Structure and mechanism of a novel HIV assembly inhibitor

In many cases the correct assembly of multiple proteins is critical for their biological function. By interfering specifically with certain protein-protein interactions one can control the assembly and as a result their function. The proteins that encapsulate the genetic information of the HIV virus undergo structural changes that change their arrangement from a spherical to a conical macromolecular assembly. The virus is only infections if the conical assembly is obtained. However, the protein-protein interactions that determine the spherical and the conical assemblies are not well understood. Several models for the arrangement have been proposed that are compatible with range of structural and biochemical data. We have a new small peptide that can interfere with both the spherical and conical assembly of the proteins in a yet unknown way. We are proposing to determine the interaction and the mechanism by which this peptide interferes with the formation of the assemblies. We intend to determine if the inhibitor simply blocks one or more important interfaces that are required for the assembly or whether it induces a conformational change or rearrangement in the protein such that assembly is disrupted. In addition we will determine the interaction of novel classes of molecules that have the same function. This will tell us if the mode of interaction of the inhibitor in hand is unique or if there are several solutions to the assembly inhibition. This will help us to understand the structural determinants that are required for the assembly and correct function of the HIV virus.

Assembly of HIV involves trafficking of the structural precursor protein Gag to the plasma membrane, where Gag proteins accumulate and initiate a budding structure. The virus is initially released as an immature, non-infectious particle containing a spherical shell of about 5000 Gag proteins underneath the viral membrane. After release, maturation is initiated by processing of Gag proteins by the viral protease. Five sequential cleavage steps of Gag lead to the separation of the matrix (MA), capsid (CA), nucleocapsid (NC) and p6 proteins as well as two intervening linker regions SP1 and SP2. Complete maturation is only possible after the very last processing step separating CA from the C-terminal 14 residue spacer peptide SP1. The cleavage steps lead to drastic morphological changes inside the virion that are required for viral infectivity. In the mature virus particle only MA remains in a spherical layer attached to the membrane, while CA forms the characteristic conical core that encases the NC-RNA complex. The interactions driving assembly and maturation are still poorly understood. The interactions in the immature (spherical) state are not known. Analysis of in vitro assembled tubular particles as well as those of viral cones in combination with structures of single CA domains shed light on the interactions in the mature capsid, According to these data CA seems to be organized in hexameric rings based on interactions mediated by the N-terminal domain, and each hexamer is connected to six adjacent hexamers via a dimer interface of the C-terminal domain. The C-terminal domain of CA (C-CA) has a hometypic interaction in the crystal structure as well as in solution. In the mature core an additional heterotypic interaction exists between the N- and C-terminal domains of different capsid proteins. However, the current data are compatible with different arrangements of the N- and C-terminal domains of CA. Recently a new peptide was discovered that prevents formation of immature and mature particles in vitro. This provides a new tool to investigate interaction sites and mechanisms that are important for assembly. High resolution nuclear magnetic resonance (NMR) will be used to determine the structure, interaction and mechanism of inhibition of assembly using this novel HIV assembly inhibitor. We will determine the structure of the C-terminal domain of the capsid protein (C-CA) in complex this novel inhibitor and determine the structural and dynamic consequences of inhibitor binding to C-CA on larger Gag constructs, including the multi-domain mature capsid protein and the uncleaved C-CA NC fragment of Gag. We will optimise the peptide ligands and determine the thermodynamics of binding with C-CA (and/or the larger Gag constructs) We will determine the interaction of small molecule inhibitors with C-CA or with larger Gag fragments in order to determine if the mechanism of inhibition is unique to the peptide.