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HIV stiffness change in response to treatment with viral poration agents: atomic force microscopy as a mechanical testing mode for viruses
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HIV stiffness change in response to treatment with viral poration agents: atomic force microscopy as a mechanical testing mode for viruses

Charles Gotuaco Ang
Master of Science (M.S.), Drexel University
Jun 2013
DOI:
https://doi.org/10.17918/etd-4266
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Abstract

HIV (Viruses)--Research Atomic Force Microscopy Biomedical Engineering
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 [mu]l of either phosphate buffered saline or 100 [mu]l of 10 [mu]M inhibitor (KR13, HNG156, NS5a) for 30 minutes at 37°C 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 "wild 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 type. In terms of determining KR13s mechanism of action, HNG156 causing poration muddies, but does not invalidate the data, as stiffness reduction greater than KR13 is not expected even if the batch had sub-KR13 poration properties. Rather, the stiffness change in HNG156, and to a lesser extent, in KR13, may be due to their shared gp120 shedding action. Furthermore, the massively increased effect of KR13 in the cholesterol treated viruses suggests that membrane stiffness does affect poration. One possibility might be that lack of membrane fluidity allows for less freedom when premature fusion occurs, magnifying the effect of the membrane disruption.

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