Affiliations of authors: F. Mortreux, I. Leclercq, Unité 524 Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Recherche sur le Cancer de Lille, and Unité d'Oncogenèse Virale, Centre Oscar Lambret, Lille, France; A.-S. Gabet, E. Wattel, Unité 524 INSERM, Institut de Recherche sur le Cancer de Lille, Unité d'Oncogenèse Virale, Centre Oscar Lambret, and Unité d'Oncogenèse Virale, UMR5537 Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard, Centre Léon Bérard, Lyon, France; A. Leroy, Unité d'Oncogenèse Virale, Centre Oscar Lambret; E. Westhof, Institut de Biologie Moleculaire et Cellulaire-CNRS, Strasbourg, France; A. Gessain (Unité d'Epidémiologie des Virus Oncogènes), S. Wain-Hobson (Unité de Retrovirologie Moléculaire), Institut Pasteur, Paris, France.
Correspondence to: Eric Wattel, M.D., Ph.D., Unité d'Oncogenèse Virale, UMR5537-CNRS-Université Claude Bernard, Centre Léon Bérard, 28, rue Laënnec 69373 Lyon cedex 08, France (e-mail: wattel{at}lyon.fnclcc.fr).
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In addition to its positive effect on cell cycling, the Tax protein negatively interferes with some DNA repair functions of the host cells. Indeed, Tax represses the expression of the human polymerase gene (20) and disrupts other prominent cellular DNA repair pathways (21,22). Via p53, Tax influences the transition from G1 to S phase and impairs the DNA-damage sentinel at this junction (23). Furthermore, Tax functionally inhibits the human mitotic checkpoint protein Mad1 (24). These functional characteristics of Tax may explain its mutagenic effect on cellular chromosomal DNA, as evidenced in vitro (25).
Considered together, these data indicate that HTLV-1 replicates mainly through persistent host cell proliferation (26) in the context of a Tax-induced genetic instability that should be detectable in vivo. In this study, we attempt to investigate the genetic variability of HTLV-1 that might be resulting from a previously undescribed mechanism and to ascertain whether the intrapatient genetic variability of the 3' RU5 region of the long terminal repeats (LTRs) is a consequence of somatic mutations in the proviral sequence rather than of reverse transcription. Mutations within flanking cellular sequences, including one integration site that corresponded to the -enolase gene (27), a housekeeping gene, were also observed in both symptomatic and asymptomatic infected individuals.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The DNA samples from PBMCs of six HTLV-1-seropositive individuals were studied: two patients with TSP/HAM (P1 and P2), two asymptomatic carriers (P3 and P4), and two patients with ATLL (P5 and P6). The age and sex of the subjects were as follows: P1, a 40-year-old female; P2, a 46-year-old female; P3, a 32-year-old female; P4, a 57-year-old female; P5, a 55-year-old male; and P6, a 40-year-old male. The DNA samples from PBMCs from five HTLV-1-seronegative individuals were used as the negative controls. According to French law, the six HTLV-1-infected individuals and the five seronegative subjects gave their informed consent before the collection of the blood samples.
Molecular Detection and Semiquantitative Analysis of HTLV-1 Integration
Inverse polymerase chain reaction (PCR) analysis of the HTLV-1 integration sites was done for the detection of infected cellular clones (13,14,26,28,29). This method consists of the amplification of the 3' extremities of the proviruses together with their flanking sequences. It allows the detection of circulating clones of HTLV-1-bearing T cells. In addition, as detailed in the "Results" section, sequencing cloned inverse PCR products makes it possible to arrange proviral sequences into cellular clones according to their cellular flanking sequences. Accordingly, for a given sample, aligning the proviral sequences flanked by distinct integration sites allows for the detection of reverse transcription-associated substitutions. By contrast, comparing proviral sequences flanked by identical integration sites helps to detect somatic mutations.
Because there is a stochastic component to the detection of HTLV-1 integration sites within the range of 1001000 copies per clone with the use of inverse PCR, samples from the six individuals were analyzed in quadruplicate (28). In this study, the last 399 base pairs (bp) of HTLV-1 3'-LTR, together with the cellular flanking sequences, were amplified.
Two micrograms of DNA was digested by 20 U NlaIII (New England Biolabs, Montigny-Le-Bretonneux, France) in 1x NlaIII buffer for 3 hours at 37 °C. The completion of the digestion was controlled by 1% agarose gel electrophoresis. DNA was extracted with phenolchloroform (1 : 1) and precipitated with 100% ethanol. One microgram of digested DNA was circularized for 14 hours at 16 °C with 20 U of T4 DNA ligase (New England Biolabs) in 600 µL of 1x T4 DNA ligase buffer and 1 mM adenosine triphosphate. DNA was extracted with phenolchloroform (1 : 1) and precipitated with 100% ethanol. Samples were analyzed through quadruplicate experiments: 4 x 500 ng of circularized DNA was amplified for 30 cycles with the use of 200 µM of primer pair BIO6 (5'-CTCCTGCTAGTTTATTGAGCCATA-3') at position 86218598 and LTR1 (5'-TCGCATCTCTCCTTCACGCG-3') at position 86578675 (nucleotide coordinates are numbered according to the HTLV-1 reference sequence on ATK-1) (30). Amplifications were performed with the use of the proofreading Pfu DNA polymerase (Stratagene Cloning Systems, La Jolla, CA), which has one of the lowest error rate (1.3 x 10-6 error per base per duplication) (31). Amplifications were performed according to the instructions of the enzyme manufacturers. Thermal cycling parameters were as follows: 96 °C for 10 minutes and 30 times at 96 °C for 60 seconds, 58 °C for 60 seconds, and 72 °C for 3 minutes, followed by a final elongation step of 10 minutes at 72 °C. Quadruplicate inverse PCR analysis of a DNA sample from patient P5 allowed the detection of a dominant clone on a background of less abundant forms, whereas five abundant clones were detected in the DNA sample from patient P6 (see the "Results" section). To detect the most abundant clone within this sample, we performed inverse PCR with 250, 100, 150, 10, 6.6, 1, and 0.1 ng of tumor DNA from patient P6 that was diluted in DNA from PBMCs of a noninfected individual.
