Hydrophobic Nanoparticles Reduce the β-Sheet Content of SEVI Amyloid Fibrils and Inhibit SEVI-Enhanced HIV Infectivity

Article abstract of DOI:10.1021/acs.langmuir.6b04295

Semen-derived enhancer of virus infection (SEVI) fibrils are naturally abundant amyloid aggregates found in semen that facilitate viral attachment and internalization of human immunodeficiency virus (HIV) in cells, thereby increasing the probability of infection. Mature SEVI fibrils are composed of aggregated peptides exhibiting high β-sheet secondary structural characteristics. Herein, we show that polymers containing hydrophobic side chains can interact with SEVI and reduce its β-sheet content by ～45% compared with the β-sheet content of SEVI in the presence of polymers with hydrophilic side chains, as estimated by polarization modulation-infrared reflectance absorption spectroscopy measurements. A nanoparticle (NP) formulation of this hydrophobic polymer reduced SEVI-mediated HIV infection in TMZ-bl cells by 60% compared with the control treatment. Although these NPs lacked specific amyloid-targeting groups, thus requiring high concentrations to observe biological activity, the use of hydrophobic interactions to alter the secondary structure of amyloids represents a useful approach to neutralizing the SEVI function. These results could, therefore, have general implications in the design of novel materials that can modify the activity of amyloids associated with a variety of other neurological and systemic diseases.

Full text of DOI:10.1021/acs.langmuir.6b04295

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Article
Hydrophobic Nanoparticles Reduce the #-sheet content of
SEVI Amyloid Fibrils and Inhibit SEVI-Enhanced HIV Infectivity
Daniel A. Sheik, Jeffrey M. Chamberlain, Lauren R. Brooks, Melissa L. Clark, Young
Hun Kim, Geoffray Leriche, Clifford P. Kubiak, Stephen Dewhurst, and Jerry Yang
Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b04295 • Publication Date (Web): 16 Feb 2017
Downloaded from http://pubs.acs.org on February 17, 2017
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Hydrophobic Nanoparticles Reduce the βꢀsheet
Content of SEVI Amyloid Fibrils and Inhibit SEVIꢀ
Enhanced HIV Infectivity
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Daniel A. Sheik,†,§Jeffrey M. Chamberlain,‡,§ Lauren Brooks,‡ Melissa Clark,† Young Hun Kim, †
Geoffray Leriche, † Clifford P. Kubiak,† Stephen Dewhurst ‡ and Jerry Yang†,*
†Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman
‡
Drive, La Jolla CA 92093ꢀ0358, United States; Department of Microbiology and Immunology,
University of Rochester, Rochester, New York 14642, United States
KEYWORDS
SEVI, Polymer, HIV, Nanoparticles, βꢀsheet
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ABSTRACT
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SemenꢀDerived Enhancer of Virus Infection (SEVI) fibrils are naturally abundant
amyloid aggregates found in semen that facilitate viral attachment and internalization of HIV in
cells, thereby increasing the probability of infection. Mature SEVI fibrils are comprised of
aggregated peptides exhibiting high βꢀsheet secondary structural characteristics. Herein, we show
that polymers containing hydrophobic side chains can interact with SEVI and reduce its βꢀsheet
content by ~45% compared to the βꢀsheet content of SEVI in the presence of polymers with
hydrophilic side chains, as estimated by Polarization ModulationꢀInfrared Reflectance
Absorption Spectroscopy Measurements. A nanoparticle formulation of this hydrophobic
polymer reduced SEVIꢀmediated HIV infection in TMZꢀbl cells by 60% compared to control
treatment. While these nanoparticles lacked specific amyloidꢀtargeting groups, thus, requiring
high concentrations to observe biological activity, the use of hydrophobic interactions to alter the
secondary structure of amyloids represents a useful approach to neutralizing SEVI function.
These results could, therefore, have general implications in the design of novel materials that can
modify the activity of amyloids associated with a variety of other neurologic and systemic
diseases.
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INTRODUCTION
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Sexual contact is the most common mode of transmission of HIV.1,2 Previous studies
revealed that peptides found within semen are capable of aggregating into amyloid fibrils that
bind strongly to HIV virions and cell membranes.3–9 For instance, Semenꢀderived Enhancer of
Virus Infection (SEVI) is a naturally abundant amyloid found in semen and is comprised of a
peptide derived from the degradation of prostatic acid phosphatase, PAP (248ꢀ286).3,10,11 The
binding of SEVI to viral and mammalian cell surfaces is believed to serve as a mediator for viral
cell attachment and internalization, with the capability of increasing the rate of HIV infection
through sexual contact by up to 400,000ꢀfold.3,6,10,12 SEVI, therefore, represents a potentially
important biological target for developing novel microbicide supplements to combat the spread
of HIV.13–19
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Previous work in our lab has shown that highꢀaffinity amyloidꢀbinding small molecules,
oligomers, and polymers can inhibit SEVIꢀmediated enhancement of HIV infectivity.20–22 These
synthetic materials putatively create bioresistive coatings23,24 on the surface of SEVI fibrils that
are capable of inhibiting the interaction of SEVI with various biologics (e.g., viral capsid
proteins and cellular lipid membranes). These previous studies revealed that increased SEVIꢀ
binding affinity and size of the amyloidꢀtargeting agent resulted in increased efficacy for
inhibiting SEVIꢀmediated HIV infection. Here, we explore a fundamentally new mechanism for
inhibiting SEVI function by examining whether hydrophobic nanoparticles that lack amyloidꢀ
targeting moieties can disrupt the secondary structure of SEVI amyloid fibrils, and whether this
structural deformation is accompanied by inhibition of SEVIꢀmediated enhancement of HIV
infectivity (Figure 1).
