Specific Residues in the 2009 H1N1 Swine-Origin
Influenza Matrix Protein Influence Virion Morphology
and Efficiency of Viral Spread In Vitro
Kristy M. Bialas, Emily A. Desmet¤, Toru Takimoto*
Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, United States of America
In April 2009, a novel influenza virus emerged as a result of genetic reassortment between two pre-existing swine strains.
This highly contagious H1N1 recombinant (pH1N1) contains the same genomic background as North American triple
reassortant (TR) viruses except for the NA and M segments which were acquired from the Eurasian swine lineage. Yet,
despite their high degree of genetic similarity, we found the morphology of virions produced by the pH1N1 isolate, A/
California/04/09 (ACal-04/09), to be predominantly spherical by immunufluorescence and electron microscopy analysis in
human lung and swine kidney epithelial cells, whereas TR strains were observed to be mostly filamentous. In addition, nine
clinical pH1N1 samples collected from nasal swab specimens showed similar spherical morphology as the ACal-04/09 strain.
Sequence analysis between TR and pH1N1 viruses revealed four amino acid differences in the viral matrix protein (M1),
a known determinant of influenza morphology, at positions 30, 142, 207, and 209. To test the role of these amino acids in
virus morphology, we rescued mutant pH1N1 viruses in which each of the four M1 residues were replaced with the
corresponding TR residue. pH1N1 containing substitutions at positions 30, 207 and 209 exhibited a switch to filamentous
morphology, indicating a role for these residues in virion morphology. Substitutions at these residues resulted in lower viral
titers, reduced growth kinetics, and small plaque phenotypes compared to wild-type, suggesting a correlation between
influenza morphology and efficient cell-to-cell spread in vitro. Furthermore, we observed efficient virus-like particle
production from cells expressing wild-type pH1N1 M1, but not M1 containing substitutions at positions 30, 207, and 209, or
M1 from other strains. These data suggest a direct role for pH1N1 specific M1 residues in the production and release of
spherical progeny, which may contribute to the rapid spread of the pandemic virus.
Citation: Bialas KM, Desmet EA, Takimoto T (2012) Specific Residues in the 2009 H1N1 Swine-Origin Influenza Matrix Protein Influence Virion Morphology and
Efficiency of Viral Spread In Vitro. PLoS ONE 7(11): e50595. doi:10.1371/journal.pone.0050595
Editor: Yi Guan, The University of Hong Kong, China
Received August 30, 2012; Accepted October 24, 2012; Published November 27, 2012
Copyright: ? 2012 Bialas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health (NIH) R21-A1078130 and the New York Influenza Center of Excellence (NYICE), a member of
the NIAID CEIRS network, under NIH contract HHSN266200700008C. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
¤ Current address: Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States of America
Influenza viruses belong to the orthomyxoviridae family consisting
of negative sense, single-stranded, RNA viruses with segmented
genomes . Even with the availability of annual vaccines against
human influenza infection, seasonal epidemics still result in nearly
500,000 deaths worldwide . Additionally, influenza has been
the cause of several global pandemics, most notably the 1918
Spanish flu which killed between 20 to 50 million people, and
more recently the 2009 H1N1 swine-origin outbreak (pH1N1) .
This novel pH1N1 virus that emerged in April 2009, was declared
a pandemic just two months later, already having spread to more
than 70 countries. Sequence analysis of its gene segments revealed
that several reassortment steps led to the emergence of this virus,
as it contains HA, NP, and NS genes from the classical swine
lineage, NA and M from the Eurasian swine lineage, a human PB1
that was seeded from an avian virus in approximately 1968, and
avian PA and PB2 genes . Six of the eight pH1N1 gene
segments are shared with North American triple reassortant (TR)
swine viruses that, despite their longtime establishment in the
swine population, rarely infect humans and exhibit limited human-
to-human transmission [5,6,7]. Because the two remaining gene
segments, NA and M, were acquired from the Eurasian swine
lineage, it is possible that these Eurasian swine genes and/or
additional mutations created after the reassortment, contribute to
the enhanced transmission of the pH1N1 virus among humans.
Both the NA and M segments encode viral proteins with key
roles in the influenza assembly, budding and release processes,
which are required for efficient viral transmission. NA encodes the
type II transmembrane protein, neuraminidase, which is found as
a tetramer on the surface of the host-derived lipid envelope. It
functions during the final stage of viral budding where it cleaves
sialic acid containing receptors to allow for the release of progeny
virions from infected cell surfaces . A recent study investigating
the contribution of the Eurasian-origin NA and M segments to the
enhanced spread of the pH1N1 strain reported a direct role for the
novel NA protein in efficient transmission between ferrets . In
this report, all naı ¨ve animals exposed to ferrets which had been
infected intranasally with the pH1N1 isolate, A/California/07/09
(Rec pH1N1), tested positive for influenza by neutralization assays,
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indicating 100% transmission efficiency. However, upon re-
placement of Rec pH1N1 NA and M with segments derived
from the TR lineage, transmission efficiency was reduced to 50%,
similar to that observed by wild-type TR and Eurasian viruses.