Runoff Analysis of Amplified Products
The length polymorphism generated by PCR amplification of HTLV-1 flanking sequences was analyzed by making a runoff, as described previously (13,14,26,28). This method consists of the linear PCR amplification of both the 3' extremity of the provirus and its flanking sequence. Two microliters of amplified product was submitted to 10 cycles of linear PCR with 2 µM of 5'-32P-radiolabeled primer BIO5 (5'-TGGCTCGGAGCCAGCGACAGCCCAT-3') (position 89959020), 1 U of the Stoffel fragment of the Taq DNA polymerase (Perkin-Elmer Applied Biosystems, Courtaboeuf, France), and 200 µM of each deoxynucleoside triphosphate in a final volume of 20 µL. The thermal cycling parameters were as follows: 95 °C for 10 minutes and 10 times at 95 °C for 60 seconds, 58 °C for 60 seconds, and 72 °C for 3 minutes, followed by a final elongation step of 10 minutes at 72 °C. After boiling in deionized water, 2 µL of runoff products was analyzed on a 6% sequencing gel.
Southern Blot Analysis
Approximately 10 µg of high-molecular-weight DNA originating from PBMCs from patients P5 and P6 was digested with EcoRI or PstI and then subjected to electrophoresis through a 0.6% agarose gel. After Southern blotting to a nylon membrane, the filter was hybridized with the randomly primed 32P-labeled PMT-23 probe, which corresponds to the 1.7-kilobase (kb) fragment of the gag-pol region obtained after digestion of the MT2 cell line DNA by PstI.
Cloning and Sequencing HTLV-1 3' RU5 Sequences Together With Their Integration Site
Purified products from inverse PCR experiments were phosphorylated by the T4 polynucleotide kinase (Pharmacia, Uppsala, Sweden) and then ligated with the SmaI-digested (Pharmacia) and dephosphorylated M13mp18 replicative-form DNA (New England Biolabs), as described previously (12,32). After transformation of Escherichia coli XL1 by electroporation, recombinant M13 plaques were screened by hybridization with the HTLV-1 LTR-specific 32P-labeled oligonucleotide BIO5. Single-stranded templates were sequenced with the use of fluorescent dideoxynucleotides (Perkin-Elmer Applied Biosystems). The products were resolved on a 377A DNA sequencer (Perkin-Elmer Applied Biosystems) with 377A software (Perkin-Elmer Applied Biosystems). Sequence alignments were performed with Sequence Navigator Software (Perkin-Elmer Applied Biosystems).
Control PCR
To check the accuracy of the inverse PCR and the absence of PCR-associated recombination, we used as controls three cloned 3' HTLV-1 RU5 sequences flanked by their integration sites and harboring distinct mutations. Two hundred fifty copies of each of these three cloned sequences were mixed with 1 µg of uninfected DNA. Five hundred nanograms of the DNA mixture was amplified for 30 cycles with the use of 200 µM of the BIO6 and LTR1 primer pair under the same conditions as those used in the analysis of DNA samples from patients. Purified PCR products were cloned and sequenced as described below. Forty-one molecular clones were obtained, sequenced, and then analyzed by CLUSTAL alignment with Sequence Navigator Software.
PCR amplification of the-enolase gene fragment was performed by classical PCR. Five hundred nanograms of DNA from patient P1 was amplified by Pfu DNA polymerase with the primer pair HA-ENO-S (5'-GGGGTTAAGGAAGAAAAGCA-3') at position 10381058 and HA-ENO-AS (5'- TTGGAACTGGAATTTCACACA-3') at position 14041383 (nucleotide coordinates are numbered according to the
-enolase GenBank reference: HSENOAL1) (27). Amplification and cycling were performed as described for inverse PCR. After the PCR products were cloned and sequenced, seven sequences were analyzed by CLUSTAL alignment with Sequence Navigator Software.
