Fullerene derivatives strongly inhibit HIV-1 replication by affecting virus maturation without impairing protease activity

Article abstract of DOI:10.1128/AAC.00341-16

Three compounds (1, 2, and 3) previously reported to inhibit HIV-1 replication and/or in vitro activity of reverse transcriptase were studied, but only fullerene derivatives 1 and 2 showed strong antiviral activity on the replication of HIV-1 in human CD4

Full text of DOI:10.1128/AAC.00341-16

AAC Accepted Manuscript Posted Online 18 July 2016
Antimicrob. Agents Chemother. doi:10.1128/AAC.00341-16
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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Fullerene Derivatives Strongly Inhibit HIV-1 Replication by Affecting Virus Matura-
tion without Impairing Protease Activity.
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Zachary S. Martinez,a Edison Castro,b Chang-Soo Seong,a Maira R. Cerón,b Luis Eche-
goyen,b and Manuel Llanoa#
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Department of Biological Sciences a, Department of Chemistry b, University of Texas at
El Paso, El Paso, TX, United States.
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Running Head: Fullerene derivatives block HIV-1 maturation
#Address correspondence to Manuel Llano, mllano@utep.edu
Z.S.M. and E.C. contributed equally to this work.
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L.E. and M.Ll. are co-corresponding authors and contributed equally to this work.
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ABSTRACT
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Three compounds (1, 2, and 3) previously reported to inhibit HIV-1 replication and/or in
vitro activity of reverse transcriptase were studied, but only fullerene derivatives 1 and 2
showed strong anti-viral activity on the replication of HIV-1 in human CD4+ T cells.
However, these compounds did not inhibit infection by single-round infection VSV-G
pseudotyped viruses, indicating no effect on the early steps of the viral life cycle. In con-
19 trast, analysis of single-round infection VSV-G pseudotyped HIV-1 produced in the
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presence of 1 or 2 showed a complete lack of infectivity in human CD4+ T cells, sug-
gesting that the late stages of the HIV-1 life cycle were affected. Quantification of virion-
associated viral RNA and p24 indicates that RNA packaging and viral production were
unremarkable in these viruses. However, Gag and Gag-Pol processing was affected, as
evidenced by immunoblot analysis with an anti-p24 antibody and the measurement of
virion-associated reverse transcriptase activity, ratifying the effect of the fullerene deriv-
26 atives on virion maturation of the HIV-1 life cycle. Surprisingly, fullerenes 1 and 2 did not
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inhibit HIV-1 protease in an in vitro assay at the doses that potently blocked viral infec-
tivity, suggesting a protease-independent mechanism of action. Highlighting the poten-
tial therapeutic relevance of fullerene derivatives, these compounds block infection of
HIV-1 resistant to protease and maturation inhibitors.
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INTRODUCTION
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Advances in anti-Human Immunodeficiency Virus (anti-HIV) retroviral drugs have
led to a significant reduction in acquired immune deficiency syndrome (AIDS) related
35 deaths, delayed disease progression, and diminished the rates of HIV transmission
36 (12). Current therapeutic treatments for effective repression of HIV replication are ad-
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ministered in a cocktail regimen known as highly active antiretroviral therapy. The an-
tiretroviral activity of these drugs is mainly due to their inhibition of HIV reverse tran-
39 scriptase and protease, essential enzymes for HIV replication. Drugs targeting protease
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prevent the cleavage of the Gag and Gag-Pol polyprotein, leading to immature virions.
These therapies efficiently suppress the spread of HIV in patients; however, the emer-
gence of drug-resistant viruses is a continuous challenge to the effectiveness of these
interventions. In addition, these anti-retroviral drugs have important side effects that limit
44 their use (28, 40, 44). Therefore the development of new and safer anti-HIV compounds
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is a critical need (11, 12, 23).
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Fullerenes consist of carbon atom cages, some, like C60, have the shape of a
hollow sphere, similar to a soccer ball (27). Due to their ability to be extensively derivat-
ized, functionalized fullerenes have shown several biological applications (2, 3). It has
been hypothesized that fullerene derivatives are capable of efficiently crossing the cell
membrane due to their hydrophobic core while water solubility can be achieved by at-
taching hydrophilic moieties (2, 3, 13). The first fullerene derivatives that exhibited anti-
HIV activity were reported in 1993 (15, 46). However, the lack of comprehensive char-
acterization of the antiviral mechanisms of fullerene derivatives has hindered their fur-
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ther development into therapeutic drugs (3, 15, 17, 25, 33, 36, 47, 50, 53). Since the
original report, it has been assumed that the anti-HIV activity of fullerene derivatives is
mediated mainly, if not exclusively, by inhibition of the viral protease. Evidences sup-
porting this mechanism are mostly based molecular docking simulations that predict the
binding of these compounds to the active site of HIV-1 protease due to the size and
conformational complementarity (8, 15, 17, 35, 63). However, this model lacks support
from empirical evidences. In addition, in vitro assays indicate that some fullerene deriva-
tives possess anti-reverse transcriptase activity (37).
