Download High frequency precise multiple-base reversion of

Proc. Nadl. Acad. Sci. USA
Vol. 89, pp. 2531-2535, April 1992
Genetics
High frequency of single-base transitions and extreme frequency of
precise multiple-base reversion mutations in poliovirus
(polymerase errors/hypermutatlon/RNA virus variability)
JUAN CARLOS DE LA TORRE*t, CRISTINA GIACHETrIt, BERT L. SEMLERt, AND JOHN J. HOLLAND*§
*Department of Biology and Center for Molecular Genetics, University of California, San Diego,
9500 Gilman Drive, La Jolla, CA 92093-0116; and
tDepartment of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, CA 92717
Communicated by E. Peter Geiduschek, December 11, 1991
quency of poliovirus type 1 to a high degree of resistance to
guanidine indicated specific single-site mutation frequencies
for poliovirus of the order of 10-3 to 10-4 (32, 33). We have
reported (34) a very high frequency (per single-base site) of
reversion [(2.1 ± 1.9) x 10-4] to guanidine resistance in
clonal pools of guanidine-dependent type 1 poliovirus.
The existence of poliovirus cDNA clones that produce
infectious virus upon transfection of primate cells (35, 36)
makes it possible to genetically manipulate the RNA genome
of poliovirus. During molecular genetic studies of the role of
polypeptide 3AB in poliovirus RNA synthesis, we constructed a type 1 poliovirus mutant, designated 3AB-310/4.
This mutant was generated by site-directed mutagenesis, and
it displayed a strong temperature-sensitive (ts) phenotype
(37, 38). We describe below the use of this genetically and
phenotypically well-characterized ts mutant of poliovirus to
quantitate specific reversion frequencies at which non-ts
revertants arose during the replication of ts 3AB-310/4 in
HeLa cells at the permissive temperature of 330C. We have
demonstrated (37) that a single amino acid substitution (Thr
-- Ile at amino acid residue 67) within the poliovirus polypeptide 3AB [produced by a single C -> U base transition at
nucleotide (nt) 5310] is responsible for the highly ts phenotype displayed at 39"C by ts 3AB-310/4 (37). The availability
of this well-characterized nonleaky mutant of poliovirus
provides reliable selection and quantitation of single-base
revertants at 390C. It thereby allows a valuable experimental
approach to determine the frequency at which the poliovirus
polymerase causes a single specific U -- C base transition at
genome site 5310. We show below that revertants selected for
this transition also exhibit a remarkable frequency of specific
multisite reversions nearby.
We employed independent clones of a temABSTRACT
perature-sensitive mutant of type 1 poliovirus, 3AB-310/4, to
quantitate the frequency of specific U -+ C transitions at
nucleotide 5310, within the genomic region encoding polypeptide 3AB, which is involved in the initiation of RNA replication.
Only this U -- C base substitution restores the wild-type
phenotypic ability to form plaques at 39C; the other two base
substitutions at this site are lethal. The observed frequency of
this specific transition averaged 2 x i0S, and all revertant
viruses forming plaques at 39°C contained the expected cytidine
at nucleotide 5310. Incredibly, only 3 of 10 revertants exhibited
this one specific U -- C transition whereas 7 of 10 exhibited this
same transition plus four additional base substitutions that
precisely reverted temperature-sensitive 3AB-310/4 to wildtype poliovirus sequence (these latter four mutations had been
introduced into 3AB-310/4 as silent third base mutations to
provide new restriction sites in infectious cDNAs). No other
mutations were detected in this polypeptide 3AB domain in
either the single-base or the precise 5-base revertants. No
intermediates were seen; all revertants exhibited either the
single U -- C transition at nucleotide 5310 or the same
transition plus four precise reversions to the wild-type sequence
at sites 8, 11, 43, and 46 bases distant from nucleotide 5310.
Similar results were obtained after transfection of cDNAderived transcripts. We discuss possible mechaniss for our
data. These include (but may not be limited to) error-prone
polymerase activity, sequential RNA recombination events
joining independent mutations, or some unusual RNA editing
process.
