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Journal of Virology, October 2000, p. 9525-9531, Vol. 74, No. 20
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
In Vivo Analysis of Human T-Cell Leukemia Virus
Type 1 Reverse Transcription Accuracy
Louis M.
Mansky*
Department of Molecular Virology, Immunology,
and Medical Genetics, Center for Retrovirus Research, The Arthur
James Cancer Hospital and Solove Research Institute, and Comprehensive
Cancer Center, Ohio State University Medical Center, Columbus, Ohio
43210
Received 11 May 2000/Accepted 17 July 2000
 |
ABSTRACT |
Several studies have indicated that the genetic diversity of human
T-cell leukemia virus type 1 (HTLV-1), a virus associated with adult
T-cell leukemia, is significantly lower than that of other
retroviruses, including that of human immunodeficiency virus type 1 (HIV-1). To test whether HTLV-1 variation is lower than other
retroviruses, a tractable vector system has been developed to measure
reverse transcription accuracy in one round of HTLV-1 replication. This
system consists of a HTLV-1 vector that contains a cassette with the
neomycin phosphotransferase (neo) gene, a bacterial origin
of DNA replication, and the lacZ
peptide gene region
(the mutational target). The vector was replicated by
trans-complementation with helper plasmids. The in vivo
mutation rate for HTLV-1 was determined to be 7 × 10
6 mutations per target base pair per replication cycle.
The majority of the mutations identified were base substitution
mutations, namely, G-to-A and C-to-T transitions, frameshift mutations,
and deletion mutations. Mutation of the methionine residue in the conserved YMDD motif of the HTLV-1 reverse transcriptase to either alanine or valine (i.e., M188A or M188V) led to a factor of two increase in the rate of mutation, indicating the role of this motif in
enzyme accuracy. The HTLV-1 in vivo mutation rate is comparable to that
of bovine leukemia virus (BLV), another member of the HTLV/BLV genus of
retroviruses, and is about fourfold lower than that of HIV-1. These
observations indicate that while the mutation rate of HTLV-1 is
significantly lower than HIV-1, this lower rate alone would not explain
the low diversity in HTLV-1 isolates, supporting the hypothesis that
HTLV-1 replicates primarily as a provirus during cellular DNA
replication rather than as a virus via reverse transcription.
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INTRODUCTION |
Human T-cell leukemia virus type 1 (HTLV-1) is a member of the human T-cell leukemia virus/bovine leukemia
virus (HTLV/BLV) genus of the Retroviridae family. HTLV-1
has been shown by epidemiology to be associated with adult T-cell
leukemia, HTLV-1-associated myelopathy-tropical spastic paraparesis,
and polymyositis (5). Infection of HTLV-1 is endemic in
Melanesia, Japan, the Caribbean, and sub-Saharan Africa. There is a
remarkable amount of homogeneity among HTLV-1 isolates (12, 13,
15, 25, 35, 41, 43-45, 52). For example, isolates from Japan
have close to 99% homology, and isolates from Japan, the Caribbean,
and Africa can also share as much as 99% homology. It has been
suggested that HTLV-1 isolates endemic in different races may be of
utility in studying the movement of ancient human populations or in
anthropologic studies (12). HTLV-1 isolates from Melanesia
would not be as useful, since there is not as much sequence homology to
the original Japanese isolate (11). This suggests that
HTLV-1 may have originated in the Pacific Rim rather than in Africa.
Genetic diversity among isolates of HTLV-2 is equally low
(48).
The low level of genetic diversity in HTLV-1 has been speculated to be
due to oligoclonal expansion of infected cells and very low levels of
virus replication in infected individuals (6, 7, 54). The
low levels of virus replication observed in cell culture has been used
to support this hypothesis. Replication of the viral genome primarily
as a provirus during cellular DNA replication would provide a
higher-fidelity mode of virus replication than viral nucleic acid
replication via reverse transcription. An advantage for the virus in
doing this is that HTLV may be able to escape immune selection. It has
been shown that the HTLV envelope protein becomes nonfunctional with a
limited number of mutations, which is in contrast to the human
immunodeficiency virus type 1 (HIV-1) envelope (38).
