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

Area of Science:

• Virology
• Structural Biology
• Molecular Biology

Background:

• The human immunodeficiency virus type 1 (HIV-1) capsid protein (CA) is crucial for viral core formation.
• Three key phosphorylation sites (S109, S149, S178) on CA have been identified, but their functional impact remains unclear.

Purpose of the Study:

• To investigate the effects of phosphorylation at CA's S109, S149, and S178 sites on CA hexamer organization, plasticity, and HIV-1 assembly.
• To evaluate the biological relevance of in silico findings through in vitro and in vivo experiments.

Main Methods:

• In silico molecular modeling to predict the impact of phosphorylation on CA hexamer structure and stability.
• In vitro assembly assays using bacterially expressed CA mutants (S109D, S149D, S178D) mimicking constitutive phosphorylation.
• Analysis of HIV-1 mutant core morphology and infectivity in vivo.

Main Results:

• Constitutive negative charge at S109 or S149 significantly impaired in vitro CA assembly and led to altered core morphology in vivo.
• S149 phosphorylation caused inter-monomer repulsions within the CA hexamer; S109 phosphorylation led to bond cleavage, destabilizing the hexamer.
• S178 phosphorylation facilitated in vitro and in vivo CA assembly into core-like structures and enhanced hexamer stability in silico.
• All studied HIV-1 mutants exhibited a complete loss of infectivity due to impaired proviral DNA production.

Conclusions:

• Phosphorylation, mimicked by negative charges, modulates the assembly capacity of HIV-1 CA.
• These phosphorylation events significantly affect the structural properties of CA hexamers and the overall HIV-1 core.
• Phosphorylation at these sites interferes with HIV-1 core organization and the viral replicative cycle, impacting infectivity.