Influenza A Virus M1 Oligomerization Figure 7. The structural characteristics of M1C analyzed by CD and Fluorescence Spectroscopy. (A) Secondary structure studies on M1C at pH 7.4. The CD spectra of M1C at the concentrations of 5 mM (dash dot line), 30 mM (dot line), 60 mM (dash line), and 100 mM (solid line) are shown. (B) Thermal denaturation of M1C at 60 mM protein concentration was recorded at 222 nm at pH 7.4. (C) Far-UV CD analyses of M1C incubated at room temperature (solid line), at 37uC for 10 min (dash line), and at 60uC for 10 min (dot line) are shown. (D) Fluorescence spectra of ANS binding to M1C at pH 7.4. The fluorescence spectra of ANS (dot line), ANS in presence of M1C at 25uC (dash line) and ANS bound with M1C at 37uC (solid line) are shown. doi:10.1371/journal.pone.0037786.g007 leading the N-terminal domain completely dissociates into a monomer, we conclude that M1 forms a stable dimer at low pH. In summary, we investigated the oligomerization of M1 of influenza A virus, and found that the N-terminal domain contributes to the pH-dependent oligomerization characteristic of M1. The C-terminal domain is reported for the first time to be a stable dimer. Both N- and C-terminal domains are involved in the oligomerization of M1, which is important in the influenza A virus life cycle. The present data may provide us with new insights into the mechanism of the formation of matrix layer, and help us to better understand the dissociation of the matrix layer of influenza A virus in the uncoating process. Materials and Methods Reagents, Escherichia coli strains and antibodies Escherichia coli (E. coli) strains DH5a and BL21 (DE3) pLysS were obtained from Novagen. High purity chemicals were purchased from Sigma-Aldrich. Other suppliers were: DNA polymerase, T4 DNA ligase and restriction enzymes (New England Biolabs); pET30a vector (Novagen); pGEX-6p-1 vector (GE Healthcare); Yest Extract and Tryptone needed for Luria-Bertani (LB) media (Merck); All kits and devices for protein purification (GE Healthcare); His probe antibody and HRP labeled goat anti- mouse antibody (Santa Cruz Biotechnology); Chemiluminescent for Western blot (Pierce). Mouse anti-M1 monoclonal antibody and rabbit anti-M1 polyclonal antibody were prepared as described previously [57]. Recombinant plasmids construction Influenza A virus A/chicken/Jiangsu/1/1998 (H9N2) M1 genes encoding full-length M1 (252 residues) and an N-terminal fragment (1–170 aa) were amplified by PCR. The amplified DNAs were purified and digested with NdeI/XhoI enzymes. Then cloned into the corresponding sites of pET30a vector (Novagen) with a His6-tag coding sequence (LEHHHHHH) fused at the carboxyl terminus. Recombinant plasmids pET30a-M1 and pET30a-M1N were obtained. The purified PCR product of M1C fragment (165– 252 aa) was amplified and cloned into pGEX-6p-1 vector using BamHI and XhoI restriction sites to obtain recombinant plasmid pGEX-6P-1-M1C. Plasmids pGEX-6p-1-GST-M1 and pET30a- hisM1 were also constructed following the similar method. All the above constructs were transformed into E. coli strain DH5a and inserted genes were confirmed by DNA sequencing. Protein expression and purification Plasmids pET30a-M1, pET30a-M1N, pET30a-hisM1 and pGEX-6P-1-M1C were transformed into E. coli strain BL21 (DE3) and expressed M1, M1N, hisM1 as His6-tag proteins, while PLoS ONE | www.plosone.org 9 May 2012 | Volume 7 | Issue 5 | e37786