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Therefore, to better understand the mechanism of action of fullerene derivatives
in HIV replication, we investigated the effect of these compounds on the different steps
64 of the HIV-1 life cycle in human CD4+ T cells. Our data demonstrate for the first time
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that the viral maturation process is the step of the HIV-1 life cycle affected. After 23
years of assuming that inhibition of HIV-1 was due to the interaction between the fuller-
ene and the hydrophobic pocket of the protease, unexpectedly we discovered that full-
erene derivatives do not inhibit HIV-1 protease at doses that potently inhibit HIV-1 infec-
tion. Furthermore, we observed that these compounds are effective in blocking replica-
tion of viruses that are resistant to the clinically approved protease inhibitors.
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MATERIALS AND METHODS
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Synthesis of fullerene derivatives. Compounds 1, 3 and the regioisomeric mixture 2
have been previously reported (10, 32, 36, 39), here we report slight modifications for
the synthesis of compounds 1, 2, 3 and the synthesis of compound 4 (24) (Figure 1).
Please, refer to the supporting information for details on the synthesis and physico-
chemical characterization of these compounds.
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Plasmids. The plasmids used to generate retroviral vectors were described previously
(31). HIV-1-derived vectors were produced using pHIV Luc and pMD.G. pHIV Luc was
80 derived from pNL4-3.Luc.R–E– (22) by introducing a deletion in the env open reading
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frame (diagrammed in figure 4). pHIV Luc containing multi-protease inhibitors resistant
to protease mutants were constructed by swapping a 4.3 Kb Sal I/Spe I fragment in
pHIV Luc with this fragment from pNL4-3 containing the mutant proteases. pMD.G en-
codes the Vesicular Stomatitis Virus glycoprotein G (VSV-G). HIV-1NL4-3 was pro-
duced from the corresponding expression plasmid whereas multi-protease inhibitor re-
sistant viruses were produced with plasmids obtained from the NIH AIDS Reagent Pro-
gram (56).
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Cell lines. SupT1 and HEK293T cells were grown in RPMI 1640 and in DMEM, respec-
tively. All culture media were supplemented with 10% of heat-inactivated fetal calf se-
rum, 2 mM L-glutamine and 1% penicillin/streptomycin.
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Generation of retroviruses. Procedures previously described were followed (19).
Briefly, 3 x 106 HEK293T cells were plated in a T75 cm2 tissue culture flask and co-
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transfected the next day with the corresponding plasmids by the calcium-phosphate
precipitation method. 18 h later the transfection medium was replaced with fresh medi-
um containing no drug, fullerene derivatives, Indinavir, or dimethyl sulfoxide (DMSO,
vehicle control). The cells were cultured for 48 h until the viral supernatant was harvest-
97 ed and filtered. Single-round infection viral vectors were further concentrated by ultra-
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centrifugation at 124750 g for 2 h on a 20% sucrose cushion. Viral preparations were
stored at -80 °C until used.
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VSV-G-pseudotyped HIV-derived reporter virus expressing firefly luciferase (HI-
Vluc) and harboring wild type or multi-protease inhibitor resistant protease mutants were
prepared by co-transfection of 15 µg of the corresponding pHIV luc and 5 µg of pMD.G.
HIV-1 wild type viruses were produced by transfection of 15 µg of the corresponding
expression plasmids.
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Single-round infectivity assay. SupT1 cells were plated at 1 x 105 cells in 500 µL of
RPMI 1640 culture medium in 24-well plates and infected with HIVluc. 4 days post-
infection, cells were collected by centrifugation at 1000 g for 6 min and the pellet resus-
108 pended in 200 μL of phosphate-buffered saline (PBS). Half of the sample was mixed
109 with 100 μL of luciferase substrate (Bright-Glow™ Luciferase Assay System, Promega)
110 and the other half with 100 μL of cell viability substrate (CellTiter-Glo® Assay,
111 Promega). Cell lysates were incubated for 10 min at room temperature in the dark and
112 then luminescence was measured in triplicate in 50 μL-samples using a microplate lu-
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minometer reader (Thermo Scientific, Luminoskan Ascent).
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HIV-1 replication assays. SupT1 cells (0.25 x 106 cells in 3 mL RPMI 1640) were in-
fected with HIV-1NL4-3 harboring protease wild type or mutant resistant to multiple pro-
tease inhibitors (2.1 ng of HIV-1 p24) in the presence of fullerene derivatives or DMSO,
or in the absence of any with HIV-1NL4-3 containing CA mutations L363F and
V362L/L363M (VL/LM) that were produced in the presence of DMSO or fullerene de-
rivatives. 24 h after infection, the cells were washed 3 times by centrifugation in 10 mL
120 (total 30 mL) of culture medium to remove the input virus and compounds. Cell superna-
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tant was then collected at different days post-infection and used for HIV-1 p24 quantifi-
cation by ELISA.