Considerable data now document the extensive genetic variability and potential for rapid evolution of RNA viruses
(1-19). The molecular basis for this variation is extremely
high mutation frequencies per average site in RNA virus
genomes (ranging between 10-3 and 10-6 and usually of the
order of 10' to 10-5). Such high mutation frequencies dictate
that even clones of RNA viruses are not homogeneous
populations but consist of complex "mutant swarms" or
"quasispecies" populations (20-26). By virtue of their extreme heterogeneity, quasispecies populations can adapt
rapidly to new environments by selection of variants preexisting within the population. Some very low polymerase error
values have been reported for poliovirus. Thus, Parvin et al.
(27) have estimated a poliovirus mutation rate of <10-6 per
site for viable neutral or quasineutral mutants of poliovirus.
A similar low error frequency of 2.5 x 10-6 was estimated for
the reversion of a poliovirus amber mutant (28). In contrast,
frequencies of resistance to monoclonal antibody neutralization for poliovirus are in the range 10-3 to 10-5 base substitutions per site (29, 30). In vitro measurements of poliovirus
polymerase error frequencies (31) and the mutation fre-
MATERIALS AND METHODS
Cells and Virus. HeLa cell monolayers with Eagle's minimal essential medium (MEM) were used throughout this
study. The generation, isolation, and characterization of
poliovirus mutant Sel-3AB-310/4 have been described (37).
Phenotypically, mutant 3AB-310/4 is highly ts for replication, plaque formation, and RNA synthesis at 390C (37, 38).
Virus Plaque Isolation and Titration. Samples (0.2 ml) of
serial virus dilutions in MEM containing 5% (vol/vol) heatinactivated (600C for 30 min) fetal bovine serum were adsorbed to HeLa cell monolayers in triplicate. After a 45-min
adsorption period, the cells were overlaid with 7 ml of MEM
containing 7% heat-inactivated fetal bovine serum and 0.4%
agarose. The flasks were incubated at 33TC (5 days) or 390C
(48 hr) for plaque development, after which the cells were
Abbreviations: ts, temperature sensitive; pfu, plaque-forming unit(s);
nt, nucleotide(s).
tPresent address: Department of Neuropharmacology, Scripps
Clinic and Research Foundation, La Jolla, CA 92037.
§To whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
2531
2532
Proc. Natl. Acad. Sci. USA 89 (1992)
Genetics: De La Torre et al.
fixed with 7% (wt/vol) paiaformaldehyde for 15 min, and
plaques were visualized by staining with 2% (wt/vol) crystal
violet (39).
For plaque purification, viruses in individual plaques were
isolated by removing the cells within and around the edge of
the plaque with a sterile Pasteur pipette. The cells and
agarose medium were resuspended in 1.0 ml of MEM/5%
heat-inactivated fetal bovine serum. After three cycles of
freezing-thawing, cell debris was removed by centrifugation,
and the virus-containing supernatants were emulsified with
chloroform (5 ,ul of chloroform per 0.5 ml of supernatant) and
further clarified by centrifugation for 15 min at 12,000 x g.
Clonal pools were prepared by plating 0.2 ml (20%o) of the
virus on HeLa cell monolayers and incubating at 330C until
cell pathology was complete. The mutant 3AB-310/4 virus
preparation picked directly from plaques after incubation at
33°C (for 5-7 days after infection) contains at most 10'-106
plaque-forming units (pfu). Thus, expansion to clonal pools is
necessary for accurate determination of reversion frequencies.
Sequencing of RNAs from. Mutant and Revertant Viruses.
Viral RNAs were prepared by infection of 60-mm diameter
dishes of HeLa cell monolayers with mutant or revertant
viruses at a multiplicity of infection of 50 except where
otherwise stated. Mutant-infected cells were incubated at
330C, and revertant-infected cells were incubated at 39°C. At
12 hr after infection for mutant virus or at 6 hr after infection
for revertants, total intracellular RNA was extracted as
described (40). The viral RNA was sequenced by extension
of a 20-nt synthetic primer complementary to nt 5401-5420 of
poliovirus RNA using [a-32P]dATP, dideoxynucleotides, and
reverse transcriptase (41, 42).