To help dissect the basis of genetic variation of retroviruses, the
mutation rate per replication cycle has been studied extensively. This
work was initiated by designing systems to determine the mutation rate
per base pair per replication cycle for spleen necrosis virus (SNV), an
avian C-type retrovirus similar to the murine type C retroviruses,
using an amber codon reversion assay with an SNV vector (9,
10). A similar reversion assay was used to determine the mutation
rate of murine leukemia virus (MLV) (53). The in vivo
forward mutation rates for various types of mutations were calculated
with the lacZ peptide gene as a reporter gene for
mutations and the blue-white colony color selection method for
identifying mutant proviruses in Escherichia coli (36,
37). The major types of mutations found were base-pair
substitutions, frameshifts, simple deletions, and deletions with
insertions. The overall in vivo mutation rate of SNV in this system was
determined to be 105 mutations/target base
pair/replication cycle.
These studies have been extended to BLV (32). BLV was found
to have a mutation rate of 4 × 106, which is 2.5 times less than that for SNV. A similar distribution of mutation types
was found with BLV relative to that of SNV, indicating that a common
property of reverse transcriptase (RT) is responsible for all of these
error processes. Temin speculated that this common property was the
strand-transfer process (50). The mutation rate of HIV-1 was
determined with a vector containing the lacZ peptide gene
(26, 31). The mutation rate of HIV-1 in this system was
determined to be 3 × 105 mutations per target base
pair per cycle. The Vpr protein of HIV-1 influences the mutation rate
and involves the interaction of Vpr with the cellular DNA repair enzyme
uracil DNA glycosylase (27, 30).
Phylogenetic analysis of RTs has indicated several conserved domains
and structures that are important for RT function (34). For
example, the fingers, palm, and thumb subdomains of the HIV-1 RT
catalytic domain are thought to be well conserved among other RTs
because of the importance of this region of the enzyme for contacting
the primer-template, and binding the incoming deoxynucleoside triphosphate (20). The conserved YXDD motif is important
because it has been associated with resistance to nucleoside analogs, decreases in enzymatic activity and viral infectivity, and changes in
the positioning of the primer in the template-primer complex (39).
The objectives of this study were to determine the accuracy of HTLV-1
reverse transcription and to see if amino acid substitutions in the
conserved YMDD motif of HTLV-1 RT would influence the rate of HTLV-1
mutation. To do this, a tractable genetic system was developed to
measure the forward rate of mutation using the lacZ peptide gene as a reporter for mutations. The mutation rate of HTLV-1
was determined to be 7 × 106 mutations/target base
pair/replication cycle. This rate is comparable to that of BLV, but is
about fourfold lower than the mutation rate of HIV-1. Mutation of the
YMDD motif doubled the mutation rate of HTLV-1.
| |
MATERIALS AND METHODS |
HTLV-1 vector construction.
The HTLV-1 shuttle vector pH1sh
(Fig. 1) was constructed from
pHTLV-CMVNEO (kindly provided by David Derse, National Cancer Institute) (8) in a two-step process. First, a deletion of the pol region was done by deletion of a
HindIII fragment. Second, a cassette containing the
simian virus 40 (SV40) promoter driving expression of the neomycin
phosphotransferase (neo) gene, a bacterial origin of DNA
replication, and the lacZ peptide gene region from a
previously described HIV-1 vector (31) was inserted in place of the CMVNEO cassette to create pH1sh. The plasmid pCMV-HT1 (kindly provided by David Derse) was used as a helper plasmid to
trans-complement the HTLV-1 shuttle vector with
gag, pol, tax, and rex
(8). The plasmid pSV-A-MLV-env was also used as a helper
plasmid and has been previously described (24). The HTLV-1
RT variants M188A and M188V were made in pCMV-HT1 by a
primary-combinatorial two-step PCR protocol (17).
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FIG. 1.
HTLV-1 vector used for reverse transcription accuracy
studies. (A) HTLV-1 vector. The vector is shown in the proviral DNA
form and has a cassette containing the SV40 promoter driving expression
of the neomycin phosphotransferase gene (neo), a bacterial
plasmid origin of DNA replication (pACYC ori), and the
lacZ peptide gene region (lacZ) that
includes the lac operator sequence. (B) Protocol for one
cycle of HTLV-1 vector virus replication. The steps going from a
parental shuttle vector provirus in the step 2 cell to a vector
provirus in the step 3 cell constitute a single cycle of replication.
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Transfections, infections, and cocultivations.