This observation suggests an important role for at least one of the
Eurasian segments in enhanced viral transmission of Rec pH1N1.
Furthermore, in this same study, NA originating from the
Eurasian lineage was shown to exhibit higher neuraminidase
activity in vitro compared to NA with TR origin. Moreover, strains
containing Eurasian origin NA with increased enzymatic activity
released more viral particles, thereby proposing a mechanism by
which the NA segment may be contributing to enhanced viral
transmission of the pH1N1 virus.
The M segment of influenza encodes two viral proteins, matrix
protein (M1) and the M2 ion channel. While M2 is essential for
uncoating of the virus during entry, M1 is known to be the key
component for both assembly and budding . Accumulation of
M1 at the plasma membrane, and its interaction with the
cytoplasmic tails of the viral surface proteins, are thought to
initiate bud formationby inducing
[10,11,12]. Additionally, M1 interaction with both newly synthe-
sized viral ribonucleoproteins (vRNPs) and the viral nuclear export
protein (NEP) are known to be required for translocation of the
viral genome from the host cell nucleus [13,14,15,16,17,18,19].
These essential functions of M1 during the production of progeny
virions support a role for the novel M segment in the enhanced
transmission of the pH1N1 strain. Strengthening this hypothesis,
Chou et al. recently showed that A/Puerto/Rico/8/34 (PR8)
expressing the M segment from A/California/04/2009 (ACal-04/
09) has increased transmission efficiency in vivo using the guinea
pig model . In this study, no viral transmission occurred
between guinea pigs infected with wild-type PR8 and naı ¨ve
animals, whereas 50% transmission efficiency was observed
between guinea pigs infected with the PR8 recombinant expres-
sing ACal-04/09 M. Though these data implicate a role for the
pH1N1 M segment, specifically the M1 protein, in the spread of
influenza, the exact mechanism and key residue(s) that determine
efficient transmission remain unknown.
Specific M1 residues are also known to affect virion morphol-
ogy. Influenza virions can range from 100 nm spheres to
filamentous particles reaching several micrometers in length
. Evidence for M1 influence on virion morphology was first
provided in a study which showed that replacement of the M gene
of A/WSN/33, a spherical producing strain, with that of the
filamentous virus A/Udorn/72 resulted in filamentous virus
morphology . Specific amino acids within the M1 protein
were later found to be required for the production of filamentous
particles, including residues 41, 95, 102, 204, and 218 [22,23,24].
Though it is known that influenza morphology affects virus
production, its role in viral transmission is still unclear. Early
reports showed that most influenza strains isolated from humans
are predominantly filamentous and, upon continual passage in egg
or tissue culture, adopt a more uniformly spherical morphology
[25,26]. This switch in morphology correlates with an increase in
virus titer [26,27]. Therefore, it is likely that the increased levels of
virus production by spherical influenza strains results in more
efficient viral transmission.
In this study, we characterized the morphology of pH1N1
isolates, and found that they are predominantly spherical in our
cultured cells. This was in contrast to the filamentous phenotype
we observed with the closely related, but poorly transmissible, TR
swine viruses. In addition, we found that, unlike other strains,
pH1N1 M1 by itself can efficiently induce virus-like particles from
transfected cells. To evaluate the contribution of M1 mutations in
virus morphology, we rescued various pH1N1 virus containing
mutations at M1 residues different between pH1N1 and TR. Our
results revealed that pH1N1 M1 residues 30, 207 and 209 are
involved in regulation of virus morphology and enhanced viral
spread in vitro. These data suggest that a few mutations present in
pH1N1 contribute to morphological change and efficient trans-
mission of influenza viruses.
Virion Morphology of the 2009 H1N1 Swine-origin
Human infection with TR swine viruses prior to 2009 was a rare
incidence, and mostly self limiting. Conversely, pH1N1, while
sharing most of the same gene segments with TR swine viruses,
transmitted readily from person-to-person and hence began the
first influenza pandemic of the 21stcentury. Although multiple
factors are likely to be involved in the emergence of the pH1N1,
efficient virion production is among the requirements for the
outbreak. Because efficient virion production has previously been
shown to correlate with spherical rather than filamentous particle
formation, we first determined the morphology of pH1N1 and TR
viruses. Initially, we investigated the presence or absence of
filamentous protrusions at the surface of human lung epithelial
A549 cells infected with the viruses by immunufluorescence (IF)
analysis. A549 cells were infected with either the TR strain A/
Wisconsin/87/2005 (H1N1, AWisc05), or a pH1N1 virus A/
California/04/2009 (ACal-04/09). At 18 h, the cells were fixed
and processed for visualization of viral surface proteins using anti-
H1N1 mouse serum. Noticeably, cells infected with AWisc05
exhibited long filamentous structures on their surface (Fig. 1). This
phenotype was not unique to AWisc05, as four additional TR
isolates tested, A/Iowa/01/2006 (H1N1), A/Iowa/02/2006
(H1N1), A/Ohio/02/2007 (H1N1) and A/Minnesota/03/2008
(H1N1) were also found to induce filament formation (data not
shown). In contrast to TR viruses, infection with ACal-04/09
resulted in a staining pattern consisting of small punctae covering
the entire cell surface that is typically seen by spherical influenza
strains. Morphological differences between AWisc05 and ACal-
04/09 were also observed in swine kidney fibroblast LLC-PK1
cells, indicating that virion morphology is conserved between these
cell types (Fig. 1).