RESULTS
High-Frequency Reversion to the Wild-Type Phenotype of
Virus rrom Independent Clonal Pools of Mutant ts 3AB-310/4.
Mutant
3AB-310/4 contains
a
single
C
U base transition at
nt 5310 that is lethal at 39°C but that allows replication at 330C
at rates approximately equal to wild type (37). This allows
reliable
quantitation of reversion frequencies for
U
--
C
transitions at nt 5310. Virus 3AB-310/4 was generated from
monolayers at 330C, and three well-isolated plaques were
picked. Virus recovered from each plaque was clarified after
chloroform extraction, again diluted 1:5, and seeded on HeLa
cells to produce three independent primary clonal pools
designated C1, C2, and C3 (Table 1, experiment A). Plaque
assays ofprimary pools C1, C2, and C3 at 330C and 390C again
demonstrated non-ts revertant frequencies 2-3 x 10-5 (Table
1, experiment A). Clonal pools C1, C2, and C3 were then
diluted 1:105 to eliminate revertants and 12 x 103, 8 x 103, and
14 X 103 pfu, respectively, were seeded onto three HeLa cell
monolayers to produce secondary clonal pools. The same
amounts of each (C1, C2, and C3) pool plated at 39°C in
triplicate HeLa cell plaque assays produced no revertant
plaques. We carefully determined the revertant frequency in
each of the resulting secondary clonal pools and again (Table
1, experiment B) observed frequencies of 2-3 x 10-s (mean
= 2.3 x 10-5). Thus, the revertant frequency was rather
constant whether the clonal pools were produced by expansion of consecutively cloned well-isolated plaques (Table 1,
experiment A and original twice-cloned pool) or by virus
diluted from three primary clonal pools and replicated to
produce secondary clonal pools (Table 1, experiment B).
Properties of Revertant Plaques Isolated at 39°C During the
Replication of Clonal Pools of 3AB-310/4. Some of the (revertant) plaques that developed at 390C were isolated and
labeled as CiR1 (clone 1, revertant 1), C1R2, C1R3, C2R1,
etc., and their growth properties at 330C vs. 390C were
examined. We compared one-step growth curves at 330C and
390C for the 3AB-310/4 C1 clonal pool (Fig. 1A) with one
representative non-ts revertant isolated from this pool (Fig.
1B}. Similar results were obtained with two other clonal pools
of 3AB-310/4 (C1 and C2 pools) and their revertants. Revertant viruses were able to grow efficiently at 390C displaying
typical wild-type one-step growth curves at both temperatures. In contrast, the corresponding mutant 3AB-310/4
clonal pools showed the strongly defective growth at 390C
that is typical of mutant 3AB-310/4 (37).
Sequence Analysis of the ts 3AB-310/4 Clonal Pools and of
the Isolated Revertant Viruses. As shown in Fig. 2A, sequence
analysis of relevant regions of the three clonal pools isolated
from mutant virus 3AB-310/4 (C1, C2, and C3) showed that
each has the same sequence as the original mutant virus
3AB-310/4. All of these ts 3AB-310/4 viral RNAs contain
five nucleotide changes with respect to the original wild-type
virus (type 1 poliovirus) sequence. Among these mutations,
four at nt 5299,5302,5353, and 5356, correspond to third-base
(silent) changes, originally introduced into viral cDNAs to
create new restriction sites (ref. 38 and C.G., unpublished
data). The kinetics of viral growth and RNA and protein
synthesis at 330C and 390C exhibited by mutants containing
only these four mutations were indistinguishable from those
a cDNA clone transfected into HeLa cells, and then the
resulting virus was plaque-purified twice on HeLa cells. A
