JOURNAL OF VIROLOGY, Sept. 2010, p. 8765–8776
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 17
Tyrosines in the Influenza A Virus M2 Protein Cytoplasmic Tail Are
Critical for Production of Infectious Virus Particles?
Michael L. Grantham,1Shaun M. Stewart,1,2Erin N. Lalime,1and Andrew Pekosz1*
W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University, Bloomberg School of
Public Health, 615 North Wolfe Street, Suite 5132, Baltimore, Maryland 21205,1and Division of Biology and Biomedical Sciences,
Washington University in Saint Louis, Campus Box 8226, 660 South Euclid Avenue, Saint Louis, Missouri 631102
Received 21 April 2010/Accepted 11 June 2010
The cytoplasmic tail of the influenza A virus M2 protein is required for the production of infectious virions.
In this study, critical residues in the M2 cytoplasmic tail were identified by single-alanine scanning mutagen-
esis. The tyrosine residue at position 76, which is conserved in >99% of influenza virus strains sequenced to
date, was identified as being critical for the formation of infectious virus particles using both reverse genetics
and a protein trans-complementation assay. Recombinant viruses encoding M2 with the Y76A mutation
demonstrated replication defects in MDCK cells as well as in primary differentiated airway epithelial cell
cultures, defects in the formation of filamentous virus particles, and reduced packaging of nucleoprotein into
virus particles. These defects could all be overcome by a mutation of serine to tyrosine at position 71 of the M2
cytoplasmic tail, which emerged after blind passage of viruses containing the Y76A mutation. These data
confirm and extend our understanding of the significance of the M2 protein for infectious virus particle
Influenza A virus is a member of the Orthomyxoviridae and
contains a segmented, negative-sense RNA genome that codes
for 10 or 11 proteins (10). The integral membrane protein M2
is the viral ion channel protein and is required during virus
entry (23) as well as for the production of infectious virus
particles (8, 11, 12). The M2 protein forms a disulfide-bonded
tetramer and contains 97 amino acids, 54 of which are pre-
dicted to be in the cytoplasmic tail of the protein (5, 9).
After endocytosis of a virus particle, the M2 protein in the
envelope of the virion is believed to shuttle hydrogen ions from
the lumen of the endosome into the interior of the virion. This
disrupts interactions between viral proteins and allows for the
dissociation of the viral ribonucleoprotein complexes (vRNPs)
from the site of virus-endosome membrane fusion. The re-
leased vRNPs can then associate with the nuclear transport
machinery and translocate to the nucleus, where RNA repli-
cation occurs (10).
During virus assembly, sequences in the M2 cytoplasmic tail
are required for incorporation of nucleoprotein (NP) and
vRNPs into progeny virions. When the M2 cytoplasmic tail is
truncated by 28 amino acids, progeny virions contain reduced
amounts of NP, reduced amounts of some viral RNA (vRNA)
segments, and drastically reduced infectivity (12). Using a
trans-complementation system consisting of a functionally M2-
null virus and cell lines that stably express M2 proteins con-
taining four adjacent alanine substitutions, the region of the
M2 cytoplasmic tail important for infectious virus production
was narrowed to an 8-amino-acid region (residues 70 to 77) or
a 4-amino-acid region (residues 74 to 77), depending on the
strain of virus used (11). Consistent with previous experiments
using truncated variants of M2, the progeny virions that con-
tained M2 with scanning alanine mutations in these regions
also exhibited reduced incorporation of NP and vRNA. To-
gether, these data suggested that one or more of the amino
acids from residues 70 to 77 of the M2 cytoplasmic tail were
required for incorporation of RNPs into progeny virions and
therefore required for infectivity.
Using single-alanine scanning mutagenesis, an individual
amino acid (tyrosine 76) was identified as critical for the pro-
duction of infectious virus and the incorporation of NP into
progeny virions. A revertant virus encoding a tyrosine for
serine substitution at position 71 of the M2 cytoplasmic tail was
identified which was able to restore the infectivity of recombi-
nant viruses encoding Y76A, reaffirming the importance of the
M2 cytoplasmic tail during the production of infectious influ-
enza virus particles.
MATERIALS AND METHODS
Plasmids and mutagenesis. Mutagenesis of the M2 cDNA was achieved
through overlap PCR and ligation of the product into the vector pCAGGS (18).
Individual codons were changed to create single amino acid substitutions at each
position from 70 to 77. The plasmids pHH21-M-Udorn-M2Y76A (where
M2Y76A is the M2 protein with the Y76A mutation) and pHH21-M-WSN-
M2Y76A were generated by site-directed mutagenesis using the plasmids
pHH21 M-Udorn and pHH21 M-WSN (16, 23), respectively, as a template.
Primer sequences are available upon request. The open reading frames (ORFs)
of all plasmids were confirmed by sequencing.
Cells. Madin-Darby canine kidney (MDCK) cells and human embryonic kid-
ney cells (293T) were cultured in Dulbecco’s modified Eagle medium (DMEM;
Sigma) containing 10% fetal bovine serum (Atlanta Biologicals, Inc.), 100 U of
penicillin/ml (Invitrogen), 100 ?g of streptomycin/ml (Invitrogen), and 1 mM
sodium pyruvate (Sigma) at 37°C and 5% CO2.
All cell lines that stably express M2 or M2 mutants were cultured in medium
identical to that used for wild-type (wt) MDCK cells except that it was supple-
mented with puromycin (7.5 ?g/ml; Sigma) and amantadine HCl (5 ?M; Sigma).
Stable cell lines expressing M2 were generated by cotransfecting MDCK cells
* Corresponding author. Mailing address: W. Harry Feinstone De-
partment of Molecular Microbiology and Immunology, Johns Hopkins
University, Bloomberg School of Public Health, 615 North Wolfe St.,
Suite 5132, Baltimore, MD 21205. Phone: (410) 502-9306. Fax: (410)
955-0105. E-mail: email@example.com.