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Amyloids are well known to adopt structures with significant βꢀsheet character,25–32
which generates highly stable structures that are often poorly soluble in aqueous environments
and are resistant to enzymatic digestion.29,33,34 We and others have previously shown that
amyloid fibrils can lose significant βꢀsheet content when they come in contact with hydrophobic
surfaces.35–37 For instance, we showed that preformed aggregates of the Alzheimerꢀrelated
amyloidꢀβ (Aβ) peptides exhibited only ~37% βꢀsheet content when deposited on a hydrophobic
polystyrene surface compared to ~60% βꢀsheet content when deposited on a hydrophilic plasmaꢀ
oxidized polystyrene surface.35 Several other groups have also shown that hydrophobic materials
can inhibit fibrillization of Aβ, further supporting the possibility of using hydrophobic materials
to disrupt the structural characteristics of Aβ aggregates.36–40 Since there is evidence that the
amyloidꢀform of PAP (248ꢀ286), and not the unaggregated peptide itself, enhances HIV
infection,3 we hypothesized that disrupting the βꢀsheet content of SEVI using hydrophobic
materials could lead to a new mechanism for inhibiting SEVIꢀmediated enhancement of HIV
infectivity.
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EXPERIMENTAL
Poly(octyl acrylate)ꢀcoꢀpoly(hydroxyethylacrylamide) (POAPHEAm, 4)
Octyl Acrylate (1, 381 mg, 2.1 mmol) and HEAm (2, 26 mg, 0.21 mmol) were dissolved in
dimethylformamide (DMF, 2.5 mL). Azobisisobutyrylnitrile (AIBN, 9 mg, 0.06 mmol) was
added and the reaction mixture was degassed by gently bubbling dry N2 gas into the solution
with stirring for 30 min. After degassing, the reaction vial was sealed under N2 atmosphere and
placed in an oil bath at 65ºC for 16 h. After cooling to room temperature (r.t.), the cloudy
solution was transferred to a centrifugation vial and the polymerization vessel was rinsed twice
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with dichloromethane (DCM, 2 mL). The polymer was precipitated by addition of methanol
(MeOH, 40 mL), centrifuged to separate the solid polymer, and then the supernatant was
decanted. The polymer was dissolved in DCM (5 mL) and again precipitated with MeOH (40
mL), followed by centrifugation and isolation of the solid. The final product was dried under
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vacuum to afford a colorless oil (83 mg, 20%). H NMR (400 MHz, CDCl3): δ 4.00 (bs, 2H),
3.67 (bs, 2H), 2.27 (bs, 2H), 1.90 (bs, 1H), 1.60 (bs, 2H), 1.29 (bs, 10H), 0.88 (bs, 3H).
Integration of the peaks at 4.00 and 3.67 ppm were used to determine the incorporation of
monomer 1 (see the Supporting Information for details).
Poly(hexaoxanonadecanyl acrylate) (PHONA, 5)
Hexaethylene glycol monomethyl ether acrylate (3, 284 mg, 0.81 mmol) and AIBN (3 mg, 0.02
mmol) were dissolved in butyl acetate (1 mL). The reaction mixture was degassed by gently
bubbling dry N2 gas with stirring for 30 min. After degassing, the reaction vial was sealed under
N2 atmosphere and placed in an oil bath at 65ºC for 16 h. After being cooled to r.t., the cloudy
solution was transferred to a centrifugation vial. The polymerization vessel was rinsed with
DCM (2 x 2 mL) to facilitate complete transfer of polymer. The polymer was precipitated by
addition of hexanes (40 mL), centrifuged to separate the polymer, and the supernatant was
decanted. The polymer was dissolved in DCM (5 mL) and again precipitated with hexanes (40
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mL). The product was dried under vacuum to afford a colorless oil (132 mg, 46%). H NMR
(400 MHz, CDCl3): δ 4.17 (2H, bs), 3.68 (22H, bm), 3.38 (3H, bs), 2.31 (1H, bs), 1.78 (2H, bs),
1.62 (1H, bs).
Nanoparticle Formulation
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Hydrophobic polymer 4 (2 mg) was dissolved in 2 mL of tetrahydrofuran (THF), which was
subsequently added dropwise to an equal volume of deionized (DI) water while stirring
vigorously. After stirring for 30 min, the THF was removed in vacuo and the resulting
nanoparticle solution (1 mg total polymer per mL of DI water) was analyzed via dynamic light
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scattering. H NMR analysis of this same preparation of nanoparticles using D2O instead of DI
water supported that this method efficiently removed THF from the nanoparticle suspension (see
supporting information for 1H NMR spectrum).
Polarization ModulationꢀInfrared Reflectance Absorption Spectroscopy (PMꢀIRRAS)
Measurements
PMꢀIRRAS was conducted according to a previously described method.35 Briefly, 10 ꢁL of a
SEVI amyloid fibril sample (10 mg/mL relative to peptide concentration) was deposited onto a
goldꢀplated glass slide coated with either polymer 4 or 5, ensuring that material spread across the
1 cm height of the slide. The coated fragments were incubated in the dark for at least 6 h before
the excess water was removed under a flow of N2. Each spectrum was recorded at 4 cmꢀ1
resolution and is an average of at least 50 scans. Spectral analysis was conducted using OMNIC
Series Software (ThermoFisher). The background IR spectrum, polymer coated slide without
SEVI incubation, was subtracted from the corresponding spectrum of SEVI amyloid fibrils
incubated on the polymer coated slide. The spectra were then baseline corrected between 1720ꢀ
1580 cmꢀ1 and smoothed at a 9 cmꢀ1 resolution. Second derivative analysis provided by the
OMNIC Series Software allowed for the resolution of conformational bands. The spectra were
fitted between 1700ꢀ1600 cmꢀ1 using least square iterative curve fitting with a mixture of
Gaussian and Lorentzian line shapes. Secondary structures were quantified by calculating the
areas of each component peak with respect to the original spectrum between 1700 and 1600 cmꢀ1.
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Estimation of Amyloid Content Using a Thioflavin T (ThT) Fluorescence Assay
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Fibril formation was monitored via Thioflavin T (ThT) fluorescence as described previously.10
Briefly, fresh fibril (10 mg/mL) was diluted 1:100 in 25 µM ThT working solution and
fluorescence (Ex/Em 465/535, 20 nm bandwidth) was measured in opaque 96ꢀwell black
microtiter plate in DTX 880 multimode plate reader.