well-isolated plaque was picked, diluted, and directly plaquepurified a second consecutive time on HeLa cells. Once
again, a well-isolated plaque was picked into 1 ml of MEM,
and 0.2 ml was plated on a HeLa cell monolayer at 330C( to
produce a clonal pool. This twice-cloned virus pool exhibited
a non-ts revertant frequency of 2-3 x 10-1. This twice-cloned
pool was diluted and plaque-purified a third time on HeLa cell
Table 1. Quantitation of mutation frequencies from mutant ts 3AB-310/4 to non-ts wild-type phenotype
Experiment B
Experiment A
Titer, pfu/ml
Titer, pfu/ml
390C
39"C/33-C
390C
330C
Clonal pool
330C
± 1.1) x 10-5
±
±
(2.7
x
x
x
109
(2.3
105
x
(8.7
1.2
3.1
0.3)
Cl
109
1.3)
104
3AB-310/4
(1.9 ± 0.2) x 10-5
(1.1 ± 0.01) x 105
2.5 x 104
(5.5 ± 0.3) x 109
8.0 x 108
3AB-310/4 C2
(2.2 ± 0.9) x 10-5
(2.4 ± 0.5) x 10
(11 ± 0.1) X 108
4.8 x 104
1.4 x 109
3AB-310/4 C3
In experiment A, each of the 3AB-310/4 clonal pools (Cl, C2, and C3) was prepared by infecting HeLa cell monolayers
(-107 cells) with 0.2 ml of a 1-ml suspension of virus recovered directly from three well-isolated plaques. Each of these
plaques had been produced on HeLa cell monolayers using a mutant 3AB-310/4 clonal pool that had been prepared after
two consecutive plaque-to-plaque clonings of virus produced after transfection of an infectious cDNA clone into HeLa cells
(38). Thus, pools Cl, C2, and C3 were independently produced by a third consecutive cloning procedure.
In experiment B a 1:105 dilution of each 3AB-310/4 clonal-pool viral stock (12 x 103 pfu of Cl, 8 x 103 pfu of C2, and
14 x 103 pfu of C3) was used to infect HeLa cells at 330C. After the complete cytopathic effect developed, the infected cells
and supernatants were processed. The frequency at which ts 3AB-310/4 virus reverted to wild-type virus phenotype was
determined based upon the ratio of pfu at 39°C/pfu at 330C. The results shown are the average (± standard deviation) of
two experiments using the same 3AB-310/4 clonal stocks.
Proc. Natl. Acad. Sci. USA 89 (1992)
Genetics: De La Torre et al.
2533
that single-base revertants do not quickly undergo further
mutation and selection to five-base revertants (Fig. 2B).
Competition experiments in which cells were infected with
various ratios of revertant viruses C3/R6 (one-base reversion) and C3/R9 (five-base reversions) and mixed infections
with wild-type poliovirus and a virus that contained the four
silent changes showed that they all have approximately equal
growth rates and that they compete rather equally. Both the
single-base revertant and the five-base revertant replicated- at
equal rates whether replicating alone or in competition with
each other (or wild-type virus) in mixed growth (data not
shown). Thus, there is no evident growth advantage of the
original wild-type virus (nor of the five-base revertant virus)
as compared to single-base revertants at nt 5310.
Multiple-Base Revertants Do Not Exhibit a Hypermutable
Phenotype. It seemed possible, though unlikely, that we had
selected for variant viruses with a hypermutable phenotype.
We tested this possibility by analyzing the frequency at which
guanidine-resistant variants (32, 34) arose from our revertant
pools. However, we obtained resistant variants at similar
frequencies from ts 3AB-310/4 C1-C3 and from several
independent clonal pools of the corresponding non-ts revertants. We observed guanidine-resistance frequencies ranging
from 3.3 x 10-8 to 4.1 x 10-7 (data not shown). These values
are similar to those reported for poliovirus and support the
documented requirement (32, 33) for several additive mutational events.