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TZM-bl cells were also used for infection assays with replicating competent HIV-
124 1. This HeLa-derived indicator cell line expresses CD4, CXCR4, CCR5 and has lucifer-
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ase and ß-galactosidase expression cassettes driven by the HIV-1 promoter stably in-
serted in the genome (7, 41, 42, 49, 58). TZM-bl cells (1x105 cells/well) were plated in a
p24-wells plate and the next day infected with p24-normalized HIV-1, respectively. Sev-
enty-two hours post-infection cells were lysed in PBS-1% Triton X-100 and luciferase
activity measured as described above.
130 HIV-1 p24 ELISA. HIV-1 p24 levels were determined by a sandwich ELISA following
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manufacturer instructions. Briefly, 200 μL of the viral samples were diluted appropriately
and incubated on the ELISA wells overnight at 37 °C. Unbound proteins were removed
by washing the wells 6 times with 200 μL of washing buffer, and bound HIV-1 p24 was
134 detected by incubating each well with 100 μL of the anti-HIV-1 p24 secondary antibody
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for 1 h. Unbound antibodies were removed by washing as described above and bound
antibodies were detected by incubating each well with 100 μL of substrate buffer for 30
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min at room temperature until the reaction was stopped by adding 100 μL of stop solu-
tion into each well. The absorbance of each well was determined at 450 nm using a mi-
139 croplate reader (Molecular Devices, Versa max microplate reader).
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Cellular viability assay. 1 x 104 SupT1 cells were plated in a 96-well plate in 100 µL
RMPI1640 culture media and left untreated or treated with fullerene derivatives, DMSO
(control), or 2 mM hydrogen peroxide (positive control). Fullerene derivatives were eval-
uated at a concentration that ranged from 3 µM to 32 µM. The cells were cultured in the
presence of the indicated compounds for 24 h and then 20 µL of the MTS were added
to each well of cells. Incubation with the reagent for an additional 3 h was allowed. The
colored formazan product was measured by absorbance at 490 nm with a reference
147 wavelength of 650 nm using a microplate reader (SpectraMax 190, Molecular Devices).
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Control wells containing the same volumes of culture medium and MTS reagent were
used to subtract background absorbance.
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Exogenous Reverse Transcriptase assay. Reverse transcriptase levels in HIV-1 re-
porter viruses produced in the presence of DMSO, fullerene derivatives 1 and 2, and
Indinavir were measured using the EnzChek® Reverse Transcriptase Assay following
the manufacturer instructions. HIV-1 p24-normalized amounts (4.34 μg) were analyzed
for each compound treated virus.
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HIV-1 protease in vitro activity assay. The effect of DMSO, compounds 1 and 2 (3
and 10 μM), and Indinavir (0.1 μM) on HIV-1 protease activity was measured using the
ProAssayTM HIV-1 Protease Assay Kit. This assay uses purified recombinant HIV-1
protease and a fluorescence resonance energy transfer (FRET) peptide derived from
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the native p17/p24 cleavage site of HIV-1 protease on Gag. Briefly, HIV-1 protease (0.2
µL) and FRET peptide (final concentration 0.5 μM) were mixed in HIV-1 protease buffer
supplemented with 1 mM DTT (final concentration) on ice and protected from light, and
immediately transferred into a black 96-wells plate that contain the compounds being
evaluated. The reaction was measured by determining the Relative Fluorescing Intensi-
164 ty (RFI) with a fluorometer at excitation/emission wavelengths of 490 nm / 530 nm every
165 5 min during 90 min.
166 Quantification of virion associated ribonucleic acid (RNA). Real-time quantitative
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reverse transcription-PCR was used to quantify virion associated RNA. RNA was puri-
fied from HIV-1 p24-normalized compound-treated virions (5 ng) after their treatment
with 1 μg of RNase and 2 units of DNase (Qiagen® RNAEasy™ Miniprep kit). cDNAs
were generated from all the viral RNA extracted with random oligonucleotides using the
171 High Capacity RNA-to-cDNA Kit from Applied Biosystems®. cDNA (1 μg) was then ana-
172 lyzed using qPCR using Gag-hybridizing primers (iQTM SYBR® Green Supermix BIO-
173 RAD®). The sequences of the oligonucleotides used in the qPCRs are available upon
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request.
Immunoblotting. Proteins of HIV-1 p24-normalized amounts of virions (0.38 μg) were
176 resolved by 13% SDS-PAGE and transferred overnight to PVDF membranes at 100
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mAmp at 4 °C. Membranes were blocked in TBS containing 10% milk for 1 h and then
incubated in the corresponding primary antibody diluted in TBS-5% milk-0.05% Tween
20 (antibody dilution buffer) overnight at 4 °C. HIV-1 p24 was detected with anti-p24
obtained from the NIH AIDS Reagent Program (Catalog # 1513). HIV-1 integrase was
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detected with anti-integrase (Catalog # sc-69721, Santa Cruz Biotechnology). Primary
antibody-bound membranes were washed in TBS-0.1% Tween 20 and all bound anti-
183 bodies detected with goat anti-mouse IgG-HRP (1/2000, KPL, 074-1806) followed by
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chemo-luminescence detection.
