The great advantage of virus-like particle (VLP) nano-vaccines is their structural identity to wild-type viruses, ensuring that antigen-specific B-cells encounter viral proteins in their natural conformation. “Wild-type” viral nanoparticles can be further genetically or biochemically functionalized with biomolecules (antigens and adjuvants). Flagellin is a potent inducer of innate immunity and it has demonstrated adjuvant effectiveness due to its affinity for toll-like receptor 5 (TLR5). In contrast to most TLR ligands, flagellin is a protein and can induce an immune response against itself. To avoid side-effects, we incorporated a less inflammatory and less immunogenic form of flagellin as an adjuvant into HIV-based nanoparticle B-cell-targeting vaccines that display either the HIV-1 envelope protein (Env) or a model antigen, hen egg lysozyme (HEL). While flagellin significantly enhanced HEL-specific IgG responses, anti-Env antibody responses were suppressed. We demonstrated that flagellin did not activate B-cells directly in vitro, but might compete for CD4+ T-cell help in vivo. Therefore, we hypothesize that in the context of VLP-based B-cell nano-vaccines, flagellin serves as an antigen itself and may outcompete a less immunogenic antigen with its antibody response. In contrast, in combination with a strong immunogen, the adjuvant activity of flagellin may dominate over its immunogenicity.
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.
cART is highly efficient in suppressing HIV replication in HIV-infected patients and has resulted in a dramatic reduction in morbidity and mortality in HIV-infected people, yet it does not cure HIV infection. In addition, cART has several disadvantages.
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