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Vol. 66, No. 6
JOURNAL OF VIROLOGY, June 1992, p. 3683-3689
0022-538X/92/063683-07$02.00/0
Copyright ©3 1992, American Society for Microbiology
Direct Determination of the Point Mutation Rate
of a Murine Retrovirus
RAYMOND J. MONK, FRANK G. MALIK, DAVID STOKESBERRY,
AND LEONARD H. EVANS*
Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute
of Allergy and Infectious Diseases, Hamilton, Montana 59840
Received 21 October 1991/Accepted 12 March 1992
rate of replication of the virus, and the ability of the virus to
The rate of evolution is the rate at which mutations
accumulate in a genome and is frequently measured in terms
of nucleotide substitutions per site per year. RNA viruses
exhibit very high rates of evolution compared with those
observed in eukaryotic DNA genomes (46, 49). For instance,
it has been estimated that 1 to 2% of the nucleotides in the
poliovirus genome have been altered by mutation during a
period of 1 year of active spread (33). Similarly, the neuraminidase and hemagglutinin genes of influenza virus have
been estimated to evolve at rates of 0.4 to 0.7% of their
nucleotides per year (2, 28, 41, 43). These values compare
with an average rate of evolution of approximately 10-7%
per year for eukaryotic genes (3, 27).
Retroviruses are reportedly among the most rapidly evolving viruses (9, 20-22, 34). The Moloney murine sarcoma
virus genome contains a viral oncogene which originated
from a homologous gene in the mouse genome, enabling a
comparison of the evolution rate of the same gene in
different genomic contexts. The rate of evolution of this viral
oncogene (v-mos) was found to exceed the rate of evolution
of its cellular homolog (c-mos) by greater than 1 millionfold
(20, 21). The env gene of the human immunodeficiency virus
(HIV) has been estimated to evolve at a rate of 0.1 to 1 % per
year (22), which also exceeds the average rate of evolution of
eukaryotic DNA genes by about 1 millionfold.
While the rate of evolution refers to the accumulation of
mutations, usually expressed on an annual basis, the mutation rate refers to the number of mutations which occur
during a replication cycle. The mutation rate of a virus, the
*
sustain mutations without elimination from the population
are all important factors in virus evolution. The mutation
rate is determined by the fidelity of the polymerases involved
in viral replication, the operation of repair mechanisms for
the polymerases, and perhaps the cellular environment in
which replication is taking place.
Retroviruses are unique among RNA viruses in that they
utilize RNA-dependent DNA, DNA-dependent RNA, and
DNA-dependent DNA synthesis in their replicative cycle,
and it has been suggested that the high rate of evolution of
retroviruses may reflect a high rate of mutation during
replication. Although nearly all mutation rate estimates of
retroviruses have been high, almost all such estimates do not
allow a precise determination of the point mutation rate in
terms of misincorporation per base copied during a replication cycle. Coffin et al. (9) calculated the mutation rate of the
avian Rous sarcoma virus (RSV), based on changes observed in RNase Tl-resistant oligonucleotides after repeated
cell-free passage of virus. Although a precise mutation rate
was not arrived at from these studies, a rate of 10-' to 10-4
was estimated, suggesting that each progeny virus differed
from its parent by 1 to 10 bases. Leider et al. (26) determined
a point mutation rate for RSV under conditions in which the
number of replication cycles was carefully controlled and
several defined regions of the RNAs of progeny viruses were
examined. They reported a rate of 1.4 x 10-4 mutations per
nucleotide during a single replication cycle for RSV, corroborating the estimate of Coffin et al. (9).
Almost all studies of in vivo viral mutation rates have
examined the rate in progeny viruses which are viable in
terms of infectivity and replication. Temin and coworkers
Corresponding author.
