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• W3201609907 abstract "Despite powerful and effective control drugs, HIV remains a worldwide epidemic, and new methods of halting its advance are always under development. Previous work in the Chaiken Lab has identified an entry inhibitor targeting HIVs Env spikes, KR13, that additionally has been observed to have porating effects on HIV, rendering the virus non-infectious. The exact mechanism of action has not yet been elucidated for KR13, but present theory suggests premature triggering of the fusion process results in membrane disruption and thus leakage. In order to further characterize this membrane disruption, Atomic Force Microscopy (AFM) has been used in order to both image and measure force changes in HIV in response to KR13 and other inhibitors. HNG156 is a structurally similar precursor to KR13, but notably does not share its porating action (later review would reveal that this batch of HNG156 does cause poration at lower rates than KR13). NS5a is known to cause poration of HIV without interacting with Env, and provides a positive control.AFM force mode enables nanoscale probing of samples by a piezo motor lowering a cantilever into a sample (here, HIV). The force measured from this descent can then be turned into a composite spring constant between the cantilever and the virus, and then, with the cantilevers spring constant known, the virus spring constant, its stiffness, can be calculated.BaL.01 pseudovirus was treated with 100 μl of either phosphate buffered saline or 100 μl of 10 μM inhibitor (KR13, HNG156, NS5a) for 30 minutes at 37oC in order to push any reaction to completion. Following treatment, viruses were then imaged; individual viruses were identified before centering the cantilever over the virus and attempting to descend 50 nm downward, measuring the force response curve as this occurred. Once obtained, using the cantilevers known spring constant, viral stiffness was calculated both with and without inhibitor effects. The base stiffness (using cantilevers with nominal stiffness of 0.06 N/m) of unmodified pseudovirus was 0.8 ± 0.2 N/m (n=27), with KR13 causing a stiffness drop to 0.4 ± 0.2 N/m (n=12), 50%. Testing of HNG156 and NS5a utilized a slightly stiffer cantilever (nominal stiffness of 0.24 N/m), resulting in different results: unmodified pseudovirus at 60 ± 20 N/m, HNG156 treated virus at 20 ± 8 N/m (n=14) (roughly 67% reduction), and NS5a treated virus at 30 ± 10 N/m (n=25) (50% reduction).Additionally, BaL.01 pseudovirus that had been exposed to an additional 2.5 mM of cholesterol was also tested by AFM, with and without KR13 exposure in order to determine if altering the membrane stiffness would change KR13s effect. The control sample of the cholesterol added pseudovirus proved significantly stiffer than the type viruses, with a stiffness of 2 ± 1 N/m (n=12) as measured by the 0.06 N/m cantilevers. Treatment with KR13 resulted in a measured stiffness of 0.05 ± 0.04 N/m (n=8) (97.5% reduction), significantly lower than was seen in the wild type, or, in fact, with any of the inhibitors in the wild…" @default.
• W3201609907 created "2021-09-27" @default.
• W3201609907 creator A5034115043 @default.
• W3201609907 date "2021-07-16" @default.
• W3201609907 modified "2023-09-24" @default.
• W3201609907 title "HIV Stiffness Change in Response to Treatment with Viral Poration Agents: Atomic Force Microscopy as a Mechanical Testing Mode for Viruses" @default.
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• W3201609907 doi "https://doi.org/10.17918/etd-4266" @default.
• W3201609907 hasPublicationYear "2021" @default.
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