Analysis of the Molecular Mechanisms Controlling HIV-1 Capsid Assembly and Capsid's Potential as a Drug Target

Abstract

The HIV-1 core is made up of capsid proteins (CA) which house the NC-RNA complex, the viral integrase (IN), and reverse transcriptase (RT). Studies show viral infectivity is dependent on proper capsid assembly, stability, and the ability of capsid to disassemble. Despite this, the molecular mechanisms controlling HIV-1 capsid assembly and disassembly are poorly understood. Sequence analysis of CA reveals two highly conserved cysteine residues Cys198 and Cys218, which can be found in most retroviruses. A mutation of either cysteine impairs viral assembly or infectivity. It has been speculated that CA assembly or disassembly is oxidation dependent and regulated by the oxidation or reduction of the intramolecular disulfide formed between Cys198 and Cys218. We investigated the role of these conserved cysteine residues and the impact their intramolecular disulfide bond had on capsid stability and assembly. We observed that oxidized C-terminal domain (CTD) is less stable than the reduced CTD but that disulfide bond formation promotes dimerization. In the full-length CA the oxidized and double Cys-Ser mutant both assemble at the same rate, but the mutant incapable for forming a disulfide, disassembles faster. These findings suggest HIV-1 CA disassembles in a redox dependent manner. The importance of CA in HIV pathogenesis makes it an attractive therapeutic target. In addition to our evaluation of the CA on a molecular level, we also investigated the use of the CTD dimer system as a tool for screening small molecules that inhibited CA oligomerization in-vitro. We describe in full, an optimized and validated high-throughput screen based on fluorescence polarization that is able to identify small compounds that prevent CTD dimerization, and also inhibit CA oligomerization in-vitro. In-depth structural, biochemical and biophysical studies that deepen our understanding of this important viral protein are instrumental in unveiling new therapies.