We further examined the structure of budding virions by
scanning electron microscopy. Using this method, we observed
a similar induction of filaments in the AWisc05-infected A549 cells
as was seen by IF analysis. In contrast, the presence of small
spherical structures was detected on the surface of cells infected
with the ACal-04/09 virus (Fig. 2A). We also characterized the
morphology of ACal-04/09 virions released from infected A549
cells by transmission electron microscopy. In order to avoid
distortion of particle shape, culture supernatants were harvested
after 24 h and concentrated by low speed centrifugation through
a filter unit and absorbed onto carbon-coated grids. The diameters
of 50 negatively stained virions were measured, after which they
were characterized as either spherical or filamentous. From this
data, we found more than 98% to be spherical, defined as any
particle having a length that is less than twice its width (Table 1).
The electron micrograph in Figure 2B represents the majority of
ACal-04/09 virions. Collectively, these data suggest ACal-04/09
as a predominantly spherical virion producing strain.
Because our data is in disagreement with previously published
work [3,9,28,29], we characterized additional pH1N1 isolates by
IF microscopy. In this experiment, A549 cells were infected with
nasal swab specimens obtained from patients with confirmed
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pH1N1 infection, which had no prior passage history in egg or
tissue culture. While we did observe some variation between the
isolates, most appeared to exhibit a similar punctae staining
pattern as ACal-04/09 suggesting that the pH1N1 strains, in
general, produce spherical virions in human lung cells (Fig. 3).
Further characterization of the clinical isolates in MDCK cells
yielded comparable results (data not shown), suggesting that viral,
rather than cellular factors are playing a central role in the
determination of virion morphology. Additionally, sequence
analysis of the M1 proteins from each of the isolates was
performed, revealing no amino acid differences between clinical
samples and the ACal-04/09 virus, making ACal-04/09 a suitable
strain for further study.
Specific Residues in the pH1N1 M1 Protein are Required
for Spherical Virion Morphology
A variety of influenza viral proteins have been shown to affect
virion morphology including each of the transmembrane proteins,
Figure 1. TR swine virus but not the pH1N1 strain induces filament formation from infected human lung and swine kidney cells.
Human A549 and swine LLC-PK1 cells infected with the ACal-04/09 or AWisc05 were processed for visualization of viral surface proteins 18 hpi by IF
microscopy using anti-H1N1 mouse serum followed by anti-mouse IgG-Texas Red.
Figure 2. Electron microscopy analysis of TR or pH1N1-infected A549 cells. (A) Mock infected A549 cells, and A549 cells infected with
AWisc05 or ACal-04/09 were fixed at 18 h and processed for scanning electron microscopy. Bars measure 10 mm (top) and 1 mm (bottom). Arrows
point to virions budding from infected cell surfaces. (B) Electron micrograph of negatively stained ACal-04/09 virion released from infected A549 cells.
PLOS ONE | www.plosone.org3 November 2012 | Volume 7 | Issue 11 | e50595
the nucleoprotein, and M1 [22,23,24,30,31,32,33,34,35]. Because
pH1N1 acquired the M gene from a Eurasian swine virus, we
hypothesized that the difference in virion morphology between
TR and pH1N1 was due to variations in the M1 protein sequence.
To test this, we compared 153 classical swine and 130 Eurasian
swine M1 sequences collected between the years of 2000 and 2008
with ACal-04/09 M1 (Table 2). From this analysis, we identified
four amino acid differences between TR and ACal-04/09 M1
proteins at positions 30, 142, 207, and 209. Two of these residues,
142 and 209, were shared between Eurasian swine and pH1N1
viruses, and are conserved among members of the Eurasian swine
lineage. pH1N1 M1 residues Ser at 30 and Asn at 207 were rarely
found in swine isolates. Moreover, extensive analysis of all
available pH1N1 M1 sequences revealed each of these residues
to be highly maintained throughout the 2009, 2010, and 2011
To determine if these M1 residues affect the morphology of
pH1N1, we rescued ACal-04/09 viruses in which one or more of
the pandemic M1 residues were substituted with the correspond-
ing TR swine residues (Table 3). Rescued viruses were plaque
cloned and stock viruses were prepared in MDCK cells. M genes
of the rescued viruses were sequenced and confirmed to contain
only the designed mutations. First, we characterized the
morphology of budding virions by IF in A549 cells. Replacement
of residue 142 had no affect on virion morphology, resulting in
virus with a similar punctae staining pattern as ACal-04/09
(Fig. 4). Single substitution of residue 30, or 209 slightly altered
virion morphology, producing short filaments in infected cells. In
sharp contrast, replacement of 207 or both 207 and 209 to those of
TR residues, caused an induction of filamentous structures
resembling those of AWisc05 (Fig. 4). Consistent results were
obtained in primary human lung epithelium (data not shown).