?Published ahead of print on 23 June 2010.
with plasmids expressing a puromycin resistance gene (pBABE) (13) and an M2
cDNA expression vector in six-well plates. Two days posttransfection, the cells
were trypsinized, placed under puromycin selection (7.5 ?g/ml), and cloned
either by limiting dilution or by plating cells in a 100-mm dish and picking
individual colonies. Cells were screened for M2 expression either by Western
blot analysis of whole-cell lysates or by indirect immunofluorescence of live cells
for cell surface-expressed M2, followed by detection of positive clones with a
fluorescent plate reader.
Viruses. Wild-type viruses were propagated on MDCK cells in DMEM con-
taining 4 ?g/ml N-acetyltrypsin (Sigma), 100 U of penicillin/ml, 100 ?g of strep-
tomycin/ml, and 0.3% bovine serum albumin (BSA; Calbiochem or Sigma). The
wild-type viruses used in this study were rUdorn (a recombinant virus derived
from A/Udorn/72) and rWSN (a recombinant virus derived from A/WSN/33) and
have been described previously (16, 23).
Three of the functionally M2-null viruses (rUdorn M2Stop, rWSN M2Stop,
and rWSN M1Udorn M2Stop) have been described previously (11). rUdorn
M1WSN M2Stop virus contains seven segments from the A/Udorn/72 strain,
segment 7 from the A/WSN/33 strain, and stop codons at residues 25 and 26 of
the M2 ORF. All of the functionally M2-null viruses were propagated on MDCK
cells that express an amantadine-sensitive mutant of WSN M2 (M2WSN-N31S).
The recombinant viruses that encode amino acid substitutions in M2 were gen-
erated by replacing the plasmid that codes for the wild-type M segment with ones
that encode M2 with the desired mutation in the 12-plasmid reverse genetics
system (23). Viruses were rescued as described previously (12, 23) with the
exception that transfected cells were cocultured with M2WSN-N31S-expressing
MDCK cells (MDCK-M2WSN-N31S cells) when the resultant virus was sus-
pected to have a defect in replication. Viruses were plaque purified on MDCK-
M2WSN-N31S cells, and the coding region of the M segment was sequenced to
ensure the presence of the desired mutations and the absence of any spurious
Infection of tissue culture cells. Low-multiplicity growth curves were carried
out in triplicate by infecting confluent cell monolayers in 24-well plates at a
multiplicity of infection (MOI) of 0.001 50% tissue culture infective doses
(TCID50) per cell. Cells were incubated with virus diluted in DMEM containing
4 ?g/ml N-acetyltrypsin, penicillin, streptomycin, and 0.3% BSA for 1 h at room
temperature, the inoculum was aspirated, and the medium was replaced. At the
times indicated in the figures, the supernatant was removed and stored at ?80°C.
The amount of infectious virus in each sample was determined by TCID50assay
as previously described (12) using MDCK cells for samples that contain wild-type
viruses and MDCK-M2WSN-N31S-expressing cells for viruses encoding mutated
High-multiplicity infections were carried out in T75 flasks (MOI of 3) as
described previously (3). Cells were incubated with virus diluted in DMEM
containing penicillin and streptomycin at room temperature for 1 h. The inoc-
ulum was removed, and the cells were washed three times with phosphate-
buffered saline (PBS), DMEM containing penicillin and streptomycin was added,
and the flasks were incubated at 37°C in 5% CO2for 15 h. Supernatants were
centrifuged at low speed to remove cell debris. Virus particles were pelleted
through a 20% sucrose solution in a Sorvall TH641 rotor at 118,000 ? g for 1 h
at 4°C. The pellets were resuspended in PBS, an aliquot was removed for
determination of infectious titer, and 4? SDS-PAGE loading buffer was added
to the remaining sample for Western blot analysis.
Western blotting. Samples were analyzed by Western blot analysis as described
previously (3, 12). Polypeptides were separated by SDS-PAGE and transferred to
polyvinylidene fluoride membrane (Immobilon-FL; Millipore). All antibodies
were diluted in PBS with 5% skim milk powder and 0.3% Tween 20. The
antibodies used in this study were 14c2 (anti-M2 monoclonal antibody at 1:1,000)
(25) and anti-A/Udorn/72 virus goat serum (1:500) (26). Antibodies were de-
tected with species-specific secondary antibodies conjugated to Alexa Fluor 647
(1:500; Invitrogen), and the blots were imaged using an FLA-5000 phosphorim-
ager (FujiFilm). Bands were quantitated using Multi Gauge software, version 3.0
(FujiFilm), and the amount of each protein was determined relative to the
amount of hemagglutinin (HA) in the sample.
Immunofluorescence microscopy. MDCK cells were infected and stained for
immunofluorescence microscopy as described previously (3). MDCK cells were
grown on tissue culture-treated glass coverslips and infected on the fifth day after
reaching confluence with 5?105TCID50per coverslip. At 15 h postinfection
(hpi) the cells were placed on ice and incubated with goat anti-H3 serum raised
against HA from A/Aichi/2/68 (1:500; V-314-591-157; National Institute of Al-
lergy and Infectious Diseases), followed by donkey anti-goat IgG conjugated with
Alexa Fluor 555 (1:500; Invitrogen). All antibodies were diluted in DMEM with
5% fetal bovine serum. Cells were fixed with 2% paraformaldehyde, and cover-
slips were mounted using ProLong Gold antifade reagent (Invitrogen).
Samples were imaged with a Nikon Eclipse 90i microscope. In each experi-
ment, images were taken of 10 nonoverlapping, adjacent fields of view (magni-
fication of ?20). Infected cells were then scored for whether they showed fila-
ments on their cell surfaces, and the percentage of infected cells with filaments
in each field of view was calculated.