Quantification of HTLV-1 Proviral Load
The HTLV-1 proviral load was assessed by real-time quantitative PCR with the use of a dual-labeled fluorescent probe (ABI PRISM 7700 Sequence Detection System; Perkin-Elmer Applied Biosystems). Standard curves for the albumin and HTLV-1 tax genes were generated with the use of DNA extracted from HTLV-1-negative PBMCs for the former and an HTLV-1 plasmid for the latter. It was estimated that 10 ng of high-molecular-weight DNA (equivalent to roughly 1500 PBMCs) would contain 3000 copies (two copies per PBMC) of the albumin gene. The primer set for the HTLV-1 tax gene was PXF (5'-GAAACCGTCAAGCACAGCTT-3') positioned at 71637182 and PXR (5'-TCTCCAAACACGTAGACTGGGT-3') positioned at 73857364. The primer set for albumin was 5'-GCTGTCATCTCTTGTGGGCTGT-3' positioned at 1628316304 and 5'-ACTCATGGGAGCTGCTGGTTC-3' positioned at 1644216421 (nucleotide coordinates are numbered according to the albumin GenBank reference: HUMALBGC) (33). The TaqMan probe consisted of an oligonucleotide with a 5'-reporter dye and a 3'-quencher dye. The fluorescent reporter dye 6-carboxy-fluorescein was covalently linked to the 5' end of the oligonucleotide. The reporter was quenched by 6-carboxy-tetramethyl-rhodamine at the 3' end. The probe for the HTLV-1 tax gene was PXT (5'-TTCCCAGGGTTTGGACAGAGTCTTCT-3') positioned at 73317355; the probe for albumin was ALB (5'-CCTGTCATGCCCACACAAATCTCTCC-3') positioned at 1634016366. TaqMan amplification was carried out in reaction volumes of 50 µL, with the use of the TaqMan PCR core Reagent Kit (Perkin-Elmer Applied Biosystems). Each reaction contained the following: 1x TaqMan buffer; 300 nmol/L of each primer; 200 nmol/L of each corresponding fluorescent probe; 3.5 mmol/L MgCl2; 200 µmol/L deoxyadenosine triphosphate, deoxycytidine triphosphate, and deoxyguanosine triphosphate; 400 µmol/L deoxyuridine triphosphate; 1.25 U of AmpliTaqTM Gold (Perkin-Elmer Applied Biosystems); and 0.5 U AmpEraseTM uracil N-glycosylase (Perkin-Elmer Applied Biosystems). Each sample was analyzed in triplicate with the use of 500 ng of DNA in each reaction. Thermal cycling was initiated with a 2-minute incubation at 50 °C, followed by a first denaturation step of 10 minutes at 95 °C and then by 45 cycles at 95 °C for 15 seconds and 58 °C for 1 minute (for tax) or 60 °C for 1 minute (for albumin).
Rex-Responsive Element and Two-Dimensional Structure Analysis
The secondary structure analysis of the Rex-responsive element (RXRE) variants was performed with ESSA-SAPSSARN software, as described previously (34,35). The probabilities of the pairing of each base with each other were computed with McCaskill's program (36).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
DNA samples were obtained from six HTLV-1-infected individualstwo patients with TSP/HAM (P1 and P2), two asymptomatic carriers (P3 and P4), and two patients with ATLL (P5 and P6). The frequency of HTLV-1 DNA-positive PBMCs was estimated by real-time quantitative PCR to be 1% for the asymptomatic carriers; 3% and 2% for patients P1 and P2, respectively (TSP/HAM); and 41% and 62% for patients P5 and P6, respectively (ATLL). This distribution reflects the general finding of proviral load in the order ATLL > TSP/HAM > asymptomatic carriers. Fig. 1 represents the pattern of HTLV-1-infected T-cell clones circulating in the peripheral blood of all six individuals as determined by inverse PCR. Because there is a stochastic element to inverse PCR amplification of low-frequency HTLV-1 integration sites (28), quadruplicate inverse PCR analysis was performed (4 x 0.5 µg, approximately 4 x 75 x 103 cell equivalents). A signal present in all four samples corresponded to a clonal frequency of one or more in 150 cells, while a single positive amplification would correspond to a frequency of one or fewer in 1500 (13,28).
|
HTLV-1 Genetic Variability: Somatic Mutations Versus Reverse Transcription-Associated Errors in the Provirus
The primary aim of this study was to test the hypothesis that somatic mutations of the provirus could account for the HTLV-1 genetic variability. To examine in detail the possible HTLV-1 mutation process in vivo, we cloned inverse PCR products from the six patient samples without size selection. A total of 208 clones were sequenced, encompassing the 3' RU5 sequences of HTLV-1 (379 bp, approximately 80 kb of data) together with their flanking cellular sequences (mean, 119 bp; range, 7362 bp; approximately 23 kb of data). The sequences could be arranged into 29 cellular clones based on cellular flanking sequences. The HTLV-1 regions of 29 cellular clones were aligned with respect to clone P1-1C, which was taken arbitrarily as the reference (Fig. 2).