3683
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The point mutation rate of a murine leukemia virus (MuLV) genome (AKV) was determined under
conditions in which the number of replicative cycles was carefully controlled and the point mutation rate was
determined by direct examination of the RNA genomes of progeny viruses. A clonal cell line infected at a low
multiplicity of infection (2 x 10-3) was derived to provide a source of virus with high genetic homogeneity.
Virus stocks from this cell line were used to infect cells at a low multiplicity of infection, and the cells were
seeded soon after infection to obtain secondary clonal cell lines. RNase TI-oligonucleotide fingerprinting
analyses of virion RNAs from 93 secondary lines revealed only 3 base changes in nearly 130,000 bases analyzed.
To obtain an independent assessment of the mutation rate, we directly sequenced virion RNAs by using a series
of DNA oligonucleotide primers distributed across the genome. RNA sequencing detected no mutations in over
21,000 bases analyzed. The combined fingerprinting and sequencing analyses yielded a mutation rate for
infectious progeny viruses of one base change per 50,000 (2 x 10-5) bases per replication cycle. Our results
suggest that over 80% of infectious progeny MuLVs may be replicated with complete fidelity and that only a
low percentage undergo more than one point mutation during a replication cycle. Previous estimates of
retroviral mutation rates suggest that the majority of infectious progeny viruses have undergone one or more
point mutations. Recent studies of the mutation rates of marker genes in spleen necrosis virus-based vectors
estimate a base substitution rate lower than estimates for infectious avian retroviruses and nearly identical to
our determinations with AKV. The differences between mutation rates observed in studies of retroviruses may
reflect the imposition of different selective conditions.
3684
J. VIROL.
MONK ET AL.
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FIG. 1. Fingerprint analysis of the RNase T1-resistant oligonucleotides of virion RNA from the progenitor clone of AKV. RNA extracted
from 32P-labeled virions released from the progenitor clonal cell line was digested with RNase T1, and the oligonucleotides were resolved by
fingerprinting. (A) Autoradiograph of the fingerprint. (B) Schematic depiction of the fingerprint with the number or letter designations of the
oligonucleotides considered in the mutation rate analyses. The oligonucleotides which have numerical designations have been identified in the
nucleotide sequence of AKV, while the oligonucleotides which have one-letter designations (A to N) are oligonucleotides which are present
in the viral RNA genome but have not been unambiguously identified in the published sequence of AKV.
(10, 11, 36, 37) have studied mutation rates of selectable
marker or reporter genes contained in spleen necrosis virus
(SNV)-based vectors and report base substitution rates near
10-5 per base pair per replication cycle. The selective
conditions in the SNV system differ substantially from
conditions which select genes obligatory for the generation
of viable retroviruses; thus, the significance of differences in
mutation rates observed with SNV-based vectors and those
observed with viable progeny retroviruses is difficult to
assess.
The point mutation rate of a mammalian retrovirus in
terms of mutations per nucleotide per replication cycle has
not been previously determined. In the present study, we
determined the point mutation rate of a murine retrovirus by
direct examination of RNAs from viruses isolated after a
single replication cycle. Our results indicate that the mutation rate for murine retroviruses is quite high compared with
that for eukaryotic genes but is somewhat lower than estimates of the mutation rate of infectious progeny RSV.
MATERIALS AND METHODS
Cells and viruses. Mus dunni cells (25) were obtained
originally from M. Cloyd, and NIH 3T3 cells were originally
obtained from E. M. Scolnick. Both cell lines were maintained on minimal essential medium containing penicillin and
supplemented with 5% fetal calf serum. The ecotropic murine leukemia virus (MuLV) AKR 2A (7) was obtained from
M. Cloyd. Following infection, clonal cell lines were obtained by diluting the cells to approximately 20 cells per ml
and plating 5 ml of the cell suspension on a 100-mm tissue
culture dish. After attachment of the cells, the medium was
replaced by fresh medium containing 0.4% Bactoagar
(GIBCO) at 40°C and allowed to solidify at ambient temperature. The dishes were incubated undisturbed at 37°C until
colonies of cells became macroscopically visible (ca. 1 mm
in diameter). Our initial experiments to obtain a chronically
infected cell line at a low multiplicity of infection were done
with NIH 3T3 cells. However, during the course of this
work, technical difficulties were encountered in establishing
NIH 3T3 cells as single-cell colonies. Thus, M. dunni cells
were used for the subsequent isolation of progeny viruses in
our protocol (see below).