These data suggest that residues 30S, 207N and 209T in pH1N1
M1 play a key role in spherical virion formation.
Morphological Change Correlates with Efficiency of Viral
Spread in vitro
Next, we examined the growth rates and plaque size of the wild-
type and mutant viruses to determine efficiency of viral spread
in vitro. Data from our multi-cycle infection showed that single
replacement of residue 30, 142, 207, or 209 in the ACal-04/09
M1 protein reduced overall viral production as well as growth
kinetics albeit to different extents (Fig. 5A). ACal-04/09 M1
A142V virus, which retained spherical virion morphology, grows
as fast as ACal-04/09 WT until 24 h. In contrast, growth rate of
viruses containing substitutions at position 207 or both 207 and
209 was slowest and the maximum virus titers of the viruses were
25 times less than that of WT (Table 3). Coinciding with our
multi-cycle growth curve analysis, the plaque size of ACal-04/09
M1 A142V following 4 day incubation was only slightly reduced
compared to wild-type (Fig. 5B). Of the remaining viruses, those
with substitutions at residues 207 and 209 formed barely visible
Expression of the ACal-04/09 M1 Protein is Sufficient for
Virus-like Particle Production
Data described above suggest a direct role for the pH1N1 M1
protein, specifically residues 30, 207 and 209, in determining
influenza morphology, enhancing viral production, and mediating
efficient viral spread in vitro. To explore the possible mechanism by
which the pH1N1 M1 protein contributes to efficient release of
highly transmissible virions, we further characterized the ACal-
04/09 M1 for its ability to induce virus-like particles (VLPs) by
itself in cultured cells. Previous studies have shown that expression
of influenza NA, M2, or HA alone, but not M1, is sufficient for
efficient VLP production [36,37,38]. We quantified VLP pro-
expressing wild-type or mutant ACal-04/09 M1, or with plasmids
expressing avian or human virus M1. In agreement with previous
reports, we found trace amounts of M1 protein released into the
supernatant of 293T cells transfected with A/chicken/Nanchang/
3-120/01 (H3N2, Nan), A/WSN/33 (H1N1, WSN), and A/
Aichi/2/68 (H3N2, Aichi) M1 plasmids (Fig. 6A). However, cells
expressing ACal-04/09 M1 induced high levels of VLP pro-
duction. Release of ACal-04/09 M1 was 7.7-, 18.9-, and 10.3-
times more efficient than that of Nan, WSN and Aichi,
respectively. Interestingly, VLP production was reduced by 76%
with S30A mutation, and 87% with N207S plus T209A mutations
(Fig. 6B). In contrast, the A142V mutation, which did not affect
virus morphology, showed no reduction in VLP production, but
even more efficiently produced VLP from transfected cells. These
results indicate a unique feature of the pH1N1 M1 protein to
induce and complete budding at the plasma membrane by itself,
which was not observed with other influenza virus M1 protein
A recent study showed that increased membrane association of
M1 fused with membrane targeting peptide enhanced production
of VLP from transfected cells, suggesting that efficiency of plasma
membrane association affect the VLP production . To address
whether ACal-04/09 M1 can more readily associate with the
plasma membrane compared to other influenza M1 proteins, we
performed membrane flotation analysis on 293T cells transfected
with ACal-04/09 or WSN M1 proteins (Fig. 7A). In addition, we
included ACal-04/09 M1 207/209 to determine if any observed
differences were due to the pH1N1 specific residues. Our results,
however, indicate no measurable difference in M1 association with
the membrane when comparing either ACal-04/09 construct with
WSN M1. All of them showed that 46 to 56% M1 association with
membrane as determined by protein contents of fractions 2–4
During viral assembly at the plasma membrane, M1 and other
viral proteins localize to lipid raft domains which are composed
mainly of glycosphingolipids and are known to be resistant to
extraction with ice-cold Triton X-100. To determine if WT ACal-
04/09 M1 localizes more efficiently within the specialized lipid raft
compartments than other influenza M1 proteins, we performed
35S-labeled 293T cells transfected with plasmids
Table 1. Measurement of ACal-04/09 viral particles.