|
Fig. 3 summarizes the different steps at which a mutation can occur during the synthesis of the HTLV-1 provirus. Accordingly, the distribution of the 46 RU5 mutations indicates that they did not correspond to minus-strand, synthesis-associated reverse transcription errors, which are expected to result in a homogeneous population of RU5 sequences (Fig. 3
, A). Similarly, the presence of at least one patient consensus sequence within each of the 29 clones demonstrated that none of the 46 RU5 substitutions corresponded to a plus-strand, synthesis-associated mutation corrected before the newly infected host cell divided. These substitutions, all of which were harbored by only a subset of sequences within clones, might have theoretically resulted from somatic mutations or from reverse transcription-associated substitutions during a single cycle of the plus-strand synthesis of the provirus, in the absence of DNA mismatch repair before the first division of the newly infected CD4+ T cell (Fig. 3
). However, two aspects of our results rule out the second possibility. First, the actual RU5 mutation frequency is incompatible with such reverse transcription-associated errors. Indeed, if the mutations shown in Fig. 2
corresponded to plus-strand, synthesis-associated errors, they all would necessarily be the result of a single cycle of reverse transcription. Accordingly, the average RU5 mutation frequency was an approximately 4.2 x 10-3 substitution per replication cycle per base (46 distinct substitutions in 29 RU5 sequences of 379 bp), which is about 600 times higher than that for the HTLV-1 reverse transcription (39) and 100 times higher than that for the human immunodeficiency virus reverse transcription (40). Second, some RU5 sequences harbored two to four substitutions, a mutation frequency not observed to date for a single step of reverse transcription-mediated DNA elongation. Therefore, the majority of substitutions appear to result from somatic mutations during cellular replication and not from plus-strand, synthesis-associated reverse transcription errors or from PCR artifacts or recombinations. Fig. 2
shows that, for about 60% of the HTLV-1-positive clones, 8%80% of the infected cells harbored a somatically mutated HTLV-1 provirus. The 46 somatic mutations are detailed in Table 1
. As can be seen, the ratio of the transition to the transversion was one, while substitutions from C and G were more frequent (which is in keeping with the high GC content of the RU5 region [approximately 60%]).
|
|
Somatic mutations were also found in the flanking sequences of two clones (Fig. 4). For the first example, P5-1 from an ATLL patient (Fig. 1
), the cellular DNA sequence did not correspond to anything in the current databases (sequence was analyzed as described by comparison to the nonredundant human sequence database, the human complementary DNA [dbEST] database, and the MONTH [June 2000] database by use of BLASTN with Search Launcher, FASTA, and Repeat Masker) (32). Three sequences encoded four single-base substitutions with respect to the most abundant sequence, which was assumed not to be somatically mutated (Fig. 4
, A). For clone P1-7, derived from a TSP/HAM sample, the proviral flanking sequences showed virtual identity with the promoter region of the human
- (or non-neuronal) enolase gene (27). Proviral integration occurred 146 bp 5' to the TATA box and, as a result, uncoupling the TATA box from a number of transcriptional motifs, such as the CCAAT box, AP1, and PEA2 sites. As Fig. 4
, B, shows, all five sequences harbored a T
C transition at position 1217 with respect to the published sequence, while one carried two additional purinepurine transitions. The T1217C transition could represent a single nucleotide polymorphism in the patient's P1
-enolase gene or a somatic mutation acquired during clonal expansion.
|
A total of seven substitutions (Table 1) were found in approximately 24 kb of cellular DNA, representing a frequency of 2.8 x 10-4/bp sequenced. This frequency is remarkably similar to that for the HTLV-1 RU5 region (5.8 x 10-4/bp sequenced [Table 1
]), particularly in light of the very different base composition of the two regions (HTLV-1, 40% AT [i.e., adenosine and thymidine]; cellular, 59% AT). These values are mutation frequencies, not mutation rates, because it is not possible to estimate the average number of mitoses in any lineage. If the same mutation frequency applied across the whole genome, one would predict approximately 1.7 x 106 mutations per diploid cell (approximately 2.8 x 10-4 x 6 x 109), or approximately one mutation per 3.5 kb (a remarkable mutation load).
Distribution of Somatic Mutations Within RU5
The mutations appear to be generally distributed randomly across the HTLV-1 RU5 region, with no evidence of hot spots (Fig. 5). About 70% of all CpG dinucleotides in the genome of vertebrates are methylated (41), and the HTLV-1 provirus has been found to be heavily methylated in cell lines (42). Five of 14 G
A and C
T transitions (36%, positions 8708, 8781, 8797, 8813, and 8842) occurred within CpG dinucleotides, suggesting that they might have arisen as a result of deamination of 5mCpG.
|
Of the 46 mutations, 22 mapped to 21 sites in the RXRE. Some mapped to the Rex-binding site or else disrupted RNA secondary structures (Fig. 6), which was evidenced by de novo calculations using the variant sequences (not shown). Because RXRE plays a crucial role in the expression of HTLV-1, it is likely that a number of these variants will have impaired expression of viral proteins.