Detection of infected clones. Infected colonies of cells were
detected by a previously described immunofluorescence
assay (45). Briefly, when the colonies became macroscopically visible, the agar-containing medium was removed by
inverting the dish and the cells were rinsed with PBBS (5)
containing 2% fetal calf serum. The dishes were then incubated at 37°C for 30 min with 200 ,ul of monoclonal antibody
83A25 (hybridoma culture supernatant), which is reactive
with cell-surface MuLV envelope proteins (16). The dishes
were rinsed twice and incubated with a fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin serum
(Sigma) at 37°C for 30 min. Finally, the dishes were rinsed
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9
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MuLV POINT MUTATION RATE
VOL. 66, 1992
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three times and examined for fluorescence under a Leitz
orthoplan fluorescence microscope. Infected colonies, as
evidenced by cell surface fluorescence, were removed from
the tissue culture dish and subcultured to obtain infected
clonal cell lines.
RNA isolation and TI-resistant oligonucleotide fingerprinting. Metabolic labeling of virion RNA with 32P and details of
the fingerprinting procedure have been previously described
(14). Virions were sedimented from tissue culture media in a
Beckman SW 28 at 25,000 rpm at 4°C for 90 min. The pellet
was resuspended in 100 mM Tris-10 mM NaCl-1 mM
EDTA, pH 7.4 (TSE), and extracted with phenol-chloroform
(9:1) several times until no precipitate was visible at the
interface. The resultant aqueous phase was precipitated by
the addition of an equal volume of n-propanol after adjusting
to 100 mM NaCl. The nucleic acid precipitate was collected
by sedimentation and dissolved in TSE containing 0.1%
sodium dodecyl sulfate (SDS), layered onto a 12-ml 15 to
40% glycerol gradient in TSE-0.1% SDS, and then sedimented at 39,000 rpm in a Beckman SW 41 rotor at 20°C for
3.25 h. The gradient was fractionated, and the peak of
radioactivity sedimenting at approximately 70S was precipitated with n-propanol as described above. The resultant
precipitate was collected by sedimentation, washed two
times with 80% ethanol, resuspended in 10 mM Tris-1 mM
NaCI-1 mM EDTA, pH 7.4, and stored at -20°C until
fingerprinting.
Virion RNA for use as a template for sequencing was
prepared as described above except, after phenol extraction
of the pelleted virions, the aqueous phase was applied to an
oligo(dT)-cellulose column to obtain polyadenylated RNA as
previously described (14). The polyadenylated RNA was
precipitated with n-propanol, washed twice with 80% ethanol, dried, dissolved in water, and stored at -20°C. The
RNA was thawed and used directly in the RNA sequencing
procedure as described below.
RNA sequencing. Retroviral RNAs were sequenced by
using the protocol described by Mierendorf and Pfeffer (29)
and the GemSeq Transcript Sequencing System (Promega
Biotec). Oligonucleotides utilized as sequencing primers
were synthesized on the Applied Biosystems Inc. model
380B. The sequencing gels were prepared and processed as
described by Williams et al. (51), and the RNA sequencing
data were analyzed by using the MBUG (NIH molecular
biology users group integrated access system) computer
program (40).
RESULTS
Establishment of a genetically homogeneous virus population and isolation of viruses after a single replicative cycle.
NIH 3T3 cells were infected at a low multiplicity of infection
with the ecotropic MuLV AKR 2A and seeded as single cells
2 h after infection, prior to the appearance of progeny
viruses. The resultant colonies were examined by the immunofluorescence assay for cell surface expression of the viral
envelope glycoprotein to identify those infected with MuLV.
