Clustered Lysine Residues of the Canine Distemper Virus Matrix Protein Regulate Membrane Association and Budding Activity

Abstract

The canine distemper virus (CDV) matrix (M) protein is multifunctional; it orchestrates viral assembly and budding, drives the formation of virus-like particles (VLPs), regulates viral RNA synthesis and may support additional functions. CDV M may assemble into dimers, where each protomer is constituted by N-terminal and C-terminal domains (NTD and CTD, respectively). Here, to investigate whether electrostatic interactions between CDV M and the plasma membrane (PM) may contribute to budding activity, selected surface-exposed positively charged lysine residue, which locate within a large basic patch of CTD, were replaced by amino acids with selected properties. We found that some M-mutants harboring amino acid with neutral and positive charge (methionine and arginine, respectively) maintained full functionality, including proper interaction and localization with the PM as well as intact VLP and progeny virus production, as demonstrated by employing a cell exit-complementation system. Conversely, while the overall structural integrity remained mostly unaltered, most of the nonconservative M-variants (carrying a glutamic acid; negatively charged) exhibited a cytosolic phenotype secondary to the lack of interaction with the PM. Consequently, such M-variants were entirely defective in VLP production and viral particle formation. Furthermore, the proteasome inhibitor Bortezomib significantly reduced wild-type M-mediated VLP production. Nevertheless, in absence of the compound, all engineered M lysine-variants exhibited unaffected ubiquitination profiles, consistent with other residues likely involved in this functionally-essential post-translational modification. Altogether, our data identified multiple surface-exposed lysine residues, located within a basic patch of CDV M-CTD, critically contributing to PM association and ensuing membrane budding activity. IMPORTANCE Although vaccines against some morbilliviruses exist, infections still occur, which can result in dramatic brain disease or fatal outcome. Post-exposure prophylaxis with antivirals would support global vaccination campaigns. Unfortunately, there is no efficient antiviral drug currently approved. The matrix (M) protein of morbilliviruses coordinates viral assembly and egress through interaction with multiple cellular and viral components. However, molecular mechanisms supporting these functions remain poorly understood, which preclude the rationale design of inhibitors. Here, to investigate potential interactions between canine distemper virus (CDV) M and the plasma membrane (PM), we combined structure-guided mutagenesis of selected surface-exposed lysine residues with biochemical, cellular and virological assays. We identified several lysines clustering in a basic patch microdomain of the CDV M C-terminal domain, which contributed to PM association and budding activity. Our findings provides novel mechanistic information of how morbilliviruses assemble and egress from infected cells, thereby delivering bases for future antiviral drug development.

Mutation of Basic Residues R283, R286, and K288 in the Matrix Protein of Newcastle Disease Virus Attenuates Viral Replication and Pathogenicity

“…In addition, the K258 residue of Nipah virus M protein is also responsible for targeting the E3-ubiquitin ligase TRIM6 to inhibit the IKKε kinase-mediated type-I interferon antiviral response [ 30 ]. In addition, a recent study reported that several lysines clustering in a basic patch microdomain of the C-terminal domain of canine distemper virus M protein contribute to plasma membrane association and viral budding activity [ 31 ]. Up until now, only a small number of basic residues, such as R36, R42, K247, and two basic amino acid clusters of NLS in the NDV M protein have been investigated [ 9 , 11 , 12 , 13 ].…”

Measles and Nipah virus assembly: Specific lipid binding drives matrix polymerization

“…5B ). Other paramyxovirus M proteins also encode positively charged residues in homologous sites; mutations to these residues in canine distemper virus, a close relative of MeV, inhibit M-induced VLP formation ( 45 ), suggesting that this site is structurally similar across paramyxovirus M proteins. Similarly, the importance of the C terminus in paramyxovirus M-driven budding has also been recently highlighted for PIV ( 46 ).…”

Oligomerization and Cell Egress Controlled by Two Microdomains of Canine Distemper Virus Matrix Protein

“…In a recent functional study, we identified several surface-exposed lysine residues located within a large basic patch of M-CTD that were likely involved in electrostatic membrane interaction. These data enabled us to propose a model contributing to explaining how M may be positioned toward the PM ( 48 ). Based on this model, the location of both helices may provide supporting ground for dimer-dimer contacts and consequent formation of a 2D-M array.…”

“…Generation of recombinant viruses. Rescue of mutated recombinant viruses were conducted by initially transfecting Bsr-T7 cells seeded in 6-well plates with the plasmid encoding the A75/17 antigenome (harboring the mutation in an M gene) together with helper (pTM-N, P and L [48]) plasmids, as well as pMD2.G (addgene), which encoded the VSV-G glycoprotein. The latter was added in order to restore viral budding in case of defective M-carrying recombinant viruses.…”