The Role of Lipid Rafts in Virus Assembly. Identification and Characterization of Microdomain Partitioning Factors of the HIV-1 Glycoprotein gp41 using Flim-FRET and Fluorescence Anisotropy Microscopy

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 composition remains unknown.We produced different variants of the HIV transmembrane protein gp41 labelled 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 reflects 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. Whereas localization and trafficking of the fusion proteins resembled reported wildtype behaviour, FACS experiments revealed a remarkable influence of CRAC mutations on plasma membrane perturbation properties of gp41. Furthermore, using fluorescence polarization anisotropy microscopy it could be shown, that wildtype gp41 oligomerization occurs at the plasma membrane. Oligomerization of CRAC mutants was found to be significantly impaired. This suggests a pooling function of lipid rafts not only for interactions with other viral components but also for assemblies of functional homo-oligomers.This study is to our knowledge the first live cell approach characterizing gp41 raft partitioning factors and relating lateral plasma membrane sorting to distinct protein functions and properties.The reported raft dependent oligomerization might be representative for general mechanisms of microdomain-facilitated protein interactions.