An MuLV-positive colony was subcultured from an infection in which only one fluorescent colony was observed
among over 500 examined. The cell line derived from this
colony has been designated the progenitor cell line. Since the
integrated provirus in the progenitor cell line replicates via
cellular DNA mechanisms, the errors introduced during
replication are negligible (10-' to 10-12 per replication cycle)
(3, 27) compared with rates reported for retroviruses (10' to
10-') (9, 26). Thus, virus released from the progenitor line
should be of the highest obtainable genetic homogeneity.
A precise knowledge of the number of replication cycles is
necessary to accurately estimate the mutation rate. In this
study we chose to analyze numerous progeny viruses isolated after a single round of replication. M. dunni cells were
infected with progeny virus from the progenitor cell line,
seeded 2 h after infection, and allowed to grow to macroscopic colonies. The colonies were assayed for infection,
and the MuLV-positive colonies were subcultured as described above for the derivation of the progenitor cell line. A
total of 93 infected clonal cell lines were isolated from
cultures infected at a multiplicity of infection of 5 x 10-3. It
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kilobases
FIG. 2. Genomic locations of RNase TI-resistant oligonucleotides and DNA primers employed in mutation analyses. The RNA genome
of AKV is represented by the bar diagram. Gene boundaries and lengths are indicated by arrows and the scale aligned with the bar diagram,
respectively. The locations of each of the numbered RNase T1 oligonucleotides and each of the DNA primers are indicated by lines drawn
from the RNase T1 oligonucleotide numbers (above the bar diagram) or from the DNA primers which are designated by the base position of
their 3' termini (below the bar diagram).
3686
J. VIROL.
MONK ET AL.
A J%
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AKV
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B
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AKV- M1
AKVFM2
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AKV - M3
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FIG. 3. Comparison of RNase Tl-resistant oligonucleotide fingerprints of mutant virion RNAs with virion RNA from the progenitor cell line. The virion RNAs from the progenitor cell line and the
three cell lines which harbored mutant viruses (AKV-Ml, AKV-M2,
and AKV-M3) were analyzed by fingerprinting as described in the
legend to Fig. 1. The oligonucleotides which are missing in the
mutant RNAs (no. 75, no. 1, and no. 50 for AKV-Ml, AKV-M2, and
AKV-M3, respectively) are circled on the fingerprint of the progenitor wild-type AKV (A). The original position of the missing
oligonucleotides are indicated by the empty circles in the fingerprints of the mutants AKV-M1 (B), AKV-M2 (C), and AKV-M3 (D).
In the cases of AKV-M2 and AKV-M3, a derivative of the original
oligonucleotide was identified and is indicated by an arrow directed
from the original location (empty circle) to the location of the
derivative (circled oligonucleotide).
is likely that each of the secondary lines was infected with an
individual virus which had undergone a single replication
cycle consisting of transcription by the cellular RNA polymerase in the progenitor cell line and reverse transcription to
the proviral DNA by the viral polymerase in the secondary
cell line. Mutations which occurred during these processes
would be fixed in the provirus and present in the vast
majority of virion RNA released from the secondary line.
Any subsequent mutations which occurred randomly in the
RNA transcribed from the provirus would not be detectable
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t~~~
't
in analyses of total virion RNA released from the secondary
cell line.
RNase Tl-resistant oligonucleotide analyses of RNAs from
virions released from the progenitor and secondary clonal cell
lines. The MuLV AKV was chosen for these analyses
because of the detailed knowledge of the genomic structure
of the virus, in terms of both the complete nucleotide
sequence (12, 23) and the precise genomic location and
sequence of a large number of RNase T1-resistant oligonucleotides that are resolved by the fingerprinting procedures
(12, 15). Eighty-nine oligonucleotides representing a total of
about 1,380 bases were considered in the analyses (Fig. 1).
