Mechanistic Investigation of HIV-1 Gag Association with Lipid Membranes

Abstract

An extensive investigation into the initial association of HIV-1 Gag with lipid membranes was conducted with second harmonic generation. The roles of the lipid phase, phospholipid 1,2-dioleoyl-sn-glycero-3-phospho-(1-myo-inositol-4,5-bisphosphate) [PI­(4,5)­P2], the presence of the myristoyl group on Gag, the C-terminus of Gag, and the presence of transfer ribonucleic acid (tRNA) in Gag–membrane association were examined using the physiologically most relevant full-length Gag protein studied thus far. The tighter packing of a bilayer composed of gel-phase lipids was found to have a lower relative amount of membrane-bound Gag in comparison to its fluid-phase counterpart. Rather than driving membrane association of Gag, the presence of PI­(4,5)­P2 and the myristoyl group were found to anchor Gag at the membrane by decreasing the rate of desorption. Specifically, the interaction with PI­(4,5)­P2 allows Gag to overcome electrostatic repulsion with negatively charged lipids at the membrane surface. This behavior was verified by measuring the binding properties of Gag mutants in the matrix domain of Gag, which prevented anchoring to the membrane either by blocking interaction with PI­(4,5)­P2 or by preventing exposure of the myristoyl group. The presence of tRNA was found to inhibit Gag association with the membrane by specifically blocking the PI­(4,5)­P2 binding region, thereby preventing exposure of the myristoyl group and precluding subsequent anchoring of Gag to the membrane. While Gag likely samples all membranes, only the anchoring provided by the myristoyl group and PI­(4,5)­P2 allows Gag to accumulate at the membrane. These quantitative results on the kinetics and thermodynamics of Gag association with lipid membranes provide important new information about the mechanism of Gag–membrane association.

Rendezvous at Plasma Membrane: Cellular Lipids and tRNA Set up Sites of HIV-1 Particle Assembly and Incorporation of Host Transmembrane Proteins

“…Therefore, hydrophobic interactions mediated by the myristoyl moiety may also be regulated by RNA binding to MA-HBR. A similar observation was also made in another in vitro study that used a purified Gag and yeast tRNA [ 76 ]. Of note, myristoylated MA under conditions that favor the myristate exposure has a weakened affinity to tRNA compared to when the myristate moiety is sequestered [ 90 ].…”

“…Supporting the charge-based preference of Gag for PI(4,5)P2, a modeling study on MA-membrane interactions predicted that nonspecific electrostatic interactions between MA and PI(4,5)P2 were sufficient to significantly enhance membrane binding [ 34 ]. However, in vitro studies showed that HIV-1 Gag or MA binds more efficiently to PI(4,5)P2-containing lipid membranes than to charge-matched liposomes containing PS, suggesting that PI(4,5)P2 enhances Gag-membrane binding by specific interaction beyond mere electrostatic attraction [ 38 , 41 , 44 , 45 , 73 , 74 , 75 , 76 , 77 ]. Membrane flotation-based studies showed that PI(3,5)P2 and PI(3,4,5)P3 were also able to enhance the binding of Gag to liposomes, although the former is not as efficient [ 38 ].…”

“…This observation suggests that Gag binding to PI(4,5)P2 can be susceptible to the RNA-mediated inhibition depending on the condition. Consistent with this notion, while in one in vitro study, tRNA below typical cellular concentrations suppressed binding of Gag to PC + PS but not PI(4,5)P2-containing membranes [ 93 ], in other in vitro systems where purified MA or Gag are added to lipid membranes, the addition of tRNAs at similar tRNA concentrations reduced membrane binding of MA/Gag to various degrees even when the membranes contain PI(4,5)P2 [ 74 , 76 , 90 ]. Therefore, the balance between MA binding to PI(4,5)P2 and tRNA can be affected by factors that differ among experimental systems.…”

Second Harmonic Generation Interrogation of the Endonuclease APE1 Binding Interaction with G-Quadruplex DNA

“…The ability to measure and quantify APE1 binding in a label-free manner is due to resonant enhancement of the SHG signal. Specifically, the UV absorption due to the π to π* electronic transitions of the aromatic amino acids in the APE1 protein are commensurate with the second harmonic emission. − Triplicate binding isotherms were obtained using the normalization procedure described previously …”

Relationship between HIV-1 Gag Multimerization and Membrane Binding

“…In vitro experiments demonstrate that Gag binds more efficiently to membranes containing PI(4,5)P 2 than those containing other acidic phospholipids such as PS [ 25 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 ]. Increasing the proportion of non-PI(4,5)P 2 acidic phospholipids to compensate for the greater negative charge of PI(4,5)P 2 does not result in a similar increase in Gag membrane binding, suggesting that Gag recognition of PI(4,5)P 2 -containing membranes is not simply charge-based but likely involves some recognition of the PI(4,5)P 2 headgroup structure [ 25 , 38 , 39 , 40 ].…”