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Surface features of a Mononegavirales matrix protein indicate sites of membrane interaction Victoria A. Moneya,b, Helen K. McPheec,1, Jackie A. Moselyc,1, John M. Sandersonc,2, and Robert P. Yeob,d,2 aThe Maurice Wilkins Centre for Molecular Biodiscovery and the School of Biological Sciences, University of Auckland, Thomas Building, 3a Symonds Street, Auckland Central 1010, New Zealand; cDepartment of Chemistry and bCentre for Bioactive Chemistry, University Science Laboratories, Durham University, South Road, Durham, DH1 3LE, United Kingdom; and dSchool for Medicine and Health, Queen’s Campus, Durham University, Stockton-on-Tees, TS17 6BH, United Kingdom Edited by Robert A. Lamb, Northwestern University, Evanston, IL, and approved January 14, 2009 (received for review June 13, 2008) The matrix protein (M) of respiratory syncytial virus (RSV), the related function of the M. M are also implicated in host cell prototype viral member of the Pneumovirinae (family Paramyxo- transcriptional cut-off (10), possibly via a direct interaction with viridae, order Mononegavirales), has been crystallized and the RNA (11). structure determined to a resolution of 1.6 Å. The structure com- A number of matrix-like proteins are known to bind membranes prises 2 compact ␤-rich domains connected by a relatively unstruc- or lipid vesicles in vitro, most likely through a combination of tured linker region. Due to the high degree of side-chain order in hydrophobic and electrostatic interactions (12–14). Expression of the structure, an extensive contiguous area of positive surface certain Ms in eukaryotic cells in the absence of other viral proteins charge covering Ϸ600 Å2 can be resolved. This unusually large can induce formation of virus-like particles (VLPs). The efficiency patch of positive surface potential spans both domains and the of VLP generation can be increased if the M is coexpressed with a linker, and provides a mechanism for driving the interaction of the viral glycoprotein (15–18). Ms share a tendency to oligomerize, a protein with a negatively-charged membrane surface or other feature likely to be important in the self-assembly and budding virion components such as the nucleocapsid. This patch is comple- processes (19). In tissue culture, RSV induces formation of long mented by regions of high hydrophobicity and a striking planar slender projections from the surface of the cell known as viral arrangement of tyrosine residues encircling the C-terminal domain. filaments. It was found that removal of the lipid membrane from Comparison of the RSV M sequence with other members of the viral filaments left an M containing sheath (5); it is possible that M Pneumovirinae shows that regions of divergence correspond to oligomerization and self-assembly is the driving force behind the surface exposed loops in the M structure, with the majority of viral formation of viral filaments. In this article we report the structure species-specific differences occurring in the N-terminal domain. of the full-length RSV M protein solved at a resolution of 1.6 Å, and discuss the implications of this structure for the function of the CD ͉ crystal structure ͉ respiratory syncytial virus ͉ sequence alignment protein. espiratory syncytial virus (RSV) is the prototype member of Results and Discussion Rthe Pneumovirinae, a subfamily of the Paramyxoviridae (or- Structure of the RSV M. The RSV M was purified by using nickel der Mononegavirales). Morphologically, the extracellular virion affinity chromatography. During the cloning process, a methi- consists of a lipid bilayer envelope, within which are embedded onine to arginine change occurred, and we refer to the resultant 3 glycoproteins, 2 of which (F and G) are important in cell formofMasM254R. The crystal structure of M254R was solved by attachment and viral entry into target cells. The third, the SH using MIRAS techniques to a resolution of 1.6 Å, representing protein, contributes to pathology in the host (1). Internally, the first example of an intact M from the Mononegavirales. virions contain helical nucleocapsids that consist of N protein Despite evidence for higher order oligomers [supporting infor- tightly bound to the negative-sense nonsegmented genomic mation (SI) Fig. S1] such as dimers, tetramers, and hexamers, in RNA. The nucleocapsid in turn is associated with components of solution, the crystallized form is monomeric. Crystallographic the viral RNA-dependent RNA polymerase (L, P, M2-1, and data are presented in Table S1. The overall fold consists of 2 M2-2 proteins), forming the holo-nucleocapsid (2–4). Between clear domains connected by a 13-residue linker region. The the holo-nucleocapsid and the outer envelope there is a layer of N-terminal domain comprises residues 1 to 126, whereas the matrix protein (M), which is associated peripherally with the C-terminal domain consists of residues 140 to 255 (Fig. 1 A and membrane (5). The other family members of the Mononegavi- B). Only