The Influenza C Virus CM2 Protein Can Alter Intracellular pH, and
Its Transmembrane Domain Can Substitute for That of the Influenza
A Virus M2 Protein and Support Infectious Virus Production
Shaun M Stewarta,band Andrew Pekosza
W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University, Bloomberg School of Public Health, Baltimore, Maryland, USAa;
Division of Biology and Biomedical Sciences, Washington University in St. Louis, St. Louis, Missouri, USAb
The influenza C virus CM2 protein and a chimeric influenza A virus M2 protein (MCM) containing the CM2 transmem-
brane domain were assessed for their ability to functionally replace the M2 protein. While all three proteins could alter
cytosolic pH to various degrees when expressed from cDNA, only M2 and MCM could at least partially restore infectious
virus production to M2-deficient influenza A viruses. The data suggest that while the CM2 ion channel activity is similar to
that of M2, sequences in the extracellular and/or cytoplasmic domains play important roles in infectious virus production.
the viral life cycle (24). The proton-selective ion channel activity
migration of vRNPs to the nucleus (9, 15, 23), and also raises the
pH of the exocytic pathway, thereby preventing premature low-
pH-induced conformational changes in hemagglutinin (HA) (5,
26). The cytoplasmic tail of M2 is required for efficient genome
packaging into budding virus particles (8, 16, 17). M2 has also
been proposed to mediate membrane scission; however, in the
and virus-like particles, respectively (2, 3, 8, 14, 25).
The influenza C virus CM2 protein is an integral membrane
protein generated by proteolytic cleavage of an internal signal
peptide of the p42 protein (10, 13, 22). CM2 forms disulfide-
linked homotetramers (12, 21) and has been shown to conduct
involved in the release of vRNPs during virus uncoating and in
raise the pH of the exocytic pathway; however, the role of this
activity during influenza C virus replication is currently unclear
chimeric M2 protein containing the CM2 transmembrane do-
main could functionally substitute for the influenza A virus M2
CM2 can be expressed efficiently from cDNA using the vac-
cinia virus-bacteriophage T7 polymerase expression system (21),
transcription machinery. Efficient plasmid-based expression of
the pCAGGS expression plasmid (20), most likely because of the
elimination of an mRNA splice donor site present in the RNA
corresponding to the signal peptide (Fig. 1A). The glycosylation
site was eliminated by mutating Thr at residue 37 to Ala, and
epitope tags for the antibodies 3F10 (antihemagglutinin epitope)
and 14C2 (anti-M2 epitope) were added at the carboxy terminus
in order to facilitate detection of the protein (Fig. 1A). In order to
determine if the ion channel activity of CM2 can substitute for
that of M2, a construct was generated with the extracellular and
he influenza A virus M2 protein is a homotetrameric, type III
integral membrane protein that functions at several stages of
cytoplasmic tail of M2 and the transmembrane domain (TM) of
CM2 (MCM) (Fig. 1A).
Because some reports show that M2 and CM2 have differ-
of the secretory pathway (1, 5, 6, 11, 19), the ion channel activ-
ities of the two proteins were compared in an assay that mea-
sures pH-dependent changes in enhanced yellow fluorescent
protein (eYFP) fluorescence (8). Whereas low-pH treatment of
no change in mean fluorescence intensity (MFI) over time,
low-pH treatment of cells cotransfected with eYFP and M2
from A/Udorn/72 (M2Ud) resulted in an ?40% decrease in
MFI (Fig. 1B and C). Low-pH treatment of cells cotransfected
with eYFP and CM2 resulted in only an ?25% decrease in MFI
(Fig. 1D), indicating that the CM2 protein was able to modu-
late the cytoplasmic pH of transfected cells but not to the same
extent as the M2 protein. The MCM protein induced a reduc-
tion in eYFP fluorescence which resembled that of CM2 (Fig.
1E), suggesting that substitution of the CM2 TM for that of M2
conferred ion channel activity that resembled that of CM2. The
cells were comparable (Fig. 1C to E), indicating that the de-
creased pH changes induced by CM2 and MCM compared to
M2 were not attributable to differential protein levels but most
likely reflected altered ion channel activities.