Contamination by Wild-Type Virus Cannot Explain These
Revertants. A trivial explanation for the above results would
be contamination of our clones by wild-type poliovirus. This
is extremely unlikely for the reasons listed below: (i) During
consecutive cloning procedures with mutant 3AB-310/4,
small to moderate plaques (up to 2 mm) require at least 5 days
of incubation at 330C after infection. In contrast, wild-type
virus produces large plaques in 2-3 days at 330C, so contamination would be readily apparent if it occurred. (ii) The three
clonal pools employed here were derived from virus that had
Q
-
a)
t2
Time, hr
FIG. 1. Comparison of one-step virus growth curves of clonal
pool 3AB-310/4 C1 at 33°C and 39°C (A) with those of a representative revertant (ClR1) from the same pool (B). HeLa cells were
infected at a multiplicity of 10 pfu per cell and virus yields were
determined at each time point as described (37, 38).
of the original wild-type virus. The fifth change exhibited by
the original mutant 3AB-310/4 virus, and all the clonal pools
employed in this study, is the lesion at nt 5310 (C U). This
introduced mutation is responsible for an amino acid change
(Thr-67 -- Ile) within the sequence of polypeptide 3AB and
for the ts defect of these viruses.
All of the non-ts revertants isolated (Cl/Ri, C1/R2, C1/R8,
etc.) had reverted the mutation at nt 5310 to the original
wild-type sequence (U -+ C). It is important to note that the
other two possible changes of nt 5310: U -- G or U -* A would
encode arginine or lysine, respectively, at this position. Both
of these changes have been demonstrated (37) to be lethal
mutations. Incredibly, 7 of 10 revertants isolated from the
three clonal pools (Cl/Ri, C1/R2, C2/R1, C2/R2, C3/R1,
C3/R2, and C3/R9) also showed reversion of all four of the
silent mutations at nt 5299, 5302, 5353, and 5356 (Fig. 2A). In
no case were we able to isolate viruses with intermediate
changes (i.e., viruses with genomic RNAs containing one,
two, or three of the four possible revertant mutations at these
four positions). Seven serial passages of single-base revertant
viruses (C1/R8, C2/R6, and C3/R6) maintained the mutated
silent changes, indicating that these mutations are stable and
#
.~
Cl
c
A
PVl
CTA CAA GCG GTG ACA ACC TTC GCC GCA GTG GCT GGA GTT GTC TAT GTC ATG TAT AAA
3AB-310/4
--G --G
---
---
-U-
---
---
---
---
---
-U-
---
---
---
---
---
CTG
---
---
---
---
---
---
--G
*
--U
c1
--G --G
---
---
___
---
---
---
---
---
---
--G
--u
Cl/Ri
Cl/R2
C1/R8
--G --G
--- --- --- --- --- ---
___
---
---
---
---
---
---
--G
--U
C2
--G --G ---
---
-U-
C2/Rl
C2/R2
C2/R6
--G --G ---
---
---
C3
C3/Rl
C3/R2
C3/R6
C3/R9
B
ur
---
u
--G--G
--- ---
-U- ___
--G --G
---
---
---
---
---
---
---
---
---
---
--G
___
---
---
--G
--u
--U
--U
Ci/R8
--G --G ---
---
---
---
---
---
---
---
---
--G --G
---
---
___
___
---
Cl/R8-P7
---
---
---
---
---
---
---
---
--G
--G
--u
C2/R6
--G --G ----G --G ---
---
___
---
---
---
---
---
---
---
---
--G
--U
--G --G ----G --G ---
---
---
---
---
---
---
---
---
---
---
---
---
---
---
---
---
---
---
---
---
---
---
---
---
---
---
---
---
---
---
--G
--G
C2/R6-P7
C3/R6
C3/R6-P7
*
*
--U
---
silent change
amino acid change
__U
FIG. 2. (A) Partial nucleotide
sequence of polypeptide 3AB of
wild-type poliovirus type 1
(PV1), its mutant ts 3AB-310/4,
the clonal pools (C1, C2, and C3)
derived from the mutant virus,
and the non-ts revertants isolated from each clonal pool. (B)
Partial nucleotide sequence of
the polypeptide 3AB of some of
the revertants isolated from each
ts clonal pool after seven serial
passages at 39°C. Dashes indicate identity with the wild-type
sequence. An asterisk and solid
diamond indicate the mutations
introduced into engineered mutant 3AB-310/4: An asterisk below the wild-type sequence at nt
5299, 5302, 5353, and 5356 indicates mutations introduced to
create new restriction sites.