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Real time PCR analysis of early steps of the HIV-1 life cycle. Procedures previously
described were followed (56). Briefly, 1 x 106 SupT1 cells were challenged with DNase-
treated single-round infection HIVluc virus produced in the presence of fullerenes or
DMSO and 24 h later 90% of the cells were used for DNA extraction (High pure PCR
template preparation kit, Roche) whereas 10% were cultured for four days to evaluate
infectivity. Extracted DNA (20 ng) was used for the detection of total HIV-1 cDNA, mito-
191 chondrial DNA, and 2LTR circles while 0.2 ng of DNA were used for the Alu-LTR junc-
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tions PCR. Total HIV-1 cDNA, Alu-LTR junctions, and 2LTR circle products were nor-
malized to mitochondrial DNA. Real time PCRs were performed in a MiniOpticon sys-
tem (Bio-rad) with primers and conditions previously described (56). Fold change was
195 calculated using the ∆Ct method as recommended in the thermo-cycler manual.
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RESULTS
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Effect of fullerene derivatives on HIV-1 replication in human CD4+ T cells. C60 full-
erene derivatives were previously shown to inhibit HIV-1 replication, but its effect on the
different steps of the viral life cycle was not evaluated (26, 34, 45, 51). In particular,
compounds 1, 2, and 3 (Figure 1) have been shown to inhibit HIV-1 reverse transcrip-
tase or protease in in vitro assays (18, 37, 45, 52). Therefore, to fully characterize the
anti-HIV-1 activity of fullerene derivatives in biologically relevant environments, com-
pounds 1, 2, and 3 were evaluated for their ability to affect HIV-1 replication in human
CD4+ T cells (Figure 2). SupT1 cells were infected with HIV-1NL4-3 in the presence of
compounds 1, 2, and 3 at 1, 3, and 10 μM concentrations, and compounds and input
viruses were removed 24 h later. Infected cells were cultured for approximately 2 weeks
and HIV-1 p24 levels in the cell supernatant were measured by ELISA.
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Data in figure 2 indicate that compounds 1 and 2 potently inhibited viral replica-
tion at 3 μM. The effect of compound 1 on HIV-1 replication at 3 and 10 μM were indis-
tinguishable and showed a 300-fold inhibition of HIV-1 viral replication (Data not
shown). The regioisomeric compound mixture 2 at 3 μM inhibited HIV-1 replication to a
similar extent as compound 1, and was still active at 1 μM (Figure 2b); however, com-
pound 1 was inactive at 1 μM (Figure 2a). Contrary to the effect of compounds 1 and 2
on HIV-1 replication, no effect was observed when cells were treated for 24 h at the
time of infection with compound 3 at 1, 3 and 10 μM (data not shown). Therefore, our
data support the anti-HIV-1 activity reported for compounds 1 and 2 (26, 34, 45, 51) but
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failed to validate in human cells the effect reported for compound 3 using in vitro assays
(37).
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Function/structure analysis of the fullerene derivatives characterized (Figure 1)
indicate that similar chemical addends present in the regioisomeric mixture 2 and the
pure regioisomer (trans-3) compound 1 most likely explain their comparable inhibitory
effect on HIV-1 replication. However, when the quaternized nitrogen in 1 and 2 was
eliminated and the pyrrolidine ring was modified with carboxylic acids, compound 3, the
anti-HIV activity was completely lost, indicating the relevance of the addends in the ac-
tivity of the fullerene derivatives.
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Effect of fullerenes on cellular viability. We have observed that compounds 1 and 2
potently inhibit HIV-1 infection (Figure 2). A possibility is that these compounds com-
promise cellular viability affecting viral infection nonspecifically. To rule out this possibil-
ity, the effect of compounds 1 and 2 on cell viability was evaluated by the tetrazolium
dye reduction assay. In these experiments, SupT1 cells were treated with compounds 1
and 2 at concentrations of 3 and 6 μM, or similar amounts of DMSO, for 24 h and then
cell viability was measured. At these concentrations none of these compounds showed
cytotoxic activity (Figure 3). Therefore, the effects on HIV-1 infection observed with
compounds 1 and 2 at 3 μM most likely are not due to cell toxicity.
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To further define the cytotoxic activity of these compounds the drug concentra-
tion that kills approximately 50% of SupT1 cells (LC50) was determined for compounds
1 and 2 after 24 h of treatment. In these experiments, LC50 for compound 1 and 2 was
22.9 μM and 38.3 μM respectively, as calculated by regression analysis of data in figure
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3. These results also indicate that compounds 1 and 2 inhibit HIV-1 infection at doses
that are not toxic to the target cells.