YFP fluorescence assay. M2 proton channel activity was tested using a previ-
ously published method (15) with modifications. Briefly, the pH-dependent flu-
orescence of yellow fluorescent protein (YFP) was used as an indicator of
cytoplasmic pH (22). Plasmids encoding cDNA constructs for YFP and M2 were
cotransfected into 293T cells. The next day, the cells were detached from the
plate, resuspended in neutral (pH 7.4) or acidic buffer (pH 5.5), and immediately
analyzed by flow cytometry. Fluorescence readings of 10,000 cells were taken
every 15 s. The percent change in the mean fluorescence intensity (MFI) of each
sample was then plotted versus time. The ion channel activity was blocked with
the addition of amantadine (50 ?M) to the medium at approximately 6 h post-
transfection, and amantadine was included during the analysis by flow cytometry.
Generation and infection of primary mTEC cultures. Primary mouse trachea
epithelial cell (mTEC) cultures were generated and infected as described previ-
ously (3, 17, 21). Cultures were infected with 3,300 TCID50/well, and at the times
indicated in the figures, the apical supernatant in each well was harvested and
stored at ?80°C. The amount of infectious virus in each sample was determined
as described above for experiments using MDCK cells. The basolateral medium
in each well was not harvested but was replaced with fresh medium every 2 days.
Statistical analysis. Low-multiplicity growth curves were compared to wild-
type control curves using two-way analysis of variance (ANOVA) with repeated
measures. Western blot analyses, infectious virus production from high-multi-
plicity infections, and the percentage of infected cells with filaments were com-
pared using t tests. All statistical analyses were done using Prism, version 4.0
(GraphPad Software Inc.).
Generation of cell lines stably expressing M2 mutants. Pre-
vious work indicated that a region from amino acid 70 to 77
was important for the production of infectious virus of the
Udorn strain (A/Udorn/72). A smaller set of amino acids
within this region (amino acids 74 to 77) was also important for
the production of infectious virus of the WSN strain (A/WSN/
33) (11). To identify individual key amino acids within this
region a trans-complementation system was used. Single amino
acid substitutions were made in the Udorn M2 of a cDNA
expression vector, and stable cell lines were established for
The cell lines were characterized for total M2 expression by
Western blot analysis using an anti-M2 monoclonal antibody
(Fig. 1A and B). Infectious virus production requires minimal
amounts of M2 expression (12), and although expression levels
showed some variation, all cell lines expressed more than the
threshold amount required. Furthermore, flow cytometric
analysis indicated that for each cell line, more than 90% of the
cells exhibited M2 expression on the cell surface (data not
shown). M2 oligomerization was examined by Western blot
analysis carried out under nonreducing conditions (Fig. 1C and
D). Each of the scanning alanine mutants was able to form
disulfide-linked dimers and tetramers to an extent that was
similar to wild-type M2.
Complementation of viruses that do not express M2. Amino
acids 70 to 73 in the M2 cytoplasmic tail were critical for
supporting infectious virus production of rUdorn M2Stop but
not rWSN M2Stop (11). Cell lines expressing Udorn M2 pro-
teins in which amino acids 70 to 73 were mutated individually
to alanine were used for low-MOI growth curves with rUdorn
M2Stop or rWSN M2Stop (MOI of 0.001). Supernatants from
cell lines expressing wild-type M2 and infected with rUdorn
M2Stop contained more than 1 ? 107TCID50/ml of infectious
8766 GRANTHAM ET AL.J. VIROL.
virus by 2 days postinfection (dpi), while cells that did not
express M2 produced little (if any) detectable virus (Fig. 2A).
Mutation of amino acid 70 had no adverse effect on the
amount of infectious virus produced, while mutation of amino
acid 71, 72, or 73 reduced infectious virus production (Fig. 2A).
The mutation that had the greatest effect was the M72A sub-
stitution. At 2 dpi, cells expressing this mutant produced
greater than 1,000-fold less infectious virus than cells that
expressed wild-type M2. When these cell lines were infected
with rWSN M2Stop, none of mutations between amino acids
70 and 73 had a statistically significant effect on the amount of
infectious virus produced. This result is consistent with previ-
ous experiments examining strain-specific effects on infectious
virus production of mutations in this region of the M2 protein
M2 amino acids 74 to 77 were shown to be required for
infectious virus production in a strain-independent manner
(12). Alanine substitutions at positions 75, 76, and 77 resulted
in significant decreases in infectious virus production com-
pared to wild-type M2 after infection with rUdorn M2Stop
(Fig. 2C). The reduction was greatest with a Y76A substitu-
tion, leading to an almost complete loss in infectious virus
production. In contrast, an E74A substitution had no detect-
Similar results were observed after infection with rWSN
M2Stop (Fig. 2D), but the magnitude of the reduction in in-
fectious virus production was not as great as that seen after
infection with rUdorn M2Stop. Since the M2Y76A mutation
resulted in the most severe phenotype, subsequent work con-
centrated on this mutation.
