Ambroggio, E. E. | bioRxiv Science

bioRxiv2022

HIV-1 GAG SPECIFICITY FOR PIP2-CONTAINING MEMBRANES MIGHT BE DRIVEN BY MACROMOLECULAR ELECTRIC PROPERTIES RATHER THAN MOLECULAR AFFINITIES

The HIV-1 assembly occurs at the plasma membrane, where the GAG polyprotein plays a crucial role. The GAG-membrane association is directed by the matrix domain (MA), which is myristoylated and has a highly basic region that interacts with the anionic lipids. Several evidence suggests that the presence of phosphatidylinositol-(4,5)-bisphosphate (PIP2) highly influence this binding. In addition, MA also interacts with nucleic acids, which is proposed to be important for the specificity of GAG for PIP2-containing membranes. It is proposed that RNA could have a chaperone function when interacting with the MA domain, preventing GAG from associating with unspecific lipid interfaces. Here, we study the interaction of MA with monolayer and bilayer membrane systems, focused on the specificity for PIP2 and on the possible effects of a GAG N-terminal peptide to impair the binding for either RNA or membrane. We found that RNA decreases the kinetics of the protein association with lipid monolayers but without any effect on the selectivity for PIP2. Interestingly, for bilayer systems, this selectivity increases in presence of both the peptide and RNA, even for highly negative charged compositions, where MA by itself doesnt discriminate between membranes with or without PIP2. Therefore, we propose that the specificity of MA for PIP2-membranes might be related to the electrostatic properties of both membrane and protein local environments, rather than a simple difference in molecular affinities. This scenario gives a new understanding of the regulation mechanism with a macromolecular view instead of considering molecular interactions within a ligand-receptor model. ImportanceHIV-1 virions are formed at the PM of infected cells through a direct interaction of the viral GAG protein with lipids. This is a finely regulated process governed by the GAG N-terminal matrix domain, MA. Here, we obtained compelling evidence on how this process depends on the local dielectric environments of both, the membrane and MA. Using bio-membrane mimicking systems, we found how MA myristoylation is involved in the interfacial absorption and anchoring of the protein, where the interaction with RNA negatively regulates this process but in a lesser extent when traces of the PIP2 lipid are present. Additionally, an N-terminal GAG-derived peptide competes with MA for the nucleic acid binding and impair the protein-membrane interaction when PIP2 is absent. All these data allowed us to propose a model for MA association with lipid interfaces and how it depends on oligonucleotide binding, lipid composition and competing peptide presence.

cell biology↗

bioRxiv2021

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

Liquid-liquid phase separation is prone to occur when positively charged proteins interact with nucleic acids. Here, we studied biophysical properties of Dengue (DENV) and Zika (ZIKV) virus capsid proteins to understand the process of RNA genome encapsidation. In this route, the capsid proteins efficiently recruit the viral RNA at the 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 or how the nucleocapsid is recruited and wrapped by the ER membrane during particle morphogenesis. Here, we measured relevant interactions concerning the viral process using purified DENV and ZIKV capsids proteins, membranes mimicking the ER lipid composition and nucleic acids at in vitro conditions. We found that both ZIKV and DENV capsid proteins bound to liposomes at liquid-disordered phase regions and docked exogenous membranes and RNA molecules. When the proteins bound nucleic acids, droplet liquid-liquid phase separation was observed. We characterized these liquid condensates by measuring nucleic acid partition constant 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 supports 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 understand the viral nucleocapsid formation as a stable liquid-liquid phase transition, which could be relevant for Dengue and Zika gemmation, opening new avenues for antiviral intervention.

biophysics↗