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Effect of fullerene derivatives on single-round infection. We have provided evi-
dence that fullerene derivatives, specifically compounds 1 and 2, inhibit HIV-1 replica-
244 tion; however the viral step implicated in this effect is unknown. In order to determine
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the step in the viral life cycle that is affected, the early phase of HIV-1 infection was first
analyzed. The effect of compounds 1, 2 and 3 on the infection of VSV-G pseudotyped
HIV-1 single-round infection viruses expressing luciferase was evaluated in SupT1 cells.
248 Although compound 3 showed no signs of inhibition of HIV-1 replication, it was evaluat-
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ed as a control. In addition, we analyzed the activity of compound 4 (Figure 1), an iso-
merically pure regioisomer (cis-2, endo-endo) (24) with two pyrrolidinium rings connect-
251 ed by a benzene bridge. This compound was incorporated in the analysis to provide
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additional information about the involvement of different addends attached to fullerene
and their anti-HIV-1 activity. Cells were exposed to 1, 3, and 10 μM of each compound
and infected with the reporter virus. 24 h later, drugs and input virus were removed and
four days post-infection luciferase and ATP levels were measured. Luciferase was nor-
malized to ATP to standardize for cell viability and number. Data in figure 4a clearly in-
dicate that none of the fullerene derivatives affect the early steps of the viral life cycle.
These results also demonstrated that LTR-driven transcription and translation of viral
proteins, or cellular viability, was not affected by fullerene derivatives. Combined analy-
sis of data in figures 2 and 4a suggest that the late phase of the viral life cycle is target-
ed by compounds 1 and 2.
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Effect of fullerene derivatives on the late phase of the HIV-1 life cycle. Compounds
1, 2, 3 and 4 were further evaluated to determine their effect on the late phase of the
264 HIV-1 life cycle. VSV-G pseudotyped, HIV-1 single-round infection viruses expressing
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luciferase were produced in the presence of DMSO, compounds 1, 2, 3 (3 μM) and/or
Indinavir (0.1 μM) in HEK293T cells cotransfected with plasmids expressing these retro-
viruses and plasmids encoding eGFP. Then, produced viruses were concentrated by
268 ultracentrifugation on a sucrose cushion and HIV-1 p24 levels were measured by
269 ELISA. Transfection efficiency was quantified by FACS analysis of eGFP.
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In order to analyze the effect of fullerene derivatives on HIV-1 infectivity, SupT1
cells were infected with p24-normalized single round infection viruses and luciferase
and ATP levels were determined four days later. Data in figure 4b indicate that infectivi-
273 ty of viruses produced in the presence of compounds 1 and 2 was dramatically reduced
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by more than 99%. This inhibitory effect was similar to the impairment caused by Indi-
navir (95%). In contrast, compounds 3 and 4 did not affect the infectivity of the viruses
produced, again highlighting the functional relevance of the addends modifying the full-
erene cage.
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HIV-1 p24 levels were similar among viruses produced in the presence or ab-
sence of fullerene derivatives or Indinavir, indicating that these compounds did not af-
fect LTR transcription, translation, and viral budding, nor cellular viability (Figure 5a).
These observations also correlate with the lack of an effect for compounds 1, 2, 3 and 4
on the expression of luciferase in the single-round infection assays (Figure 4a). The
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similar levels of HIV-1 p24 also correlated with equivalent eGFP expression in the pro-
ducer cells (Data not shown), indicating comparable transfection efficiency.
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To further investigate the effect of fullerene derivatives on viral assembly, we de-
termined whether compounds 1 and 2 as well as Indinavir affected the amount of virion-
associated viral RNA. HIV-1 RNA was extracted from p24-normalized amounts of com-
pound-treated virions and then converted into cDNA that was quantified by real time
PCR with primers hybridizing to Gag. Using this method, similar RNA levels were found
in virions produced in the presence of DMSO, compounds 1, 2, and Indinavir, indicating
that these compounds do not affect RNA packaging (Figure 5b). Altogether, the data
shown in figures 4b and 5a-b show that fullerene derivatives target the viral maturation
process.
294 Characterization of the early steps of the viral life cycle of virions produced in
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fullerene derivative-treated cells. We have shown that compounds 1 and 2 impair the
maturation step of HIV-1 drastically reducing virion infectivity. Using real time PCR
analysis we determined the competence of fullerene derivative-treated viruses to com-
plete the different steps of the early phase of the HIV-1 viral life cycle. SupT1 cells were
infected with DNase-treated HIV-1 p24-normalized amounts of concentrated HIV-1 pro-
duced in the presence of DMSO, Indinavir, or compounds 1 and 2. DNA was extracted
from these cells 24 h and 4 days after infection and total HIV-1 cDNA that is formed only
after efficient reverse transcription, 2LTR circles that is synthesized upon nuclear import
of the linear HIV-1 cDNA, and Alu-LTR junctions, products indicative of HIV-1 DNA inte-
gration, were quantified by real-time PCR.
