Effect of multimerization on membrane association of Rous sarcoma virus and HIV-1 matrix domain proteins

In most retroviruses, plasma membrane (PM) association of the Gag structural protein is a critical step in viral assembly, relying in part on interaction between the highly basic Gag MA domain and the negatively charged inner leaflet of the PM. Assembly is thought to begin with Gag dimerization followed by multimerization, resulting in a hexameric lattice. To directly address the role of multimerization in membrane binding, we fused the MA domains of Rous sarcoma virus (RSV) and HIV-1 to the chemically inducible dimerization domain FK506-binding protein (FKBP) or to the hexameric protein CcmK4 from cyanobacteria. The cellular localization of the resulting green fluorescent protein (GFP)-tagged chimeric proteins was examined by fluorescence imaging, and the association of the proteins with liposomes was quantified by flotation in sucrose gradients, following synthesis in a reticulocyte extract or as purified proteins. Four lipid compositions were tested, representative of liposomes commonly reported in flotation experiments. By themselves, GFP-tagged RSV and HIV-1 MA proteins were largely cytoplasmic, but both hexamerized proteins were highly concentrated at the PM. Dimerization led to partial PM localization for HIV-1 MA. These in vivo effects of multimerization were reproduced in vitro. In flotation analyses, the intact RSV and HIV-1 Gag proteins were more similar to multimerized MA than to monomeric MA. RNA is reported to compete with acidic liposomes for HIV-1 Gag binding, and thus we also examined the effects of RNase treatment or tRNA addition on flotation. tRNA competed with liposomes in the case of some but not all lipid compositions and ionic strengths. Taken together, our results further underpin the model that multimerization is critical for PM association of retroviral Gag proteins. In addition, they suggest that the modulation of membrane binding by RNA, as previously reported for HIV-1, may not hold for RSV.