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Toxicity Assays
An Alamar Blue Cell Viability assay was performed in 96ꢀwell plate format according to
manufacturer’s instructions (Invitrogen). Briefly, TZMꢀbl cells were plated at 7.5 x 103 cells/well
and allowed to adhere overnight. Cells were then treated with either NP 4 or polymer 5 at 200
µg/mL (final concentration) in growth media for 2 h. Fresh growth media was added along with
the alamar blue reagent and fluorescence (Ex/Em 535/595) was measured using a DTX 880
multimode detector plate reader (Beckman Coulter).
Measurement of SEVIꢀmediated enhancement of HIV infection in TZMꢀbl cells
TZMꢀbl cells were plated at 7.5 x 103 cells/well and allowed to adhere overnight. DMEM
growth media supplemented with 10% FBS and 10 mM HEPES was adjusted to pH 6, and then
filter sterilized through a 0.2 µm syringe filter. Nanoparticles (final concentration 200 µg/mL)
were preꢀincubated with or without SEVI fibril (final concentration 15 µg/mL) for 1h at room
temp in growth media. HIVIIIB virions (final p24 concentration of 85 ng/mL) were then added to
the sample solutions and incubated for additional 15 min. Treatments were added to cells in
triplicate (100 µL/well) and incubated for 2 h in humidified 37ºC incubator to allow for
infection, after which initial inoculum were removed and fresh media of pH 7.5 was added. Cells
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were incubated for additional 72 h at 37ºC. Cells were lysed by addition of 50 µL/well passive
lysis buffer (Promega) and incubation at 37ºC for 5 min. Lysates were clarified by centrifugation
for 5 min at 300 x g in vꢀbottom 96ꢀwell plates. 5 µL of clarified lysate was combined with 200
µL of Quick Start Bradford Reagent (BioꢀRad) to measure total cellular protein. 25 µL of
clarified lysate was used to measure luciferase activity by mixing with firefly substrate (Promega
cat# E151A, 1:1 ratio) in opaque white plates. Bradford and luciferase were measured using a
DTX 880 multimode detector plate reader (Beckman Coulter) and luciferase values were
normalized to total cellular protein.
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RESULTS AND DISCUSSION
Design, Synthesis and Characterization of Polymeric Materials
In order to test the effect hydrophobic materials impose on SEVIꢀmediated HIV
infection, we synthesized an acrylateꢀbased polymer carrying octyl hydrocarbon sidechains
through coupling of octanol and acryloyl chloride under basic conditions followed by freeꢀ
radical polymerization (Scheme 1). We found it necessary to incorporate a small percentage of a
hydrophilic monomer, Nꢀhydroxyethyl acrylamide (HEAm, 2), into this polymer in order to
prevent uncontrolled aggregation and precipitation of this polymer from aqueous solution. Using
a feed ratio of 1:10 of monomers 2 and 1, respectively, we generated poly(octyl acrylate)ꢀcoꢀ
poly(hydroxyethylacrylamide) (POAPHEAm, 4) polymers with a number average molecular
weight (Mn) of 6.5 kDa and PDI of 1.84 (Table 1 and SI Figure S1A). Polymer 4 was
synthesized using uncontrolled freeꢀradical polymerization with azobisisobutyronitrile (AIBN)
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and isolated by precipitation from methanol in ~20% overall yield. H NMR analysis revealed
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that polymer 4 contained only 5% of HEAm monomer 2 (i.e., only half of the original feed ratio
of monomer 2 was incorporated into the isolated polymer).
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Since we observed that this hydrophobic material slowly aggregated in aqueous solution,
we then generated nanoparticles from this polymer in order to increase the homogeneity of the
aggregate size and shape of the material (which we found significantly affects reproducibility in
HIV infection assays). Although it was possible to increase the percentage of hydrophilic groups
into these polymers by increasing the feed ratio of monomer 2 (data not shown), we found that
incorporating higher percentages of hydrophilic monomers rendered these polymers too soluble
in aqueous solution to be formulated into nanoparticles. Using polymer 4, we generated spherical
nanoparticles with average diameter of 265 nm in solution and average polydispersity of 0.16
using a previously described solvent evaporation technique (Table 1 and SI Figure S2).22,41,42
While this technique made it possible to reproducibly generate nanoparticles with similar size (±
37 nm over 5 preparations), other methods for nanoparticle preparation43–45 could potentially be
explored to predictably generate a set of nanoparticles with a range of different diameters.
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As a control hydrophilic polymer, we also synthesized a previously reported
poly(hexaoxanonadecanyl acrylate) (PHONA, 5) polymer containing 100% monomethyl
hydroxyethylene glycol sidechains in ~45% yield using the same freeꢀradical polymerization
procedure used to generate polymer 4 (Scheme 1, Table 1, and SI Figure S1B).22 We analyzed
the effects of polymer 5 in subsequent SEVI structure and function assays without further
changes to its formulation, as the high solubility of this polymer in aqueous solutions precluded
the possibility for its conversion to stable nanoparticles.
Quantification of βꢀsheet and Amyloid Content in Mature SEVI Fibril Preparations
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In order to determine the effects of polymers 4 and 5 on the structure of SEVI amyloid
fibrils, we used a previously described spinꢀcoating procedure35,46 to create a thin film (~100 nm
thick) of polymer on the surface of goldꢀplated microscope slides. We then used polarization
modulationꢀinfrared reflectionꢀabsorption spectroscopy (PMꢀIRRAS) to analyze the βꢀsheet
content of 5 preformed SEVI fibril samples after deposition on surfaces coated with either the
hydrophobic or hydrophilic polymer. The capability to estimate βꢀsheet content from small
quantities (10 ug) of SEVI is a distinct advantage of PMꢀIRRAS compared to techniques such as
circular dichroism, which would require impractical quantities of aggregated peptide to
determine secondary structural characteristics for this study.8,35,47,48 Examination of the Amide I
region between 1600ꢀ1700 cmꢀ1 of the peptides (Figure 2A and 2B, black curves) in SEVI
revealed that polymer 4 decreased the βꢀsheet content (red curves) of SEVI by an average of
~45% compared to the βꢀsheet content of SEVI deposited on a thin film of polymer 5 (Figure
2AꢀC, also see SI Figure S3 for representative PMꢀIRRAS spectra of all SEVI samples).
