Inhibitory effects of archetypical nucleic acid ligands on the interactions of HIV-1 nucleocapsid protein with elements of Psi-RNA

Disrupting the interactions between human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein and structural elements of the packaging signal (Ψ-RNA) could constitute an ideal strategy to inhibit the functions of this region of the genome leader in the virus life cycle. We have employed electrospray ionization (ESI) Fourier transform mass spectrometry (FTMS) to assess the ability of a series of nucleic acid ligands to bind selected structures of Ψ-RNA and inhibit their specific interactions with NC in vitro. We found that the majority of the ligands included in the study were able to form stable non-covalent complexes with stem–loop 2, 3 and 4 (SL2–4), consistent with their characteristic nucleic acid binding modes. However, only aminoglycosidic antibiotics were capable of dissociating preformed NC•SL3 and NC•SL4 complexes, but not NC•SL2. The apparent specificity of these inhibitory effects is closely dependent on distinctive structural features of the different NC•RNA complexes. The trends observed for the IC₅₀ values correlate very well with those provided by the ligand binding affinities and the dissociation constants of target NC•RNA complexes. This systematic investigation of archetypical nucleic acid ligands provides a valid framework to support the design of novel ligand inhibitors for HIV-1 treatment.     

Differential contribution of basic residues to HIV-1 nucleocapsid protein’s nucleic acid chaperone function and retroviral replication

The human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein contains 15 basic residues located throughout its 55-amino acid sequence, as well as one aromatic residue in each of its two CCHC-type zinc finger motifs. NC facilitates nucleic acid (NA) rearrangements via its chaperone activity, but the structural basis for this activity and its consequences in vivo are not completely understood. Here, we investigate the role played by basic residues in the N-terminal domain, the N-terminal zinc finger and the linker region between the two zinc fingers. We use in vitro ensemble and single-molecule DNA stretching experiments to measure the characteristics of wild-type and mutant HIV-1 NC proteins, and correlate these results with cell-based HIV-1 replication assays. All of the cationic residue mutations lead to NA interaction defects, as well as reduced HIV-1 infectivity, and these effects are most pronounced on neutralizing all five N-terminal cationic residues. HIV-1 infectivity in cells is correlated most strongly with NC’s NA annealing capabilities as well as its ability to intercalate the DNA duplex. Although NC’s aromatic residues participate directly in DNA intercalation, our findings suggest that specific basic residues enhance these interactions, resulting in optimal NA chaperone activity.     

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.