Interplay of Cholesterol and Env Protein in the Lytic Deformation of HIV-1 by Peptide Triazoles

Background: HIV-1 Env spike consists of gp120 involved in binding receptors and gp41 which is embedded in a lipid envelope that is mainly cholesterol (45-mol %) and involved in fusion. Envelope cholesterol affects infectivity of the virus and interacts with gp41. Our lab has developed peptide triazoles that inhibit gp120 binding to CD4 and CCR5/CXCR4 and cause leakage of capsid p24, from the viral lumen. Examining the effects of cholesterol on p24 release allows us to investigate lipid-protein interplay at the Env spike. Methodology: Cholesterol was extracted from BaL.01 (HIV-1) and Vesicular Stomatitis Virus (VSV-G) spike psuedotyped viruses containing the Luciferase gene using methyl β-cyclodextrin (MβCD). Infectivity was detected by chemiluminescence from Luciferase expression in cells while p24 release and gp41 were measured by spinning virus and testing supernatant and pellet fractions respectively by ELISA. Fluidity was measured with Laurdan. Shed gp120 was detected from western blots. Morphological analysis was done on fixed virus by transmission electron microscopy (TEM). Cholesterol was quantified using Cholesterol Oxidase and the fluorescent Amplex Red dye. Results: Initial depletion of viral membrane cholesterol strikingly enhanced both KR-13-induced p24 capsid protein leakage and infectivity and decreased virus size (TEM). Further depletion of cholesterol arrested both processes and caused complete shedding of gp120 but gp41 and the virus size remained intact and membrane fluidity increased though this wasn’t specific to HIV-1. Under similar conditions, the non-virolytic parent peptide HNG-156 did not induce p24 release and the VSV pseudotyped viruses did not see any enhanced infectivity. Conclusions: Light cholesterol depletion enhances both KR-13 triggered p24 release and infectivity while heavy depletion arrest both processes, possibly due to shedding of gp120. While fluidity increases during cholesterol depletion, it is not specific to the HIV-1 spike. Correlated trends between infectivity and leakage during cholesterol depletion leading up to the gp 120 shedding suggest lipid-protein interplay. To determine the mechanism of enhancement, we will test protease-treated viruses and Env mutations that disrupt the cholesterol interacting (CRAC), transmembrane, and cytoplasmic tail domains interacting with the lipid envelope. Together, this work will help determine the impact of lipid-protein interplay and define approaches to inactivate the virus.