Additionally, we found a good correlation (Pearson correlation coefficient, r, of 0.86)49,50
between the βꢀsheet content of these SEVI fibril preparations (estimated from PMꢀIRRAS
measurements of SEVI deposited onto a film of hydrophilic polymer 5) and the fluorescence
emission of Thioflavin T (ThT) incubated with the SEVI fibril solution (Figure 2D); ThT
fluorescence is typically used to estimate the abundance of amyloid fibrils in solution.11,51,52 In
these experiments, aliquots from the same SEVI solutions were analyzed by either PMꢀIRRAS or
ThT fluorescence measurements separately in order to eliminate any potential interference of
ThT with the interaction of the polymers and SEVI. For ThT fluorescence measurements, no
polymers or NPs were present in the SEVI samples. These studies support that polymer 4
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significantly disrupts the βꢀsheet content of SEVI compared to hydrophilic polymer 5, and that
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the βꢀsheet content is a reasonable measure of the amyloid abundance of SEVI in solution.
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In order to examine whether the βꢀsheet content of SEVI had an effect on the
enhancement of HIV infectivity mediated by SEVI, we examined the same 5 preparations of
SEVI used in the PMꢀIRRAS experiments in an HIV infection assay.12,20–22 Briefly, this assay
employs TMZꢀbl cells that express a luciferase reporter upon infection with HIV virions. Figure
3 shows plots of the observed ThT fluorescence values or % βꢀsheet content of SEVI versus the
enhancement of HIV infectivity induced by SEVI (shown as fold enhancement relative to the
measured HIV infectivity in the absence of SEVI). This analysis reveals a good correlation
between ThT fluorescence values (r = 0.97) or the percentage of βꢀsheet character of the SEVI (r
= 0.74) sample versus the observed activity of SEVI for enhancing HIV infection. Taken
together, these results further demonstrate that the capability of SEVI to enhance HIV infectivity
is strongly dependent on its amyloid content, which can be approximated by its relative
percentage of βꢀsheet character. We also conducted atomic force microscopy (AFM) experiments
to characterize the interaction between nanoparticles generated from polymer 4 and mature SEVI
fibrils. While these AFM studies support that the nanoparticles indeed interact with the SEVI
fibrils (See Figure S4 in the supporting information), we did not find any clear evidence of gross
morphological changes to the SEVI fibrils as a result of these interactions.
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Hydrophobic Nanoparticles Inhibit SEVIꢀmediated HIV Infection
In order to evaluate the effects of hydrophobic nanoparticles comprised of polymer 4 (NP
4) or hydrophilic polymer 5 on SEVIꢀmediated HIV infection, we first examined the toxicity of
these synthetic materials in TZMꢀbl cells and their effects on HIV infection in the absence of
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SEVI. Figure 4A shows that both NP 4 and polymer 5 were not toxic to these cells at
concentrations equal to or below 400 µg/mL, which was twice the concentration of NP 4 or
polymer 5 used in the infectivity assays. While we did not observe any effect of NP 4 on HIV
infection (in the absence of SEVI) at this concentration of nanoparticles, we were surprised to
observe that a 200 µg/mL concentration of polymer 5 reduced HIV infection by ~50% compared
to treatment with buffer (vehicle) control (Figure 4B). Polymers containing polyethylene glycol
groups (such as the sidechain groups in polymer 5) are well known to surround membrane
surfaces and block interactions of various biologics with cells.53,54 Results from a viral cell
attachment assay20 confirmed that HIV virions exhibit a reduced capability to attach to cells in
the presence of polymer 5 (Figure 4C), supporting that the effects of this polymer on HIV
infection (in the absence of SEVI) was likely due to known effects of similar hydrophilic
polymers on stabilization of cells53,54 and not likely due to any inherent antiviral activity. In
contrast to polymer 5, NP 4 did not interfere with viral attachment in the absence of SEVI
(Figure 4C).
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In the presence of SEVI, a 200 µg/mL concentration of the hydrophobic nanoparticles
(NP 4) significantly reduced SEVIꢀenhanced HIV infection, resulting in a 66% reduction in the
enhancement of HIV infection by SEVI compared to SEVIꢀmediated HIV infection in the
absence of the polymer (Figure 4D). A high concentration of NP 4 was necessary to observe
inhibition of SEVIꢀmediated infection, since these particles do not contain any amyloidꢀtargeting
group that could increase their binding interaction with SEVI (which we omitted purposely to
exclude mechanisms other than disruption of SEVI βꢀsheet content that lead to inhibiting SEVIꢀ
enhanced HIV infections20–22). For hydrophilic polymer 5, we did not observe any effect on
SEVIꢀmediated enhancement of HIV infection compared to the effects of this polymer on HIV
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infection alone (i.e., in the absence of SEVI). Collectively, these results support the hypothesis
that hydrophobic nanoparticles can significantly affect the function of SEVI for enhancing HIV
infection through reducing its βꢀsheet content.
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CONCLUSION
In conclusion, we demonstrated a new mechanism for inhibiting SEVIꢀmediated HIV
infection through the disruption of SEVI amyloid fibril secondary structure using hydrophobic
nanoparticles. PMꢀIRRAS measurements show that these hydrophobic polymeric materials
reduce the βꢀsheet content of SEVI fibrils compared to hydrophilic materials. Comparison of
PMꢀIRRAS measurements and ThT fluorescence of different SEVI preparations revealed a
strong correlation between βꢀsheet content and amyloid characteristics (as estimated by ThT
fluorescence) of SEVI fibrils. The βꢀsheet content and amyloid characteristics of SEVI fibrils
also corresponded well with the capability of SEVI to enhance HIV infection. While studies on
the effects of hydrophobic materials and aggregated forms of Alzheimerꢀrelated amyloidꢀβ (Aβ)
peptides have been previously reported,35–39 the results reported here represent the first
demonstration of using hydrophobic materials to alter the βꢀsheet content of SEVI amyloid
fibrils and affect their natural biological activity. While the present study involves using
hydrophobic interactions to inhibit the function of a potentially key amyloid mediator for sexual
transmission of HIV, we anticipate that this fundamentally different approach to altering the
function of amyloids through modulation of its secondary structure could be applied in the
design of novel strategies to combat other amyloidꢀassociated diseases. For instance, prion
diseases, such as CreutzfeldtꢀJakob disease, are characterized by infectious amyloid structures
that are notoriously resistant to protease degradation.55–57 Current efforts are aimed at using
hydrophobic materials to affect the secondary structure of prion amyloids and to increase their
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susceptibility to enzymatic processing, thereby decreasing the chance of infection and spread of
the disease. Other efforts include incorporation of amyloidꢀtargeting agents into hydrophobic
materials in order to improve their potency for disrupting amyloid secondary structure and for
inhibiting concomitant biological activity.