To determine whether the genetic background of the mu-
tated M2 protein affects the complementation of M2Stop vi-
ruses, a cell line was generated that expresses the M2 protein
from A/WSN/33 in which tyrosine 76 was replaced with ala-
nine. In this case, the protein also contains a mutation which
confers amantadine sensitivity (substitution of asparagine to
serine at position 31) (4, 23) so that amantadine could be
added during routine cell culture to reduce the potentially
toxic effects of constitutive M2 expression. The surface expres-
sion and oligomerization of M2WSN-N31S/Y76A was con-
firmed and was similar to that of M2WSN-N31S (data not
shown). As shown in Fig. 2E and F, the phenotype of the
M2WSN-N31S/Y76A mutant was consistent with that seen
with M2Udorn-Y76A, indicating that the Y76A mutation had
a similar effect in either the Udorn- or WSN-derived M2 pro-
teins. When infected with rUdorn M2Stop, the cells produced
little infectious virus at any time point tested. When this cell
line was infected with rWSN M2Stop, more infectious virus
was produced than when cells were infected with rUdorn
M2Stop; however, the supernatants still contained less infec-
tious virus than supernatants from infected M2WSN-N31S-
Previous experiments suggest that at least some of the ob-
served differences in the M2 cytoplasmic tail sequence require-
ments between the Udorn and WSN strains of virus can be
mapped to the M1 protein (1, 11). MDCK-M2Udorn-Y76A
cells were infected either with a rUdorn M2Stop virus that
encodes the M1 protein from the WSN strain (rUdorn
M1WSN M2Stop) (Fig. 3A) or with an rWSN M2Stop virus
that encodes the M1 protein from the Udorn strain (rWSN
M1Udorn M2Stop) (Fig. 3B). Replacing the Udorn M1 pro-
tein with the WSN M1 protein led to increased virus replica-
FIG. 1. Expression of mutated M2 proteins in stably transfected MDCK cells. The expression of mutated M2 proteins was analyzed by Western
blotting of cell lysates from stably transfected MDCK cells under reducing (A and B) and nonreducing (C and D) conditions. Molecular mass
markers are indicated on the left of each blot, and antibodies to ?-actin served as loading controls.
VOL. 84, 2010INFLUENZA A VIRUS M2 AND INFECTIOUS VIRUS PRODUCTION8767
tion (compare Fig. 3A and 2E), while replacing the WSN M1
protein with the Udorn M1 protein reduced infectious virus
production (compare Fig. 3B and 2F). These data indicate that
the extent of the defect in infectious virus production for the
M2Y76A mutant is dependent upon sequences present within
the M1 protein.
Ion channel activity of M2Y76A. Previous data indicated
that many mutations in the cytoplasmic tail of the M2 protein
(e.g., truncation of M2) had no deleterious effect on ion chan-
nel activity (24). In order to determine if the M2Y76A protein
displayed altered ion channel activity, changes in YFP fluores-
cence induced by cytosolic pH fluxes were assessed.
FIG. 2. Single alanine substitutions in the M2 cytoplasmic tail alter the production of infectious virus particles. MDCK cells stably transfected
with M2Udorn proteins containing single alanine substitutions at amino acids 70 to 73 (A and B) or 74 to 77 (C and D) or with amantadine-
sensitive (N31S) M2WSN proteins encoding either Y or A at position 76 (E and F) were infected with the indicated M2Stop viruses at an MOI
of 0.001, and infectious virus titers in the cell supernatants were quantified at the indicated times postinfection. The finely dotted lines indicate the
limits of detection, and asterisks indicate statistically significant differences compared to wild-type control curves (**, P ? 0.01;***, P ? 0.001).
8768 GRANTHAM ET AL.J. VIROL.
As shown in Fig. 4A, exposure of cells expressing the M2
protein to low pH resulted in a decrease in the MFI over time,
reaching a maximum within 7.5 min. This decreased MFI was
not seen in the absence of M2 expression. The decrease in MFI
was not as great in M2-expressing cells treated with amanta-
dine, indicating that the effect was specific to the M2 ion
channel activity. When the ion channel activity of M2Y76A
was tested (Fig. 4B), the change in MFI was pH dependent,
and the magnitude of the change in MFI was indistinguishable
from that of wt M2 Udorn, indicating that there is no discern-
ible defect in the ion channel activity of M2Y76A compared to
wt M2, and therefore the loss of infectious virus production
due to this mutation was not the result of defective ion channel
Generation of M2Y76A viruses. To verify the effect of
M2Y76A that was observed in the trans-complementation sys-
tem, the mutation was introduced into the genomes of both the
Udorn and WSN strains by reverse genetics, and the resulting
viruses were propagated on cells that express wild-type M2.
MDCK cells were infected at a low multiplicity, and the
amount of infectious virus in infected cell supernatants was
determined (Fig. 5A and B). Consistent with the results from
complementation assays, viruses carrying the M2Y76A muta-
tion had a statistically significant reduction of infectious virus
production in MDCK cells. The defect seen in rUdorn-
M2Y76A was particularly severe since infectious virus was not
detected at any of the time points tested. The rWSN-M2Y76A
virus replicated better than rUdorn-M2Y76A, and the extent
of attenuation was on the order of that seen when M2WSN-
N31S/Y76A-expressing cells were used to complement the rep-
lication of rWSN M2Stop (Fig. 2F).
To verify that the defect in infectious virus production was
due to the introduction of the M2Y76A mutation and not to
the presence of an unidentified spontaneous mutation else-
where in the virus genome, growth of the recombinant viruses
was examined on cells that express M2WSN-N31S (Fig. 5C and
D). For both virus strains, the M2Y76A mutant replicated as
well as the wild-type control on the M2-complementing cell
line, indicating that the defect in infectious virus production
for both of these mutants was due solely to the presence of the
M2Y76A mutation in the protein and was not a result of
changes in the genomic RNA that may have disrupted RNA
Tyrosine 76 is required for incorporation of NP into virus
particles. To determine whether the Y76A mutation affected
the incorporation of proteins into progeny virus particles,
MDCK cells were infected (MOI of 3) in the absence of tryp-
FIG. 3. The M1 protein is a viral determinant that controls the magnitude of the defect in infectious virus production produced by M2Y76A.
MDCK cells expressing wt M2Udorn or M2Udorn-Y76A were infected with rUdorn M1WSN M2Stop (A) or rWSN M1Udorn M2Stop (B) at an
MOI of 0.001, and infectious virus production was assessed by TCID50assay at the indicated times postinfection. The dotted lines indicate the limits
of detection, and asterisks indicate statistically significant differences compared to wild-type control curves (***, P ? 0.001).
