Journal of General Virology (1994). 75, 389 393. Printed in Great Britain
389
An estimation of the nucleotide substitution rate at defined positions in the
influenza virus haemagglutinin gene
P a u l a S u d r e z - L 6 p e z ~ and Juan Ortin*
Centro Nacional de Biotecnologia ( C S I C ) , Universidad Aut6noma de MadrM, Cantoblanco, 28049 Madrid, Spa#z
The mutation rates to a viable m a r (monoclonal
antibody-resistant mutant) genotype of wild-type influenza (A/Victoria/3/75; H3N2) virus or its mutator
variant strains have been previously determined. In
order to estimate the mutation rates per nucleotide
position, the sequence alterations present in 44 m a r
mutants isolated from either the wild-type or the rout43
mutator strain have been determined. These m a r mutants
were selected with either of two non-overlapping,
haemagglutinin-specific, monoclonal antibodies (2G10
and p7). Most of the protein changes were identified as
substitutions of large, charged amino acids for glycine
residues, as the result of G to A transitions. Particularly
interesting amino acid changes, not previously reported,
were observed in the p7 monoclonal antibody-specific
mutants, in which only Gly to Ser and Gly to Asp at
position 226 were detected. The identification of the
nucleotide substitutions responsible for the m a r phenotype has allowed the calculation of approximate values
for the total mutation rates at these positions.
The influenza viruses have been considered a paradigm
of a genetically and antigenically variable virus (for
reviews, see Holland et al., 1982; Palese & Young, 1982;
Webster et al., 1992). Their genome consists of eight
RNA segments of negative polarity, encoding a set of 10
proteins (Lamb, 1989), whose transcription and replication take place on ribonucleoprotein particles comprising the three subunits of the polymerase (PB1, PB2
and PA) and the nucleoprotein (Krug et al., 1989). The
genetic heterogeneity of the influenza viruses is a general
property affecting every one of the viral genes, albeit to
different extents (Ortin et al., 1980; Young et al., 1979).
This is reflected in a general phenotypic diversity which
includes not only the antigenic characteristics of the virus
(Air et al., 1985; Air & Laver, 1986; Van Wyke et al.,
1980; Wiley & Skehel, 1987; Wiley et al., 1981), but also
the receptor binding specificity (Rogers et al., 1983;
Underwood et al., 1987) and viral pathogenicity (Klenk
& Rott, 1988), among other properties. The frequency of
mutants in virus populations has been estimated by
several means: Fields & Winter (1981) described sequence heterogeneity among cDNA clones of influenza
virus genes; the proportion of mutants resistant to
haemagglutinin (HA)-specific monoclonal antibodies
(MAbs) (mar mutants) in virus populations has been
reported (Lubeck et al., 1980; Portner et aI., 1980;
Yewdell et al., 1979), as well as the frequency of
temperature-sensitive mutants in wild-type (wt) virus
stocks (Oxford et al., 1980; Sugiura et al., 1972).
Therefore, the influenza viruses conform to the quasispecies model for a biological population (Eigen &
Schuster, 1979), i.e. each virus stock, even those recently
cloned, becomes extremely heterogeneous and is able to
evolve rapidly to adapt itself to a change in the
environmental conditions (for reviews, see Domingo et
al., 1985; Holland et al., 1992).
An important factor in this context is the mutation
rate of the virus, i.e. the frequency at which nucleotide
changes are introduced in the course of RNA replication.
The average value for viable mutation rate at any
position in the NS segment of influenza virus was
determined by random sequencing of viral clones (Parvin
et al., 1986) and the mutation rate to the mar genotype
at two independent epitopes of HA was estimated by the
fluctuation test (Valc~ircel & Ortin, 1989). Although not
providing mutation rates per nucleotide position, the
latter approach allowed the comparison of different virus
clones and hence was instrumental in the isolation of
influenza virus mutator strains, i.e. mutants with a
mutation rate higher than that of the wt virus (Su~irez et
al., 1992). In this report we describe the characterization
of mar mutants obtained in either the wt or the mutator
genetic background and use this information to evaluate
the viral mutation rates at certain nucleotide positions of
the HA gene.
