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.
1. Palese P, Shaw M (2007) Orthomyxoviridae : The Viruses and T heir
Replication; Knipe DM, Howley PM, editors. Philadelphia, PA: Lippincott
Williams & Wilkins.
2. Fauci AS (2006) Emerging and re-emerging infectious diseases: influenza as
a prototype of the host-pathogen balancing act. Cell 124: 665–670.
3. Neumann G, Noda T, Kawaoka Y (2009) Emergence and pandemic potential of
swine-origin H1N1 influenza virus. Nature 459: 931–939.
4. Garten RJ, Davis CT, Russell CA, Shu B, Lindstrom S, et al. (2009) Antigenic
and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses
circulating in humans. Science 325: 197–201.
5. Dawood FS, Jain S, Finelli L, Shaw MW, Lindstrom S, et al. (2009) Emergence
of a Novel Swine-Origin Influenza A (H1N1) Virus in Humans. New England
Journal of Medicine 360: 2605–2615.
6. Newman AP, Reisdorf E, Beinemann J, Uyeki TM, Balish A, et al. (2008)
Human case of swine influenza A (H1N1) triple reassortant virus infection,
Wisconsin. Emerg Infect Dis 14: 1470–1472.
7. Shinde V, Bridges CB, Uyeki TM, Shu B, Balish A, et al. (2009) Triple-
reassortant swine influenza A (H1) in humans in the Uni ted States, 2005–2009.
N Engl J Med 360: 2616–2625.
8. Nayak DP, Hui EK, Barman S (2004) Assembly and budding of influenza virus.
Virus Res 106: 147–165.
9. Lakdawala SS, Lamirande EW, Suguitan AL Jr, Wang W, Santos CP, et al.
(2011) Eurasian-origin gene segments contribute to the transmissibility, aerosol
release, and morphology of the 2009 pandemic H1N1 influenza virus. PLoS
Pathog 7: e1002443.
10. Enami M, Enami K (1996) Influenza virus hemagglutinin and neuraminidase
glycoproteins stimulate the membrane association of the matrix protein. J Virol
11. Jin H, Leser GP, Zhang J, Lamb RA (1997) Influenza virus hemagglutinin and
neuraminidase cytoplasmic tails control particle shape. Embo J 16: 1236–1247.
12. Ali A, Avalos RT, Ponimaskin E, Nayak DP (2000) Influenza virus assembly:
effect of influenza virus glycoproteins on the membrane association of M1
protein. J Virol 74: 8709–8719.
13. Ye Z, Liu T, Offringa DP, McInnis J, Levandowski RA (1999) Association of
influenza virus matrix protein with ribonucleoproteins. J Virol 73: 7467–7473.
14. Baudin F, Petit I, Weissenhorn W, Ruigrok RW (2001) In vitro dissection of the
membrane and RNP binding activities of influenza virus M1 protein. Virology
15. Noton SL, Medcalf E, Fisher D, Mullin AE, Elton D, et al. (2007) Identification
of the domains of the influenza A virus M1 matrix protein required for NP
binding, oligomerization and incorporation into virions. J Gen Virol 88: 2280–
16. Yasuda J, Nakada S, Kato A, Toyoda T, Ishihama A (1993) Molecular assembly
of influenza virus: association of the NS2 protein with virion matrix. Virology
17. Akarsu H, Burmeister WP, Petosa C, Petit I, Muller CW, et al. (2003) Crystal
structure of the M1 protein-binding domain of the influenza A virus nuclear
export protein (NEP/NS2). Embo J 22: 4646–4655.
18. Bui M, Wills EG, Helenius A, Whittaker GR (2000) Role of the influenza vir us
M1 protein in nuclear export of viral ribonucleoproteins. J Virol 74: 1781–1786.
19. Huang X, Liu T, Muller J, Levandowski RA, Ye Z (2001) Effect of influenza
virus matrix protein and viral RNA on ribonucleoprotein formation and nuclear
export. Virology 287: 405–416.
20. Chou YY, Albrecht RA, Pica N, Lowen AC, Richt JA, et al. (2011) The M
segment of the 2009 new pandemic H1N1 influenza virus is critical for its high
transmission efficiency in the guinea pig model. J Virol 85: 11235–11241.
