associated pathogenesis but also on the
fundamental processes of long-term virus-cell coexistence.
This study shows that the majority of HTLV-1-infected individuals
studied have defective integrated proviral genomes in white blood cells
and that several of these genomes have potential polypeptide coding
capacity. The goal of our research was to detect and characterize defective proviruses with large internal deletions and a Gag-pX structure in ATLL, HAM/TSP, and asymptomatic HTLV-1 carriers. Complete
proviruses were found in 45 of the 51 DNA samples analyzed. Since
genomic Southern hybridization and/or long-range PCR do not detect
small genomic changes and point mutations, it is likely that many of
these full-length proviruses are not replication competent.
We detected numerous defective proviruses with
gag-env-pX0 structure in most DNA
samples from ATLL patients, HAM/TSP patients, asymptomatic HTLV-1
carriers, and PBMC-derived cell lines from these individuals.
Proviruses without a 5' LTR were frequent, indicating a likely
block to transcription of viral genes. The amplimers with a 5' LTR
ranged in size from nearly 200 to 1,300 bp. Evidently, amplimers
without the 5' LTR were shorter, ranging from 180 to nearly 800 bp.
Direct sequence analysis of 17 deleted proviruses amplified by nested
PCR indicated that most of these proviruses, besides the truncated MA
and pX0 sequence, contain fragments of CA, SU, and TM. While most of
the sequenced proviruses contained numerous stop codons, several
proviruses had relatively long ORFs, starting from the ATG of MA
(position 804) and capable of coding for proteins of up to 12 kDa.
While the N-terminal parts of these proteins belong to MA (from 41 to
109 amino acids), the C-terminal parts (because of deletions) were
usually out of frame and did not correspond to any known protein. One
provirus with the ATG of MA has an ORF that because of a mutation in
the second codon gives rise to a completely out of frame 4.2-kDa
protein. Thus, these ORFs have a chimeric structure and represent a
potential new class of HTLV-1-related proteins composed of truncated MA and variable out-of-frame polypeptides. We examined three such proviruses in an in vitro transcription-translation system and detected
proteins with N-terminal p19 (MA) epitopes.
When DNA samples from asymptomatic HTLV-1 carriers were examined by
PCR, 18 of 20 DNA samples were found to have defective proviruses with
large internal deletions and a MA-TM-pX structure. By RT-PCR
analysis, corresponding deleted transcripts were detected. Direct
nucleotide sequencing of two RT-PCR-amplified transcripts demonstrated
that both amplimers contained exactly the same mutations as detected in
the proviral DNA.
The detection of deleted transcripts in cells of HTLV-1-infected
individuals raises not only a possible role of chimeric proteins in
infected cells but also the question of whether they are incorporated into particles, and transmitted, as shown for the defective proviruses in MT-2 cells (1, 25). The RNAs detected in asymptomatic carriers are likely to contain a slip site (UUAAAAU) with a
potential pseudoknot structure between gag and
env. Since we did not estimate the frameshift efficiency of
this sequence, it is difficult to make any firm conclusions, although
the UUUAAAC sequence present in coronavirus MHV-A59 yielded
a frameshift efficiency of nearly 40% when tested in a rabbit
reticulocyte lysate (2). Several further points need to be
investigated. First, how frequently and how efficiently is deleted RNA
(if 
is intact) packaged inside the virion? Second, is there any
competition between deleted and complete RNA for packaging? Third, if
copackaging is possible, are heterozygous particles infectious? Are
chimeric MA-pX proteins expressed in vivo? If so, do they interface
with cellular processes, with HTLV-1 replication, or with host immune responses?
Taken together, our results indicate that HTLV-1 proviruses with large
internal deletions are present in more than 80% of HTLV-1-infected
individuals, with no special association of these proviruses with one
of the HTLV-1-associated pathologies. However, defective proviruses
with long ORFs were detected only in cells (or cell lines) from TSP/HAM
patients. It may be informative to test further HAM/TSP patients
and examine whether defective proviral load and the ratio of complete
and defective proviruses differ between categories of patients.
Expression of chimeric proteins from defective proviruses might
contribute to the autoimmune features of HAM/TSP.
| |
ACKNOWLEDGMENTS |
We thank C. Patience, D. Griffiths, and H. King for fruitful
discussions and help.
V.A.M. was supported by a UICC Yamagiwa-Yoshida Research Fellowship.
This work was funded in part by Medical Research Council and was
supported by the European Union HTLV European Research Network.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Wohl Virion
Centre, Windeyer Institute of Medical Sciences, University College
London, 46 Cleveland St., London W1P 6DB, United Kingdom. Phone: 44 (207) 679 9554. Fax: 44 (207) 679 9555. E-mail:
r.weiss{at}ucl.ac.uk.
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Journal of Virology, May 2000, p. 3933-3940, Vol. 74, No. 9
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Abstract
Introduction