Length/width ratio# of particles
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lipid raft isolation on 293T cells expressing ACal-04/09 M1,
ACal-04/09 M1 207/209, or WSN M1 (Fig. 7B). Similar to the
membrane flotation analysis, lipid raft isolation revealed no
difference in M1 association between influenza strains. Thus,
association of M1 with lipid membrane or raft compartment is
unlikely to be the major factor that enhances VLP production by
The pH1N1 strain emerged from swine in Mexico and rapidly
spread to more than 200 countries . This novel pandemic virus
arose from a reassortment event between a North American TR
swine virus and a Eurasian swine virus. Both TR and Eurasian
swine viruses had previously caused sporadic infections in humans,
but failed to spread from person-to-person [6,7]. Therefore, it is
likely that genes introduced by the reassortment and/or mutations
which occurred after the reassortment caused emergence of the
highly transmissible pH1N1 viruses. In fact, a recent study
suggested the contribution of NA and M genes, which were
derived from the Eurasian swine lineage, to virus transmissibility of
pH1N1 in the ferret model . For efficient transmission among
human hosts, multiple factors are involved including efficient virus
attachment to upper respiratory tract, replication in these tissues,
and release and aerosolization of virus particles . Aerosolized
droplets range in size and thus the capacity to travel over long
distances. Large droplets measuring greater than 6 mm can reach
only several meters and may therefore contribute to transmission
mainly by direct contact, whereas smaller droplets less than 1mm
are carried much further and with greater propensity to transmit
virus via inhalation . Ferrets infected with the pH1N1 were
found to release a higher number of submicron particles contain-
Figure 3. Direct infection of A549 cells with clinical pH1N1 nasal swabs does not induce long filaments. (A–I) A549 cells were directly
infected with nasal swab specimens collected from nine patients with confirmed pH1N1 infection. At 18 h, cells were processed for surface staining of
viral proteins as described for Fig. 1.
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ing viral RNA and exhibited more efficient viral transmission than
either TR or Eurasian swine viruses , suggesting a possible role
for small aerosolized droplets in enhancing viral transmission.
Moreover, these submicron droplets likely contain spherical rather
than filamentous virions due to obvious size constraints, thereby
strengthening the hypothesis that strains producing spherical
virions have the potential to transmit more efficiently.
In this study, we determined the role of the pH1N1 M1 protein
in virus morphology and growth in vitro. We report that the
majority of purified pH1N1 viruses grown in A549 cells formed
spherical virions. IF analysis of cells infected with pH1N1 revealed
punctae structures at the cell surface, which clearly differ from the
filamentous protrusions observed in TR virus-infected cells. Our
data is in accord with the initial structural analysis performed by
members of the CDC (http://www.cdc.gov/h1n1flu/images.htm),
yet disagrees with an investigation which reported the pH1N1
strain as predominantly filamentous when analyzed using MDCK
or primary human airway epithelial cells  or with a study
reporting that 60% of purified A/California/07/2009 grown in
MDCK cells was filamentous . Possible explanations for these
discrepancies include differences in passage history of the viruses
and cell types used in these studies. To address the first issue, we
infected cells with nasal swab samples from patients which had
RT-PCR confirmed pH1N1 infection, and determined the cell
surface structures by IF. These viruses showed punctae or very
short filament protrusions from cell surfaces, similar to the ACal-
04/09 strain (Figs. 1 and 3). Secondly, characterization of
influenza in different cell types may yield contrasting results.
However, in our cell cultures, virion morphology was found to be
consistent between A549 cells and primary human lung epithe-
lium. Although it is difficult to predict the virion morphology
produced in the human respiratory tract, our data suggest that
pH1N1 viruses contains a phenotype that readily produces
spherical or very short filamentous virions from infected human
respiratory epithelial cells.
Table 2. Amino acid differences between classical swine, Eurasian swine and the 2009 pH1N1 M1 proteins.
30142 207 209
Classical SwineD (153/153)V (152/153)
Eurasian Swine G (126/130)
A (130/130) S (119/130)
(human 2009 isolates)
(human 2010–2012 isolates)
N (721/721)T (721/721)
Number of isolates containing the indicated residues/total number of isolates is indicated.
Figure 4. Substitutions at positions 30, 207 and 209 in the ACal-04/09 M1 protein resulted in filament formation in infected cells.
A549 cells were infected with ACal-04/09 viruses containing single or multiple substitutions at positions 30, 142, 207, and 209 in the M1 protein as
indicated. Cells were fixed and processed for visualization of viral surface proteins 18 h by IF microscopy as described for Fig. 1.
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By introducing TR M1 residues into pH1N1 M1, we identified
three residues at 30, 207 and 209, which affected virion
morphology and virus growth. Single mutations at these residues,
reduced plaque size, growth rate and overall virus production
(Fig. 5 and Table 3). pH1N1 mutants containing 207S or both
207S and 209A induced especially long filamentous protrusions at
the cell surface, possibly reflecting inefficient fission and release of
budding virions (Fig. 4). Sequence analysis revealed position 209 to
be a highly conserved lineage-specific residue at which all Eurasian
swine viruses between 2000 and 2008 possessed Thr, which differs
from Ala found in the classical swine viruses (Table 2). Though
residue 142 was also found to be lineage-specific, it did not appear
to affect virion morphology by itself in our study. In contrast, Ser
and Asn found at positions 30 and 207 in the pH1N1 M1
sequence rarely exist in other swine strains. Based on these data, it
is possible that additional mutations at residue 30 and/or 207
introduced to the Eurasian M segment of the precursor reassortant
virus contribute to the efficiency of spherical virion production and
transmission of pH1N1.
