New Insights on the Versatile Role of the Cholesterol Binding Motif of the HIV-1 Glycoprotein Gp41

Abstract

Recent experimental results indicate that host cell invasion as well as assembly and budding of the Human Immunodeficiency Virus (HIV) are highly cholesterol dependent. Supposably, cholesterol enriched plasma membrane microdomains, so called rafts, play an important role in different steps of the virus lifecycle. However, the exact function and molecular background of this sensitivity to bilayer compositions remains unknown.We produced different variants of the HIV transmembrane protein gp41 labeled with a yellow fluorescent protein. Fluorescence lifetime imaging microscopy was used to report Forster Resonance Energy Transfer (FRET) between a raft marker labelled with a cyan fluorescent protein and gp41 chimeras in living cells. Since it is highly distance dependent, occurring FRET reports a co-clustering of both fluorescent protein species in microdomains. By comparison of FRET efficiencies from different truncation and mutation variants of gp41, the Cholesterol Recognition Amino Acid Consensus (CRAC) was identified as main determinant of the protein's raft partitioning in the plasma membrane and interestingly in the Golgi apparatus as well. Moreover, using fluorescence polarization anisotropy microscopy we found indications, that wildtype gp41 oligomers are stabilized in plasma membrane microdomains. However, oligomerization of CRAC mutants was found to be significantly impaired, suggesting a pooling function of the lipid rafts for the assembly and sustainment of functional homo-oligomers. Moreover, flow cytometer experiments revealed a remarkable influence of CRAC mutations on the well-known plasma membrane perturbation properties of gp41. Finally, different biophysical methods were applied to further reveal the role of the cholesterol-CRAC interaction in more details.Our data provide further insight into the molecular basis and biological implications of the cholesterol dependent lateral protein sorting for the virus assembly processes at cellular membranes.