In order to determine if CM2 and MCM were able to comple-
ment an M2-null influenza A virus, clonal MDCK cell lines stably
expressing the proteins were generated. Total expression of CM2
or M2 from A/WSN/33 with a mutation conveying amantadine
Received 13 July 2011 Accepted 3 September 2011
Published ahead of print 14 September 2011
Address correspondence to Andrew Pekosz, email@example.com.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
0022-538X/12/$12.00Journal of Virology p. 1277–1281jvi.asm.org
sion level was only 2% of the levels of M2. However, even trace
amounts of M2 protein can functionally complement an M2-null
influenza A virus (17), so the levels of CM2 expression are ex-
pected to be sufficient for the complementation assay. MCM was
able to form disulfide-linked oligomers, similar to the M2 and
CM2 proteins (Fig. 2B). In order to determine if constitutive ex-
pression of CM2 or MCM prevented replication of influenza A
binant A/WSN/33 (H1N1, rWSN) were determined on the cell
lines (Fig. 2C). Both rUd and rWSN had similar titers on all cell
lines tested, suggesting that expression of CM2 or MCM does not
inhibit influenza A virus infection. To assess the ability of CM2
viruses rUd M2Stop and rWSN M2Stop (8, 27) were determined
on the cell lines (Fig. 2C). Both rUd M2Stop and rWSN M2Stop
showed decreased titers on CM2-expressing cells compared to
M2-expressing cells, indicating that CM2 cannot functionally re-
place M2. In comparing MCM- to M2-expressing cells, rUd
in titer, demonstrating that the MCM protein can complement
ion channel activity can functionally substitute for that of M2 in
some influenza A virus strains. However, given the low level of
CM2 expression, it remains to be determined if overexpression of
the CM2 protein to levels much higher than those needed for M2
The ability of MCM to substitute for M2 was also tested in
rWSN M2Stop was able to grow in CM2-expressing cells despite
the fact that the level of CM2 expression was higher than that
needed to complement these viruses with M2 protein (8, 16, 17).
growth of rWSN M2Stop was more attenuated than that of rUd
M2Stop. Since CM2 expression could not complement while
MCM expression could partially complement two different M2-
M2 mutations that cause strain-specific defects in growth of
8, 16). The ability of MCM expression to complement rUd
M-WSN M2Stop (expressing 7 segments from Ud, M1 from
WSN, and no M2) growth was comparable to its ability to com-
plement rUd M2Stop (Fig. 2D and F). Likewise, the ability of
MCM expression to complement rWSN M-Ud M2Stop (express-
ing 7 segments from WSN, M1 from Ud, and no M2) growth was
comparable to its ability to complement rWSN M2Stop (Fig. 2E
and G). Together, these data indicate that the decreased ability of
MCM to complement rWSN M2Stop maps to viral sequences
outside the M1 protein.
FIG 1 Schematic and ion channel activities of M2, CM2, and MCM. (A)
Schematic of the ectodomain (Ecto), transmembrane domain (TM), and cy-
toplasmic tail (Cyto) of wild-type M2, CM2, and MCM. Amino acid numbers
are indicated. Sequences recognized by the HA (black box) and M2 (hatched
conduct ions was indirectly measured using the pH-sensitive fluorescent pro-
tein eYFP. 293T cells were transiently transfected with plasmids encoding
expressing the indicated protein. The next day, cells were detached and resus-
The mean fluorescence intensity (MFI) of eYFP for 10,000 cells was deter-
mined every 15 s. The change in eYFP MFI versus time is plotted from a
fits to one-phase exponential-decay models, and dotted lines represent no
change in eYFP MFI. The expression levels of M2, CM2, and MCM in eYFP-
a goat anti-mouse antibody conjugated to Alexa Fluor 647. The MFI for M2,
CM2, and MCM and standard deviation are included in each panel.
