Membrane-induced alterations in HIV-1 gag and matrix protein-protein interactions

The matrix (MA) domain of human immunodeficiency virus type 1 (HIV-1) contains sequences that direct association with the nucleus at early times in the virus replication cycle and with the plasma membrane at late times in the cycle. Localization to these sites is critical for functions related to the establishment of the infecting provirus and viral assembly, respectively. Mutational and structural analyses indicate that the opposing targeting signals which mediate these subcellular localization events include the same basic residues found in the N-terminal region of the protein. Here, we examined protein multimerization as a determinant of membrane association. Under high ionic strength conditions, Gag, but not MA, binds phospholipid membranes with high affinity. The oligomerization state of the protein per se did not appear to be a prerequisite for stable membrane binding, as Gag and MA were both capable of forming oligomers in high ionic strength buffer. To determine the fate of Gag and MA multimers in the presence of phospholipid membranes in real time, we measured resonance energy transfer between oligomer subunits in the presence and absence of lipid. The presence of phospholipid significantly increased the efficiency of resonance energy transfer between Gag molecules, consistent with enhanced Gag multimerization. This suggests that Gag oligomers assembled on the membrane surface and correlated with the observed stability of membrane binding. In contrast, the efficiency of resonance energy transfer between MA molecules decreased, indicating that MA oligomers dissociated in the presence of membrane, consistent with observed unstable binding. Identical results were obtained whether the probes were covalently attached to a Lys residue in Gag or to residues specifically within the MA domain of Gag; whether the fluorophore was rhodamine or fluorescein; or whether hetero- or homotransfer was measured. The results suggest that phospholipid induces alterations in Gag and MA protein-protein interactions that may contribute to the puzzling ability of MA to direct targeting functions requiring alternately membrane binding and membrane dissociation. The results also suggest that regions downstream of the MA domain in the precursor, or conformations formed after maturation of MA, play a critical role in oligomerization-modulated membrane binding.