|
It has been clearly established that genomic instability is characteristic of some cancers (51). Accordingly, tumor clones P5-1 and P6-4 from the two ATLL patients harbored a high mutation frequency (Fig. 2). However, Fig. 2
shows that somatic mutations were not restricted to ATLL samples. If somatic mutation were associated with DNA replication, then its extent should be generally related to the number of rounds of mitosis, which may be reflected in the clonal frequency in vivo. It is possible to get an approximate estimate of clonal frequencies from quadruplicate inverse PCR (compare Fig. 1
) (14,28). Fig. 7
, A, shows that the average number of mutations per kilobase in 57 RU5 sequences belonging to six HTLV-1 clones with a detection frequency of one or more per 150 PBMCs was more than twice that of the remaining 151 sequences derived from 23 clones with a detection frequency of fewer than one per 150 PBMCs. This difference was statistically significant (two-sided P = .037; Student's t test for unpaired samples). Indeed, since the frequency of abundant, circulating clones is in the order ATLL > TSP/HAM > asymptomatic carriers, the number of acquired somatic mutations was higher in HTLV-1-associated disease than in the virus carriers. As Fig. 7
, B, shows, PBMC DNA from ATLL patients, TSP/HAM patients, and asymptomatic carriers harbored a mutation frequency of 0.71, 0.52, and 0.22 substitution per kilobase of sequence (RU5 sequences plus integration sites), respectively. The difference between asymptomatic carriers and ATLL patients was statistically significant (two-sided P = .048; Student's t test for independent samples). However, the somatic mutation frequency of the 45 sequences derived from the six nonmalignant ATLL clones was identical to that of TSP/HAM sequences: 0.5 substitution per kilobase of sequence (RU5 sequences plus integration sites).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The average mutation frequency of HTLV-1 flanking sequences is remarkably high, approximately 1.7 x 106 mutations per diploid cell (approximately 2.8 x 10-4 x 6 x 109), or approximately one mutation per 3.5 kb. For some clones, the mutation frequency is even higher. Since mutations are accumulated in a stepwise manner (Fig. 4, B), it is not possible to estimate the rate of somatic mutation per mitosis. However, a few approximate calculations may be made for that number. Since the host genome mutation rate is something of the order of 10-10 to 10-9 per base per mitosis, attaining a mutation load of several million substitutions per genome would require an unrealistic number of rounds of replication for any one cloneapproaching 106. Note that this calculation is based on mutation rates. Because a fraction of the mutations are probably deleterious, the number of rounds of replication is underestimated. However, if some arms of mismatch repair were inactivated, then this number could be reduced considerably. Given the data showing that the HTLV-1 Tax protein interferes with the DNA mismatch repair systems in vitro (21,22), it may be assumed that the Tax protein may do the same thing in vivo.
Given a novel source of mutation, to what extent is this exploited, if at all, by the infected cell? It is probably safe to assume that, with such a mutation pressure, there is considerable negative selection, although the associated cell loss might well be masked by clonal expansion. Expression of HTLV-1 proteins marks out the cell as non-self and a target for cell-mediated immunity, which is particularly intense (52). Although deletions of only single nucleotides were noted, if they occurred in the viral open reading frames, they could considerably limit protein expression. Negative selection will ensure maintenance of the Tax+ phenotype. Because Tax is a target for cytotoxic T lymphocytes (CTLs), it is possible that some mutations in human leukocyte antigen (HLA) class I-restricted CTL epitopes are compatible with Tax function, which, therefore, allows the cellular clone to escape cell-mediated immunity (53). This mutation in HLA class I-restricted epitope that is compatible with Tax function may occur as long as most of the other HTLV-1 proteins were inactivated first by somatic mutations. This speculation follows from the observation that cases of CTL escape seem to occur only when the cellular immune responses are highly focused on a single target (54).
It has been demonstrated that Tax expression is observed frequently in CD4+-infected cells in vivo (55). By contrast, HTLV-1 infection in vivo is characterized by a very low viremia, which is usually undetectable. One can explain this paradoxical association of Tax expression with the absence of viremia by the fact that the genetic organization of HTLV- 1 (especially the robust inactivation of Tax expression by Rex) precludes an intense expression of Tax that is needed for viral production. By contrast, the level of Tax expressed in the cell appears to be sufficient to interfere with the numerous cellular pathways leading to clonal expansion. Is it possible that some of the mutations are incorporated into packaged virion RNA? Since RNA viruses manifest mutation rates of one to two per genome per cycle (56), which can only be slightly increased by chemical mutagenesis (57), it seems, by extrapolation, that an HTLV-1 provirus could absorb between one and two substitutions without suffering irreversible loss. However, the low fixation rate of amino acid substitutionsof the order of 0.1% per century (58)would suggest that the majority of somatically mutated proviruses are without virus progeny.
In view of the similarities among the HTLV/bovine leukemia virus (BLV) group of retroviruses, somatic mutations are expected to be identified for simian T-cell leukemia virus, HTLV-2, and BLV. It is of note that both a replication via mitosis and a defective DNA repair in infected cells have been evidenced for HTLV-2 and BLV (22). It will be interesting to see if high-frequency somatic mutations pertain to other viruses having a replication cycle that can include integration and host cell proliferation. In this context, it may be noted that the hepatitis B virus X protein has been found to disrupt cellular DNA repair (59).
In conclusion, the in vivo HTLV-1 genetic variability results predominantly from somatic mutations of the proviral sequence rather than from reverse transcription-associated substitutions. Furthermore, it appears that somatic mutation accompanies clonal expansion. The degree of somatic mutation is so great that it is consistent with the in vitro findings of a Tax-associated mutator phenotype. Not all somatic mutations were deleterious to the HTLV-1-bearing cellular clone, for there was evidence of sequential mutations. These findings suggest that somatic mutations after integration, presumably coupled with selection, help move the cellular clones toward a transformed phenotype, of which ATLL is the end point. A conundrum would appear to remain: Given the mutation pressure due to the high level of somatic mutations, why is the lifetime risk of ATLL as low as 3%5%? The expression of HTLV-1 proteins would mark the infected cell as non-self. Furthermore, the frequency of neoantigen formation could be higher than that typically found in other malignancies. Together, these features may allow robust control by host cell-mediated immunity. In support of this hypothesis, clinical observations and experimental investigations have shown that suppression of HTLV-1-specific cellular immune response led to the development of ATLL (6064).