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ASSOCIATED CONTENT
Supporting Information. Materials and procedures used in the synthesis and characterization of
all monomers, PMꢀIRRAS measurement procedures, nanoparticle formulation s, and in vitro
assays can be found in the supporting information. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
Eꢀmail: jerryyang@ucsd.edu
Tel: 858ꢀ534ꢀ6006; Fax: 858ꢀ534ꢀ4554
Author Contributions
§D.A.S. and J.M.C. contributed equally. D.A.S., J.M.C., L.B., M.C., C.P.K., S.D., and J.Y.
designed the experiments and analyzed the data. D.A.S, J.M.C., L.B., M.C. executed the
experiments, D.A.S. and J.Y. wrote the manuscript. All authors have given approval to the final
version of the manuscript.
Funding Sources
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This work was supported, in part, by NIH Grant Nos. R21AI094511 and R33AI094511, a
California HIV/AIDS Research Program grant (ID10ꢀSDꢀ034), and by the University of
Rochester Center for AIDS Research (NIH P30AI078498).
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Notes
The authors declare no competing financial interests.
ABBREVIATIONS
HIV, Human Immunodeficiency Virus; SEVI, Semenꢀderived Enhancer of Virus Infection;
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REFERENCES
5
6
7
8
(1)
(2)
(3)
Royce, R. A.; Sena, A.; Cates, Jr., W.; Cohen, M. S. Sexual Transmission of HIV. N.
Engl. J. Med. 1997, 336, 1072–1078.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Galvin, S. R.; Cohen, M. S. The Role of Sexually Transmitted Diseases in HIV
Transmission. Nat. Rev. Microbiol. 2004, 2, 33–42.
Münch, J.; Rücker, E.; Ständker, L.; Adermann, K.; Goffinet, C.; Schindler, M.; Wildum,
S.; Chinnadurai, R.; Rajan, D.; Specht, A.; et al. SemenꢀDerived Amyloid Fibrils
Drastically Enhance HIV Infection. Cell 2007, 131 (6), 1059–1071.
(4)
(5)
(6)
(7)
Roan, N. R.; Müller, J. A.; Liu, H.; Chu, S.; Arnold, F.; Stürzel, C. M.; Walther, P.; Dong,
M.; Witkowska, H. E.; Kirchhoff, F.; et al. Peptides Released by Physiological Cleavage
of Semen Coagulum Proteins Form Amyloids That Enhance HIV Infection. Cell Host
Microbe 2011, 10 (6), 541–550.
Arnold, F.; Schnell, J.; Zirafi, O.; Stürzel, C.; Meier, C.; Weil, T.; Ständker, L.;
Forssmann, W.ꢀG.; Roan, N. R.; Greene, W. C.; et al. Naturally Occurring Fragments
from Two Distinct Regions of the Prostatic Acid Phosphatase Form Amyloidogenic
Enhancers of HIV Infection. J. Virol. 2012, 86 (2), 1244–1249.
Roan, N. R.; Münch, J.; Arhel, N.; Mothes, W.; Neidleman, J.; Kobayashi, A.; Smithꢀ
McCune, K.; Kirchhoff, F.; Greene, W. C. The Cationic Properties of SEVI Underlie Its
Ability to Enhance Human Immunodeficiency Virus Infection. J. Virol. 2009, 83 (1), 73–
80.
Easterhoff, D.; Ontiveros, F.; Brooks, L. R.; Kim, Y.; Ross, B.; Silva, J. N.; Olsen, J. S.;
ACS Paragon Plus Environment
16

Page 17 of 31
Langmuir
1
2
3
4
5
6
7
8
9
Feng, C.; Hardy, D. J.; Dunman, P. M.; et al. SemenꢀDerived Enhancer of Viral Infection
(SEVI) Binds Bacteria, Enhances Bacterial Phagocytosis by Macrophages, and Can
Protect against Vaginal Infection by a Sexually Transmitted Bacterial Pathogen.
Antimicrob. Agents Chemother. 2013, 57 (6), 2443–2450.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(8)
Brender, J. R.; Hartman, K.; Gottler, L. M.; Cavitt, M. E.; Youngstrom, D. W.;
Ramamoorthy, A. Helical Conformation of the SEVI Precursor Peptide PAP248ꢀ286, a
Dramatic Enhancer of HIV Infectivity, Promotes Lipid Aggregation and Fusion. Biophys.
J. 2009, 97 (9), 2474–2483.
(9)
Hartman, K.; Brender, J. R.; Monde, K.; Ono, A.; Evans, M. L.; Popovych, N.; Chapman,
M. R.; Ramamoorthy, A. Bacterial Curli Protein Promotes the Conversion of PAP248ꢀ286
into the Amyloid SEVI: CrossꢀSeeding of Dissimilar Amyloid Sequences. PeerJ 2013, 1,
e5.
(10) Olsen, J. S.; DiMaio, J. T. M.; Doran, T. M.; Brown, C.; Nilsson, B. L.; Dewhurst, S.
Seminal Plasma Accelerates SemenꢀDerived Enhancer of Viral Infection (SEVI) Fibril
Formation by the Prostatic Acid Phosphatase (PAP248ꢀ286) Peptide. J. Biol. Chem. 2012,
287 (15), 11842–11849.
