Analysis of Small Molecule Ligands Targeting the HIV-1 Matrix Protein-RNA Binding Site

Abstract

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.

Trimer Enhancement Mutation Effects on HIV-1 Matrix Protein Binding Activities

“…WT myristoylated and unmyristoylated [designated Myr(Ϫ)] MA C-terminally His-tagged proteins were expressed in Escherichia coli strain BL21(DE3)/pLysS (Novagen) from pET-11a-based vectors and purified and analyzed as described previously (9,11,13,38,39). The WT MAGFP protein was purified as described above.…”

“…Fluorescence anisotropy (FA) binding assays were performed as described previously (11,13). Briefly, 10 nM fluorescein isothiocyanate (FITC)-labeled Sel25 RNA oligomer (5= FITC-G GACA GGAAU UAAUA GUAGC UGUCC-3=; Invitrogen) samples were incubated in 25 mM sodium phosphate (pH 6.0), 50 mM NaCl with successive additions of 30 M protein to achieve final concentrations of 0 to 5,120 nM protein.…”

“…This function is accomplished in part by virtue of an amino-terminal myristate moiety and, in part, through direct binding to PI(4,5)P2 (1). Evidence indicates that the PI(4,5)P2-binding site on MA overlaps a binding site for RNA, which suggests a chaperone model in which MA-RNA binding protects the MA domain of Pr55Gag from associating with inappropriate intracellular membranes prior to delivery to the PI(4,5)P2-rich PM assembly sites (9)(10)(11)(12)(13)(14)(15)(16). In some experimental systems, it has been shown that Pr55Gag proteins with MA deletions (⌬MA) or swaps of MA with other membrane binding domains are capable of directing the assembly of conditionally infectious viruses, but in these cases, assembly and replication efficiencies have tended to be compromised (17)(18)(19)(20)(21).…”

Analysis of HIV-1 Gag Protein Interactions via Biotin Ligase Tagging

“…Infections were tracked by immunoblot detection of infected-cell CA levels as described below. For TZM-bl infections, cells were infected with virus dilutions for 48 h and collected, lysed, and monitored for ␤-galactosidase reporter activity as described previously (10).…”

“…PrGag itself is cotranslationally myristoylated at its N-terminal MA residue and travels via a still incompletely elucidated pathway to plasma membrane (PM) assembly sites that are enriched in cholesterol and phosphatidylinositol-(4,5)-bisphosphate (PI [4,5]P2) (1)(2)(3)(4)(5)(6)(7)(8). The PrGag MA domain binds PI(4,5)P2, which, in part, explains how PM targeting occurs (4,(9)(10)(11)(12). MA also binds to RNA, and models suggest that MA-RNA binding serves a chaperone function, preventing PrGag from binding to intracellular membranes prior to arrival at the PI(4,5)P2-rich PM (3, 4, 9-11, 13, 14).…”