envelope [42]. It has been reported that the N-terminal domain mediates the oligomerization of M1. The crystal structure of the N-terminal domain showed that it dimerizes through the interaction interfaces [31,32], and that the 91–158 aa region is the main determinant of M1 self-oligomerization [10]. Interestingly, Noton et al. reported that the C-terminal domain also plays an important role in oligomerization by interacting with the N-terminal domain, but not with other C-terminal domains [10]. Furthermore, Ruigrok et al. found that the C-terminal domain is involved in M1 oligomerization, for the C-terminal alone could cause aggregation [43]. Studies of the behavior of the C-terminal domain have produced conflicting results, which lead to future investigation into fully elucidating the role of C-terminal domain in M1 oligomerization. During the uncoating process of virus infection, the virion is acidified by the influx of H+ [44,45,46]. A structural transition of the matrix layer has been observed when the virus was incubated at low pH [42]. Recent research by cryo-electron tomography further showed that the intermolecular interactions in the M1 layer are affected when the virions were incubated at pH 4.9, and the matrix layer was no longer seen in the virions [47]. The interaction between M1 and vRNP has also been shown to be disrupted by low pH [9]. Zhirnov found that M1 extracted from M1-vRNP complexes at an acidic pH is in a monomeric form and does not aggregate after pH neutralization [9,15]. But the crystal structure of the N-terminal domain solved by Harris at pH 4.0 suggested that this domain is a dimer [32]. Therefore, it is not certain which oligomerization state of M1 forms in acidic pH, and how the influence of pH affects the oligomerization of M1. In order to resolve these aforementioned issues, we investigated the oligomerization of M1 and determined the individual contribution of the N- and C-terminal domains. We found that the oligomerization of M1 is pH-dependent. M1 can form multiple-ordered oligomers at neutral pH, and those oligomers dissociate at acidic pH to dimeric form. Further studies revealed that pH-dependent oligomerization characteristic of M1 is due to the N-terminal domain. The C-terminal domain exists as a stable dimer in solution, independent of pH and concentration. Results The oligomerization of M1 is dependent on pH It has been reported that M1 forms an organized structure adjacent to the envelope in virus particles [42]. Ruigrok et al. extracted M1 oligomers from influenza A virus [41] and Zhao et al. reported that M1 tends to oligomerize soon after synthesis in BHK-21 cells [48]. To investigate the oligomerization of M1 in vitro, we constructed recombinant M1 with a C-terminal His6-tag, which was produced in soluble form in E. coli. M1 was purified by nickel affinity chromatography and showed a single band at about 28 kDa on SDS-PAGE (Fig. S1A, lane 3). CD spectroscopy analysis showed that purified M1 was a typical a-helical protein (Fig. S1B). Consistent with the previous report [32], the result showed that recombinant M1 was not stable and that the protein could degrade Purified M1 from nickel affinity chromatography at a concen- tration of 0.8 mg/ml was eluted from the gel filtration column in four major fractions at pH 7.4, whose elution volumes were 8.8 ml, 11.2 ml, 13.5 ml, and 15.8 ml, respectively (Fig. 1A). The apparent molecular mass of the 15.8-ml fraction was estimated to be 52 kDa, approximately double of that of the M1 monomer (28.9 kDa) (Fig. S2). The result indicated that the smallest oligomerization state of M1 is a dimer. The oligomerization state of 15.8-ml fraction of M1 was further confirmed by cross linking Influenza A Virus M1 Oligomerization assay. After treating with glutaraldehyde, the sample showed two bands on Tricine-SDS-PAGE. One band was at the same position with the untreated sample (28 kDa), while the other ran to the position of about 56 kDa, which is the molecular mass of an M1 dimer (Fig. 2A). The dimeric M1 eluted from a gel filtration column was collected and concentrated to different concentrations by Ultra filtration, and then re-applied to the gel filtration column at pH 7.4. In this process, M1 exhibited concentration-dependent oligomerization (Fig. 1B). When the loaded protein reached a concentration of 0.1 mg/ml, M1 was eluted from the gel filtration column as a single fraction of 15.8 ml. However, with an increase of protein concentration to 0.5 mg/ml, M1 yielded four fractions with the elution volumes of 10.1 ml, 11.2 ml, 13.5 ml, and 15.8 ml, corresponding to the molecular masses of 580 kDa, 460 kDa, 155 kDa, and 52 kDa, respectively. An 8.8-ml fraction was observed on the gel filtration column when the loaded protein concentration reached 1.0 mg/ ml, indicating M1 is able to form higher molecular mass oligomers which beyond the detection limits of the column. The proportion of 10.1-ml, 11.2-ml, and 13.5-ml fractions also increased with increasing of sample concentration. We further collected 10.1-ml, 11.2-ml, and 13.5-ml fractions, respectively, and re-applied the samples to the gel filtration column at pH 7.4. The proteins continued to elute at the same volumes (Fig. 1D). Even if the 10.1-ml, 11.2-ml, and 13.5-ml fractions were diluted to the concentration of 0.05 mg/ml, their elution volumes did not change and no other oligomerization state formed on the gel filtration column at pH 7.4 (data not shown). These results indicated that M1 can form stable oligomers at neutral pH, and that the oligomerization process is irreversible. The effect of acidity on oligomerization of M1 was investigated at pH 5.0. M1 purified by nickel affinity chromatography was exchanged into a buffer at pH 5.0. 1.0 mg/ml M1 was loaded onto the gel filtration column, and only the 15.8-ml fraction was observed (Fig. S3A). The fraction was collected and concentrated to higher concentrations by ultrafiltration, but it continued to elute at 15.8 ml on the column (Fig. 1C). The result indicated that the dimeric protein underwent no detectable oligomerization during concentration. Furthermore, it was ascertained that increasing the pH environment of M1 by changing the buffer to pH 7.4 did not change the oligomerization state of M1. M1 dimer alone was observed on the gel filtration column after pH neutralization (Fig. S3C). The pH influence on the high molecular mass oligomers was also examined. We collected the 10.1-ml, 11.2-ml, and 13.5-ml fractions from the gel filtration column at pH 7.4 and exchanged the samples into buffer of pH 5.0. Each sample was loaded on the column at pH 5.0, and all the samples eluted at 15.8 ml. The 15.8-ml fraction generated from acidification of 11.2-ml fraction was shown in Fig. 1D. The result indicated the interactions between dimeric M1 molecules were destroyed by low pH. Taken together, these results indicated that the oligomerization of M1 is dependent on pH, and that the dimeric form of M1 is stable at acidic pH. The analysis of direct interactions between M1 molecules was carried out by a GST pull-down assay. We found that hisM1 could bind to GST-M1 (Fig. 2B, lane 3), but not to GST alone at pH 7.4 (Fig. 2B, lane 2). The hisM1 was pulled down by GST- M1 at pH 7.4 and was then washed with an acidic buffer (pH 5.0), but no hisM1 was detected by the anti-His antibody (Fig. 2B, lane 4). As M1 forms dimers at pH 5.0, this result indicated that the direct interaction between dimeric M1 could be inhibited by low pH. PLoS ONE | www.plosone.org 2 May 2012 | Volume 7 | Issue 5 | e37786