FIG. 4. Effect of the Y76A mutation on the M2 protein ion channel
activity. 293T cells were transiently transfected with plasmids encoding
the indicated proteins along with a plasmid encoding YFP. (A) The
transfected cells were incubated with or without 50 ?M amantadine
and then exposed to a pH 5.5 solution. Changes in YFP fluorescence
due to the altered pH of the cytoplasm were monitored by flow cy-
tometry. (B) The pH-dependent changes in YFP fluorescence were
measured by flow cytometry after the transiently transfected 293T cells
were incubated with a solution at either pH 7.4 or pH 5.5.
VOL. 84, 2010INFLUENZA A VIRUS M2 AND INFECTIOUS VIRUS PRODUCTION8769
sin, and the supernatant was collected at 15 hpi. Virus particles
were concentrated by ultracentrifugation, and Western blot
analysis was used to examine the viral structural proteins.
The amount of HA did not vary greatly between the rUdorn
and rUdorn-M2Y76A viruses, suggesting that there was no
defect in the total amount of virus particles produced (Fig.
6A). The amount of infectious virus was reduced by more than
100-fold, indicating that the rUdorn-M2Y76A virus was capa-
ble of producing virus particles, but these particles were less
infectious than those of rUdorn.
The amounts of NP, M1, and M2 shown in Fig. 6A were
quantified after normalization to total HA (Table 1). Although
there was a tendency for less M1 to be incorporated into
rUdorn-M2Y76A particles, this difference was not always sta-
tistically significant between experiments. Both NP and M2
were seen as doublet bands in Western blot analyses. This may
be due to the cleavage of these two proteins by cellular
caspases, as described previously (27). The slower-migrating
form of NP (representing full-length NP, which we designated
NPa) was present at 2.6-fold-lower levels (P ? 0.05) in the
rUdorn-M2Y76A virus particles than in rUdorn virus. Further-
more, the total amount of NP incorporated into rUdorn-
M2Y76A virus particles was less than half the amount incor-
porated into wild-type virus particles. The total amount of M2
incorporated was similar, indicating that the change in virus
infectivity was not a result of reduced incorporation of the
M2Y76A protein into virions. Viral proteins, in particular
NPa, were present at equivalent amounts in virus-infected cell
lysates (Fig. 6A), suggesting that the packaging, not the ab-
sence of NP, was the limiting factor in rUdorn-M2Y76A-in-
To determine if expression of M2Y76A in trans could alter
NP protein packaging, MDCK cells expressing either wild-type
M2 or M2Udorn-Y76A were infected with rUdorn M2Stop in
the absence of trypsin. At 15 hpi, the supernatants were har-
vested and analyzed for viral protein incorporation and infec-
tious virus titer. However, virus particles containing M2Y76A
still packaged nearly 2-fold less NPa than virus particles com-
plemented with wild-type M2 (Fig. 6C and Table 1). Consistent
with the reduced incorporation of NPa, the amount of infec-
tious virus produced by the cells expressing M2Y76A was 10-
fold less than that produced by cells expressing wild-type M2
(Fig. 6D). Interestingly, rUdorn M2Stop virus particles har-
vested from MDCK cells also displayed reduced incorporation
of NPa, indicating that the absence of M2 also led to altered
incorporation of NPa. In contrast to the data with recombinant
viruses, the levels of M1 protein incorporation into rUdorn
M2Stop viruses grown on M2Y76A-expressing cells was con-
sistently less than that observed in virions purified from cells
expressing wild-type M2 (Table 1). These data suggest that
incorporation of large amounts of M2Y76A is not sufficient to
overcome the defect in the production of infectious rUdorn-
To determine whether wild-type M2 could complement the
FIG. 5. Recombinant viruses expressing M2Y76A have reduced replication kinetics in MDCK cells. MDCK cells (A and B) or MDCK-
M2WSN-N31S cells (C and D) were infected at a low MOI with recombinant viruses in the rUdorn (A and C) or rWSN (B and D) genetic
backgrounds. Infectious virus titers were determined at the indicated times postinfection. The dotted lines indicate the limits of detection, and
asterisks indicate statistically significant differences compared to wild-type control curves (***, P ? 0.001).
8770GRANTHAM ET AL. J. VIROL.
defect in NPa incorporation of rUdorn-M2Y76A, virus was
grown on MDCK cells expressing M2Udorn (MDCK-
M2Udorn), and Western blot analysis was carried out as de-
scribed above (Fig. 6E). After normalization to the HA0 pro-
tein, there were no significant differences in the amounts of
viral proteins packaged into virions for either virus strain (Ta-
ble 1). Furthermore, there was a slight but significant increase
in the amount of infectious rUdorn-M2Y76A produced com-
FIG. 6. Mutations in the M2 protein cytoplasmic tail alter virion polypeptide composition. The indicated viruses were used to infect MDCK
cells at a high MOI. At 15 hpi, cell lysates were harvested for Western blotting (A), and virus particles purified from infected cell supernatants were
analyzed for polypeptide composition by Western blotting (A) and for infectious virus by TCID50assay (B). The indicated cells were infected with
rUdorn M2Stop virus at a high MOI for 15 h, and virus particles were purified from infected cell supernatants and analyzed for polypeptide
composition by Western blotting (C) and for infectious virus by TCID50assay (D). The indicated viruses were used to infect MDCK-M2Udorn
cells at a high MOI. At 15 hpi, virus particles were purified from infected cell supernatants and analyzed for polypeptide composition by Western
blotting (E) and for infectious virus by TCID50assay (F).**, P ? 0.01;***, P ? 0.001;****, P ? 0.0001.