residues 99 and 100 could not be assigned a clear rales (Rhabdoviridae, Filoviridae, and Bornaviridae) all subscribe location within the electron density. The N-terminal domain to this basic arrangement of the virion, although the overall consists of a twisted ␤-sandwich comprised of 2 ␤-sheets, 1 of 3 morphology can vary between the families. For example, and 1 of 4 strands, positioned almost perpendicular to each Paramyxoviridae virions are pleiomorphic, whereas the Rhab- other. The overall topology of this domain is of a curved doviridae have a regular bullet shape structure, and the Filov- horseshoe-like arrangement with ␤-sheet 1 forming the concave, iridae have a more filamentous shape. Extracellular RSV virions form by a budding process that occurs at the plasma membrane within specialized lipid domains Author contributions: V.A.M., J.M.S., and R.P.Y. designed research; V.A.M., H.K.M., J.A.M., J.M.S., and R.P.Y. performed research; V.A.M., H.K.M., J.A.M., J.M.S., and R.P.Y. analyzed (5, 6) and M appears to drive the final assembly process, which data; and V.A.M., J.M.S., and R.P.Y. wrote the paper. is the incorporation of the holo-nucleocapsid and initiation of The authors declare no conflict of interest. the budding process (7, 8). Before budding, there is a coordi- This article is a PNAS Direct Submission. nated assembly of viral components; and it is evident that the Data deposition: The atomic coordinates and structure factors for the RSV M protein have glycoproteins and M proteins are important determinants of the been deposited in the Protein Data Bank, www.rcsb.org [PDB ID codes 2vqp (coordinates) location on the plasma membrane at which the virus buds (9). It and r2vqpsf (structure factors)]. is also possible that the interaction between M and the glycop- 1H.K.M. and J.A.M. contributed equally to this work. roteins, possibly mediated with the cytoplasmic tails, are impor- 2To whom correspondence may be addressed. E-mail: [email protected] or tant in the budding process. Genome silencing to prevent [email protected]. transcription and replication by the viral polymerase before This article contains supporting information online at www.pnas.org/cgi/content/full/ MICROBIOLOGY incorporation of the holo-nucleocapsid in the nascent virion is a 0805740106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0805740106 PNAS ͉ March 17, 2009 ͉ vol. 106 ͉ no. 11 ͉ 4441–4446 Downloaded by guest on September 30, 2021 Fig. 1. Three-dimensional structure of the RSV M protein. The crystal structure of M254R (resolution 1.6 Å) shows 2 domains composed largely of ␤-sheets. Statistical information for the X-ray data are provided in SI Materials and Methods.(A) Divergent (wall eyed) stereoview of M254R colored according to domain with the linker shown in cyan, the N-terminal domain in blue and the C-terminal domain in red. Residue R254 is shown in ball-and-stick representation. (B)A topology diagram of the protein. The linker between the N- and C-terminal domains is shown in magenta. Residues (numbers refer to Met as ϩ1) in ␤-sheets are represented by broad arrows and helices as cylinders. (C) Electrostatic surface potential (calculated with APBS) for M254R, presented in a color range from red to blue (Ϫ5toϩ5 kT/e); uncharged residues are uncolored. inner face flanked by loop regions, and ␤-sheet 2 forming the acids Thr-136 and Leu-137 (see Fig. S1). These weak interdo- convex, outer face. The C-terminal domain consists of a flat- main interactions further suggest that the protein may exist in tened ␤-barrel, comprising 2 3-stranded anti-parallel ␤-sheets. alternative quaternary structures in solution, and that the inter- The regions linking the sheets between strands 2 and 3, and domain packing observed in the crystal may be metastable, strands 5 and 6, are largely helical in nature (Fig. 1B). There is driven mainly by sequestering of the hydrophobic residues, which no evidence for any complexed metal ions or for a potential zinc form the major part of the interface, away from the bulk solvent. finger motif. The 254R substitution in the protein lies at the very Flexibility of this type has been speculated as being important for end of the C-terminal domain in an area largely devoid of the M of the influenza virus, as well as for EBOV VP40 and VSV secondary structure; therefore, it is unlikely to have a significant M, and is thought to be necessary to accommodate the different effect on the protein structure or function. functions of the M throughout the viral life cycle. The linker region is largely lacking in secondary structure features, with the exception of a short helical region. The Structure Comparison. The tertiary structure of M254R is globally presence of this linker is consistent with structures obtained for similar to that of the EBOV VP40 protein (24), showing the same fragments of the Ebola virus (EBOV) VP40 and vesicular overall fold with a Z score of 6.6 and a rmsd of 3.7 Å.

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