It is unclear at this stage how pH1N1 M1 mutations at 30, 207
and 209 affect virus morphology. Influenza virus assembly and
budding is a complex, multi-step process involving interactions
between viral and cellular proteins. Many viral protein interactions
are known to affect virus assembly and morphology, such as M1-
HA, M1-NA, M1-M2, and M1-vRNP interactions . The exact
site of M1 interaction with HA and NA is not yet identified,
however, crosslinking between the cytoplasmic tails of HA and NA
with M1 is postulated. Viruses containing mutations in the
cytoplasmic tails of both HA and NA have greatly altered
morphology and, as seen with viruses harboring deletions in the
M2 cytoplasmic tail, exhibit reduced incorporation of M1 and
vRNP. This is suggestive that a structural change in M1 induced
by interaction with the cytoplasmic tails of viral transmembrane
proteins affects the interaction with vRNP and stabilizes the
morphology of progeny virions [11,43,44]. However, based on our
data, it is likely that pH1N1 M1 itself has a unique phenotype that
facilitates virion production because of its ability to produce VLP
efficiently unlike other virus strains tested (Fig. 6). Mutations at
residues 30, 207 and 209, which resulted in enhanced formation of
filamentous protrusion in infected cells, greatly reduced VLP
production from transfected cells. In contrast, a mutation at
residue 142 barely affected infected cell surface structure and did
Table 3. Virus titer and plaque phenotypes of recombinant ACal-04/09 viruses.
Viruses Residues 30/142/207/209
A/California/04/2009 WTS/A/N/T 7.4 0.7
A/California/04/2009 M1 S30DD/A/N/T 6.9 0.3
A/California/04/2009 M1 A142T S/V/N/T 6.60.5
A/California/04/2009 M1 N207SS/A/S/T6.00.3
A/California/04/2009 M1 T209A S/A/N/A 6.60.4
A/California/04/2009 M1 207/209 S/A/S/A6.00.3
Figure 5. Substitutions at positions 30, 207 and 209 in the ACal-04/09 M1 protein reduce viral growth and spread in vitro. (A) Growth
kinetics of WT and mutant ACal-04/09 viruses were measured in MDCK cells infected at MOI 0.01. Virus titer was determined by TCID50at the
indicated time points. Error bars signify standard deviation. (B) Plaque phenotypes of WT and recombinant ACal-04/09 viruses were characterized in
MDCK cells 4 dpi following crystal violet staining.
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not reduce VLP production. A previous study suggested that an
association of membrane targeting peptide to M1 increased VLP
production in transfected cells, suggesting that plasma membrane
association of M1 is critically important for VLP formation .
However, we did not detect any major difference in membrane
association between ACal-04/09 M1, ACal-04/09 M1 207/209,
and WSN M1 (Fig. 7A), indicating that efficient production of
VLP by ACal-04/09 M1 is not due to enhanced membrane
association. Additionally, we determined M1 association with lipid
raft domains in the plasma membrane, which are considered to be
the sites of influenza virus assembly and budding [45,46].
However, we found similar levels of M1 co-localization with lipid
raft domains following Triton6100 extraction between the viruses
tested, suggesting comparable targeting to known sites of influenza
virus assembly. Thus, the unique feature of the pH1N1 M1
protein that allows for efficient VLP production is unlikely to be
due to its localization to budding sites at the plasma membrane.
Recent cryo-electron tomography data indicate that M1 forms
a helical net under the viral membrane. The pitch of the helical
turn of the M1 protein differs between spherical and filamentous
virions, suggesting that M1-M1 interaction and its structure
determine virion morphology . A helix formation provides
a structural mechanism by which assembly of M1 subunits at the
plasma membrane drive budding. Therefore, M1-M1 interaction
sites are likely to be the key determinant of virion morphology.
Another possibility that alters virion morphology and VLP
Figure 6. Mutations of ACal-04/09 M1 at residues 30 or 207 and 209 reduced VLP production from transfected cells. (A) 293T cells
were transfected with expression plasmids containing WT or mutant ACal-04/09, or other M1 genes. Purified radiolabeled VLPs in culture
supernatants were analyzed by SDS-PAGE and total M1 in cell lysates was determined by Western blot analysis. (B) Band intensities were quantified
using BioRad software. VLP production is shown as the amount of M1 released into cell culture supernatant normalized to M1 produced in cell
lysates. ACal-04/09 WT was set to 1. Data shown are an average of three individual experiments. Error bars represent standard deviation.