VOL. 84, 2010INFLUENZA A VIRUS M2 AND INFECTIOUS VIRUS PRODUCTION8771
pared to wild-type (Fig. 6F). These data indicate that expres-
sion of wild-type M2 is able not only to complement the defect
in infectious virus production but also to restore the incorpo-
ration of NPa into virus particles.
Together, these data indicate that tyrosine 76 is required
for incorporation of NP into virions and to maintain infec-
tious virus titers. Specifically, only for NPa did the amount
of the protein consistently correlate with changes in infec-
Generation and characterization of an M2Y76A suppressor
mutant. To determine if suppressor mutations that could re-
store infectivity to rUdorn-M2Y76A viruses could be identi-
fied, MDCK cells were infected with rUdorn-M2Y76A. At 60
hpi with an MOI of 0.1, approximately 50% of the cells exhib-
ited cytopathic effect (CPE). The supernatant contained a low
level of infectious virus (4.6 ? 103TCID50/ml; referred to as
rUdorn-M2Y76AP1, where P1 is passage 1) that was used to
infect fresh MDCK cells (MOI of 0.001). At 48 hpi, 100% of
the cells exhibited CPE, and infectious virus titers in the su-
pernatant were substantially increased (rUdorn-M2Y76AP2;
3.2 ? 106TCID50/ml). The resulting virus was able to form
plaques on MDCK cells, unlike rUdorn-M2Y76A. The M-
segment coding region of five plaque-purified viruses was se-
quenced, and in addition to the Y76A mutation, a serine-to-
tyrosine mutation was present at position 71 of the M2 ORF of
each virus. Two plaque-purified viruses were expanded and
used in low-MOI growth curve experiments on MDCK cells
(Fig. 7A). Both viruses had growth kinetics and reached peak
titers that were nearly identical to those of the wild-type virus.
To determine whether the M2S71Y mutation was sufficient
to restore replication of rUdorn-M2Y76A, recombinant vi-
ruses containing the suppressor mutation were generated and
characterized. Virus containing either the M2S71Y mutation
alone or both S71Y and Y76A mutations (M2S71Y/Y76A)
were generated and analyzed by a low-MOI growth curve (Fig.
7B). M2S71Y-encoding viruses replicated to significantly
greater titers than the rUdorn M2Y76A virus, indicating that
the S71Y mutation alone could restore the replication of that
virus on MDCK cells. rUdorn-M2S71Y replicated slightly
faster than wild-type virus and produced about 1 log more
infectious virus at 1 dpi. rUdorn-M2S71Y/Y76A replicated
slightly more slowly than rUdorn and produced about 1 log less
infectious virus at 1 dpi.
The structural proteins packaged into virions were examined
following infection of MDCK cells at a high MOI, and viruses
encoding S71Y were indistinguishable from wild-type virus, indi-
cating that the packaging of NPa was restored in recombinant
viruses bearing this mutation (Fig. 7C; quantitation in Table 2).
As observed previously (Fig. 6), the amount of full-length NP
(NPa) incorporated into virus particles correlated with changes in
the amount of infectious virus (Fig. 7D) since the presence of the
S71Y mutation restored infectious virus titers to levels seen with
wild-type virus. Taken together, the data indicate that the S71Y
mutation can restore NP packaging and infectious virus produc-
tion in the presence of the Y76A mutation.
The M2S71Y mutation enhances filament production. Most
clinical isolates of influenza virus and some laboratory strains
(including rUdorn) are able to form filamentous virus particles
TABLE 1. Quantitation of viral proteins incorporated into influenza A virus particles
Amount of protein by cell linea
MDCK M2Udorn M2Udorn-Y76A
0.11 ? 0.02
0.050 ? 0.005*
0.10 ? 0.02
0.021 ? 0.003*
0.8 ? 0.1
0.5 ? 0.1**
0.026 ? 0.002
0.023 ? 0.002
0.014 ? 0.001
0.0086 ? 0.0009
0.079 ? 0.009*
0.034 ? 0.004**
0.9 ? 0.3*
0.10 ? 0.01
0.064 ? 0.008
1.4 ? 0.3
0.5 ? 0.1
0.3 ? 0.1
0.091 ? 0.007
0.036 ? 0.002*
0.35 ? 0.08**
0.39 ? 0.04
0.09 ? 0.02
6ETotal NP rUdorn
0.13 ? 0.03
0.10 ? 0.01
0.10 ? 0.02
0.074 ? 0.007
1.0 ? 0.3
0.6 ? 0.1
0.81 ? 0.01
0.7 ? 0.1
0.329 ? 0.009
0.21 ? 0.03
aVirus proteins were quantified, and amounts are expressed relative to the amount of HA protein. Three to six independent experiments were averaged, and the
standard error is shown. NA, not applicable; M2a, full-length M2; ?, P ? 0.05; ??, P ? 0.01.
8772GRANTHAM ET AL. J. VIROL.
in tissue culture (14, 19, 20). To determine whether the
M2Y76A and M2S71Y mutations affected the formation of
filamentous influenza, MDCK cells were infected and analyzed
by immunofluorescence microscopy. Qualitatively, cells in-
fected with rUdorn-M2Y76A produced filaments that ap-
peared shorter and less abundant on a per-cell basis than those
produced by rUdorn (Fig. 8A). However, rUdorn-M2S71Y
and rUdorn-M2S71Y/Y76A both produced filaments that were
very similar to those seen in rUdorn-infected cells. As shown in
Fig. 8B, rUdorn-M2Y76A infections produced detectable fil-
aments on the surface of a smaller proportion of infected cells
than rUdorn, rUdorn-M2S71Y, or rUdorn-S71Y/Y76A. These
data indicate that, along with a defect in NPa incorporation
into progeny virions, rUdorn-M2Y76A exhibits a defect in
filament formation on the surface of infected cells and that this
defect can be overcome by the M2S71Y mutation.
