Electrostatic repulsion between HIV-1 capsid proteins modulates hexamer plasticity and in vitro assembly

Abstract

Capsid protein (CA) is the major component of the human immunodeficiency virus type 1 (HIV-1) core. Three major phosphorylation sites have been identified at positions S(109), S(149) and S(178) in the amino-acid sequence of CA. Here, we investigated the possible consequences of phosphorylation at these sites on the CA hexamer organization and plasticity using in silico approaches. The biological relevance of molecular modeling was then evaluated by analyzing the in vitro assembly properties of bacterially expressed CA bearing S(109)D, S(149)D, or S(178)D substitutions that mimic constitutive phosphorylation at these sites. We found that a constitutive negative charge at position 109 or 149 impaired the capacity of mature CA to assemble in vitro. In vivo, HIV-1 mutants bearing the corresponding mutation showed dramatic alterations of core morphology. At the level of CA hexamer, S(149) phosphorylation generates inter-monomer repulsions, while phosphorylation at position 109 resulted in cleavage of important bonds required for preserving the stability of the edifice. Addition of a negative charge at position 178 allowed efficient assembly of CA into core-like structures in vitro and in vivo and significantly increased CA hexamer stability when modeled in silico. All mutant viruses studied lacked infectivity since they were unable to produce proviral DNA. Altogether our data indicate that negative charges, that mimic phosphorylation, modulate assembling capacity of CA and affect structural properties of CA hexamers and of HIV-1 cores. In the context of the assembled core, phosphorylation at these sites may be considered as an event interfering with core organization and HIV-1 replicative cycle.

Phosphorylation of the HIV-1 capsid by MELK triggers uncoating to promote viral cDNA synthesis

“…To determine whether the viral core interacts with MELK, we purified One-STrEP-FLAG-(OSF)-tagged MELK protein expressed in HeLa cells, and viral cores from cell-free virions. The HIV-1 envelope was removed from virions and envelope-stripped cores were enriched by ultracentrifugation through a discontinuous 10% and 30% sucrose gradient with 0.1% Triton X-100 in the 10% sucrose layer, as previously reported [20] (Fig 2D). The envelope-stripped cores were characterized by transmission electron microscopy (TEM) showing recognizable ~100 nm cone-shaped structures similar to authentic HIV-1 cores (Fig 2E).…”

“…Envelope-stripped HIV-1 cores were prepared as described previously [20]. Briefly, HIV-1-containing culture supernatants were prepared by transiently transfecting HeLa cells with pNL4-3 using LipofectAMINE LTX PLUS (Invitrogen Corp., Carlsbad, CA).…”

Kinetics of Surface-Driven Self-Assembly and Fatigue-Induced Disassembly of a Virus-Based Nanocoating

“…However, in the latter case assembly typically requires relatively high protein concentrations and very high ionic strength (51,52,54) that may screen electrostatic repulsions in CA (63)(64)(65), a macromolecular crowding agent (53,66), or use of fused NC protein and added nucleic acid (67). In contrast, fast and efficient self-assembly of the CA lattice as a nanocoating on mica takes place even at CA concentrations <1 mM at close to physiological ionic strength and pH in the absence of crowding agents, NC and RNA.…”

Cellular kinases incorporated into HIV-1 particles: passive or active passengers?

“…Our approach developed in silico to model the consequences of phosphorylation at the level of a p24 hexamer has revealed that negative charges generated by phosphate conjugation at each serine position favors inter-monomer repulsion or cleavage of important inter-monomeric bonds required for preserving the stability of the hexameric ring of p24 [90] (Figure 3). According to these results, p24 phosphorylation may be considered as an event that could modify organization of p24 hexamers and at a higher order impact the organization of the viral core edifice.…”

“…Changes observed in the internal diameter of the CTD of the non phosphorylated (left) and S 149 -phosphorylated CA hexamer (right) at 4500 ps. For clarity, only the CTD is represented in ribbons and one color is assigned per monomer [90]. …”