Seventy-five of these oligonucleotides, totaling 1,228 bases,
have been unambiguously identified in published AKV RNA
sequences (12, 23) and are identified in Fig. 1. These
oligonucleotides represent a sampling of many short (8- to
30-base) sequences across the entire genome (Fig. 2). The
remaining resolved oligonucleotides (designated by the letters A to N in Fig. 1), representing about 150 bases, are
present at stoichiometric levels but could not be unambiguously located in the published sequence.
In the oligonucleotide fingerprinting analyses, the positions of oligonucleotides in the fingerprint are very sensitive
to base composition. In most cases a single-base change
within any of the large oligonucleotides results in an obvious
change in the migration of the oligonucleotide. However,
A-to-C and C-to-A base substitutions result in small changes
in migration and are more difficult to detect, particularly if
they occur in one of the larger oligonucleotides. Assuming
that each of the 12 possible base exchanges occurs with
equal probability and correcting for the average base composition of RNase T1-resistant oligonucleotides (ca. 31% A,
31% U, 31% C, and 7% G), 80% of the point mutations would
be easily detected by the fingerprinting analyses even if all
A-to-C and C-to-A changes went undetected.
The fingerprint of the cloned isolate of AKV from the
progenitor cell line was identical to that found in previous
analyses of AKR 2A (13) (Fig. 1), indicating that mutations
which affect the migration of the large oligonucleotides had
not occurred during the initial cloning of the progenitor
provirus. The fingerprints of virion RNAs from 90 of 93
secondary clonal cell lines analyzed were identical to that of
the progenitor virus, while the fingerprints of 3 isolates were
altered (Fig. 3). Each of these mutants differed from the
progenitor MuLV by a small sequence difference within a
single oligonucleotide, and in each case a different oligonucleotide was altered, indicating that the mutants were
unique. Two of the mutations occurred in gag gene sequences, and the third occurred in the pol gene (Fig. 4).
DNA primers complementary to sequences in the genome
near the mutations were constructed, and the RNA sequences of the regions containing the altered oligonucleotides were determined for each of the mutants. These
analyses (Fig. 5) indicated that each altered oligonucleotide
differed from those of the progenitor virus by a single-base
change which, in each case, resulted in a coding change to a
different amino acid. Two of the changes were transition
mutations, and the third was a transversion mutation. The
observed change of oligonucleotide migration in the fingerprint of each mutant was consistent with the change predicted from the altered base sequence. The fingerprint analyses of 93 isolates represented a total of nearly 130,000 bases
with only three detectable point mutations.
Sequencing of RNAs from virions released from secondary
clonal cell lines. The fingerprinting analyses suggested a
mutation rate substantially lower for the MuLV than esti-
Vol. 66, 1992
AKV-M2
5ED=
1
3687
MuLV POINT MUTATION RATE
AKV-M1
AKV-M3
1
I
3*
AKV
(Oligo. # 75)
A C- A A A A A
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AKV M1
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A
A G
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1
2
3
4
5
6
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8.37
kilobases
FIG. 4. Genomic location of detected mutations. The RNA genome of AKV 2A is represented by the bar diagram. Gene boundaries and lengths are indicated by horizontal arrows and the scale
aligned with the bar diagram, respectively. Arrows pointing to the
bar diagram indicate the locations of the mutations detected in the
three mutant progeny viruses.
DISCUSSION
Estimates of the mutation rate of infectious progeny avian
retroviruses suggest that the virus may sustain 1 to 10 point
mutations during a single replication cycle (9, 26), corresponding to an estimated mutation rate of 1.4 x 10-4. Our
estimate for the point mutation rate of AKV (2 x 10-5) is
somewhat lower and suggests that nearly 85% of the viable
progeny viruses may be replicated with complete fidelity
with few viruses (ca. 3%) sustaining more than one point
mutation per replicative cycle. Poisson statistics predicted
that the actual point mutation rate for AKV is between 1 in
17,000 and 1 in 240,000 bases replicated (39). Application of
the same Poisson statistics to the mutation rate data obtained
for RSV by Leider et al. (26) predicts that the point mutation
rate for the avian virus is between 1 in 3,700 and 1 in 16,000.