(11) Easterhoff, D.; DiMaio, J. T. M.; Liyanage, W.; Lo, C.ꢀW.; Bae, W.; Doran, T. M.;
Smrcka, A.; Nilsson, B. L.; Dewhurst, S. Fluorescence Detection of Cationic Amyloid
Fibrils in Human Semen. Bioorg. Med. Chem. Lett. 2013, 23 (18), 5199–5202.
(12) Easterhoff, D.; DiMaio, J. T. M.; Doran, T. M.; Dewhurst, S.; Nilsson, B. L. Enhancement
of HIVꢀ1 Infectivity by Simple, SelfꢀAssembling Modular Peptides. Biophys. J. 2011, 100
ACS Paragon Plus Environment
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Langmuir
Page 18 of 31
1
2
3
4
(5), 1325–1334.
5
6
7
8
9
(13) Roan, N. R.; Sowinski, S.; Münch, J.; Kirchhoff, F.; Greene, W. C. Aminoquinoline
Surfen Inhibits the Action of SEVI (SemenꢀDerived Enhancer of Viral Infection). J. Biol.
Chem. 2010, 285 (3), 1861–1869.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(14) Hartjen, P.; Frerk, S.; Hauber, I.; Matzat, V.; Thomssen, A.; Holstermann, B.; Hohenberg,
H.; Schulze, W.; Schulze Zur Wiesch, J.; van Lunzen, J. Assessment of the Range of the
HIVꢀ1 Infectivity Enhancing Effect of Individual Human Semen Specimen and the Range
of Inhibition by EGCG. AIDS Res. Ther. 2012, 9 (1), 2.
(15) Hauber, I.; Hohenberg, H.; Holstermann, B.; Hunstein, W.; Hauber, J. The Main Green
Tea Polyphenol Epigallocatechinꢀ3ꢀGallate Counteracts SemenꢀMediated Enhancement of
HIV Infection. Proc. Natl. Acad. Sci. U. S. A. 2009, 106 (22), 9033–9038.
(16) Pavot, V.; Rochereau, N.; Lawrence, P.; Girard, M. P.; Genin, C.; Verrier, B.; Paul, S.
Recent Progress in HIV Vaccines Inducing Mucosal Immune Responses. AIDS 2014, 28
(12), 1701–1718.
(17) Abdool Karim, S. S.; Baxter, C. Overview of Microbicides for the Prevention of Human
Immunodeficiency Virus. Best Pract. Res. Clin. Obstet. Gynaecol. 2012, 26 (4), 427–439.
(18) Castellano, L. M.; Shorter, J. The Surprising Role of Amyloid Fibrils in HIV Infection.
Biology 2012, 1 (1), 58–80.
(19) Popovych, N.; Brender, J. R.; Soong, R.; Vivekanandan, S.; Hartman, K.; Basrur, V.;
Macdonald, P. M.; Ramamoorthy, A. Site Specific Interaction of the Polyphenol EGCG
ACS Paragon Plus Environment
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Page 19 of 31
Langmuir
1
2
3
4
with the SEVI Amyloid Precursor Peptide PAP(248ꢀ286). J. Phys. Chem. B 2012, 116
5
6
(11), 3650–3658.
7
8
9
(20) Olsen, J. S.; Brown, C.; Capule, C. C.; Rubinshtein, M.; Doran, T. M.; Srivastava, R. K.;
Feng, C.; Nilsson, B. L.; Yang, J.; Dewhurst, S. Amyloid Binding Small Molecules
Efficiently Block SEVI and Semen Mediated Enhancement of HIVꢀ1 Infection. J. Biol.
Chem. 2010, 285, 35488–35496.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(21) Capule, C. C.; Brown, C.; Olsen, J. S.; Dewhurst, S.; Yang, J. Oligovalent Amyloidꢀ
Binding Agents Reduce SEVIꢀMediated Enhancement of HIVꢀ1 Infection. J. Am. Chem.
Soc. 2012, 134 (2), 905–908.
(22) Sheik, D. A.; Brooks, L.; Frantzen, K.; Dewhurst, S.; Yang, J. Inhibition of the
Enhancement of Infection of Human Immunodeficiency Virus by SemenꢀDerived
Enhancer of Virus Infection Using AmyloidꢀTargeting Polymeric Nanoparticles. ACS
Nano 2015, 9 (2), 1829–1836.
(23) Inbar, P.; Yang, J. Inhibiting ProteinꢀAmyloid Interactions with Small Molecules: A
Surface Chemistry Approach. Bioorg. Med. Chem. Lett. 2006, 16 (4), 1076–1079.
(24) Inbar, P.; Li, C. Q.; Takayama, S. A.; Bautista, M. R.; Yang, J. Oligo(ethylene Glycol)
Derivatives of Thioflavin T as Inhibitors of ProteinꢀAmyloid Interactions. Chembiochem
2006, 7 (10), 1563–1566.
(25) Serpell, L. C. Alzheimer’s Amyloid Fibrils: Structure and Assembly. Biochim. Biophys.
Acta 2000, 1502 (1), 16–30.
ACS Paragon Plus Environment
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Langmuir
Page 20 of 31
1
2
3
4
5
6
7
8
9
(26) Khurana, R.; IonescuꢀZanetti, C.; Pope, M.; Li, J.; Nielson, L.; RamírezꢀAlvarado, M.;
Regan, L.; Fink, A. L.; Carter, S. A. A General Model for Amyloid Fibril Assembly Based
on Morphological Studies Using Atomic Force Microscopy. Biophys. J. 2003, 85 (2),
1135–1144.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(27) Walsh, D. M.; Hartley, D. M.; Kusumoto, Y.; Fezoui, Y.; Condron, M. M.; Lomakin, A.;
Benedek, G. B.; Selkoe, D. J.; Teplow, D. B. Amyloid βꢀProtein Fibrillogenesis. Structure
and Biological Activity of Protofibrillar Intermediates. J. Biol. Chem. 1999, 274 (36),
25945–25952.