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In these analyses, we found that total HIV-1 cDNA was dramatically reduced in
viruses produced in the presence of compounds 1 and 2, and this effect was of a higher
magnitude than in virions produced in Indinavir-treated cells (Figure 6a I). As a conse-
308 quence, 2LTR circles and Alu-LTR junctions were also markedly diminished in cells in-
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fected with fullerene-treated than DMSO-treated viruses (Figure 6a II and III, respective-
ly). As expected, this inhibitory effect of fullerene derivatives was also observed in DNA
extracted four days after infection (Figure 6b). Importantly, findings in figure 6 correlated
312 with the lack of infectivity reported in figure 4b, indicating a severe defect in the infectivi-
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ty of viruses produced in the presence of compounds 1 and 2.
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HIV-1 Gag processing in fullerene-treated virions. The fact that fullerene severely
blocks infectivity of viruses produced in their presence (Figure 4b and 6) but not mature
viruses (Figure 4a) suggests that these compounds affect virion maturation. This step of
the viral life cycle is initiated by the required proteolytic processing of Gag and Gag-Pol
318 polyproteins by HIV-1 protease and the further assembly of the viral components. To
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formally evaluate the effect of fullerenes on maturation we determined Gag-Pol pro-
cessing in virions treated or not with fullerene derivatives. HEK293T cells were trans-
fected with pHIV luc and pMD.G and the next day the transfection medium was re-
placed with culture medium containing DMSO, compounds 1 and 2 (3 μM), or Indinavir
(0.1 μM). Produced viruses were concentrated by ultracentrifugation on a sucrose cush-
324 ion and used for quantification of HIV-1 p24 by ELISA. Then, HIV-1 p24-normalized
325 amounts of viruses were used for Gag and Pol processing analysis. As shown in figure
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7a, Gag processing was affected by compounds 1 or 2 at 3 μM to a similar extent of
Indinavir at 0.1 μM. These three inhibitors blocked Gag processing at different cleavag-
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es sites including MA-CA-SP1-NC (~50 kDa), MA-CA (~41 kDa), and CA-SP1-NC (~33
kDa) (Figure 7b). However, integrase processing was not altered by fullerene deriva-
330 tives at 3 μM or Indinavir at 0.1 μM (Figure 7b) although at these doses these drugs
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significantly affected HIV-1 infection (Figure 2 and 4b). Nevertheless, Indinavir at higher
doses (10 μM) severely impaired Gag and integrase processing (Data not shown).
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Therefore, data in figures 7a and b indicate that fullerene derivatives, similar to
334 indinavir, impair Gag processing. To further evaluate the effects of fullerene derivatives
335 (3 μM) and Indinavir (0.1 μM) on HIV-1 maturation, we also determined the virion-
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associated reverse transcriptase activity of p24-normalized viruses using an exogenous
ssDNA template (exogenous reverse transcription assay). Although Indinavir impaired
reverse transcriptase activity, the effect of fullerenes, and in particular of the regioiso-
meric mixture 2, were more pronounced (Figure 7c), indicating a defect in HIV-1 matu-
ration in these viruses. The fact that the inhibitory effect on reverse transcriptase activity
of the regioisomeric mixture 2 was more potent than compound 1 (Figure 7c) highlights
the relevance of the fullerene addend groups in their activity. Future work will address
the effects of specific regioisomers in 2.
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Effect of fullerene derivatives on HIV-1 protease activity. In silico analysis suggest
that fullerene derivatives 1 and 2 could bind to the active site of HIV-1 protease (8, 15,
17, 25, 35, 63). This enzyme is essential for viral maturation, the step of the viral life
347 cycle that we have identified to be affected by fullerenes. However, fullerenes 1 and 2
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did not affect integrase processing (Figure 7b) although this event also depends on the
HIV-1 protease activity. Therefore, to further characterize the specific mechanism of
17

350
351
352
353
354
355
356
357
action of these compounds, we determined their effect on the in vitro activity of HIV-1
protease using a FRET peptide-based assay. Data in figure 7a indicated that indinavir
and compounds 1 and 2 block the processing of the native MA-CA (p17/p24) cleavage
site of HIV-1 protease on Gag, therefore a peptide containing this site was selected for
evaluation of the effect of these compounds on the activity of protease. Recombinant
protease was incubated with the substrate, an HIV-1-derived FRET peptide containing
the MA-CA cleavage site, in the presence of DMSO, fullerene derivatives, or Indinavir,
and fluorescence emission was tracked for 90 mins (Figure 8).
358
As expected, Indinavir completely blocked the activity of protease but surprising-
359 ly, compounds 1 and 2 were inactive at 3 μM (Figure 8a), a concentration that severely
360
361
362
363
364
365
366
impairs HIV-1 infection (Figures 2 and 4b). In further support, even at 10 μM, fullerene
derivatives only minimally affect protease activity (Figure 8b). Only at toxic concentra-
tions (Figure 3), above 40 μM, fullerenes 1 and 2 inhibited protease activity (Data not
shown). Therefore, our data definitively demonstrate that fullerene derivatives fail to
inhibit HIV-1 protease at doses that potently block HIV-1 maturation. These results con-
clusively show that fullerenes 1 and 2 are strongly anti-HIV active, but not via HIV-1 pro-
tease, contradicting the multiple studies previously reported (8, 15, 17, 35, 45, 52, 63).