In the course of the determination of mutation rates of
wt A/Victoria/3/75 (H3N2) influenza virus (VIC) and
mutator mutant rout43, a number of mar mutants specific
]" Present address: Centro de BiologfaMolecular (CSIC-UAM),
Universidad Autdnoma de Madrid, Cantoblanco, 28049 Madrid,
Spain.
0001-1975 © 1994 SGM
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390
Short communication
for MABs 2G10 and p7 were obtained (Smirez et al.,
1992). In order to identify the epitopes recognized by
these MAbs, the sequence of the HA1 gene region of
several mutants was determined. Three overlapping PCR
products were generated by reverse transcriptase-PCR
using virion RNA as template and three sets of
oligonucleotides: HA 1.20 (5' AGCAAAAGCAGGGGATAATT 3') plus HA466.447 (5' CCAGTCCAATTGAAGCCTTC 3"); HA365. 384 (5' CAACTGTTACCCTTATGATG 3') plus HA772. 753 (5' CTTATTCTACTAGACAGACC 3'); HA660.679 (5' CTATATGTTCAAGCATCAGG 3') plus HA1150.1131 (5' TTTTGATGCCTGAAACCGTA 3') (Fig. 1a). The amplification
reactions were carried out in duplicate and one of the
oligonucleotides used in each reaction was previously
phosphorylated. The amplified strand containing a
phosphorylated 5' end was eliminated by digestion with
exonuclease I as described (Tabor, 1987) and the
complementary strand was sequenced by the dideoxynucleotide method using Sequenase and the conditions
recommended by the supplier. In this way, the HA1 gene
regions of mar mutants G10Rll, G10R32, G10R45,
p7R8 and p7R20 were sequenced on both strands. The
nucleotide and amino acid changes observed are summarized in Fig. 1 (b and c). The mar mutants G10R11
and G10R32 showed a single nucleotide change (positions 483 and 517, respectively) leading to an amino acid
change at position 136 or 147. Mutant G10R45 contained two nucleotide substitutions at positions 505 and
507 which led to exchange of the amino acids at positions
143 and 144 (Fig. 1b). Therefore, the epitope recognized
by MAb 2G10 would include at least amino acids Gly
136, Gly 147, Gly 143 and/or Pro 144 and should be
considered to be within antigenic area A (Wiley et al.,
1981). On the other hand, p7R8 and p7R20 mar mutants
each contained a single nucleotide change (positions 753
and 754 respectively), causing an amino acid exchange at
position 226 (Fig. 1 c). The assignment of this epitope to
any of the antigenic areas of the HA is not clear,
although position 226 in X31 virus (which corresponds
to position 227 in the VIC strain) has been proposed to
belong to region D (Wiley et at., 1981).
Once the gene regions at which mar mutations
accumulated were determined, a collection of mar
mutants was studied by the same experimental strategy
but limiting the sequence determination to regions of
nucleotides 420 to 550 (for mar mutants selected with
MAb 2G10) and 660 to 820 (for mar mutants selected
with MAb p7). Each of the mar mutants in the collection
was isolated from an independent virus plaque, either wt
or rout43 mutator mutant, to avoid the selection of sister
mutants. The results obtained for 26 mar mutants
isolated from wt virus (16 2G10-specific and 10 p7specific) and 18 mar mutants obtained from the mut43
(a)
1
H A gene
500
1000
1500
I
466
HA1
I
466
365
HA365
772
772
i
I
XXy~
66~
HA660
!
1150
(b)
480
VIC
G10RII
G10R32
500
520
GGG GGA AGC AAT GCT TGC AAA J~T4& GGA CCT GAT ATC GGT TTT
A
A
A
G10R45
135
vlc
GIOR11
A
140
145
GIy GIy Set ASh Ala Cys LyS Arg Gly Pro Asp Ile Gly Phe
Arg
A~p
G10R32
G10R45
G I u Thr
(c)
750
VIC
TGG GTAAGG
pTR8
pTR20
760
G G T CTG TCT AGE AGA
A
A
225
VZC
230
T r p V a l Arg Gly Leu Set Set Arg
p7R8
p7R20
Asp
Ser
Fig. 1. Localization of the p7 and 2G10 epitopes in the HA molecule.