These mutations resulted in no
amino acid changes. A solid diamond above the sequence at nt
5310 indicates the mutation
made to generate the change Thr
-- Ile at amino acid residue 67
within the sequence of 3AB.
This mutation is responsible for
the ts phenotype of the mutant.
2534
Genetics: De La Torre et al.
been twice-cloned previously. (iii) The reversion frequencies
observed in the twice-cloned 3AB-310/4 clonal pool agreed
closely with those observed in the three primary clonal pools
(and in the secondary clonal pools derived from each).
Wild-type virus would have had to contaminate each pool at
almost precisely the same frequency (~_10-5). This is not only
highly improbable but also nearly impossible, because contaminating virus quickly forms large plaques (as mentioned
above). To be present at 10-5 levels, wild-type virus would
have had to enter tightly capped T-25 culture flasks (but not
our mock-infected control flasks) during the fourth or fifth
day of plaque development of mutant 3AB-310/4 at precisely
the time when the single-base revertants were being produced
by mutation since they were also present at =10-5 frequency
in all three clones (C1, C2, and C3). This is improbable
because clones C1, C2, and C3 and their clonal pools were
produced and reverted in a laboratory where wild-type
poliovirus has not been handled for more than a decade.
Guanidine-dependent poliovirus obtained from E. Wimmer
(State University of New York at Stony Brook) has been
employed (as have its guanidine-resistant revertants), but
contamination by either of these is ruled out by the guanidine
data above. (iv) Transfection experiments produced similar
results.
After RNA transfections with in vitro-synthesized transcripts derived from full-length poliovirus cDNAs containing
the identical four silent mutations in ts 3AB-310/4 plus either
one of two lethal base transversions at nt 5310 [either C -+ A
(Thr -- Lys) or C -* G (Thr -* Arg)], infectious virus was
sometimes produced very late after transfection. When virus
was produced late after transfections, recovered virus frequently exhibited precise reversion to wild-type poliovirus
sequence at all five of the same sites (C.G., unpublished
data). Even more remarkable are the results of RNA transfection experiments in which precise six-base revertants
were produced. Mutations were introduced into the hydrophobic domain of 3AB using the cDNA construct that was
employed for mutant 3AB-310/4. This construct contains the
same four silent mutations plus two lethal mutations [U -- A
(Thr -- Lys) at nt 5310 and G -- A (Gly -+ Glu) at nt 5331].
It is designated 3AB/5-61. When in vitro-transcribed full-size
poliovirus RNA of 3AB/5-61 was transfected into HeLa
cells, no plaques had appeared at 37°C by 40 hr or at 33°C by
64 hr (by which time transfected wild-type controls showed
numerous plaques with a linear dose-response curve to RNA
input). However, after 7 days, two small plaques and one
large plaque had developed in the HeLa cell monolayers
incubated at 33°C (none appeared at 37°C). Sequencing of
these three revertant virus clones showed that one of the
small plaques was a two-base revertant with an A -+ C
transversion at nt 5310 and an A -+ G transition at nt 5331.
The other two revertants had also reverted these same two
lethal mutations, but they had in addition reverted the four
silent mutations; i.e., they were precise six-base revertants to
wild-type sequence.
It was important to learn whether the four silent mutations
in 3AB-310/4 exhibit a high probability of four-base reversions in the absence of reversions in the nt 5310 coding site.
Therefore, we picked a number of plaques grown at 33°C
from the secondary clonal pools (C1, C2, and C3) from which
revertants had been obtained at 39°C. Virus recovered from
each of these well-isolated plaques was then expanded into
independent clonal pools at 33°C and the (non-ts revertant)
virus recovered from each was sequenced in the relevant
3AB region. Nine plaques were picked (three from pool C1,
two from C2, and four from C3), and clonal pools were
prepared at 330C on HeLa cell monolayers of 3 x 106 cells.