The CEM-A
cell line used was obtained from the NIH AIDS Reagent Program and was
maintained in RPMI 1640 supplemented with 2 mM L-glutamine
containing 10% calf serum. The HTLV-1 vector and expression plasmids
were transfected into CEM-A cells by use of either dimethyl
sulfoxide-Polybrene (18) or Superfect (Qiagen). CEM-A cells
were infected in the presence of Polybrene (28). Cocultivation of CEM-A target cells with virus-producing cells was also
done as previously described (29, 32). Briefly,
virus-producing cells (typically, 2.5 × 105 cells per
60-mm petri dish or 5 × 105 cells per 100-mm petri
dish or 7.5 × 105 cells per 150-mm petri dish were treated
with mitomycin C (8 µg/ml), an inhibitor of host cell DNA synthesis,
for 1.5 h at 37°C. The cells were then washed three times with
fresh medium, and CEM-A target cells equivalent to the number of
treated virus-producing cells were added. Two days after cocultivation,
selective medium containing G418 was added. Control experiments were
done with each cocultivation experiment to ensure that mitomycin
C-treated, virus-producing cells did not proliferate and no longer
adhered to the surfaces of culture dishes. Cells expressing the
neo gene were selected with the neomycin phosphotransferase
analog, G418, until the formation of colonies (typically about 3 weeks).
Analysis of HTLV-1 reverse transcription accuracy in a single
replication cycle.
The experimental protocol developed to assay a
single cycle of HTLV-1 vector replication is shown in Fig. 1. The
protocol contains three steps. In step 1, the HTLV-1 vector was
introduced into CEM-A cells by transfection and placed under G418
selection. Cell clones were then transiently transfected with the
helper plasmids. In step 2, vector virus was harvested 48 h
posttransfection from step 1 cells and used to infect fresh CEM-A
cells. G418-resistant cell clones were transiently transfected with the
helper plasmids (step 2 cells). Step 2 clones were tested by Southern
analysis to ensure that only a single vector proviral DNA was present. The lacZ peptide gene in the vector proviral DNA of step
2 clones was sequenced to confirm that no mutations were introduced. In step 3, vector virus was transferred to fresh CEM-A target cells by
cocultivation for 24 to 48 h after transient transfection of helper plasmids; cells were then placed under G418 selection (step 3 cells). Cocultivation was used to produce step 3 cells to maximize the
number of step 3 cells for analysis of the mutant frequency.
Recovery of shuttle vector proviral DNA and DNA sequencing of the
lacZ peptide region.
Purified genomic DNA
(42) from pools of step 3 clones was digested with the
restriction enzyme SalI to release the neo, pACYC
origin of replication, and lacZ peptide gene sequences from the HTLV-1 shuttle vector proviral DNA (Fig. 1). Proviral DNA was
purified with the Lac repressor protein as previously described
(32). The Lac repressor protein was purified from E. coli HB101/lac pIQ (kindly supplied from Tom Record,
University of Wisconsin-Madison) as previously described
(23). The purified proviral DNA was ligated and used to
electroporate competent E. coli XL1 Blue cells (Stratagene).
Kanamycin-resistant bacterial colonies were selected in the presence of
the isopropyl-
-D-thiogalactoside (IPTG) inducer. The
ratio of white plus light-blue bacterial colonies to total bacterial
colonies observed provided a forward mutant frequency for a single
retroviral replication cycle. Plasmid DNA was purified (42)
and sequenced in the lacZ peptide gene region in order to
determine the mutation rate.
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RESULTS |
A HTLV-1 single cycle replication assay for mutation
detection.
In order to measure HTLV-1 reverse transcription
accuracy, a replication assay was developed to measure the in vivo
mutation rate in a single cycle of replication. An HTLV-1 shuttle
vector was constructed in order to identify mutations that had occurred during HTLV-1 replication. The vector contained a cassette which included the neo gene under control of the SV40 promoter, a
bacterial origin of plasmid DNA replication, and the lacZ
peptide gene region as a mutational target. This cassette has been
previously used for analysis of reverse transcription accuracy of HIV-1
and BLV (31, 32). This vector, pH1sh, can replicate in both
mammalian cells as a virus and in bacterial cells as a plasmid.
The assay developed is shown in Fig.
1. CEM-A cells were used in these
studies because they are adherent T-lymphoid cells
that are permissive
for HTLV-1 replication (
51). The utility
of adherent cells
is that they could be placed under drug selection
when vector virus was
introduced either by transfection or infection
and form drug-resistant
colonies. The HTLV-1 shuttle vector, pH1sh,
was first introduced into
fresh CEM-A cells by transfection and
placed under G418 selection (step
1 cells). G418-resistant colonies
were pooled and then transfected with
helper plasmids. The supernatant
from these cells was used to infect
fresh CEM-A cells. Titers
of the vector virus were very low (~10
CFU/ml) but generated several
infected cell clones for further studies.
