The matrix (MA) domain of the 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]P2) assembly sites at the plasma membranes (PMs) 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)P2-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 PM 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 previously were selected 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)P2 binding sites overlap, and suggest models for Gag-membrane and Gag-RNA interactions, and for the HIV assembly pathway.
The human immunodeficiency virus (HIV) capsid (CA) protein assembles into a hexameric lattice that forms the mature virus core. Contacts between the CA N-terminal domain (NTD) of one monomer and the C-terminal domain (CTD) of the adjacent monomer are important for the assembly of this core. In this study, we have examined the effects of mutations in the NTD region associated with this interaction. We have found that such mutations yielded modest reductions of virus release but major effects on viral infectivity. Cell culture and in vitro assays indicate that the infectivity defects relate to abnormalities in the viral cores. We have selected second-site compensatory mutations that partially restored HIV infectivity. These mutations map to the CA CTD and to spacer peptide 1 (SP1), the portion of the precursor Gag protein immediately C terminal to the CTD. The compensatory mutations do not locate to the molecularly modeled intermolecular NTD-CTD interface. Rather, the compensatory mutations appear to act indirectly, possibly by realignment of the C-terminal helix of the CA CTD, which participates in the NTD-CTD interface and has been shown to serve an important role in the assembly of infectious virus.
Background:The HIV-1 matrix protein (MA) binds both RNA and phospholipids. Results: Ligands that compete with RNA for binding to MA were identified and characterized. Conclusion: Thiadiazolanes bind to residues in the HIV-1 MA -II-V cleft that mediates RNA and phospholipid binding to MA. Significance: These investigations provide new insights into MA-ligand binding and antiviral design.
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