Thus, there is a 95% expectation that the differences in
mutation rates measured for the avian and murine viruses
C
T CT
C
C
A
A
AKV M2
AKV (Oligo. # 50)
A
C
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C
C
C
C
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C
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C A
A
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AKV M3
FIG. 5. RNA sequences of the altered RNase TI-resistant oligonucleotides of the viral mutants. The RNA sequence of the altered
oligonucleotide in each of the mutants is compared with the original
sequence. Nucleotide identity is indicated by vertical lines between
the AKV and mutant AKV sequences, and the altered base of each
mutant is shown in bold lettering. The amino acids encoded by the
original AKV and the AKV mutants are indicated above and below
the sequences, respectively.
due simply to random variation in the determinations. A number of factors could influence the differences in
mutation rates observed for the avian and murine viruses.
Since the point mutation rates determined for both retrovirus
types are rates for infectious progeny viruses, mutations
which result in the loss of the ability to independently
replicate would not be detected. The ability to replicate may
vary with different selective conditions. Furthermore, the
ability to sustain mutations may vary among different rctroviruses or among different genes of a particular retrovirus.
Recently, Temin and coworkers have studied mutation
rates of genes inserted into SNV-based vectors during a
single replication cycle (10, 11, 36, 37). They report a
reversion frequency due to base substitution of an amber
mutation in the neo gene of 2 x 10-5 (11) and a forward
mutation rate resulting from base substitution of the lacZ
gene of 7 x 106 (36). Although these values are in very
close agreement with our present determination of the point
mutation rate of AKV, a comparison between the studies is
difficult. In our studies, as in nearly all point mutation rate
studies of animal viruses, viable progeny viruses were selected. Large genetic alterations such as frameshift mutations or large deletions, which are likely to be catastropic for
viral replication or infectivity, would be infrequently detected. Furthermore, the point mutation rate determined for
viable progeny viruses is expected to be lower than the
actual rate of misincorporation of bases during the replicative cycle (the in vivo polymerase error rate). Under these
conditions, the mutation rate largely reflects the incidencc of
mutations which are functionally silent with regard to replication and infectivity. The mutation rates determined for
SNV were determined at the level of the DNA provirus,
thereby eliminating selection for viable progeny retroviruses. In addition to base substitution, those studies detected frameshift mutations, deletions, and hypermutation
(an inordinately high level of base substitution on the same
polynucleotide). It is somewhat surprising that the basc
substitution rates observed in the absence of selection for
viability were lower than rates which havc been reported for
viable avian retroviruses (9, 26). However, the reversion and
forward mutation studies of the SNV vectors involved
selection for altered phenotypic functions of the neo and the
lacZ genes, respectively, and did not examine the incidence
are not
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mates of mutation rates of infectious avian retroviruscs. To
obtain an independent assessment of the mutation rate,
several DNA oligonucleotides complementary to AKV sequences were synthesized and used to prime RNA sequencing reactions with many of the secondary AKV isolates as
well as the progenitor AKV. The oligonucleotides were
chosen from regions dispersed across the genome to maximize the probability that the sequences analyzed would
reflect the mutation rate of the entire genome, rather than a
limited region which may or may not be representative (Fig.
2). Several differences were noted between the sequences
generated in our analyses and the published sequence of the
molecularly cloned endogenous MuLV of AKR mice (AKV)
(12, 23). However, all such differences were reflected in each
of the viral RNAs analyzed and indicated differences between our progenitor MuLV and the published sequence of
AKV rather than de novo mutations. With the exception of
the three oligonucleotide mutations confirmed by sequence
determinations, our RNA sequence analyses revealed no de
novo mutations in over 21,000 bases analyzed. This is
equivalent to over two complete MuLV RNA genomes
without a single mutation, corroborating the data obtained
from the fingerprinting analyses.