Virus from these clonal pools was used to infect HeLa cell
monolayers of 8 x 106 cells at an input multiplicity of 5-10 pfu
per cell. After a 12-hr infection at 330C, RNA was extracted
Proc. Natl. Acad. Sci. USA 89 (1992)
and sequenced. All nine of these ts clones propagated at 330C
were observed to have retained the exact sequence of 3AB310/4. This shows that reversions at the four silent bases do
not occur regularly in the absence of high-temperature selection for non-ts revertants at nt 5310. These results also
serve as controls to rule out frequent contamination of
wild-type virus.
DISCUSSION
The most remarkable result above was the finding that the
four silent mutations reverted together or not at all. Clearly,
precise multiple reversions have occurred at an extreme rate
that cannot be explained by sequential independent mutational events. Even if we assume a high base substitution
frequency approaching 10-4 per site, more than 1020 pfu
would have to be produced before there was a significant
probability of finding any mutants carrying these five specific
reversions if they arose sequentially. One possible explanation would be the occurrence of multiple mutations (at least
in this region of the genome) during single genome replication
events, perhaps due to localized error-prone replicasetemplate-substrate interactions. Direct sequence analysis
has previously demonstrated the occurrence of multiple
mutations near single-base sites in viral RNA oligonucleotides that had been selected for the presence of a specific base
mutation (43). Other nearby random mutations that must
have occurred could have been deleterious or lethal.
The above explanation is almost impossible to accept from
the vantage point of classical genetics, but the RNA transfection results corroborate the multiple revertant data. Alternatively, our results might be explained by multiple RNA
recombination events bringing together many single-base
changes from a number of genomes or by some unusual kind
of RNA editing. "Biased hypermutations" may occur by
RNA editing, such as RNA unwinding activity, or by repetitive monotonous replicase errors (11, 44-50), but the transitions and transversions observed here fit no presently
known pattern. The extensive secondary structure in the
region involved might facilitate RNA editing or multiple
recombination events. The predicted fold of the 150-base
region of type 1 poliovirus was computer-generated by the
method of Zuker and Stiegler (51), and the results suggest a
stable extensively base-paired stem-loop structure with a
AG°1C.1 of -38.8 (unpublished data). Possibly this structure
influenced our results by strong selective effects or by
affecting RNA recombination or RNA editing. A cloverleaf
structure in the poliovirus 5' noncoding terminus is known to
form a ribonucleoprotein complex required for replication
(52). Low-multiplicity infection may be important for generation/selection of precise multiple base revertants because
we employed low multiplicity of infection in all RNA transfection and virus reversion experiments above. In contrast,
all of our previous isolations of single-base revertants at nt
5310 (37) and at other sites in this polypeptide 3AB domain
(C.G., unpublished data) involved high multiplicity of infection infections with high-titer virus stocks. It is important to
note that sequence comparisons of the 3AB region (nt 52975356) among the three poliovirus serotypes suggest no obvious sequence constraints (but the predicted secondary structure in this region of the genome might impose structural
constraints). About 80%o of positions within this domain were
conserved, and 93% of the variable sites involved third base
positions. In particular, at the sites of the four silent mutations (nt 5299, 5301, 5353, and 5356) only three of the four
bases present in wild-type poliovirus type 1 are conserved in
poliovirus type 2 and none are conserved in type 3. Furthermore, when we performed mixed growth competition experiments (data not shown), we observed no selective advantage
of five-base revertants over single-base revertants (nor of
Genetics: De La Torre et al.
wild type over virus with the four silent mutations). Therefore, it is completely unclear why single-base revertants
should not greatly outnumber precise multiple-base revertants, but the latter obviously occurred at nearly equal
frequencies or were selected in a genetically linked manner.
These findings cannot be adequately explained at present,
and they bring additional complexity to the already very
complex field of RNA virus genetics and evolution.
J.C.T. and C.G. made equal contributions to the work presented
in this paper. This work was supported by Public Health Service
Grants A122693 (B.L.S.) and A114627 (J.J.H.). B.L.S. is supported
by Research Career Development Award A100721 and J.C.T. by a
Fullbright Fellowship. C.G. received partial support from the Irvine
Research Unit in Animal Virology.
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