Figure 7. The pH1N1 M1 protein associates as efficiency with the plasma membrane and lipid raft domains as other influenza M1
proteins. 293 T cells were transfected with WT ACal-04/09 M1, ACal-04/09 M1 207/209 and WSN M1 protein expression plasmids. At 24 hpi, cells
were (A) homogenized with a 27 gauge needle, mixed with 70% sucrose, and subject to membrane flotation analysis or (B) lysed in 0.5% Triton6100
and mixed with 70% sucrose for lipid raft isolation. Fractions were collected from the top of each gradient following ultracentrifugation, separated by
gel electrophoresis and analyzed by Western Blot for M1 protein. Bands intensities were quantified using BioRad Software.
PLOS ONE | www.plosone.org8 November 2012 | Volume 7 | Issue 11 | e50595
production could be interaction with cellular proteins that mediate
pinch-off of budding virus or VLP. Although it has not been
identified which cellular factors are responsible for the pinch off of
budding influenza viruses, efficiency of the pinch off is likely to
affect the morphology of the viruses. It is unclear if residues 30,
207 and 209 are involved in the specific helical contact sites in the
matrix layer or interaction with cellular factors required for pinch
off of budding virus. However, the unique phenotype of pH1N1
M1 in efficient VLP production by itself may reflect its ability to
induce spherical virion formation and efficient transmission
among human hosts.
Materials and Methods
Cells and Viruses
Human lung epithelial (A549), human embryonic kidney
(293T), and Madin-Darby canine kidney (MDCK) cells were
cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 8% fetal calf serum (FCS). Porcine kidney
(LLC-PK1) cells were obtained from American Type Culture
Collection and were cultured in Medium 199 supplemented with
8% FCS. Normal human bronchial epithelial (NHBE) cells
obtained from Lonza were maintained in bronchial epithelial cell
basal medium (BEBM) with the supplements provided. Triple
reassortant swine isolates A/Wisconsin/87/2005 (H1N1), A/
Iowa/01/2006 (H1N1), A/Iowa/02/2006 (H1N1), A/Ohio/02/
2007 (H1N1) and A/Minnesota/03/2008 (H1N1)  were
obtained from A. Klimov (Centers for Disease Control and
Prevention, Atlanta, GA). A/chicken/Nanchang/3-120/01, A/
WSN/33 (H1N1), and A/Aichi/2/68 (H3N2) were described
previously . Nasal swab specimens from nine patients with
PCR-confirmed pH1N1 infection were collected by J. Treanor
(University of Rochester Medical Center, Rochester, NY).
Collection of nasal swabs was approved by the Institutional
Review Board of the University of Rochester, and all subjects gave
written informed consent before participation.
cDNAs encoding Nan, Aichi and ACal-04/09 M1 protein were
synthesized by RT-PCR using SuperScript III One-step RT-PCR
Platinum Taq HiFi (Invitrogen) from total RNAs extracted from
virus-infected cells, and subcloned into pCAGGS. Mutations in
the M1 gene were created using the QuikChange II site-directed
mutagenesis kit (Agilent Technologies, Santa Clara, CA). cDNAs
used to rescue ACal-04/09 were synthesized from RNAs provided
by R. Webster and R. Webby (St. Jude Children’s Hospital,
Memphis, TN) using RT-PCR kit and subcloned into the pPolI
vector given to us by Y. Kawaoka (University of Wisconsin,
Madison, WI). Amino acid substitutions at positions 30, 142, 207,
and 209 were introduced into the pPolI-M plasmid by site-directed
mutagenesis and used for rescue of the ACal-04/09 M1 mutant
Viruses were rescued using the 12-plasmid rescue system
developed by Neumann and Kawaoka . Briefly, MDCK/
293T co-cultures were transfected with pCAGGS-ACal-04/09-
PB1, -PB2, -PA and -NP expression plasmids together with pPolI
vectors encoding each of the gene segments using lipofectamine
2000 (Invitrogen). Plaque purified stocks were propagated in
MDCK cells with DMEM containing 0.15% bovine serum
albumin (BSA) and 2 ug/ml tosylsulfonyl phenylalanyl chloro-
methyl ketone (TPCK) treated trypsin. Mutations in the M
segment were confirmed by sequence analysis, and viruses were
titered in MDCK cells by immunofluorescence analysis of the
IF and Electron Microscopy
A549 or LLC-PK1 cells were infected with the viruses at MOI 1
and incubated with DMEM containing 0.15% BSA for 18 h. For
detection of viral surface proteins by IF, cells fixed with 4%
paraformaldehyde were incubated with anti-H1N1 mouse serum
supplied by D. Topham (University of Rochester Medical Center,
Rochester, NY) followed by goat anti-mouse IgG conjugated with
Texas Red (Invitrogen). Images were taken using an Olympus
inverted microscope. For scanning electron microscopy, cells were
fixed with 2.5% glutaraldehyde and visualized under a Zeiss
Auriga supra 40VP Field Emmison microscope. For the analysis of
virions, supernatant from virus-infected A549 cells was spun
through a 100 K Amicon filter unit (Millipore) for 18 min at
3,500 rpm. A 10 ml volume of concentrated virus was absorbed to
carbon-coated 200 square mesh nickel grids for 3 min, washed
once with PBS(+), and negatively stained for 1 min with filter
sterilized 2% phosphotungstic acid. Transmission electron micro-
graphs were obtained at 6200,000.
