ABSTRACT
During HIV-1 morphogenesis, the precursor Gag protein is processed to release capsid (CA) proteins that form the mature virus core. In this process, the CA proteins assemble a lattice in which N-terminal domain (NTD) helices 1-3 are critical for multimer formation. Mature core assembly requires refolding of the N-terminus of CA into a ß-hairpin, but the precise contribution of the hairpin core morphogenesis is unclear. We found that mutations at isoleucine 15 (I15), between the ß-hairpin and NTD helix 1 are incompatible with proper mature core assembly. However, a compensatory mutation of histidine 12 in the ß-hairpin to a tyrosine was selected by long term passage of an I15 mutant virus in T cells. The tyrosine does not interact directly with residue 15, but with NTD helix 3, supporting a model in which ß-hairpin folding serves to align helix 3 for mature NTD multimerization.
Subject(s)
HIV Core Protein p24/genetics , HIV-1/genetics , Mutation, Missense , Suppression, Genetic , Amino Acid Substitution , Cell Line , HIV-1/physiology , Humans , Models, Molecular , Protein Conformation , Protein Folding , Protein Multimerization , Virus AssemblyABSTRACT
The matrix domain (MA) of the HIV-1 precursor Gag (PrGag) protein directs PrGag proteins to assembly sites at the plasma membrane by virtue of its affinity to the phospholipid, phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)). MA also binds to RNA at a site that overlaps its PI(4,5)P(2) site, suggesting that RNA binding may protect MA from associating with inappropriate cellular membranes prior to PrGag delivery to the PM. Based on this, we have developed an assay in which small molecule competitors to MA-RNA binding can be characterized, with the assumption that such compounds might interfere with essential MA functions and help elucidate additional features of MA binding. Following this approach, we have identified four compounds, including three thiadiazolanes, that compete with RNA for MA binding. We also have identified MA residues involved in thiadiazolane binding and found that they overlap the MA PI(4,5)P(2) and RNA sites. Cell culture studies demonstrated that thiadiazolanes inhibit HIV-1 replication but are associated with significant levels of toxicity. Nevertheless, these observations provide new insights into MA binding and pave the way for the development of antivirals that target the HIV-1 matrix domain.
Subject(s)
Anti-HIV Agents/chemistry , HIV-1/chemistry , Ligands , Phospholipids/chemistry , RNA/chemistry , Binding Sites , Cell Line , Cell Survival , Dose-Response Relationship, Drug , Drug Design , Humans , Magnetic Resonance Spectroscopy/methods , Microscopy, Fluorescence/methods , Models, Chemical , Models, Molecular , Molecular Conformation , Protein Binding , Retroviridae/metabolism , Thiadiazoles/chemistryABSTRACT
The matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) precursor Gag (PrGag) protein plays multiple roles in the viral replication cycle. One essential role is to target PrGag proteins to their lipid raft-associated phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] assembly sites at the plasma membranes of infected cells. In addition to this role, several reports have implicated nucleic acid binding properties to retroviral MAs. Evidence indicates that RNA binding enhances the binding specificity of MA to PI(4,5)P(2)-containing membranes and supports a hypothesis in which RNA binding to MA acts as a chaperone that protects MA from associating with inappropriate cellular membranes prior to PrGag delivery to plasma membrane assembly sites. To gain a better understanding of HIV-1 MA-RNA interactions, we have analyzed the interaction of HIV MA with RNA ligands that were selected previously for their high affinities to MA. Binding interactions were characterized via bead binding, fluorescence anisotropy, gel shift, and analytical ultracentrifugation methods. Moreover, MA residues that are involved in RNA binding were identified from NMR chemical shift data. Our results indicate that the MA RNA and PI(4,5)P(2) binding sites overlap and suggest models for Gag-membrane and Gag-RNA interactions and for the HIV assembly pathway.
Subject(s)
HIV-1/metabolism , RNA, Viral/metabolism , Viral Matrix Proteins/metabolism , gag Gene Products, Human Immunodeficiency Virus/metabolism , Anisotropy , Binding Sites , Binding, Competitive , Electrophoretic Mobility Shift Assay , Fluorescence , Humans , Magnetic Resonance Spectroscopy , Protein Binding , Viral Matrix Proteins/chemistry , gag Gene Products, Human Immunodeficiency Virus/chemistryABSTRACT
Based on structural information, we have analyzed the mechanism of mature HIV-1 core assembly and the contributions of structural elements to the assembly process. Through the use of several in vitro assembly assay systems, we have examined details of how capsid (CA) protein helix 1, ß-hairpin and cyclophilin loop elements impact assembly-dependent protein interactions, and we present evidence for a contribution of CA helix 6 to the mature assembly-competent conformation of CA. Additional experiments with mixtures of proteins in assembly reactions provide novel analyses of the mature core assembly mechanism. Our results support a model in which initial assembly products serve as scaffolds for further assembly by converting incoming subunits to assembly proficient conformations, while mutant subunits increase the probability of assembly termination events.