Mutation of Hydrophobic Residues in the C-Terminal Domain of the Marburg Virus Matrix Protein VP40 Disrupts Trafficking to the Plasma Membrane

Abstract

Marburg virus (MARV) is a lipid-enveloped negative sense single stranded RNA virus, which can cause a deadly hemorrhagic fever. MARV encodes seven proteins, including VP40 (mVP40), a matrix protein that interacts with the cytoplasmic leaflet of the host cell plasma membrane. VP40 traffics to the plasma membrane inner leaflet, where it assembles to facilitate the budding of viral particles. VP40 is a multifunctional protein that interacts with several host proteins and lipids to complete the viral replication cycle, but many of these host interactions remain unknown or are poorly characterized. In this study, we investigated the role of a hydrophobic loop region in the carboxy-terminal domain (CTD) of mVP40 that shares sequence similarity with the CTD of Ebola virus VP40 (eVP40). These conserved hydrophobic residues in eVP40 have been previously shown to be critical to plasma membrane localization and membrane insertion. An array of cellular experiments and confirmatory in vitro work strongly suggests proper orientation and hydrophobic residues (Phe281, Leu283, and Phe286) in the mVP40 CTD are critical to plasma membrane localization. In line with the different functions proposed for eVP40 and mVP40 CTD hydrophobic residues, molecular dynamics simulations demonstrate large flexibility of residues in the EBOV CTD whereas conserved mVP40 hydrophobic residues are more restricted in their flexibility. This study sheds further light on important amino acids and structural features in mVP40 required for its plasma membrane localization as well as differences in the functional role of CTD amino acids in eVP40 and mVP40.

The Minor Matrix Protein VP24 from Ebola Virus Lacks Direct Lipid-Binding Properties

“…The results exhibited lack of detectable binding by VP24 to lipids on either the PIP strip or the membrane lipid strip. In contrast, eVP40 and mVP40 bound significantly to a number of anionic lipids, as previously reported, including PS [ 27 , 29 , 37 ] and phosphoinositides [ 28 , 30 , 32 , 33 , 38 ]. Lastly, no background of lipid binding was observed using purified protein from E. coli embracing an empty expression vector ( Figure 1 ).…”

“…In contrast, the typical matrix proteins in filovirus such as EBOV VP40 (eVP40) and Marburg virus (MARV) VP40 (mVP40) are membrane associated peripheral proteins that bind to membrane lipids directly with high affinity. For instance, the association of eVP40 with phosphatidylserine (PS) and phosphatidylinositol-4,5-biphosphate (PI(4,5)P 2 ) in the inner leaflet of the plasma membrane facilitates extensive eVP40 oligomerization and virus particle budding [ 26 , 27 , 28 , 29 ], while mVP40 tends to bind anionic membrane lipids nonspecifically through electrostatic interactions [ 30 , 31 , 32 , 33 ]. The expression of either eVP40 or mVP40 in mammalian cells is also sufficient to induce VLP generation [ 13 , 34 , 35 , 36 ].…”

The Minor Matrix Protein VP24 from Ebola Virus Lacks Direct Lipid-Binding Properties

“…The results exhibited lack of detectable binding by VP24 to lipids on either the PIP strip or the membrane lipid strip. In contrast, eVP40 and mVP40 bound significantly to a number of anionic lipids as previously reported, including PS [27,29,37] and phosphoinositides [28,30,32,33,38] Lastly, no background of lipid binding was observed using purified protein from E.coli embracing an empty expression vector (Figure 1). Figure 1.…”

“…In contrast, the typical matrix proteins in filovirus such as EBOV VP40 (eVP40) and Marburg virus (MARV) VP40 (mVP40) are membrane associated peripheral proteins that bind to membrane lipids directly with high affinity. For instance, the association of eVP40 with phosphatidylserine (PS) and phosphatidylinositol-4,5-biphosphate (PI(4,5)P2) in the inner leaflet of the plasma membrane facilitates extensive eVP40 oligomerization and virus particle budding [26][27][28][29], while mVP40 tends to bind anionic membrane lipids nonspecifically through electrostatic interactions [30][31][32][33]. The expression of either eVP40 or mVP40 in mammalian cells is also sufficient to induce VLP generation [13,[34][35][36].…”

Identification of the Functional Domain of HPIV3 Matrix Protein Interacting with Nucleocapsid Protein

“…VLP is also widely used to study virus assembly, germination mechanisms, and viral protein interactions. Similar to the M of other enveloped RNA viruses, expressing M of HPIV3 alone can form VLP [ 13 , 16 – 20 ], while N cannot.…”