21. Fujiyoshi Y, Kume NP, Sakata K, Sato SB (1994) Fine structure of influenza A
virus observed by electron cryo-microscopy. Embo J 13: 318–326.
22. Bourmakina SV, Garcia-Sastre A (2003) Reverse genetics studies on the
filamentous morphology of influenza A virus. J Gen Virol 84: 517–527.
23. Elleman CJ, Barclay WS (2004) The M1 matrix protein controls the filamentous
phenotype of influenza A virus. Virology 321: 144–153.
24. Burleigh LM, Calder LJ, Skehel JJ, Steinhauer DA (2005) Influenza a viruses
with m utations in th e m1 heli x six domain display a wide variety of
morphological phenotypes. J Virol 79: 1262–1270.
25. Chu CM, Dawson IM, Elford WJ (1949) Filamentous forms associated with
newly isolated influenza virus. Lancet 1: 602.
26. Kilbourne ED, Murphy JS (1960) Genetic studies of influenza viruses. I. Viral
morphology and growth capacity as exchangeable genetic traits. Rapid in ovo
adaptation of early passage Asian strain isolates by combination with PR8. J Exp
Med 111: 387–406.
27. Choppin PW, Murphy JS, Tamm I (1960) Studies of two kinds of virus particles
which comprise influenza A2 virus strains. III. Morphological characteristics:
independence to morphological and functional traits. J Exp Med 112: 945–952.
28. Itoh Y, Shinya K, Kiso M, Watanabe T, Sakoda Y, et al. (2009) In vitro and
in vivo characterization of new swine-origin H1N1 influenza viruses. Nature
29. Nakajima N, Hata S, Sato Y, Tobiume M, Katano H, et al. (2010) The first
autopsy case of pandemic influenza (A/H1N1pdm) virus infection in Japan:
detection of a high copy number of the virus in type II alveolar epithelial cells by
pathological and virological examination. Jpn J Infect Dis 63: 67–71.
30. Roberts PC, Lamb RA, Compans RW (1998) The M1 and M2 proteins of
influenza A virus are important determinants in filamentous particle formation.
Virology 240: 127–137.
31. Liu T, Muller J, Ye Z (2002) Association of influenza virus matrix protein with
ribonucleoproteins may control viral growth and morphology. Virology 304: 89–
32. McCown MF, Pekosz A (2006) Distinct domains of the influenza a virus M2
protein cytoplasmic tail mediate binding to the M1 protein and facilitate
infectious virus production. J Virol 80: 8178–8189.
33. Iwatsuki-Horimoto K, Horimoto T, Noda T, Kiso M, Maeda J, et al. (2006) The
cytoplasmic tail of the influenza A virus M2 protein plays a role in viral
assembly. J Virol 80: 5233–5240.
34. Noton SL, Simpson-Holley M, Medcalf E, Wise HM, Hutchinson EC, et al.
(2009) Studies of an influenza A virus temperature-sensitiv e mutant identify a late
role for NP in the formation of infectious virions. J Virol 83: 562–571.
35. Rossman JS, Jing X, Leser GP, Balannik V, Pinto LH, et al. (2010) Influenza
virus m2 ion channel protein is necessary for filamentous virion formation.
J Virol 84: 5078–5088.
36. Lai JC, Chan WW, Kien F, Nicholls JM, Peiris JS, et al. (2010) Formation of
virus-like particles from human cell lines exclusively expressing influenza
neuraminidase. J Gen Virol 91: 2322–2330.
37. Wang D, Harmon A, Jin J, Francis DH, Christopher-Hennings J, et al. (2010)
The lack of an inherent membrane targeting signal is responsible for the failure
of the matrix (M1) protein of influenza A virus to bud into virus-like particles.
J Virol 84: 4673–4681.
38. Rossman JS, Jing X, Leser GP, Lamb RA (2010) Influenza virus M2 protein
mediates ESCRT-independent membrane scission. Cell 142: 902–913.
PLOS ONE | www.plosone.org 10 November 2012 | Volume 7 | Issue 11 | e50595