![]() |
NOTES |
---|
Present address: I. Leclercq, Université Mons-Hainaut, Service de Biologie Moléculaire-Pentagone aile A 3ème étage, Mons, Belgium.
Supported by grants from the Association pour la Recherche sur le Cancer, from the Fondation Contre la Leucémie, and from the Comité Départemental du Rhône de la Ligue Nationale Contre le Cancer. I. Leclercq and F. Mortreux were supported by bursaries from the Ministère de l'Enseignement Supérieur et de la Recherche. A.-S. Gabet was supported by a grant from the Centre Léon Bérard.
We thank Claudine Pique, Ali Saib, and Renaud Mahieux for their critical review of this article. We also thank Pierre Wattre and collaborators in whose laboratories we conducted the DNA extraction, digestion, ligation, and polymerase chain reaction analysis. We are grateful to Marie-Dominique Reynaud for assistance with the preparation of this manuscript and to Dr. Michel Crépin (from the plateau technique de séquençage du Centre Hospitalier Régional Universitaire de Lille) for technical assistance.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1 Poiesz BJ, Ruscetti FW, Gazdar AF, Bunn PA, Minna JD, Gallo RC. Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc Natl Acad Sci U S A 1980;77:74159.[Abstract]
2 Yoshida M, Mioshi I, Hinuma Y. Isolation and characterization of retrovirus from cell lines of human adult T-cell leukemia and its implication in the disease. Proc Natl Acad Sci U S A 1982;79:20315.[Abstract]
3 Gessain A, Barin F, Vernant JC, Gout O, Maurs L, Calender A, et al. Antibodies to human T-lymphotropic virus type-I in patients with tropical spastic paraparesis. Lancet 1985;2:40710.[Medline]
4 Osame M, Usuku K, Izumo S, Ijichi N, Amitani H, Igata A, et al. HTLV-I associated myelopathy, a new clinical entity [letter]. Lancet 1986;1:10312.[Medline]
5 Eguchi K, Matsuoka N, Ida H, Nakashima M, Sakai M, Sakito S, et al. Primary Sjogren's syndrome with antibodies to HTLV-I: clinical and laboratory features. Ann Rheum Dis 1992;51:76976.[Abstract]
6 LaGrenade L, Hanchard B, Fletcher V, Cranston B, Blattner W. Infective dermatitis of Jamaican children: a marker for HTLV-I infection. Lancet 1990;336:13457.[Medline]
7 Mattos K, Queiroz C, Pecanha-Martins AC, Publio L, Vinhas V, Melo A. Lymphocyte alveolitis in HAM/TSP patients. Preliminary report. Arq Neuropsiquiatr 1993;51:1346.[Medline]
8 Mochizuki M, Tajima K, Watanabe T, Yamaguchi K. Human T lymphotropic virus type 1 uveitis. Br J Ophthalmol 1994;78:14954.[Abstract]
9 Sato K, Maruyama I, Maruyama Y, Kitajima I, Nakajima Y, Higaki M, et al. Arthritis in patients infected with human T lymphotropic virus type I. Clinical and immunopathologic features. Arthritis Rheum 1991;34:71421.[Medline]
10 Sherman MP, Amin RM, Rodgers-Johnson PE, Morgan OS, Char G, Mora CA, et al. Identification of human T cell leukemia/lymphoma virus type I antibodies, DNA, and protein in patients with polymyositis. Arthritis Rheum 1995;38:6908.[Medline]
11 Wattel E, Cavrois M, Gessain A, Wain-Hobson S. Clonal expansion of infected cellsa way of life for HTLV-I. J Acquir Immune Defic Syndr Hum Retrovirol 1996;13 Suppl 1:S929.[Medline]
12 Wattel E, Vartanian JP, Pannetier C, Wain-Hobson S. Clonal expansion of human T-cell leukemia virus type I-infected cells in asymptomatic and symptomatic carriers without malignancy. J Virol 1995;69:28638.[Abstract]
13 Cavrois M, Gessain A, Wain-Hobson S, Wattel E. Proliferation of HTLV-1 infected circulating cells in vivo in all asymptomatic carriers and patients with TSP/HAM. Oncogene 1996;12:241923.[Medline]
14
Cavrois M, Wain-Hobson S, Gessain A, Plumelle Y, Wattel E. Adult T-cell leukemia/lymphoma on a background of clonally expanding human T-cell leukemia virus type-1-positive cells. Blood 1996;88:464650.
15 Fujii M, Niki T, Mori T, Matsuda T, Matsui M, Nomura N, et al. HTLV-1 Tax induces expression of various immediate early serum responsive genes. Oncogene 1991;6:10239.[Medline]
16 Inoue J, Seiki M, Taniguchi T, Tsuru S, Yoshida M. Induction of interleukin 2 receptor gene expression by p40x encoded by human T-cell leukemia virus type 1. EMBO J 1986;5:28838.[Abstract]
17 Suzuki T, Kitao S, Matsushime H, Yoshida M. HTLV-1 Tax protein interacts with cyclin-dependent kinase inhibitor p16INK4A and counteracts its inhibitory activity towards CDK4. EMBO J 1996;15:160714.[Abstract]
18
Santiago F, Clark E, Chong S, Molina C, Mozafari F, Mahieux R, et al. Transcriptional up-regulation of the cyclin D2 gene and acquisition of new cyclin-dependent kinase partners in human T-cell leukemia virus type 1-infected cells. J Virol 1999;73:991727.
