MCPHEE, HELEN,KENNEDY (2009) A Study of the Membrane Binding Properties of the Matrix Protein from Human Respiratory Syncytial Virus. Doctoral thesis, Durham University.
This thesis describes the structure of the matrix (M) protein from human respiratory syncytial virus (RSV), and its interactions with model membranes composed of a range of lipids, which have been characterized using a variety of techniques.
The M protein was expressed in E. coli with a histidine tag and purified by immobilised metal affinity chromatography (IMAP). The protein was found to have acquired a mutation at position 254 from methionine to arginine, but this did not appear to affect its behaviour and both the native and mutated form of the protein were used in several experiments.
Efforts to produce 2D crystals of the M protein by incubation with lipid and detergent were unsuccessful, but in several detergent-free incubations, particularly where the lipid DPPE was included, extended helical structures were formed that bore a striking resemblance to the structure of filamentous RSV virions with the viral envelope removed. These helical assemblies appeared to grow from liposomes, and demonstrate the inherent ability of RSV M to self assemble into arrays of this nature.
Samples of purified protein were provided to Dr Victoria Money who was able to crystallize M and solve its structure to a resolution of 1.6 Å, revealing two domains linked by a region with little secondary structure that is potentially flexible, and a large positively charged area on the surface extending across both domains. The overall structure is somewhat similar to the crystal structure of the Ebola virus matrix protein VP40. Circular dichroism (CD) studies of M showed that it contains significantly less secondary structure in solution than in the crystal structure. Cross-linking experiments suggested that RSV M preferentially forms dimers, tetramers and hexamers in solution, in common with several other viral matrix proteins.
Experiments performed on a Langmuir trough revealed that the M protein partitions into phospholipid monolayers, regardless of whether the lipid head group is phosphocholine or phosphoethanolamine. It interacts differently with monolayers containing sphingomyelin, which in combination with cholesterol forms phase separated liquid-ordered (Lo) microdomains. RSV M was found to bind extrinsically to the sphingomyelin-containing Lo domains. This did not occur when the protein was in contact with monolayers composed of only phospholipids and cholesterol that are also known to form Lo domains, showing that RSV M has a specific interaction with sphingomyelin. The evidence for this hypothesis obtained from Langmuir pressure-area isotherms was supported by direct visualization of the monolayer at the surface using Brewster angle microscopy (BAM), and examination of monolayers deposited on a modified silicon surface by atomic force microscopy (AFM).
The findings described herein suggest that specific interactions with sphingomyelin may provide a mechanism for localization of the matrix protein, and therefore targeting of the site of viral assembly, to particular regions of the host cell membrane. The structure of the protein, with its two domains linked by a potentially flexible region, would allow for changes in protein conformation whilst bound to the membrane: it is possible that such changes could play a key role in the function of RSV M.