(a) Sections of the HA gene that were amplified from virion RNA as
indicated in the text. (b) The nucleotide sequence (region 480 to 521)
and the amino acid sequence (residues 135 to 148) of influenza virus
strain VIC are indicated. The nucleotides and amino acids indicated
below correspond to the changes detected in m a r mutants G10Rll,
G10R32 and G IOR45, respectively. (c) The nucleotide sequence (region
744 to 767) and the amino acid sequence (residues 223 to 230) of
influenza virus strain VIC are indicated. The nucleotides and amino
acids indicated below correspond to the changes detected in m a r
mutants p7R8 and p7R20, respectively.
Table 1. Nucleotide and amino acid changes detected in
mar mutants isolated f r o m influenza virus strain VIC
Mutant
G10Rll, G10R17, G10R20,
G10R22, G10R26, G10R30
G10R14
G 10R45
G10R13, G10RI6, G10R18.
GIOR27, G10R32, G10R35,
G10R37, G10R40
p7R9, p7R11, p7R20,
p7R21, p7R39, p7R42,
p7R43, p7R44
p7R8, p7R13
Nucleotide
change
Amino acid
change
G483A
Gly136Arg
G483A
G484A
G505A
C507A
G517A
Gly 136Lys
Gly 143Glu
Pro144Thr
Gly147Asp
G753A
Gly226Ser
G754A
Gly226Asp
mutator strain (10 2G10-specific and eight p7-specific)
are summarized in Tables 1 and 2. The spectrum of
mutations at the 2G10 epitope was not very different
from that previously observed (Fig. l b): only an
additional change at nucleotide 484 was detected in
mutant G10R14, together with a substitution at nucleo-
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Table 2. Nucleotide and amino acid changes d e t e c t e d in
mar m u t a n t s isolated f r o m mut43 m u t a t o r influenza
virus strain
Mutant
G10R48, G10R49, G10R55
G10R51,*I G10R53*'~
G1OR50
GIOR56,~?G10R57,1-G10R58~
G 10R47
p7R14, p7R16, p7R29, p7R36,
p7R45, p7R46, pTR47
p7R35
Nucleotide
change
Aminoacid
change
G483A
G484A
C507T
G516A
G517A
G753A
Gly136Arg
Gly136Glu
Pro144Ser
Gly147Ser
Gly147Asp
Gly226Ser
G754A
Gly226Asp
* These mutants showed, in addition, a G/A heterogeneity at
position 483.
t Thesemutantsshowedpartial resistanceto 2G10 MAb.
tide 483 (Table 1). For those m a r mutants obtained in the
mutator background the same assortment of changes
was observed, except for mutants G10R56, G10R57 and
G10R58, in which a substitution at nucleotide 516 was
detected. It should be mentioned, however, that these
mutants showed only partial resistance. In addition, a C
to T nucleotide substitution at position 507 was detected
in mutant G10R50 (Table 2). On the other hand, the
spectrum of mutations at the p7 epitope was identical to
that obtained for the sequencing of mutants p7R8 and
p7R20 (compare Fig. 1 c with Tables 1 and 2), although
no amino acid change could be detected in the region
sequenced in four of the mutants selected.
The distribution of mutations among the different
possible sites is not the same in the wt or the mutator
background (compare Table 1 with 2), but this difference
might not be significant since no special randomization
procedure was followed in the process of plaque
isolation.
The amino acid changes G136R, P144T, P144S,
G147D and G147S had been previously reported in
natural isolates or m a r mutants (for review, see Wiley &
Skehel, 1987). It should be mentioned that the numbering
for the VIC strain is different from that of X31 virus
owing to an amino acid insertion in the former. The rest
are described for the first time in this report.