19
Neuveut C, Low KG, Maldarelli F, Schmitt I, Majone F, Grassmann R, et al. Human T-cell leukemia virus type 1 Tax and cell cycle progression: role of cyclin D-cdk and p110Rb. Mol Cell Biol 1998;18:362032.
20 Jeang KT, Widen SG, Semmes OJ 4th, Wilson SH. HTLV-I trans-activator protein, tax, is a trans-repressor of the human beta-polymerase gene. Science 1990;247:10824.[Medline]
21
Kao SY, Marriott SJ. Disruption of nucleotide excision repair by the human T-cell leukemia virus type 1 Tax protein. J Virol 1999;73:4299304.
22
Philpott SM, Buehring GC. Defective DNA repair in cells with human T-cell leukemia/bovine leukemia viruses: role of tax gene. J Natl Cancer Inst 1999;91:93342.
23
Pise-Masison CA, Choi KS, Radonovich M, Dittmer J, Kim SJ, Brady JN. Inhibition of p53 transactivation function by the human T-cell lymphotropic virus type 1 Tax protein. J Virol 1998;72:116570.
24 Jin DY, Spencer F, Jeang KT. Human T cell leukemia virus type 1 oncoprotein Tax targets the human mitotic checkpoint protein MAD1. Cell 1998;93:8191.[Medline]
25 Miyake H, Suzuki T, Hirai H, Yoshida M. Trans-activator Tax of human T-cell leukemia virus type 1 enhances mutation frequency of the cellular genome. Virology 1999;253:15561.[Medline]
26 Cavrois M, Leclercq I, Gout O, Gessain A, Wain-Hobson S, Wattel E. Persistent oligoclonal expansion of human T-cell leukemia virus type 1-infected circulating cells in patients with tropical spastic paraparesis/HTLV-1 associated myelopathy. Oncogene 1998;17:7782.[Medline]
27 Giallongo A, Oliva D, Cali L, Barba G, Barbieri G, Feo S. Structure of the human gene for alpha-enolase. Eur J Biochem 1990;190:56773.[Abstract]
28 Cavrois M, Wain-Hobson S, Wattel E. Stochastic events in the amplification of HTLV-I integration sites by linker-mediated PCR. Res Virol 1995;146:17984.[Medline]
29
Takemoto S, Matsuoka M, Yamaguchi K, Takatsuki K. A novel diagnostic method of adult T-cell leukemia: monoclonal integration of human T-cell lymphotropic virus type I provirus DNA detected by inverse polymerase chain reaction. Blood 1994;84:30805.
30 Seiki M, Hattori S, Hirayama Y, Yoshida M. Human adult T-cell leukemia virus: complete nucleotide sequence of the provirus genome integrated in leukemia cell DNA. Proc Natl Acad Sci U S A 1983;80:361822.[Abstract]
31
Cline J, Braman JC, Hogrefe HH. PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res 1996;24:354651.
32
Leclercq I, Mortreux F, Cavrois M, Leroy A, Gessain A, Wain-Hobson S, et al. Host sequences flanking the human T-cell leukemia virus type 1 provirus in vivo. J Virol 2000;74:230512.
33
Minghetti PP, Ruffner DE, Kuang WJ, Dennison OE, Hawkins JW, Beattie WG, et al. Molecular structure of the human albumin gene is revealed by nucleotide sequence within q1122 of chromosome 4. J Biol Chem 1986;261:674757.
34
Chetouani F, Monestie P, Thebault P, Gaspin C, Michot B. ESSA: an integrated and interactive computer tool for analysing RNA secondary structure. Nucleic Acids Res 1997;25:351422.
35 Gaspin C, Westhof E. An interactive framework for RNA secondary structure prediction with a dynamical treatment of constraints. J Mol Biol 1995;254:16374.[Medline]
36 McCaskill JS. The equilibrium partition function and base pair binding probabilities for RNA secondary structure. Biopolymers 1990;29:1105 19.[Medline]
37 Leclercq I, Cavrois M, Mortreux F, Hermine O, Gessain A, Morschhauser F, et al. Oligoclonal proliferation of human T-cell leukaemia virus type 1 bearing T cells in adult T-cell leukaemia/lymphoma without deletion of the 3' provirus integration sites. Br J Haematol 1998;101:5006.[Medline]
38 Meyerhans A, Vartanian JP, Wain-Hobson S. DNA recombination during PCR. Nucleic Acids Res 1990;18:168791.[Abstract]
39
Mansky LM. In vivo analysis of human T-cell leukemia virus type 1 reverse transcription accuracy. J Virol 2000;74:952531.
40 Mansky LM, Temin HM. Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase. J Virol 1995;69:508794.[Abstract]
41
Zingg JM, Jones PA. Genetic and epigenetic aspects of DNA methylation on genome expression, evolution, mutation and carcinogenesis. Carcinogenesis 1997;18:86982.
42 Saggioro D, Panozzo M, Chieco-Bianchi L. Human T-lymphotropic virus type I transcriptional regulation by methylation. Cancer Res 1990;50:496873.[Abstract]
43 Ahmed YF, Hanly SM, Malim MH, Cullen BR, Greene WC. Structure function analyses of the HTLV-I Rex and HIV-1 Rev RNA response elements: insights into the mechanism of Rex and Rev action. Genes Dev 1990;4:101422.[Abstract]
44
Askjaer P, Kjems J. Mapping of multiple RNA binding sites of human T-cell lymphotropic virus type I rex protein within 5'- and 3'-Rex response elements. J Biol Chem 1998;273:1146371.