Viral Growth Analysis
MDCKs in 6-well culture plates were infected with the viruses
at MOI 0.01 and incubated in 2 ml DMEM containing 0.15%
BSA and 2 ug/ml TPCK-treated trypsin. At various times points,
200 ml of the culture supernatant was harvested and replaced with
an equal volume of fresh media. Virus titers were determined in
MDCK cells as described by Reed and Muench . For plaque
analysis, MDCK cells infected with WT or mutant ACal-04/09
viruses were incubated at 37uC for 4 days. Cells were fixed for
10 min in 10% trichloroacetic acid (TCA). After removal of
overlay medium, cells were stained with 0.1% crystal violet in 20%
Virus-like particle production.
pCAGGS-M1 vectors were labeled for 24 h with35S-Met/Cys in
DMEM lacking methionine and cystine. Cell culture supernatants
were cleared of cell debris by low speed centrifugation, layered
onto 20% sucrose cushions and centrifuged at 35,000 rpm for 2 h
in an SW41 rotor (Beckman). Pellets containing virus-like particles
were resuspended in laemmli buffer (BioRad) and analyzed by
SDS-PAGE. Cell lysates were harvested in RIPA buffer (50 mM
Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1%
Triton X-100), clarified, and separated by SDS-PAGE. Proteins
were transferred to a PVDF membrane and subject to Western
Blot analysis with anti-M1 GA2B monoclonal antibody (Sigma).
Band intensities were quantified with BioRad Quantity One
software and the amount of M1 detected in culture supernatant
was normalized to M1 expressed in cell lysates.
293T cells transfected with
Membrane Flotation Analysis
Flotation analysis on 293 T cells transfected with pCAGGS-M1
plasmids was performed as described by Sanderson et al.  in
a 6-well format. Briefly, cells were washed 24 h post-transfection
with cold PBS(+), harvested by scraping and pelleted by low speed
centrifugation. Following resuspension in 0.7 ml hypotonic lysis
buffer (10 mM Tris (pH 7.5); 10 mM KCl; 5 mM MgCl2), cell
lysates were incubated on ice for 10 min, passaged 15 times
through a 26 gauge hypodermic needle and centrifuged 7 min at
1,0006g. Half ml aliquots of supernatant were mixed with 3.5 ml
70% sucrose in low salt buffer (LSB) (50 mM Tris HCl (pH 7.5)
25 mM KCl and 5 mM MgCl2), overlaid with 5.5 ml 55%
sucrose and 2 ml 10% sucrose in LSB and ultracentrifuged at
38,000 rpm for 18 h at 4uC in an SW41 rotor. One-ml fractions
PLOS ONE | www.plosone.org9 November 2012 | Volume 7 | Issue 11 | e50595
were collected from the top of each gradient to which 0.25 ml
100% trichloroacetic acid was added for protein precipitation.
After 10 min incubation on ice, fractions were spun at top speed
for 5 min. Protein pellets were then washed twice with cold
acetone, heated to 95uC for 5 min and resuspended in NuPAGE
sample buffer (Invitrogen) for SDS-PAGE and Western Blot
analysis with anti-M1 GA2B antibody. Band intensities within
each fraction were quantified using BioRad Quantity One
Lipid Raft Isolation
Isolation of lipid rafts from 293 T cells transfected with
pCAGGS-M1 plasmids was performed as described by Carrasco
et al.  with several modifications. Cells were washed 24 h post-
transfection with cold PBS(+) and incubated on ice for 20 min in
0.5 ml LSB containing 0.5% Triton6100. Each cell lysate (0.3 ml)
was mixed with 0.7 ml 70% sucrose in LSB and overlaid with 2 ml
30% sucrose and 1 ml 2.5% sucrose in LSB. Gradients were spun
at 28,000 rpm for 16 h at 4uC in an SW55 rotor, after which
0.4 ml fractions were collected from the top. Protein was
precipitated with TCA as described above, resuspended in
NuPAGE sample buffer, separated in a 4–12% Bis-Tris gel
(Novex), and subject to Western Blot analysis with anti-M1 GA2B.
Anti-caveolin 1 polyclonal antibody (abcam) was used as a marker
for fractions containing lipid raft associated proteins. Band
intensities within each fraction were quantified using BioRad
Quantity One software.
We thank A. Klimov, Y. Kawaoka, J. Treanor, D. Topham, R. Webster
and R. Webby for reagents, and K. Bentley (University of Rochester EM
core) for technical assistance.
Conceived and designed the experiments: KMB EAD TT. Performed the
experiments: KMB TT. Analyzed the data: KMB TT. Contributed
reagents/materials/analysis tools: KMB TT. Wrote the paper: KMB TT.
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