Analysis of Human Immunodeficiency Virus Type 1 Matrix Binding to Membranes and Nucleic Acids

Abstract

The human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein targets HIV-1 precursor Gag (PrGag) proteins to assembly sites at plasma membrane (PM) sites that are enriched in cholesterol and phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P 2 ]. MA is myristoylated, which enhances membrane binding, and specifically binds PI(4,5)P 2 through headgroup and 2 acyl chain contacts. MA also binds nucleic acids, although the significance of this association with regard to the viral life cycle is unclear. We have devised a novel MA binding assay and used it to examine MA interactions with membranes and nucleic acids. Our results indicate that cholesterol increases the selectivity of MA for PI(4,5)P 2 -containing membranes, that PI(4,5)P 2 binding tolerates 2 acyl chain variation, and that the MA myristate enhances membrane binding efficiency but not selectivity. We also observed that soluble PI(4,5)P 2 analogues do not compete effectively with PI(4,5)P 2 -containing liposomes for MA binding but surprisingly do increase nonspecific binding to liposomes. Finally, we have demonstrated that PI(4,5)P 2 -containing liposomes successfully outcompete nucleic acids for MA binding, whereas other liposomes do not. These results support a model in which RNA binding protects MA from associating with inappropriate cellular membranes prior to PrGag delivery to PM assembly sites.

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

“…Moreover, we have shown that RNA binding enhances the binding specificity of MA to PI(4,5)P 2 -containing membranes. This was evident by the fact that PI(4,5)P 2 -containing liposomes successfully competed with nucleic acids for MA binding, whereas other liposomes did not (32,33). In agreement with our results, RNase treatment of Gag synthesized in vitro using rabbit reticulocyte lysate significantly reduced the selectivity of Gag binding to PI(4,5)P 2 liposomes as opposed to phosphatidylserine-containing liposomes (34).…”

“…NMR investigations have indi-cated that HIV-1 MA preferentially binds to soluble PI(4,5)P 2 mimics through contacts with the lipid headgroup and its 2Ј-acyl chain and that binding promotes both exposure of the MA myristate group and protein oligomerization (28,45). Consistent with the above observations, we have shown that MA proteins tend to organize as hexamers of trimers on lipid membranes containing PI(4,5)P 2 (51) and that the binding specificity of MA is enhanced by cholesterol (32,33).…”

“…The proteins were desalted by three rounds of buffer exchange using dialysis buffer of 10 mM sodium phosphate (pH 7.8) and 50 mM NaCl, supplemented with ␤-mercaptoethanol (1 mM final concentration), aliquoted, and stored at Ϫ80°C under nitrogen gas. 15 N-isotopically labeled proteins were prepared by growing cells on 15 NH 4 Cl (NLM-467-5, Cambridge Isotopes) as the sole nitrogen source as described previously (27,28,32,33,43,45,56). For NMR studies, the proteins were dialyzed against 50 mM sodium phosphate (pH 5.5), 100 mM NaCl, and 5 mM dithiothreitol (DTT) and then concentrated by Millipore ultrafiltration concentrators against the same buffer.…”

“…Protein Preparation-The myristoylated HIV-1 MA protein (MyrMA) as well as the unmyristoylated MA protein (MA) were expressed in Escherichia coli strain BL21(DE3)/pLysS (Novagen) along with Saccharomyces cerevisiae N-myristyltransferase from pET-11a-based vectors kindly provided by Michael F. Summers (University of Maryland Baltimore County) as described previously (27,28,32,33,45,56). The MA protein was prepared similarly to MyrMA, except that myristic acid was excluded from the bacterial growth medium during the induction phase.…”

“…In addition to membrane and Env protein binding, several reports have ascribed nucleic acid binding properties to retroviral MAs (29 -35), and it is possible that such binding might facilitate PrGag delivery to the PM, virus assembly, and/or nuclear import of viral preintegration complexes (36 -42). How does the structure (27,28,32,(43)(44)(45) of MA accommodate all these essential functions? In addition to its N-terminal myristate, which is essential for membrane binding (8,25,46,47), MA is composed of a globular domain consisting of five helices and three ␤ sheet strands (27,28,(43)(44)(45)(46)(47).…”