Dengue and Zika virus capsid proteins bind to membranes and self-assemble into liquid droplets with nucleic acids.

Dengue virus (DENV) and Zika virus (ZIKV) capsid proteins efficiently recruit and surround the viral RNA at the endoplasmic reticulum (ER) membrane to yield nascent viral particles. However, little is known either about the molecular mechanisms by which multiple copies of capsid proteins assemble into nucleocapsids (NCs) or how the NC is recruited and wrapped by the ER membrane during particle morphogenesis. Here, we measured relevant interactions concerning this viral process using purified DENV and ZIKV capsid proteins, membranes mimicking the ER lipid composition, and nucleic acids in in vitro conditions to understand the biophysical properties of the RNA genome encapsidation process. We found that both ZIKV and DENV capsid proteins bound to liposomes at liquid-disordered phase regions, docked exogenous membranes, and RNA molecules. Liquid-liquid phase separation is prone to occur when positively charged proteins interact with nucleic acids, which is indeed the case for the studied capsids. We characterized these liquid condensates by measuring nucleic acid partition constants and the extent of water dipolar relaxation, observing a cooperative process for the formation of the new phase that involves a distinct water organization. Our data support a new model in which capsid-RNA complexes directly bind the ER membrane, seeding the process of RNA recruitment for viral particle assembly. These results contribute to our understanding of the viral NC formation as a stable liquid-liquid phase transition, which could be relevant for dengue and Zika gemmation, opening new avenues for antiviral intervention.

The influence of myristoylation, liposome surface charge and nucleic acid interaction in the partition properties of HIV-1 Gag-N-terminal peptides to membranes.

The group-specific antigen (GAG) polyprotein of HIV-1 is the main coordinator of the virus assembly process at the plasma membrane (PM) and is directed by its N-terminal matrix domain (MA). MA is myristoylated and possess a highly basic region (HBR) responsible for the interaction with the negative lipids of the PM, especially with PIP2. In addition, MA binds RNA molecules proposed as a regulatory step of the assembly process. Here we study the interaction of a synthetic peptide (N-terminal 21 amino acids of MA) and liposomes of different compositions using a variety of biophysical techniques. Particularly, we use the fluorescence properties of the single tryptophan of the peptide to analyze its partition to membranes, where we harness for first time the analytical ability of spectral phasors method to study this interaction. We found that electrostatic interactions play an important role for peptide partition to membranes and myristoylation reduces the free energy of the process. Interestingly, we observe that while the presence of PIP2 does not cause measurable changes on the peptide-membrane interaction, the interaction is favored by cholesterol. Additionally, we found that the partition process goes through a transition state involving peptide disaggregation and changes in the peptide secondary structure. On the other hand, we found that the presence of oligonucleotides competes with the interaction with lipids by increasing peptide solubility. In summary, we think that our results, in context of the current knowledge of the role of HIV-1 MA, contribute to a better molecular understanding of the membrane association process.

Self-homodimerization of an actinoporin by disulfide bridging reveals implications for their structure and pore formation.

The Trp111 to Cys mutant of sticholysin I, an actinoporin from Stichodactyla helianthus sea anemone, forms a homodimer via a disulfide bridge. The purified dimer is 193 times less hemolytic than the monomer. Ultracentrifugation, dynamic light scattering and size-exclusion chromatography demonstrate that monomers and dimers are the only independent oligomeric states encountered. Indeed, circular dichroism and fluorescence spectroscopies showed that Trp/Tyr residues participate in homodimerization and that the dimer is less thermostable than the monomer. A homodimer three-dimensional model was constructed and indicates that Trp147/Tyr137 are at the homodimer interface. Spectroscopy results validated the 3D-model and assigned 85° to the disulfide bridge dihedral angle responsible for dimerization. The homodimer model suggests that alterations in the membrane/carbohydrate-binding sites in one of the monomers, as result of dimerization, could explain the decrease in the homodimer ability to form pores.