Prior to use in the
single cycle replication assay, a few selected
clones were analyzed
for the presence of a single integrated provirus.
Data indicated
that the selected clones contained single integrated
copies of
the HTLV-1 vector (data not shown). Transfection of helper
plasmids
into these cells (step 2 cells) was done to allow for virus
production
of the vector. These virus-producing cells were treated with
mitomycin
C and cocultured with fresh CEM-A cells. Cocultivation was
used
to produce step 3 cells to maximize the number of step 3 cells
for
analysis of the mutant frequency. Following cocultivation,
cells were
placed under G418 selection. Approximately 1,000 to
1,800 G418-resistant colonies were obtained per 7.5 × 10
5
target cells cocultivated with step 2 cells. The G418 resistant
cells
(step 3 cells) were then pooled from over 40,000 colonies,
and the
total DNA was purified. The purified DNA was digested
with
SalI, and the cassette containing the
lacZ
peptide gene
region mutational target containing the Lac operator
sequence
was purified using the Lac repressor
protein.
Mutant frequency, type, and location in HTLV-1 replicated with
wild-type RT.
The mutant frequency from several parallel
experiments indicated that the average mutant frequency was 0.0009 (33/36,561) mutation per target base pair per replication cycle (Table
1). Nucleotide sequence analysis of the
lacZ peptide gene region was done to determine the types
of mutations responsible for the mutant colony color phenotype (Table
2). Of the 33 mutants recovered, 20 (61%) had base substitution mutations. Of the 20, 14 (70%) were
G-to-A and C-to-T transition mutations. One C-to-T hypermutant was
identified, which contained two C-to-T transition mutations within the
mutational target. Of the 33 mutants, 7 (21%) had frameshift mutations. Six of the seven frameshift mutations were +1 frameshifts in
runs of A's, C's, or T's. Finally, 6 of the 33 mutants (18%) had
deletion mutations. The deletion mutations were either simple deletions
(four of six) that contained base pair homology at the deletion
junctions or were more complex deletions with insertion mutations
(inserted sequences of unknown origin).
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TABLE 1.
Analysis of HTLV-1 reverse transcription accuracy by
recovery of mutant proviruses following replication with either
wild-type HTLV-1 RT or YXDD variants
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TABLE 2.
Spectrum of mutations in the lacZ peptide
gene region of recovered HTLV-1 vector proviruses after replication
with either wild-type HTLV-1 RT or by YXDD variants
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Analysis of the location of mutations indicated that the base
substitutions and frameshift mutations occurred at locations
in the
lacZ peptide gene region where substitutions and
frameshift
mutations had been previously characterized for both BLV and
HIV-1
(Fig.
2). This suggests that there
are mutational hotspots within
the
lacZ peptide gene
region that are recognized as such by many
RTs. The deletion mutations
were located throughout the
lacZ peptide gene region but
in most instances had deletion junctions
near locations that may be
mutational hotspots. The deletion junctions
for the simple deletions
had either 3- or 4-bp homology (Fig.
3).
Two deletions with insertion mutants were identified that
did not have
base pair homology at the deletion junctions.
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FIG. 2.
Plus strand nucleotide sequence of the
lacZ gene region in the HTLV-1 vector provirus. The start
and stop codons of the lacZ open reading frame (small
boxed sequences) and the lac operator sequence (large boxed
sequence) are shown. Nucleotide positions of base pair substitutions
(letters above the sequence), +1 frameshifts (letters with  above
the sequence),  1 frameshifts ( above the sequence) are indicated.
G-to-A and C-to-T hypermutants are indicated with a number sign (#),
asterisk, or apostrophe adjacent to the letter above or below the
sequence. The locations of base substitution and frameshift mutations
in the parental HTLV-1 vector provirus replicated with wild-type HTLV-1
RT are indicated above the sequence, while the mutations in the HTLV-1
provirus replicated with the M188A and M188A RT variants are indicated
below the nucleotide sequence.
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FIG. 3.
