0.5 nm. The data were obtained by averaging five repeat scans and corrected by subtraction of the buffer absorption. The resulting spectra expressed as mean molar ellipticity per residue. The secondary structure content was analyzed by DICHROWEB. Thermal denaturation of M1C was monitored by far-UV CD spectra at 222 nm in 1 mm path length cell. 30 mM M1C in 50 mM sodium phosphate, pH 7.4 was heated from 4uC to 96uC at the scan rate of 0.5uC/min. The sample was cooled down to room temperature immediately when one course complete. The far-UV CD spectrum of renatured protein was compared with that of before heating. The final data of thermal denaturation was averaged by thrice scans. DLS analysis The degree of compactness and particle hydrodynamic diam- eter of M1C was examined by dynamic Light Scattering DLS. M1C got from on gel filtration column was concentrated to the optimal reactive concentration of 1.0 mg/ml in 20 mM Tris (pH 7.4), and then the measurement was performed at 25uC. Fluorescence Spectroscopy Fluorescence emission spectra were recorded at room temper- ature by a HITACHI F-4500 fluorescence spectrophotometer in 1 cm quartz cuvette. ANS binding was detected by fluorescence spectra at an excitation wavelength of 380 nm. Emission spectra were measured from 400 nm to 600 nm with the molar ratio ANS/M1C 100:1. Each spectrum was the average of three emission scans. Supporting Information Figure S1 Expression, purification of M1, M1N, M1C from E. coli and secondary structure analysis. (A) M1, M1N and M1C produced in E. coli were purified by affinity chromatography. The purified proteins were analyzed by Tricine- SDS-PAGE. Lane M, the molecular mass marker; lane 1, total cell lysate after induction of pET30a; lane 2, total cell lysate after induction of pET30a-M1 for 11 h at 16uC; lane 3, purified M1 from nickel affinity chromatography; lane 4, total cell lysate after pET30a-M1N was induced for 12 h; lane 5, purified M1N from nickel affinity chromatography; lane 6, total cell lysate of pGEX- 6p-1-M1C prior to induction; lane 7, total cell lysate of pGEX-6p- 1-M1C after induction for 12 h at 16uC; lane 8, GST-M1C eluted from Glutathione Sepharose 4B; lane 9, M1C yielded by References 1. Enami M, Fukuda R, Ishihama A (1985) Transcription and replication of eight RNA segments of influenza virus. Virology 142: 68–77. 2. Heggeness MH, Smith PR, Ulmanen I, Krug RM, Choppin PW (1982) Studies on the helical nucleocapsid of influenza virus. Virology 118: 466–470. 3. Horimoto T, Kawaoka Y (2005) Influenza: lessons from past pandemics, warnings from current incidents. Nat Rev Microbiol 3: 591–600. 4. Lamb RA, Choppin PW (1983) The gene structure and replication of influenza virus. Annu Rev Biochem 52: 467–506. 5. Shaw ML, Stone KL, Colangelo CM, Gulcicek EE, Palese P (2008) Cellular proteins in influenza virus particles. PLoS Pathog 4: e1000085. 6. 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. 7. Ruigrok RW, Calder LJ, Wharton SA (1989) Electron microscopy of the influenza virus submembranal structure. Virology 173: 311–316. 8. Coloma R, Valpuesta JM, Arranz R, Carrascosa JL, Ortin J, et al. (2009) The structure of a biologically active influenza virus ribonucleoprotein complex. PLoS Pathog 5: e1000491. 9. Bui M, Whittaker G, Helenius A (1996) Effect of M1 protein and low pH on nuclear transport of influenza virus ribonucleoproteins. J Virol 70: 8391–8401. 10. 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: 2280–2290. Influenza A Virus M1 Oligomerization PreScission protease cleavage of GST-M1C. (B) Far-UV CD analyses of M1and M1N at the concentration of 30 mM are shown. (TIF) Figure S2 Molecular mass estimation using UNICORN Software. The apparent molecular masses of the 15.8-ml fraction of M1, the 17.6-ml fraction of M1N and the 17.3-ml of M1C on analytical gel filtration column at pH 7.4 were analyzed using UNICORN Software Analysis Module. The column was calibrat- ed by a series of standard global proteins, and the correlation of Log (Molecular weight) and Kav was plotted. The apparent molecular masses indicated by arrows were calculated from the straight line. (TIF) Figure S3 Gel filtration analyses of neutralized proteins obtained from acidic pH. (A and B) Purified M1 (A) and M1N (B) from nickel affinity chromatography were concentrated to 0.7 and 0.6 mg/ml respectively, and loaded on a Superdex 200 column. Purified proteins from the affinity chromatography and gel filtration were analyzed on SDS-PAGE. (C and D) M1 (C) and M1N (D) were purified from gel filtration chromatography at pH 5.0 (solid line), and neutralized into a buffer of pH 7.4, concentrated and reapplied to the column at pH 7.4 (dot line). (TIF) Figure S4 The molecular size of M1C measured by DLS. The degree of compactness and particle hydrodynamic diameter of M1C were examined by DLS. Essentially 100% of the scattering mass was attributed to a single species of M1C. (TIF) Acknowledgments We thank Professor Tao Jiang for critical comments of the manuscript, Ms. Xiaoxia Yu for kindly assistance in sedimentation experiments, Fei Peng for help in CD, Dr. Lei Sun, Dr. Yuhai Bi, Jing Li, Shuai Cao, Shanshan Wang, Caiwei Chen, Zhendong Zhao for helpful discussion and other assistance. Author Contributions Conceived and designed the experiments: WL BX KZ. Performed the experiments: KZ ZW XL. Analyzed the data: WL BX KZ ZW XL. Wrote the paper: KZ ZW XL CY ZB BX WL. 11. Noda T, Sagara H, Yen A, Takada A, Kida H, et al. (2006) Architecture of ribonucleoprotein complexes in influenza A virus particles. Nature 439: 490–492. 12. Elster C, Larsen K, Gagnon J, Ruigrok RW, Baudin F (1997) Influenza virus M1 protein binds to RNA through its nuclear localization signal. J Gen Virol 78(Pt 7): 1589–1596. 13. Elster C, Fourest E, Baudin F, Larsen K, Cusack S, et al. 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Chen BJ, Leser GP, Morita E, Lamb RA (2007) Influenza virus hemagglutinin and neuraminidase, but not the matrix protein, are required for assembly and budding of plasmid-derived virus-like particles. J Virol 81: 7111–7123. PLoS ONE | www.plosone.org 11 May 2012 | Volume 7 | Issue 5 | e37786