Nearly 150,000 bases were analyzed by fingerprinting and
sequencing in our experiments. The detection of only three
mutations yielded an estimated mutation rate of 2.0 x 10-5.
Assuming a Poisson distribution, the detection of three base
changes in 150,000 predicts with 95% confidence that the
actual mutation rate for AKV is between 5.85 x 10-5 and
4.13 x 10-6 (ca. between 1 mutation in 17,000 and 1 in
240,000 bases) (39). Even at the upper limit this is substantially less than one mutation per progeny virus.
AKV (Oligo. # 1)
3688
J. VIROL.
MONK ET AL.
of functionally silent mutations. The selective stringency for
base substitution imposed by selection for viability compared to that imposed by selection for altered functions of
genes carried by the SNV vectors is unknown. Such stringeny would likely depend on the characteristics of the particular gene carried by the vector.
Pathak and Temin (36) encountered a single provirus
containing 15 G-to-A transition mutations corresponding to a
mutation rate approximately 1,000-fold higher than the rate
determined for the virus population as a whole. They term
this phenomenon hypermutation and suggest that it may be
the result of an aberrant polymerase in the population.
Frequent G-to-A hypermutation has also been reported for
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HIV type 1 (50). Although we did not observe hypermutations (or other extensive genetic alterations), it is possible
that they are generated but not frequently selected as infectious progeny virus in our analyses. In this regard, it seems
likely that a population of retroviruses which has been
allowed to diverge may encode polymerases which vary in
their fidelity. In that case, the mutation rate of a virus
population would be an average rate and may differ from a
mutation rate determined for a clonal isolate. The contribution of polymerase divergence to the population mutation
rate is difficult to assess. A high degree of genetic recombination among retroviruses and the formation of viral
pseudotypes may facilitate the maintenance of aberrant
polymerases in the population.
A considerable range of mutation rates have been reported
among the RNA viruses. Steinhauer and Holland (48) observed a very high rate of mutation of about 7 x 10' base
substitutions per replicative cycle for the vesicular stomatitis
virus. Parvin et al. (35) have reported the mutation rate of
the NS gene of influenza A virus to be 1.5 x 10-', while the
rate observed for the VP1 gene of poliovirus was <2.1 x
10-6. The estimate for MuLV developed in this study
appears to be near the middle of this range and close to that
of the NS gene of influenza A virus. Parvin et al. (35) have
suggested that the higher mutation rate of influenza A virus
than of poliovirus may account for the rapid antigenic drift of
influenza A virus and the necessity to frequently revise
influenza A vaccines.
The possible role of immune escape mechanisms has
received considerable attention in the case of the lentiviruses
such as the visna virus (6, 30, 31), equine infectious anemia
virus (4, 24, 38, 44), and HIV (8, 22, 42, 47). Considering the
difference between the point mutation rates of MuLV and
RSV, it is difficult to extrapolate the data to the lentiviruses.
However, in the case of the equine infectious anemia virus,
the analyses of two isolates obtained sequentially from an
infected horse at a 30-day interval differed by only 0.25% in
their sequences (4). Ignoring in vivo virus replication which
occurred, the extensive in vitro isolation procedures required to obtain these isolates (amplification by infectious
spread in an in vitro cell line followed by multiple endpoint
dilution cloning) suggest a mutation rate nearer that of
MuLV than that of RSV.
Finally, it is noted that the mutation rate during virus
replication in fibroblastic cell lines might not extrapolate to
virus replication in various cell types of the infected host.
For example, lentiviruses, such as HIV, infect and replicate
in macrophages which may serve as a reservoir for the
viruses in an infected individual (17-19, 31, 32). Upon
under these conditions could conceivably result in
elevated virus mutation rates.
VOL. 66, 1992
3689
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MuLV POINT MUTATION RATE