The mutation G136E is present in only two mar
mutants isolated from the rout43 strain, is accompanied
by a heterogeneity in the preceding nucleotide position,
and exhibits only partial resistance to the antibody
(Table 2). However, the double nucleotide change leading
to the G136K mutation, present in mutant G10R14,
endows complete resistance to 2G10 antibody. On the
other hand, the mutation G143E is present in mutant
G10R45, which also contains the change P144T. Since
the latter has been already described in an H3 HA (Laver
et at., 1980), it is possible that the former is simply a
passenger mutation.
391
The mutations detected in epitope p7, G226S and
G226D, have never been reported previously in natural
isolates. However, a change (G225N) at the corresponding position in X31 virus HA was introduced by
site-directed mutagenesis with no apparent effect on the
binding of MAbs specific for the antigenic sites A, B, C
or D (Gallagher et al., 1988). Changes at the next amino
acid position (L226) have been reported (Wiley et al.,
1981) which affect the binding of MAbs (Moss et al.,
1980), the affinity of receptor binding (Daniels et al.,
1987; Rogers et al., 1983) and the pH of HA-mediated
cell fusion (Daniels et al., 1987). It would be interesting
to study the receptor binding and fusion properties ofp7specific mar mutants.
It is noteworthy that most of the mutations observed,
both in the wt and the mutator background, were
changes from a glycine to a different amino acid
(arginine, lysine, aspartic acid or glutamic acid or serine)
(Tables 1 and 2). These changes, which always involve
the substitution of a large amino acid for a very small
one, might be preferred because they dramatically alter
the epitope structure. In addition, other substitutions
might be excluded because of structural or functional
limitations in the HA molecule or because they would
not affect neutralization by the MAb. This result is
remarkable, in view of the fact that many other amino
acid residues have been altered in the HAs of natural
isolates and in m a r mutants (Wiley et al., 1981).
The limitation in the potential amino acid changes at
the 2G10 and p7 epitopes severely restricts the possibilities of using the m a r mutants described in this report as
probes for the analysis of the mutation spectrum of the
wt and mutator strains. Thus, almost all nucleotide
changes observed were G to A transitions, the probable
result, since glycine codons are GGX and transitions are
the most frequent nucleotide substitution mutations
(Topal & Fresco, 1976). The availability of a reverse
genetic approach in influenza viruses (Luytjes et al.,
1989) should open the way to the design of more
appropriate probes to attack this problem.
Having identified the nucleotide positions at which
m a r mutations accumulated in a large number of
mutants, an estimation could be made of the mutation
rate per nucleotide that led to a viable m a r genotype.
Among m a r mutants derived from the VIC strain, five
different nucleotide substitutions were identified in the
2G10 epitope and two in the p7 epitope. The mutation
G505A could be irrelevant for MAb resistance (see
above) and was not considered in the calculations.
Therefore, the average mutation rates per single nucleotide position that lead to a viable m a r genotype were
calculated as follows: the mutation rate to a viable m a r
genotype at the site defined by MAb 2G10 (4-20 × 10-5)
(Su~irez et al., 1992) was divided by the number of
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392
Short communication
Table 3. M u t a t i o n rates o f w t and m u t a t o r influenza viruses
Mutation rate to viable
m a r genotype*
Virus
wt
lnut43
Mutation rate per
nucleotide to viable m a r
genotypet
Total mutation rate per
nucleotide:~
2G10
p7
2G10
p7
2G10
p7
4-2 × 10-5
1"5 × 10-4
1"8 × 10-6
7"3 × 10-6
1-0 × 10-~
3"0 x 10-~
8'9 × 10-7
3'6 × 10-G
3"0 x 10 5
0"9 × 10 4
2.7 x 10.6
1.1 × 10-5
* As defined in Sufirez et al. (1992).
"~ Average value determined by division of the mutation rate to viable m a r genotype by the
number of nucleotide positions at which mutation led to the m a r genotype.