Nucleotide sequence analysis of deletions and deletions
with insertions. (A) Deletions and deletions with insertions in HTLV-1
proviruses replicated with wild-type RT. Short direct repeats at the
deletion junctions are shown in boxes. The numbers of nucleotides
deleted are indicated between the deletion junctions and are preceded
by a minus sign; the number of inserted nucleotides are preceded by a
plus sign. (B) Deletions and deletions with insertions in HTLV-1
proviruses replicated with M188A and M188V HTLV-1 RTs. Short direct
repeats and numbers of nucleotides deleted and inserted are as
described in panel A.
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Mutant frequency and characterization of HTLV-1 replicated with RT
variants in one round of replication.
To determine whether amino
acid substitutions in the HTLV-1 RT could influence the accuracy of the
reverse transcription process, the methionine residue at position 188 was changed to either alanine (M188A) or valine (M188V). M188 lies in
the highly conserved YXDD motif. The HTLV-1 vector was then replicated
in parallel with RT containing either the M188A mutation or the M188V
mutation or with wild-type RT. Following recovery of proviruses from
infected CEM-A target cells, the mutant frequency observed after
replication with the M188A RT was found to be 0.0023 (18/7,916)
mutant/cycle (Table 1). This is significantly higher
(
2 = 11; P < 0.005) than the
mutant frequency found by replication with wild-type HTLV-1 RT
(33/36,561) (Table 1). The mutant frequency observed following
replication with M188V RT was 0.0021 (20/9,741) mutant/cycle (Table 1).
The mutant frequency for M188V is also significantly higher
(2 = 9; P < 0.005) than the mutant
frequency obtained after replication with wild-type HTLV-1.
To determine whether the types of mutations that occurred when the
HTLV-1 vector was replicated with either M188A or M188V
were different
than when the vector was replicated with wild-type
RT, the 38 mutants
isolated were sequenced. Table
2 summarizes
the types of mutants
identified. A total of 39 and 35% of the
mutations identified for both
M188A and M188V were substitution
mutations (7 of 18 and 7 of 20),
respectively. For both M188A
and M188V, G-to-A and C-to-T transition
mutations were the most
common substitutions, occurring either 71% (5 of 7) or 86% (6
of 7) of the time, respectively. Frameshift mutations
were observed
for both M188A and M188V and represented 44% (8 of 18)
or 45%
(9 of 20) of the total mutants, respectively. This trend
indicates
a doubling of frameshift mutations compared to that with
wild-type
HTLV-1 RT (i.e., 7 of 33, 21%) (Table
2). M188A and M188V
led
to deletion mutations either 11% (2 of 18) or 15% (3 of 20) of
the total, respectively. The deletion mutations included both
simple
deletions and deletions with insertions. A G-to-A hypermutant
was
recovered with M188A that had two G-to-A mutations, and a
C-to-T
hypermutant was recovered with M188V that had two C-to-T
mutations in
the
lacZ peptide gene
region.
The locations of the base substitution and frameshift mutations were
compared to that observed with wild-type RT (Fig.
2).
In general, the
locations of mutations identified when HTLV-1
was replicated with
either M188A or M188V were similar to that
seen with wild-type
RT.
The deletion mutations were located throughout the
lacZ
peptide gene region but in most instances had deletion junctions
near
locations that may be mutational hotspots. The deletion junctions
for
the simple deletions had either 3- or 1-bp homology (Fig.
3).
Interestingly, one simple deletion had no base-pair homology
at the
deletion junction. One deletion with an insertion mutant
was also
identified that did not have base pair homology at the
deletion
junctions. The observation that the simple deletions
had fewer
homologous base pairs at the deletion junctions suggests
that M188A and
M188V may be less processive than wild-type HTLV-1
RT.
Relative rate of mutation for HTLV-1 to that of BLV, HIV-1, and SNV
using the lacZ peptide gene region as a mutational
target.
The characterized mutants from replicating HTLV-1 with
wild-type RT allowed for calculation of the in vivo mutation rate for HTLV-1. The mutation rate was calculated to be 7 × 106 mutation/target base pair/replication cycle (Table
3). Target nucleotides in the mutational
target have been previously described (1, 3, 36). The mutant
frequency for HTLV-1 is not significantly different
(2 = 1.2; P > 0.1) than that of
BLV, but is fourfold lower than that of HIV-1 (2 = 77; P < 0.001) and is twofold lower than the mutant
frequency of SNV (2 = 15; P < 0.005). This indicates that the HTLV-1 and BLV mutation rates are
comparable, whereas the HTLV-1 mutation rate is significantly different
from the HIV-1 and SNV mutation rates (Table 3).
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TABLE 3.