(28) Serpell, L. C.; Sunde, M.; Benson, M. D.; Tennent, G. A.; Pepys, M. B.; Fraser, P. E. The
Protofilament Substructure of Amyloid Fibrils. J. Mol. Biol. 2000, 300 (5), 1033–1039.
(29) Selkoe, D. J. Folding Proteins in Fatal Ways. Nature 2003, 426 (6968), 900–904.
(30) Rambaran, R. N.; Serpell, L. C. Amyloid Fibrils: Abnormal Protein Assembly. Prion
2008, 2 (3), 112–117.
(31) Lührs, T.; Ritter, C.; Adrian, M.; RiekꢀLoher, D.; Bohrmann, B.; Dobeli, H.; Schubert, D.;
Riek, R. 3D Structure of Alzheimer’s Amyloidꢀβ(1–42) Fibrils. Proc. Natl. Acad. Sci. U.
S. A. 2005, 102 (48), 17342–17347.
(32) Nanga, R. P. R.; Brender, J. R.; Vivekanandan, S.; Popovych, N.; Ramamoorthy, A. NMR
Structure in a Membrane Environment Reveals Putative Amyloidogenic Regions of the
SEVI Precursor Peptide PAP(248ꢀ286). J. Am. Chem. Soc. 2009, 131 (49), 17972–17979.
(33) Koo, E. H.; Lansbury, P. T.; Kelly, J. W. Amyloid Diseases: Abnormal Protein
ACS Paragon Plus Environment
20

Page 21 of 31
Langmuir
1
2
3
4
Aggregation in Neurodegeneration. Proc. Natl. Acad. Sci. U. S. A. 1999, 96 (18), 9989–
5
6
9990.
7
8
9
(34) Hardy, J. A.; Higgins, G. A. Alzheimer’s Disease: The Amyloid Cascade Hypothesis.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Science 1992, 256 (5054), 184–185.
(35) Capule, C. C.; Yang, J. EnzymeꢀLinked Immunosorbent AssayꢀBased Method to Quantify
the Association of Small Molecules with Aggregated Amyloid Peptides. Anal. Chem.
2012, 84 (3), 1786–1791.
(36) Giacomelli, C. E.; Norde, W. Conformational Changes of the Amyloid BetaꢀPeptide (1ꢀ
40) Adsorbed on Solid Surfaces. Macromol. Biosci. 2005, 5 (5), 401–407.
(37) Moores, B.; Drolle, E.; Attwood, S. J.; Simons, J.; Leonenko, Z. Effect of Surfaces on
Amyloid Fibril Formation. PLoS One 2011, 6 (10), e25954.
(38) Skaat, H.; Chen, R.; Grinberg, I.; Margel, S. Engineered Polymer Nanoparticles
Containing Hydrophobic Dipeptide for Inhibition of Amyloidꢀβ Fibrillation.
Biomacromolecules 2012, 13 (9), 2662–2670.
(39) Ghavami, M.; Rezaei, M.; Ejtehadi, R.; Lotfi, M.; Shokrgozar, M. A.; Abd Emamy, B.;
Raush, J.; Mahmoudi, M. Physiological Temperature Has a Crucial Role in Amyloid Beta
in the Absence and Presence of Hydrophobic and Hydrophilic Nanoparticles. ACS Chem.
Neurosci. 2013, 4 (3), 375–378.
(40) Yoo, S. Il; Yang, M.; Brender, J. R.; Subramanian, V.; Sun, K.; Joo, N. E.; Jeong, S. H.;
Ramamoorthy, A.; Kotov, N. A. Inhibition of Amyloid Peptide Fibrillation by Inorganic
ACS Paragon Plus Environment
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Langmuir
Page 22 of 31
1
2
3
4
Nanoparticles: Functional Similarities with Proteins. Angew. Chemie - Int. Ed. 2011, 50
5
6
(22), 5110–5115.
7
8
9
(41) Yan, C.; Yuan, X.; Kang, C.; Zhao, Y.ꢀH.; Liu, J.; Guo, Y.; Lu, J.; Pu, P.; Sheng, J.
Preparation of Carmustine‐loaded PLA Ultrasmall‐nanoparticles by Adjusting Micellar
Behavior of Surfactants. J. Appl. Polym. Sci. 2008, 110, 2446–2452.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(42) Farokhzad, O. C.; Cheng, J.; Teply, B. A.; Sherifi, I.; Jon, S.; Kantoff, P. W.; Richie, J. P.;
Langer, R. Targeted NanoparticleꢀAptamer Bioconjugates for Cancer Chemotherapy in
Vivo. Proc. Natl. Acad. Sci. U. S. A. 2006, 103 (16), 6315–6320.
(43) Rao, J. P.; Geckeler, K. E. Polymer Nanoparticles: Preparation Techniques and Sizeꢀ
Control Parameters. Prog. Polym. Sci. 2011, 36 (7), 887–913.
(44) Prabha, S.; Zhou, W.ꢀZ.; Panyam, J.; Labhasetwar, V. SizeꢀDependency of Nanoparticleꢀ
Mediated Gene Transfection: Studies with Fractionated Nanoparticles. Int. J. Pharm.
2002, 244 (1–2), 105–115.
(45) Yin Win, K.; Feng, S.ꢀS. Effects of Particle Size and Surface Coating on Cellular Uptake
of Polymeric Nanoparticles for Oral Delivery of Anticancer Drugs. Biomaterials 2005, 26
(15), 2713–2722.
(46) Hall, D. B.; Underhill, P.; Torkelson, J. M. Spin Coating of Thin and Ultrathin Polymer
Films. Polym. Eng. Sci. 1998, 38 (12), 2039–2045.
(47) Greenfield, N. Using Circular Dichroism Spectra to Estimate Protein Secondary Structure.
Nat. Protoc. 2007, 1 (6), 2876–2890.
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Page 23 of 31
Langmuir
1
2
3
4
5
6
7
8
9
(48) Elias, A. K.; Scanlon, D.; Musgrave, I. F.; Carver, J. A. SEVI, the Semen Enhancer of
HIV Infection along with Fragments from Its Central Region, Form Amyloid Fibrils That
Are Toxic to Neuronal Cells. Biochim. Biophys. Acta 2014, 1844 (9), 1591–1598.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(49) Rodgers, J. L.; Nicewander, W. A. Thirteen Ways to Look at the Correlation Coefficient.