Stewart and Pekosz
jvi.asm.org Journal of Virology
The inability of CM2 to complement M2-null viruses could
be due to a defect in particle budding. Virus particles from
high-multiplicity-of-infection (MOI) infections were concen-
trated by ultracentrifugation through 35% sucrose and ana-
lyzed by Western blotting and 50% tissue culture infective dose
(TCID50) assays (Fig. 3). As previously published, rUd M2Stop
virus particles from MDCK cells are released but are defective
in the incorporation of full-length NP (NPa) and have lower
infectious virus titers relative to total HA (Fig. 3A to C) (8).
rWSN M2Stop virus particles grown on MDCK cells also have
decreased titers relative to total HA but increased release and
cleavage of HA (Fig. 3D and E). Both M2-null viruses grown on
CM2-expressing cells showed phenotypes similar to the phe-
notypes of those grown on MDCK cells (Fig. 3). However, rUd
M2Stop virus particles grown on MCM-expressing cells incor-
expressing cells (Fig. 3A to C). rWSN M2Stop virus particles
grown on MCM-expressing cells also had structural protein
incorporation similar to that of particles from M2-expressing
cells. The ratio of titer to HA (a measure of the infectivity of the
particles) of viruses grown on MCM-expressing cells was inter-
mediate between that of those grown on MDCK or CM2-
expressing cells and that of those grown on M2-expressing cells
(Fig. 3C and E). Taken together, these data demonstrate that
expression of MCM, but not CM2, is able to complement the
defect of NPa incorporation present in viruses grown on
MDCK cells but that the particles from MCM-expressing cells
M2Stop than in rUd M2Stop.
Negative-stain electron microscopy was performed on the
purified rUd M2Stop virus particles in order to assess any mor-
phological differences in virus particles grown on the various
cell lines. rUd M2Stop particles grown on MDCK or CM2-
expressing cells had a smaller average diameter and higher par-
ticle/TCID50ratios than did those grown on M2-expressing
cells (Fig. 4). Virus particles isolated from MCM-expressing
cells were of a size similar to the size of those grown on M2-
expressing cells but had slightly higher particle/TCID50ratios
(Fig. 4F). All cell lines produced comparable numbers of par-
ticles (Fig. 4F). These data indicate that the infectivity of the
virus particles correlated with the size of the particles and that
proteins were analyzed by Western blotting in order to determine the total expression under reducing conditions (A) and the presence of disulfide-linked
oligomers under nonreducing conditions (B). The antibodies used were anti-M2 monoclonal antibody 14C2 (1:500), anti-?-actin monoclonal antibody
to M2WSN N31S, are listed below the corresponding lane. (C) Titers of the indicated recombinant virus were determined by TCID50assay on MDCK cells or
MDCK cells stably expressing the indicated protein. The mean and the standard error of the mean are graphed from two independent experiments. (D to G)
MDCK cells or MDCK cells stably expressing the indicated protein were infected at an MOI of 0.001 with the indicated recombinant influenza A viruses. The
amount of infectious virus at each time point was determined by TCID50assay on cells expressing wild-type M2. The mean and the standard error of the mean
are graphed. The limit of detection is marked by a horizontal dotted line. Infectious virus production from 24 to 72 h was analyzed using mixed analyses of
cells are indicated (???, P ? 0.001).
Influenza C Virus CM2 Protein
January 2012 Volume 86 Number 2jvi.asm.org 1279
MCM, but not CM2, could partially complement the rUd
These studies suggest that influenza A virus produced in the
absence of M2 or in the presence of CM2 has a defect in the
incorporation of NPa (and most likely viral RNA), smaller par-
ticles, an increased ratio of total to infectious particles, and
absence of growth in multistep growth curves. The level of
CM2 expression was higher than the level of M2 expression
needed for complementation; however, we cannot rule out the
fact that higher levels of CM2 expression might lead to some
degree of complementation or that the addition of C-terminal
epitope tags may be interfering with CM2 protein function.
normal level of NPa and are of normal size but have a slightly
reduced infectivity. We hypothesize that the defect in infectiv-
ity of virus particles grown on MCM-expressing cells is due to
the differential ion channel activity of the MCM protein versus
that of the M2 protein. The ion channel activity (Fig. 1) shows
that MCM does not lower the pH of the cytoplasm as efficiently
as does M2, most likely leading to a decreased ability to release
vRNPs during virus entry.
We acknowledge Michael Delannoy from the Institute for Basic Biomed-
ical Sciences Microscope Facility at the Johns Hopkins University School
of the Pekosz laboratory for helpful discussions and suggestions.
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Influenza C Virus CM2 Protein
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