:~ Values obtained assuming that the alternative, unobserved changes at the positions considered
do occur at the same rate but do not lead to a viable m a r genotype.
nucleotide positions (four) at which mutations led to the
m a r mutants and that they occur as frequently as those
m a r genotype, giving a value of 1.0 x 10-5. Likewise, a
observed. The last conjecture is unlikely, however, in
view of the fact that only G to A transitions were
observed and transversions are not as widespread.
Therefore, maximal mutation rates could be estimated at
approximately 3.0 x 10 5 and 2.7 x 10 G mutations per
nucleotide and replication round for epitopes 2G10 and
p7, respectively, for the VIC strain. These values are
similar to those reported for the average rate of mutation
to viable clones in the influenza virus NS gene (Parvin et
al., 1986) or those determined for retroviruses (Dougherty & Temin, 1988), but are much smaller than the
values reported for Qfl bacteriophage (Batschelet et al.,
1976) or vesicular stomatitis virus (Steinhauer et al.,
1989). This is not surprising, however, since different
viruses and sequence positions have been studied.
Likewise, values of 0.9 x 10 4 and 1.1 x 10 5 could be
estimated for the m u t 4 3 mutator strain at the nucleotide
positions analysed (Table 3).
In conclusion, the characterization of the mutations
selected in a large number of m a r mutants for two
independent antibodies specific for the influenza virus
H A molecule has allowed the identification of antigenically relevant mutations not previously reported.
Surprisingly, most changes detected were substitutions of
large, charged amino acids for glycine residues. Furthermore, the determination of the number and nature of
the nucleotide substitutions leading to the m a r genotypes
has allowed the calculation of approximate values for the
total mutation rates at those positions.
value of 8.9 x 10 7 (1.79 x 10 G/2) was obtained for the p7
epitope (Table 3). Using the same rationale, the values
for the mutation rate per nucleotide that led to a viable
m a r genotype in the m u t 4 3 mutator strain were calculated
and are summarized in Table 3. The possibility cannot be
excluded that, upon sequencing of a larger collection of
m a r mutants, mutations at additional nucleotide positions leading to the m a r genotype would be identified.
Therefore, the values described above should be considered as approximate maximal mutation rates.
Some of the m a r mutants sequenced showed either two
mutations or a sequence heterogeneity at a position other
than that mutated. This is not surprising in view of the
quasispecies nature of viral populations (Domingo et al.,
1985). With the calculated mutation rates, the frequency
of single mutants in the virus stock from which the m a r
mutants were isolated would be consistent with the
detection of two additional mutations in a total of
approximately 11000 nucleotides. Although the distribution of mutations among the different mutable
positions is not uniform (Tables 1 and 2), no attempt was
made to calculate the mutation rates per nucleotide at
each position, since there was no assurance that the
process of plaque isolation was really random. The
values obtained suggest that the differences in the
mutation rate at the two epitopes cannot be attributed
merely to the number of mutable positions yielding
viable m a r mutants but rather that the nucleotide
sequence context at the 2G10 epitope is more errorprone than that at the p7 region.
Using the rates of mutation to a viable m a r genotype
estimated above, the total (not only the viable) mutation
rates at the nucleotide positions can be calculated; since
at those positions only one of the three possible
nucleotide substitutions was observed, the total mutation
rates would be three times higher than the viable
mutation rates. This conclusion is based on the assumption that the other two possible changes at the
positions analysed occur but are not detected in viable
We thank L. Enjuanes and J. A. Melero for their helpful suggestions
on the manuscript and J. A. Melero for a gift of p7 MAb. The technical
assistance of A. Beloso, J. Rebelles, F. Ocafia, A. Lumbreras and
V. Blanco is gratefully acknowledged. P. Su~irez-Ldpez was a predoctoral fellow of Plan Nacional de Formacidn de Personal Investigador. This work was supported by Comisi6n Interministerial de
Ciencia y Tecnologia (grant BIO92-1044).
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(Received 21 July 1993; Accepted 17 September 19931)
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