Replication in primary, differentiated mTEC cultures. To
determine if the Y76A and S71Y mutations could alter
influenza virus replication in respiratory epithelial cells, dif-
ferentiated mouse trachea epithelial cell (mTEC) cultures
were prepared as described previously (3, 17, 21) and in-
fected with rUdorn, rUdorn-M2Y76A, rUdorn-M2S71Y,
rUdorn-M2S71Y/Y76A, rWSN, and rWSN-M2Y76A. As
shown in Fig. 9, the phenotypes seen previously in MDCK
cells were reproduced in the primary mTEC cultures but to
a much greater extent. Cultures infected with rUdorn-
M2S71Y produced greater than 100-fold more infectious
virus on day 2 postinfection than cultures infected with
rUdorn, and peak virus titers occurred considerably earlier.
Cultures infected with rUdorn-M2S71Y/Y76A reached peak
titer at a similar time as cultures infected with rUdorn but
contained about 100-fold less infectious virus. However, su-
pernatants from rUdorn-M2S71Y/Y76A-infected cells still
contained at least 100-fold more infectious virus than rU-
dorn-M2Y76A-infected cultures. Similarly, the replication
defect of rWSN-M2Y76A was greater in mTEC cultures
(Fig. 9B) than in MDCK cells (Fig. 5B). Together, these
data indicate that the phenotypes seen in MDCK cells hold
true but are significantly more pronounced in primary,
differentiated airway epithelial cell cultures. These data con-
firm a central role for M2Y76A in infectious virus produc-
FIG. 7. Identification of a suppressor mutation for M2Y76A. (A) Supernatants from rUdorn-M2Y76A-infected MDCK cells were passaged
twice on MDCK cells (rUdorn-M2Y76AP2). Viruses from the resulting infected cell supernatants were plaqued on MDCK cells (pq2 and pq4)
and then used to infect MDCK cells at a low MOI. Infectious virus titers in infected cell supernatants were determined at the indicated times.
(B) Recombinant influenza viruses bearing the indicated mutations were used to infect MDCK cells at a low MOI. Infectious virus titers in the
infected cell supernatants were determined at the indicated times. The dotted lines indicate the limits of detection, and asterisks indicate
statistically significant differences compared to wild-type control curves. MDCK cells were infected at a high MOI, and virus particles were purified
from infected cell supernatants at 15 hpi and analyzed by Western blotting (C) or for infectious virus titer (D).*, P ? 0.05;**, P ? 0.01;***,
P ? 0.001.
VOL. 84, 2010 INFLUENZA A VIRUS M2 AND INFECTIOUS VIRUS PRODUCTION8773
The influenza A virus M2 protein plays a number of key
roles in the virus life cycle. The ion channel activity of the
protein is required during virus entry to allow proper release of
vRNPs from virions, and sequences in the cytoplasmic tail of
M2 are essential for the incorporation of NP and vRNPs into
progeny virions (11, 12, 23). The data described here indicate
that amino acid 76 of the M2 cytoplasmic tail is a key residue
that is required for incorporation of NP into virus particles and
for maintaining infectious virus production. Tyrosine 76 is
highly conserved among influenza A isolates, showing conser-
vation in 8,702 of 8,706 sequences examined (data not shown),
but it is not phosphorylated (6). Among the mutations exam-
ined, the M2Y76A mutation resulted in the greatest defect in
the production of infectious virus. The effect of this mutation
is largely independent of the genetic source of the M2 protein
since cells expressing M2Udorn-Y76A or M2WSN-N31S/
Y76A exhibited similar complementation activity in the
complementation system. This key residue falls within larger
mutations made in both the Udorn and WSN genetic back-
grounds in previous publications, and the replication defect
seen here is consistent with that seen in previous studies (2,
Disruption of the protein sequences between residues 70
and 77 led to a defect in the specific incorporation of NP and
vRNPs into progeny virions in the Udorn background, and
disruption of the sequence between residues 74 to 77 led to a
similar defect in the WSN background (11). Studies using
recombinant rWSN viruses carrying mutations in M2 demon-
strated a similar defect in NP incorporation when M2 was
truncated after residue 75 (8). In addition to important func-
tions of the M2 protein, an RNA sequence that is important
for replication has also been identified in this region. Synony-
mous mutations introduced into codons 71 and 73 resulted in
reduced replication of recombinant virus (7).
While there was a trend toward reduced total incorporation
of NP into the rUdorn-M2Y76A virus, the difference was not
always statistically significant. However, there was a significant
difference in the amount of the slower-migrating species of the
NP doublet (NPa) incorporated into rUdorn-M2Y76A. The
NPa protein most likely represents the full-length protein prior
to its cleavage by cellular caspases (27). In each of the systems
examined, a defect in the production of infectious virus was
concomitant with a defect in the specific incorporation of NPa
into progeny virions. This was demonstrated for rUdorn-
M2Y76A grown on MDCK cells and in experiments using the
M2 protein trans-complementation system. Furthermore, a
suppressor mutation in the M2 cytoplasmic tail restored infec-
tious virus production and NPa incorporation of rUdorn-
M2Y76A. However, it is not yet known how the abundance of
NPa affects virion infectivity. Incorporation of a full comple-
FIG. 8. Mutations at positions 71 and 76 of the M2 cytoplasmic tail
alter the production of filamentous virus particles. (A) MDCK cells
were infected at a high MOI with the indicated recombinant viruses
and analyzed for filamentous particle formation by immunofluores-
cence microscopy at 15 hpi. The images shown represent a decon-
volved series of images acquired with a 40? objective. (B) The number
of infected cells and infected cells expressing filamentous virus projec-
tions were quantified per field imaged (magnification, ?20) and are
expressed as follows: number of infected cells with filaments/number of
infected cells ? 100.*, P ? 0.05;***, P ? 0.001.