Am. Stat. 1988, 42 (1), 59.
(50) Adler, J.; Parmryd, I. Quantifying Colocalization by Correlation: The Pearson Correlation
Coefficient Is Superior to the Mander’s Overlap Coefficient. Cytom. Part A 2010, 77 (8),
733–742.
(51) Inbar, P.; Bautista, M. R.; Takayama, S. A.; Yang, J. Assay To Screen for Molecules That
Associate with Alzheimer’s Related ꢀAmyloid Fibrils. Anal. Chem. 2008, 80 (9), 3502–
3506.
(52) Ban, T.; Hamada, D.; Hasegawa, K.; Naiki, H.; Goto, Y. Direct Observation of Amyloid
Fibril Growth Monitored by Thioflavin T Fluorescence. J. Biol. Chem. 2003, 278 (19),
16462–16465.
(53) Holmberg, K.; Bergström, K.; Brink, C.; Österberg, E.; Tiberg, F.; Harris, J. M. Effects on
Protein Adsorption, Bacterial Adhesion and Contact Angle of Grafting PEG Chains to
Polystyrene. J. Adhes. Sci. Technol. 1993, 7 (6), 503–517.
(54) Miller, C. R.; Bondurant, B.; McLean, S. D.; McGovern, K. A.; O’Brien, D. F.
Liposome−Cell Interactions in Vitro: Effect of Liposome Surface Charge on the Binding
and Endocytosis of Conventional and Sterically Stabilized Liposomes. Biochemistry 1998,
37 (37), 12875–12883.
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(55) DeMarco, M. L.; Daggett, V.; Fersht, A. From Conversion to Aggregationꢂ: Protofibril
5
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Formation of the Prion Protein. Proc. Natl. Acad. Sci. U. S. A. 2004, 101 (8), 2293–2298.
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(56) Mallucci, G.; Dickinson, A.; Linehan, J.; Klöhn, P.ꢀC.; Brandner, S.; Collinge, J.
Depleting Neuronal PrP in Prion Infection Prevents Disease and Reverses Spongiosis.
Science 2003, 302 (5646), 871–874.
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(57) White, A. R.; Enever, P.; Tayebi, M.; Mushens, R.; Linehan, J.; Brandner, S.; Anstee, D.;
Collinge, J.; Hawke, S. Monoclonal Antibodies Inhibit Prion Replication and Delay the
Development of Prion Disease. Nature 2003, 422 (6927), 80–83.
(58) Dong, A.; Huang, P.; Caughey, W. S. Protein Secondary Structures in Water from
SecondꢀDerivative Amide I Infrared Spectra. Biochemistry 1990, 29 (13), 3303–3308.
(59) Yang, H.; Yang, S.; Kong, J.; Dong, A.; Yu, S. Obtaining Information about Protein
Secondary Structures in Aqueous Solution Using Fourier Transform IR Spectroscopy.
Nat. Protoc. 2015, 10 (3), 382–396.
(60) Byler, D. M.; Susi, H. Examination of the Secondary Structure of Proteins by
Deconvolved FTIR Spectra. Biopolymers 1986, 25 (3), 469–487.
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Figure 1. Cartoon depicting the disruption of SEVI structure in the presence of hydrophobic
nanoparticles and concomitant reduction of SEVIꢀmediated enhancement of HIV infection.
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Scheme 1. Synthesis of polyacrylateꢀbased hydrophobic (4) and hydrophilic (5) polymers.
Abbreviations: triethylamine (TEA), dichloromethane (DCM), azobisisobutyrylnitrile (AIBN),
dimethylformamide (DMF), potassium hydroxide (KOH), methanol (MeOH).
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Table 1. Polymer and Nanoparticle Characterization
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Polymer
Mn (kDa)
Mw/Mn
% 1
Nanoparticle
Diameter
(nm)
Polydispersity
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6.5
1.84
1.42
95
265 ± 37
N/A
0.16 ± 0.04
N/A
4
5
12.9
N/A
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Figure 2. PMꢀIRRAS analysis of SEVI amyloid fibrils and the correlations between βꢀsheet
content and observed fluorescence of ThT in presence of SEVI. Representative PMꢀIRRAS
absorbance spectra (black lines) of preformed SEVI fibrils deposited on a goldꢀplated slide
coated with the hydrophilic polymer 5 (A) or the hydrophobic polymer 4 (B). Iterative least
square curve fitting58,59 of the Amide I region reveals the approximate abundance of βꢀsheet (red
curves), αꢀhelix/unordered (green curves), βꢀturn (purple curves), and antiꢀparallel βꢀsheet as
well as possible sidechain absorbances (blue curves) within each SEVI sample.60 C) Scatterplots
showing the observed βꢀsheet content of 5 different preparations of SEVI fibrils (as estimated by
PMꢀIRRAS measurements) deposited on surfaces coated with either hydrophilic (5) or
hydrophobic (4) polymers. D) Plot of the observed ThT fluorescence in 5 different SEVI
preparations versus the estimated βꢀsheet content of the same SEVI fibrils when deposited on
surfaces coated with the hydrophilic polymer 5. Dashed lines indicate the 95% confidence
interval. *** denotes P value < 0.001, as estimated by paired tꢀtest.
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Figure 3. Plots of ThT fluorescence in SEVI samples or relative βꢀsheet content of SEVI versus
the effects of SEVI on HIV infectivity (represented as fold enhancement compared to the level of
HIV infection in the absence of SEVI). A) Graph of ThT fluorescence versus fold enhancement
of SEVIꢀmediated HIV infection for 5 different preparations of SEVI fibrils. B) Graph of βꢀsheet
content (as estimated by PMꢀIRRAS) and fold enhancement of SEVIꢀmediated HIV infection for
the same 5 SEVI fibril samples used to generate the data in (A). Dashed lines indicate 95%
confidence intervals.
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