Dimerization Efficiency of Canine Distemper Virus Matrix Protein Regulates Membrane-Budding Activity

Abstract

Paramyxoviruses rely on the matrix (M) protein to orchestrate viral assembly and budding at the plasma membrane. Although the mechanistic details remain largely unknown, structural data suggested that M dimers and/or higher-order oligomers may facilitate membrane budding. To gain functional insights, we employed a structure-guided mutagenesis approach to investigate the role of canine distemper virus (CDV) M protein self-assembly in membrane-budding activity. Three six-alanine-block (6A-block) mutants with mutations located at strategic oligomeric positions were initially designed. While the first one includes residues potentially residing at the protomer-protomer interface, the other two display amino acids located within two distal surface-exposed ␣-helices proposed to be involved in dimerdimer contacts. We further focused on the core of the dimeric interface by mutating asparagine 138 (N138) to several nonconservative amino acids. Cellular localization combined with dimerization and coimmunopurification assays, performed under various denaturing conditions, revealed that all 6A-block mutants were impaired in selfassembly and cell periphery accumulation. These phenotypes correlated with deficiencies in relocating CDV nucleocapsid proteins to the cell periphery and in virus-like particle (VLP) production. Conversely, all M-N138 mutants remained capable of selfassembly, though to various extents, which correlated with proper accumulation and redistribution of nucleocapsid proteins at the plasma membrane. However, membrane deformation and VLP assays indicated that the M-N138 variants exhibiting the most reduced dimerization propensity were also defective in triggering membrane remodeling and budding, despite proper plasma membrane accumulation. Overall, our data provide mechanistic evidence that the efficiency of CDV M dimerization/oligomerization governs both cell periphery localization and membrane-budding activity.IMPORTANCE Despite the availability of effective vaccines, both measles virus (MeV) and canine distemper virus (CDV) still lead to significant human and animal mortality worldwide. It is assumed that postexposure prophylaxis with specific antiviral compounds may synergize with vaccination campaigns to better control ongoing epidemics. Targeting the matrix (M) protein of MeV/CDV is attractive, because M coordinates viral assembly and egress through interaction with multiple cellular and viral components. However, the lack of basic molecular knowledge of how M orchestrates these functions precludes the rational design of antivirals. Here we combined structure-guided mutagenesis with cellular, biochemical, and functional assays to investigate a potential correlation between CDV M self-assembly and virus-like particle (VLP) formation. Altogether, our findings provide evidence that stable M dimers at the cell periphery are required to productively trigger VLPs. Such stabilized M dimeric units may facilitate further assembly into robust higher-order oligomers necessary to promote plasma membrane-b...

The Functional Oligomeric State of Tegument Protein GP41 Is Essential for Baculovirus Budded Virion and Occlusion-Derived Virion Assembly

“…For RNA viruses, it is also quite common to find their matrix proteins forming oligomers. Dimerization of the canine distemper virus (CDV) matrix (M) protein governs its cell membrane periphery localization and membrane budding activity (11). Studies have shown that Ebola virus VP40 (42,43), respiratory syncytial virus M (44), vesicular stomatitis virus M (45), and influenza virus M1 (8) form oligomers or multimers in vivo or in vitro.…”

“…T he viral tegument is a proteinaceous layer sandwiched between the nucleocapsid and envelope of virus particles. Tegument proteins (also called matrix proteins for some viruses) have been demonstrated to play important roles in the replication cycles of a wide variety of enveloped viruses, including herpesviruses (1)(2)(3)(4), retroviruses (5,6), orthomyxoviruses (7,8), paramyxoviruses (9)(10)(11), filoviruses (12), and pneumoviruses (13). One of the best-studied examples in relation to tegument proteins is herpesvirus, a large double-stranded DNA virus in which more than 20 tegument proteins have been identified by mass spectrometry (MS).…”

Tropism and molecular pathogenesis of canine distemper virus

“…The attachment protein H, which lacks the neuraminidase action observed in other viruses, attaches to receptors present on the plasma membranes of host cells, such as SLAM, nectin-4, and other, in glial cells [55]. Moreover, the M protein is essential in the assembly and budding of CDV particles, and acts as an intermediate between the RNP and the glycoprotein surfaces by enabling the interaction of M with the C- and N-terminal of N and the cytoplasmic tails of H and F proteins [56]. V and C proteins are non-essential with respect to the virus replication process but critical for preventing the host immune responses.…”

“…Similar to the MeV replication cycle, it is crucial to assemble the M protein, which plays an important role in the assembly and budding of virions, considered as an intermediary between the RNP and the surface glycoproteins and orchestrating the viral particle assembly process [56, 70]. The RNP and the glycoproteins, in specific regions of the plasma membrane of the host-infected cells, form complete infectious CDV particles as the result of a coordinated interaction between viral and cellular factors [71].…”

Genetic Adaptations, Biases, and Evolutionary Analysis of Canine Distemper Virus Asia-4 Lineage in a Fatal Outbreak of Wild-Caught Civets in Thailand

“…During infection, the CDV H protein initiates the infection by attaching to the signaling lymphocyte activating molecule (SLAM) receptor of immune cells [9,10] and nectin-4 receptors in various epithelial cells of the hosts [11][12][13]. The F protein then plays a crucial role in viral fusion on the host cellular membrane after H protein binding to the host cell [14,15]. After CDV infects the local lymphoid cells, the virus spreads to distant lymphatic tissues where T-cell and B-cell lymphocytes proliferate.…”