45 Ballaun C, Farrington GK, Dobrovnik M, Rusche J, Hauber J, Bohnlein E. Functional analysis of human T-cell leukemia virus type I rex-response element: direct RNA binding of Rex protein correlates with in vivo activity. J Virol 1991;65:440813.[Medline]
46 Hanly SM, Rimsky LT, Malim MH, Kim JH, Hauber J, Duc Dodon M, et al. Comparative analysis of the HTLV-I Rex and HIV-1 Rev trans-regulatory proteins and their RNA response elements. Genes Dev 1989;3:153444.[Abstract]
47 Toyoshima H, Itoh M, Inoue J, Seiki M, Takaku F, Yoshida M. Secondary structure of the human T-cell leukemia virus type 1 rex-responsive element is essential for rex regulation of RNA processing and transport of unspliced RNAs. J Virol 1990;64:282532.[Medline]
48 Inoue J, Yoshida M, Seiki M. Transcriptional (p40x) and post-transcriptional (p27x-III) regulators are required for the expression and replication of human T-cell leukemia virus type I genes. Proc Natl Acad Sci U S A 1987;84:36537.[Abstract]
49 Baskerville S, Zapp M, Ellington AD. High-resolution mapping of the human T-cell leukemia virus type 1 Rex-binding element by in vitro selection. J Virol 1995;69:755969.[Abstract]
50 Bogerd HP, Tiley LS, Cullen BR. Specific binding of the human T-cell leukemia virus type I Rex protein to a short RNA sequence located within the Rex-response element. J Virol 1992;66:75725.[Abstract]
51 Loeb LA. Cancer cells exhibit a mutator phenotype. Adv Cancer Res 1998;72:2556.[Medline]
52 Jacobson S, Shida H, McFarlin DE, Fauci AS, Koenig S. Circulating CD8+ cytotoxic T lymphocytes specific for HTLV-I pX in patients with HTLV-I associated neurological disease. Nature 1990;348:2458.[Medline]
53 Nowak MA, Bangham CR. Population dynamics of immune responses to persistent viruses. Science 1996;272:749.[Abstract]
54 Borrow P, Lewicki H, Wei X, Horwitz MS, Peffer N, Meyers H, et al. Antiviral pressure exerted by HIV-1-specific cytotoxic T lymphocytes (CTLs) during primary infection demonstrated by rapid selection of CTL escape virus. Nat Med 1997;3:20511.[Medline]
55
Hanon E, Hall S, Taylor GP, Saito M, Davis R, Tanaka Y, et al. Abundant tax protein expression in CD4+ T cells infected with human T-cell lymphotropic virus type I (HTLV-I) is prevented by cytotoxic T lymphocytes. Blood 2000;95:138692.
56
Drake JW, Holland JJ. Mutation rates among RNA viruses. Proc Natl Acad Sci U S A 1999;96:139103.
57 Lee CH, Gilbertson DL, Novella IS, Huerta R, Domingo E, Holland JJ. Negative effects of chemical mutagenesis on the adaptive behavior of vesicular stomatitis virus. J Virol 1997;71:363640.[Abstract]
58 Gessain A, Gallo RC, Franchini G. Low degree of human T-cell leukemia/lymphoma virus type I genetic drift in vivo as a means of monitoring viral transmission and movement of ancient human populations. J Virol 1992;66:228895.[Abstract]
59 Jia L, Wang XW, Harris CC. Hepatitis B virus X protein inhibits nucleotide excision repair. Int J Cancer 1999;80:8759.[Medline]
60 D'Incan M, Combemale P, Verrier B, Garin D, Audoly G, Brunot J, et al. Transient adult T-cell leukemia/lymphoma picture during varicella infection in an HTLV-1 carrier. Leukemia 1994;8:6827.[Medline]
61
Hanabuchi S, Ohashi T, Koya Y, Kato H, Takemura F, Hirokawa K, et al. Development of human T-cell leukemia virus type 1-transformed tumors in rats following suppression of T-cell immunity by CD80 and CD86 blockade. J Virol 2000;74:42835.
62 Jenks PJ, Barrett WY, Raftery MJ, Kelsey SM, van der Walt JD, Kon SP, et al. Development of human T-cell lymphotropic virus type I-associated adult T-cell leukemia/lymphoma during immunosuppressive treatment following renal transplantation. Clin Infect Dis 1995;21:9923.[Medline]
63 Tsurumi H, Tani K, Tsuruta T, Shirato R, Matsudaira T, Tojo A, et al. Adult T-cell leukemia developing during immunosuppressive treatment in a renal transplant recipient. Am J Hematol 1992;41:2924.[Medline]
64 Zanke BW, Rush DN, Jeffery JR, Israels LG. HTLV-1 T cell lymphoma in a cyclosporine-treated renal transplant patient. Transplantation 1989;48:6957.[Medline]
Manuscript received July 26, 2000; revised December 29, 2000; accepted January 10, 2001.
This article has been cited by other articles in HighWire Press-hosted journals:
![]() |
||||
|
Oxford University Press Privacy Policy and Legal Statement |