TABLE 2. Quantitation of viral proteins incorporated into
influenza A virus particles expressing M2 proteins with
an S71Y mutation
Amount of protein in
7CTotal NP rUdorn
0.20 ? 0.04
0.10 ? 0.02*
0.16 ? 0.03*
0.15 ? 0.02
0.16 ? 0.04
0.044 ? 0.008*
0.14 ? 0.03
0.12 ? 0.02
1.5 ? 0.4
0.8 ? 0.2
1.4 ? 0.3
1.2 ? 0.3
0.030 ? 0.005
0.018 ? 0.002
0.021 ? 0.006
0.017 ? 0.004
0.014 ? 0.003
0.007 ? 0.001
0.017 ? 0.006
0.008 ? 0.002
aVirus proteins were quantified, and amounts are expressed relative to the
amount of HA protein. Three or four independent experiments were averaged
and the standard error is shown. M2a, full-length M2; ?, P ? 0.05.
8774GRANTHAM ET AL.J. VIROL.
ment of vRNPs into progeny virions could require an interac-
tion (direct or indirect) between NPa and the region of the M2
cytoplasmic tail that is centered on residue 76.
The altered packaging of NPa in the presence of the
M2Y76A mutation is not the result of disruption of an RNA
packaging signal in the M segment of the genome since the
Y76A mutation can be complemented by wild-type M2 protein
expressed in trans. In addition, synonymous mutations made in
a previous study at positions 75 to 77 did not affect virus
replication, suggesting that these codons are not critical for
RNA packaging (7).
Chen et al. also performed scanning alanine mutagenesis of
the M2 cytoplasmic tail of the Udorn strain and made recom-
binant viruses of each mutant (2). Three consecutive alanine
substitutions were introduced, and the greatest replication de-
fect was found when residues 71 to 73 and 74 to 77 were
mutated, consistent with previously published data from our
group (11). However, Chen et al. concluded that the defect in
viruses with mutations from 71 to 73 and 74 to 77 was the result
of altered incorporation of M1 into virions, altered protein-
protein interactions between M1 and M2, and altered plasma
membrane localization of M1. In our studies, although there
was an apparent trend for decreased incorporation of M1 into
virus particles, the differences in M1 incorporation were not
always statistically significant. This does not rule out the pos-
sibility that the M2Y76A mutation alters an M1-M2 interac-
tion. In fact, the reduced specific incorporation of M1 into
rUdorn M2Stop grown on cells expressing M2Y76A could be
a result of overexpression of a variant of M2 with altered
M1-M2 interactions. Furthermore, the finding that the extent
of the defect in infectious virus production depends on the
source of the M1 protein in the virus could be interpreted as
further genetic evidence for functionally important M1-M2
interactions. Although the data in this study and the one from
Chen et al. appear to be contradictory in terms of the relative
importance of M1 and NP (specifically, NPa) incorporation
into virions, the differences are most likely due to the methods
used to quantitate Western blot analyses. Chen et al. normal-
ized the amount of protein in virions to the amount of M1 in
infected cell lysates, and this measures the effects of a given
mutation on the general process of virus budding. In this study,
similar amounts of HA in samples of rUdorn and rUdorn-
M2Y76A suggest that there was no major defect in virus bud-
ding. Therefore, the amount of protein in virions was normal-
ized to that of HA in the supernatants. This method accounts
for minor differences in general virus budding and quantifies
the specific incorporation of each viral protein.
A selection for mutations that suppress the phenotype of
rUdorn-M2Y76A yielded a virus that contained an M2S71Y
substitution after only two blind passages of the parental virus
in MDCK cells. Recombinant viruses containing this mutation
and the M2Y76A mutation exhibited nearly wild-type levels of
replication in MDCK cells and at least a 100-fold increase over
the rUdorn-M2Y76A peak titer on mTEC cultures. Virus con-
taining both M2 mutations contained levels of structural pro-
teins that were indistinguishable from those of wild-type virus,
while virus containing only the M2S71Y mutation replicated
slightly better on MDCK cells and considerably better on
mTEC cultures than wild-type virus.
In addition, the M2S71Y mutation led to a statistically sig-
nificant increase in the number of infected cells that exhibited
HA-positive, cell surface filaments. The significance of this
finding is not completely clear since it has been difficult to
correlate the mechanism of filament formation with that of the
assembly of infectious virus particles. However, increased fil-
ament formation may be a sign of more efficient virus assembly
in the presence of the M2S71Y mutation. Interestingly, an
identical mutation was identified in a screen for viruses that
were resistant to the growth-inhibitory effects of the anti-M2
monoclonal antibody 14c2. The resulting virus was also able to
form filaments in the presence of 14c2, unlike the parental
A/Udorn/72 (20). The mechanism by which that might occur is
a matter of investigation but is likely to lead to new insight into
the mechanisms required for the assembly of infectious influ-
enza A virus particles.
We acknowledge and thank the members of the Pekosz laboratory
for their comments and suggestions.
This study was supported by Public Health Service grants AI007417
(M.L.G.), AI061253 (A.P.), and AI053629 (A.P.) from the National
Institutes of Allergy and Infectious Diseases. A.P. also acknowledges
support from the Eliasberg Foundation and the Marjorie Gilbert
FIG. 9. Mutations at positions 71 and 76 of the M2 cytoplasmic tail
alter virus replication in mTEC cultures. The indicated recombinant
viruses in the rUdorn (A) or rWSN (B) genetic background were used
to infect mTEC cultures. Infectious virus titers in the apical washings
were determined at the indicated times postinfection. The dotted lines
indicate the limits of detection, and asterisks indicate statistically sig-
nificant differences compared to wild-type control curves.**, P ? 0.01;
***, P ? 0.001.
VOL. 84, 2010INFLUENZA A VIRUS M2 AND INFECTIOUS VIRUS PRODUCTION8775
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