Relative rates of mutation for HTLV-1, BLV, HIV-1, and
SNV using the lacZ peptide gene region as a
mutational target
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DISCUSSION |
Determination of the HTLV-1 in vivo mutation rate.
The
accuracy of HTLV-1 reverse transcription has been determined. Using an
HTLV-1 vector containing the lacZ peptide gene region as
a mutational target, the mutant frequency was determined to be 0.0009 mutant/replication cycle. Sequence analysis of the recovered mutants
indicated substitution, frameshift, and deletion mutations had
occurred. The predominant type of mutations to occur were G-to-A and
C-to-T transition mutations. Deletion mutations were found to represent
about one-quarter of the mutants recovered. The calculated in vivo
mutation rate for HTLV-1, 7 × 106 mutation per
target base pair per replication cycle, is comparable to that
previously reported for BLV but is significantly different than that of
HIV-1 and SNV.
Possible mechanisms responsible for the creation of mutations.
The HTLV-1 vector used in these studies does not allow the
determination of whether mutations occurred during minus-strand or
plus-strand DNA synthesis, but the locations of the mutations suggest
particular mechanisms for their creation. The majority of the G-to-A
transitions characterized were in GpA dinucleotides. Transition
mutations adjacent to runs of a single nucleotide appear to occur by
the mechanism of dislocation mutagenesis (1, 22). In this
model, dislocation of the primer to the template produces an unpaired
nucleotide base; realignment occurs between the primer and the template
resulting in a mismatch, followed by elongation beyond the mismatch.
Most of the G-to-A transition mutations occurred at sites adjacent to a
run of nucleotides, which suggests that these mutations could have
occurred by dislocation mutagenesis.
The frameshift mutations characterized were mainly +1 frameshifts in
runs of A's and T's. Plus-one frameshift mutations in
runs of T's
and A's occurred with SNV in vivo (
4,
37). The
frameshift
mutations in homo-oligomeric runs suggest that these
result from
template-primer slippage (
2,
21,
46,
47).
The +1 frameshift
mutations may have occurred during minus-strand
DNA synthesis
(
4), while the
1 frameshift mutations could
have occurred
during either minus- or plus-strand DNA synthesis.
Simple deletion and
deletion with insertion mutants have been
previously identified and the
mechanisms by which they could have
occurred have been proposed
(
36,
40).
Mutation of M188 in HTLV-1 RT decreases reverse transcription
accuracy.
Two HTLV-1 RT variants, M188A and M188V, were found to
significantly increase the rate of HTLV-1 mutation by a factor of 2 and
therefore decrease the accuracy of HTLV-1 reverse transcription by
twofold. The types of mutations that occurred during replication with
these variants indicated a spectrum similar to what was observed with
wild-type RT. However, limited number of mutants characterized suggests
that the frequency of frameshift mutations doubled. Mutation of the
YXDD motif may influence the template-primer affinity and could
potentially influence frameshift fidelity.
Deletion rates.
It has been observed that defective proviruses
represent about 25 to 40% of all HTLV-1 genomes present in lymphocytes
from infected individuals (many of which are defective due to deletion mutations in gag, pol, and/or env)
(16, 19, 33, 49). A large number of deleted HTLV-1
proviruses have also been observed in cell lines infected with a
variety of HTLV-1 isolates (14). These observations could
indicate that these deletion mutations are created during the reverse
transcription process at a higher rate than that observed for other
retroviruses. However, comparison of the frequency of deletion
mutations among retroviruses in which in vivo mutation rates have been
determined using the lacZ peptide gene as a mutational
target does not support this (Table 3). Rather, it indicates that
HTLV-1 is no more prone to deletion mutations than BLV, HIV-1, or SNV.
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ACKNOWLEDGMENTS |
I thank L. Bernard, P. Pandya, M. Reinhardt, and A. Waggoner for
outstanding technical assistance. I also thank M. Williams for comments
on the manuscript.
This work was supported by the Public Health Service (GM56615), the
American Cancer Society, and the Ohio Cancer Research Associates.
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FOOTNOTES |
*
Mailing address: Department of Molecular Virology,
Immunology, and Medical Genetics, 2078 Graves Hall, 333 W. 10th Ave.,
Columbus, OH 43210. Phone: (614) 292-5525. Fax: (614) 292-9805. E-mail: mansky.3{at}osu.edu.
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Journal of Virology, October 2000, p. 9525-9531, Vol. 74, No. 20
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Abstract
Introduction
