Dissection of Influenza A Virus M1 Protein: pH- Dependent Oligomerization of N-Terminal Domain and Dimerization of C-Terminal Domain Ke Zhang1,2, Zhao Wang 3 , Xiaoling Liu1, Changcheng Yin3, Zeshan Basit1, Bin Xia4*, Wenjun Liu1,2* 1 Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, 2 Graduate University of Chinese Academy of Sciences, Beijing, China, 3 Department of Biophysics, Health Science Center, Peking University, Beijing, China, 4 Beijing Nuclear Magnetic Resonance Center, Peking University, Beijing, China Abstract Background: The matrix 1 (M1) protein of Influenza A virus plays many critical roles throughout the virus life cycle. The oligomerization of M1 is essential for the formation of the viral matrix layer during the assembly and budding process. Methodology/Principal Findings: In the present study, we report that M1 can oligomerize in vitro, and that the oligomerization is pH-dependent. The N-terminal domain of M1 alone exists as multiple-order oligomers at pH 7.4, and the C-terminal domain alone forms an exclusively stable dimer. As a result, intact M1 can display different forms of oligomers and dimer is the smallest oligomerization state, at neutral pH. At pH 5.0, oligomers of the N-terminal domain completely dissociate into monomers, while the C-terminal domain remains in dimeric form. As a result, oligomers of intact M1 dissociate into a stable dimer at acidic pH. Conclusions/Significance: Oligomerization of M1 involves both the N- and C-terminal domains. The N-terminal domain determines the pH-dependent oligomerization characteristic, and C-terminal domain forms a stable dimer, which contributes to the dimerization of M1. The present study will help to unveil the mechanisms of influenza A virus assembly and uncoating process. Citation: Zhang K, Wang Z, Liu X, Yin C, Basit Z, et al. (2012) Dissection of Influenza A Virus M1 Protein: pH-Dependent Oligomerization of N-Terminal Domain and Dimerization of C-Terminal Domain. PLoS ONE 7(5): e37786. doi:10.1371/journal.pone.0037786 Editor: Dong-Yan Jin, University of Hong Kong, Hong Kong Received November 21, 2011; Accepted April 26, 2012; Published May 24, 2012 Copyright: 2012 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Basic Research Program (973) of China (2011CB504705), Chinese Academy of Sciences Innovation projects (KSCX2-YW-N-054, KSCX2-YW-R-158), and National Natural Science Foundation of China (30972185, 30901073). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: liuwj@im.ac.cn (WL); binxia@pku.edu.cn (BX) Introduction Influenza viruses are enveloped viruses that belong to the Orthomyxovirus family. The genome of influenza A virus contains eight negative-stranded RNA segments, which code for 10 or 11 proteins [1,2,3,4,5]. The matrix 1 (M1) protein, encoded by segment 7, is the most abundant structural protein in the virions [6,7]. M1 is a multifunctional protein that plays essential roles in many aspects of the virus life cycle. It forms the matrix layer localized between the lipid membrane and the viral ribonucleo- proteins (vRNPs) [8], ensuring the stabilization of the architecture of the virion. By interacting with RNA and vRNPs [9,10,11], M1 is also involved in nuclear localization [12], RNA transcription inhibition [13,14,15], and regulation of the import/export of newly synthesized vRNPs [16,17,18]. As the major structural protein, M1 plays an important role in virus assembly and budding. It can form virus-like particles (VLPs) through collabo- ration with other viral proteins [19,20,21,22]. During budding, M1 brings viral components to the budding site [23], interacts with viral envelope proteins (HA, NA, M2) [24,25,26], and also recruits host components needed for bud completion [24,27,28]. M1 protein is made up of 252 amino acid residues [29], and consists of two domains (N-terminal domain from 1 to 164 aa and C-terminal domain from 165 to 252 aa) linked by a protease- sensitive loop. The three-dimensional structure of the N-terminal domain was determined by X-ray diffraction at pH 4.0 and pH 7.0 [30,31,32]. The structures showed that the N-terminal domain consists of two 4-helix bundles (2 to 67 aa and 91 to 158 aa) connected by a helix linker (H5). The three dimensional structure of C-terminal domain has not been obtained so far, but data from circular dichroism (CD), tritium bombardment and bioinformatics analysis suggest that C-terminal domain folds into helices and contains an appreciably unstructured region [30,33,34,35,36]. Previous investigations have also shown that M1 has a strong tendency to oligomerize [37]. In virus assembly and the budding, the oligomerization of M1 is required for the matrix layer to form under the lipid membrane [21,38,39,40,41]. Furthermore, the M1-M1 interaction facilitates membrane bending, which is required for bud initiation [38,41]. Expression of M1 alone in eukaryotic cells allows for the production of VLPs [21,22]. While in authentic virions, M1 forms an ordered structure adjacent to the PLoS ONE | www.plosone.org 1 May 2012 | Volume 7 | Issue 5 | e37786