Impact of envelope cholesterol and spike gp41 on cell-independent lytic inactivation of HIV-1 by peptide triazole thiols

Ramalingam Venkat Kalyana Sundaram

doi:10.17918/etd-6583

HIV-1 is a retrovirus that infects host cells carrying the receptor CD4 and the co-receptor CCR5/CXCR4. The process of infection is carried out by the virus specific proteins gp120 and gp41, expressed as a trimer of dimers on the virus surface. This interaction can be interrupted with the use of peptide triazole thiols (PTT). They are a family of entry inhibitors that carry dual antagonist behavior against gp120 by blocking both CD4 and co-receptor interactions. The thiol introduced into the PTT sequence by a C-terminal Cysteine adds an additional irreversible inactivating step consisting of lysis leading to the release of capsid p24 protein from the lumen of the virus. Since PTTs do not interact with the membrane as established with viral particles with no spike or particles pseudotyped with VSV-G, lysis must be a consequence of conformational changes within the spike being triggered by PTTs, resulting in membrane perturbation and the eventual mixing of viral luminal contents with the extracellular surroundings. Since there is a similar mixing of viral contents with intracellular contents after CD4/co-receptor interaction with the virus, we decided to use this lytic event as a window to study the lipid-protein interactions that take place to allow the disruption of the membrane and the eventual release of luminal contents. This study was split into two sections. In the first section, the lipids that make up the viral lipid bilayer (envelope) were investigated and a thorough survey of literature pointed to cholesterol, the major lipid constituent (ca. 45 mol %). Prior literature has shown that depletion with a chemical agent specific for cholesterol, methyl beta-cyclodextrin (M[beta]CD) from the viral envelope or from cells producing viruses resulted in a complete loss of infectivity. When tested for the impact of sterol depletion on lysis with PTT, the results were dramatic. Small amounts of M[beta]CD treatment (< 312 [mu]M) led to a stark increase in the amount of lysis (ca. 2.5x base-line lysis) before being suppressed at higher [M[beta]CD]. This correlated with a similar bell-shaped trend in infectivity of HIV-1 pseudotyped spikes but not VSV-G pseudotyped spikes suggesting it was specific to HIV-1. Further biochemical investigations showed that cholesterol content had mostly dropped (ca. 40%) within the small range of M[beta]CD treatment and majority of the spike gp120 had shed, consistent with previous reports on M[beta]CD treatment. The enhancement and suppression of lysis after cholesterol depletion could be reversed by the supplementation of exogenous cholesterol. More crucially, reversal could also be achieved with a sterol that supported rafts (cholestanol) and to a much lesser extent with a sterol that did not (coprostanol). Fluorescent investigations into the viral envelope showed a rise in membrane fluidity (> 312 [mu]M M[beta]CD) using the probe Laurdan and a bell-shaped quenching of fluorescence using the probe Dehydroergosterol with a nadir in intensity at 312 [mu]M M[beta]CD. These data suggested that the membrane was undergoing morphological changes with the depletion of cholesterol and this was affecting the lysis observed with PTTs and infectivity. The sterol data and DHE data hint at the role of rafts in the transitions observed but this has not been conclusively proven. To further the understanding, the protein involved with the membrane gp41 was investigated. Different regions of gp41 were examined for their role in lysis through site-directed mutagenesis of the BaL.01 sequence. Of the mutants created, all showed dose-dependent lytic release with PTT treatment in comparable levels and IC50s to the wild-type BaL.01 pseudovirus. While all mutants showed reduced infectivity, which was consistent with literature, mutations at the putative interface between envelope cholesterol and the spike (CRAC [right arrow] L676I, C-terminal tail truncation [right arrow] R706St) showed enhancement of lysis at [M[beta]CD] lower than that for wild type (~ 10 [mu]M). One possible reason might be that envelope cholesterol that is held tightly by viral spike interactions is more easily removed by M[beta]CD in the mutants. Mutations that targeted conserved tryptophan residues within the membrane proximal external region (MPER domain) affected the sensitivity to cholesterol depletion, with the mutant containing all Trp residues mutated (W(1-5)A) being the least sensitive. Since these residues are known to be critical for infectivity of HIV-1 and other viruses including Influenza and Ebola, the data suggest a common purpose for this region in both infectivity and lysis. Based on the mutational data collected, one may conclude the following: (1) Mutations have a much bigger effect on infectivity than on lysis. (2) Multiple regions of gp41 might be involved in the lytic mechanism, and mutations targeting single regions might not be big enough to stop lysis. (3) Alternatively, none of the regions targeted with mutations are crucial for lysis, though this is very unlikely due to the trends observed with sensitivity to cholesterol. Taken in context with the cholesterol depletion data, the findings can be explained with an energy to reaction argument. High cholesterol content (~ 45 mol %) results in low fluidity and tight packing of phospholipids, and this might benefit the spike in helping it maintain structure and conformation. However, it raises the energy barrier for the membrane-interacting gp41 protein in processes such as fusion and lysis which require large conformational changes such as the formation of the 6-helix bundle. Removing cholesterol to a limited extent might help lower the barrier, permitting these events to occur at a higher frequency and greater likelihood and this may be why we see an enhancement in lysis and infection. Investigations into the lytic mechanism with PTTs have provided a potential window into the mechanism of fusion that occurs with HIV-1. This is of critical importance, as there is a pressing need for entry inhibitors and a better understanding of the mechanism might foster a whole new generation of virus-inactivating, lytic entry inhibitors.

Impact of envelope cholesterol and spike gp41 on cell-independent lytic inactivation of HIV-1 by peptide triazole thiols

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Impact of envelope cholesterol and spike gp41 on cell-independent lytic inactivation of HIV-1 by peptide triazole thiols

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