Figure 5. GST pull-down detected the direct interaction between N-terminal domains. Glutathione-Sepharose bound GST- M1 or GST was incubated with M1N in the binding buffer (pH 7.4). After washing extensively with the buffer of different pH (7.4 or 5.0), the M1N bound to the beads was extracted and analyzed by immunoblotting with anti-His antibody. doi:10.1371/journal.pone.0037786.g005 Discussion Matrix proteins play a crucial role in virus assembly. They can interact with the RNP complex, as well as with the viral membrane. The importance of oligomerization has been observed in a number of viral matrix proteins. Several examples of this include: Nipah virus M, which has a tendency to oligomerize [49], when expressed alone is sufficient to form VLPs [50]; oligomer- ization of Measles virus M protein can promote the release of VLPs [51]; the tetrameric form of Borna virus M protein is reported to be the basic oligomerization species for the assembly of larger two-dimensional lattices [52]; and oligomerization of Ebola virus VP40 is essential for particle morphogenesis and regulation of viral transcription [53]. In influenza A virus particles, Ruigrok et al. reported that M1 is a 6 nm-long rod [7,41]. It forms an ordered helical layer adjacent to the envelope in the virus particles [42]. Schulze et al. estimated that this protein layer was about 60 A˚ thick [54]. The oligomerization of M1 has been studied from many aspects. M1 oligomers extracted from the virus were found to form flexible ribbons and small coils [41]. Furthermore, when M1 alone was expressed in cells, they found that M1 tends to oligomerize soon after synthesis [48] and produce VLPs. In our present study, we investigated the oligomerization of M1 in vitro. Recombinant M1, expressed in soluble form in E. coli, were able to form multi-ordered oligomers at pH 7.4. We determined the smallest oligomerization state of M1 to be a dimer, and we found the oligomerization of M1 to depend on pH. M1 oligomers were able to disassemble to dimeric form in acidic pH. To our knowledge, this is the first report on the detection of M1 oligomers in vitro. Previous studies showed that M1 extracted from the virus at acidic pH could not oligomerize after pH neutralization [15]. We also identified recombinant M1 purified in vitro at acidic pH that could also not oligomerize after neutralizing the buffer pH. In more in-depth studies, we dissected the contributions of the N- and C-terminal domains to the oligomerization of M1 in detail. Influenza A Virus M1 Oligomerization The crystal structure of N-terminal domain solved by Sha and Harris showed that this domain is in dimeric form [31,32]. But Arz et al. reported that the N-terminal domain is a monomer in solution [30]. In our study, we identified that the smallest oligomerization state of the N-terminal domain is a monomer. Interaction interfaces between N-terminal domains were observed in the crystal structure, and Noton et al. reported that the middle region dominates the oligomerization of M1 [10]. Here, we found that N-terminal domain can form multi-order oligomers at pH 7.4, and confirmed the direct interaction between N-terminal domains. The results indicated that self-oligomerization with the N-terminal domain is important in the oligomerization of M1. Further studies showed the oligomerization of N-terminal domain is pH-dependent. The interaction between N-terminal domains is disrupted at pH 5.0, causing the oligomers of M1N dissociate to monomeric form. Moreover, our findings indicated that the N- terminal domain contributes to the pH-dependence of oligomer- ization in the intact protein. During the uncoating process of virus infection, H+ effluxes from endosomes into virus particles and acidifies the interior of the virion. Calder et al. found that matrix layer could disassemble at low pH [42]. The recent studies further showed the matrix layer would disappear when the virions were incubated at pH 4.9 for 30 min [47]. Our findings suggest that dissociation of M1 oligomers may result from the disruption of interactions in N-terminal domain at low pH. Our data also showed that the N-terminal domain purified at acidic pH could not oligomerize after pH neutralization, which may cause the same phenomenon observed in M1. This result could be related to the release of vRNP into the nucleus from M1-vRNP complexes, during which the interaction between M1 and vRNP is destroyed under acidification [9]. As low pH induces an irreversible conformation change of M1, it would be unable to re-associate with vRNP in the cytoplasm [55]. The atomic structure of C-terminal domain has not been resolved yet. The results of tritium-bombardment experiments suggest that the C-terminal domain consists largely of a-helix [33,34,35]. Arzt et al calculated the C-terminal domain to have a structure consisting of 38% a-helix and considered that C-terminal domain contributes more size to monomeric intact protein than its molecular weight [30]. Our DLS data support the view about the size of the C-terminal domain. The diameter of the C-terminal domain is 2.6 nm, larger than the global protein with a molecular mass of 20 kDa (about 2.1 nm). We directly measured the a-helix content in the C-terminal domain to be 31%, and examined the structural plasticity by several methods. It has been proposed that the weakly structured C-terminal domain contributes more to the flexibility of M1, and considerably unstructured regions enable C- terminal domain to interact with some components of the host cell [35,36]. On the other hand, previous research has identified the important role of the C-terminal domain in virus assembly. Studies by Noton et al. showed that the N-terminal domain alone is not sufficient for virion assembly [10]. M1 SUMOylated at K242 has also been identified to be involved in the process of virus assembly [56]. The mechanisms of how the C-terminal domain participates in the oligomerization of M1 reported by Noton et al. [10] and Ruigrok et al. were inconsistent. Our results disagree with either of them. In our study, we have demonstrated that the C-terminal domain is a stable dimer independent of pH and protein concentration. There is no direct interaction between dimeric forms of C-terminal domains. Therefore, it is reasonable to infer that the smallest oligomerization state of M1 is dimer. Our results also demonstrated that the C-terminal domain does not interact with the N-terminal domain. Together with the result that the interaction between N-terminal domains is disrupted at acidic pH, PLoS ONE | www.plosone.org 7 May 2012 | Volume 7 | Issue 5 | e37786