367
368
369
370
371
Given that our results challenge the existing fullerene-protease paradigm, for
completeness we decided to study the HIV-1 protease activity of the originally reported
compound by Friedman in 1993 (scheme 4S in supplementary information, compound
5) (15). As expected, compound 5 inhibited HIV-1 viral replication (Figure A12) as com-
pounds 1 and 2 did, but was inactive in the in vitro activity of HIV-1 protease using the
18

372
373
374
FRET peptide-based assay at 3 and 10 μM (Figure A13). Therefore, our results clearly
show that fullerene derivatives are potent anti-HIV agents but their mechanistic activity
does not involve HIV-1 protease binding.
375 Effect of fullerene derivatives on the infectivity of HIV-1 resistant to protease or
376
377
378
379
380
381
382
383
384
385
386
387
maturation inhibitors. Our data indicate that fullerene derivatives do not affect HIV-1
protease; thus it is possible that these compounds could inhibit the infectivity of HIV-1
viruses that are resistant to the clinically used protease inhibitors. To test this hypothe-
sis further, we determined the sensitivity to compound 1 of multi-protease inhibitor-
resistant HIV-1 recombinant infectious molecular clones previously described (56).
Compound 1 and not 2 was chosen for these experiments because compound 1 is the
pure regioisomer (trans-3) contained in compound 2, a regioisomeric mixture. The mu-
tant viruses analyzed included 11803, 11806, 11807, 11808, and 11809 that are re-
sistant to nelfinavir, Fosamprenavir, Saquinavir, Indinavir, Atazanavir, Lopinavir, Tipra-
navir, and Darunavir; and 11805 that is also resistant to these drugs except for Tipra-
navir and Darunavir. The protease (99 amino acids) in these viruses contains between
10 and 24 point mutations (55).
388
Single-round infection HIV-1 expressing luciferase that harbor the multi-protease
389 inhibitor-resistant protease mutants were produced in HEK293T cells as described
390
391
392
above in the presence of DMSO, compound 1 (3 μM), or Indinavir (0.1 μM). The viruses
were concentrated by ultracentrifugation on a sucrose cushion, normalized for p24 con-
tent, and used to infect SupT1 cells. In these experiments we observed that compound
19

393
394
1, but not Indinavir, potently blocked the infectivity of all the mutant viruses with a similar
efficiency (Figure 9a).
395
396
397
398
To further verify these data, the effect of fullerenes on the replication of HIV-1
11803 was evaluated. This mutant was selected for analysis because, although it con-
tains 18 point mutations in protease that renders the virus resistant to eight different
protease inhibitors (55), it has a replication capacity of 63% of protease wild type HIV-1.
399 SupT1 cells were infected with NL4-3 or 11803 in the presence of DMSO or compound
400
401
402
403
404
405
406
1 (3 μM) and 24 h later the cells were washed to remove the input drugs and viruses,
and viral replication was evaluated by quantification of HIV-1 p24 in the cell supernatant
at different times post-infection. Viruses were used at p24 levels that result in robust
viral replication one week after infection to compensate for differences in viral fitness.
Data in figure 9b confirmed that compound 1 strongly inhibits viral replication of the mul-
ti-protease inhibitor-resistant virus 11803 to a similar extent than the protease wild type
NL4-3 virus. Therefore, data in figure 9 highlight the potential clinical relevance of fuller-
407 ene derivatives to block replication of multi-protease inhibitor resistant viruses.
408
409
410
411
412
413
414
In addition, we evaluated the effect of compound 1 on the infectivity of HIV-1
NL4-3 harboring mutations at the end of CA, L363F and V362L/L363M. These viruses
have been demonstrated to be resistant to the maturation inhibitor 3-O-(3’,3’-
dimethylsuccinyl) betulinic acid (DSB) (60, 62), and to fail to interact with DSB (38, 61).
Therefore, compounds blocking these mutant viruses have the potential to be clinically
relevant (57). Viruses were produced by plasmid transfection in HEK293T cells in the
presence of DMSO or compound 1 and their infectivity was determined in TZM-bl cells
20

415
416
417
418
419
420
421
422
using p24-normalized HIV-1. In these experiments compound 1 was used at a concen-
tration that only affect by 50% the infectivity of HIV-1 NL4-3 wild type, allowing detection
of small differences in the sensitivity of mutant and wild type viruses. Comparison of the
infectivity of viruses produced in the presence of DMSO or compound 1 (Figure 9d) in-
dicated that compound 1 was as active against HIV-1 NL4-3 V362L/L363M (Figure 9d
lane 1) and L363F (Figure 9d lane 2) mutants as the wild type viruses (Figure 9d lane
3). Therefore, these data indicated no cross-resistance between DSB and compound 1,
further highlighting the potential anti-HIV clinical relevance of fullerene derivatives.
423
21

424
DISCUSSION
425
426
427
428
429
430
431
432
433
Fullerene derivatives have been demonstrated to impair HIV-1 replication in hu-
man cells; (15, 25, 33, 45, 50) however, detailed virological characterization of the activ-
ity of these compounds is absent, limiting their development as anti-HIV therapeutic
agents. Defining the step in the viral life cycle affected by fullerene derivatives allows for
a better understanding of their anti-HIV mechanism. Molecular docking predictions have
proposed that fullerene derivatives could bind to the active site of HIV-1 protease while
the enzyme is in its catalytically active conformation as a homodimer (9, 15, 16, 35, 63).
Other groups have proposed that certain derivatives inhibit recombinant HIV-1 reverse
transcriptase in vitro activity (37).
434
435
Encouraged by these initial findings and the need for novel anti-HIV therapeutics,
we decided to fully characterize the anti-HIV-1 activity of fullerene derivatives 1 and 2.
436 Our data has corroborated the reported anti-HIV-1 activity of compounds 1 and 2 but do
437 not support findings or predictions indicating reverse transcriptase (37) or protease (9,
438 15, 16, 35, 45, 53, 63) as their targets. Instead, we have demonstrated for the first time
439
440
441
442
443
444
445
446
that fullerene inhibitors block HIV-1 maturation. HIV-1 is released from infected cells in
the form of immature, non-infectious virions that then proceed through maturation be-
fore gaining full infectivity. Viral maturation is triggered by proteolytic processing of Gag
and Gag-Pol polyproteins by HIV-1 protease. This processing results in the production
of functional viral proteins which assemble into the mature virion (14, 48). HIV-1 matura-
tion can be pharmacologically hindered by drugs that affect the activity of the viral pro-
tease (protease inhibitors) or that bind to the polyproteins (maturation inhibitors). The
importance of Gag- and Gag-Pol-derived proteins in every step of the viral life cycle
22

447 provides an opportunity to profoundly decrease HIV-1 infectivity by protease and matu-
448
ration inhibitors (14, 48).
449
450
451
452
453
454
455
456
457
We have evidenced that fullerene derivatives block HIV-1 maturation by inhibiting
Gag processing through a protease-independent mechanism. We speculate that inter-
action of fullerene derivatives with unprocessed Gag could modify the conformation or
assembly of these substrates, altering the specificity of protease, and thereby causing
defective processing. Similarly, binding of maturation inhibitors to CA result in structural
changes at remote regions on this protein (20, 54). Protease-mediated processing of
Gag and Gag-Pol occurs in a strictly organized manner and interfering with this highly
ordered process results in immature virions (14, 48); hence, local changes in the protein
conformation could globally impact its processing. Importantly, the physical parameters
458 that regulate the interaction of protease with its substrates is unclear (1, 29), perhaps
459
gaining an understanding of fullerene activity will shed light on of this phenomenon.
460
461
462
463
Our findings also highlight the potential therapeutic relevance of fullerene deriva-
tives to block clinically relevant resistant viruses. HIV-1 resistant to multiple clinically
used protease inhibitors, or to the maturation inhibitors DSB and PF-46396 (4, 5, 43,
59-62) were also potently blocked by compound 1.
464
465
466
467
468
We have also demonstrated that the addends on the C60 fullerenes and their re-
giochemistry have pronounced effects on their anti-HIV-1 activity, beyond simple water
solubility effects. The difference in anti-HIV-1 activity between compounds 1, 2, 3 and 4
is completely reliant on the chemical nature of their side chains. Previously it has been
reported that different regioisomers of 2 exhibit similar HIV-1 inhibitory activity and it
23

469
was concluded that the trans-3 compound 1 is more potent than the corresponding cis-3
470 isomer (33, 36). The fact that the regiochemistry influences the anti-HIV-1 activity of
471
472
these fullerene derivatives is important and is currently under investigation in our labora-
tories.
473
474
475
476
In summary, our data indicate that fullerene derivatives affect virion maturation of
HIV-1 wild type and protease and maturation inhibitors resistant viruses by impairing
viral polyprotein processing through a protease-independent mechanism, a paradigm-
shifting finding.
477
24

478
ACKNOWLEDGMENTS
479
480
481
482
We thank Diana Torres (UTEP) for technical help. The following reagents were obtained
through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: 1. From Dr.
Robert Shafer the HIV-1 expression plasmids Cat# 11803, pF190755_3, Cat# 11805,
pV38886_2, Cat# 11806, pV48334_2, Cat# 11807, pM135498_1, Cat# 11808,
483 pT450023_2, and Cat# 11809, pV16970_2. 2. From Dr. Bruce Chesebro the anti-HIV-1
484
485
p24 hybridoma (183-H12-5C). 3. TZM-bl from Dr. John C. Kappes, Dr. Xiaoyun Wu and
Tranzyme Inc. HIV-1 NL4-3 CA mutants L363F and V362L/L363M expression plasmids
486 were a gift of Dr. Christopher R. Aiken (Vanderbilt University School of Medicine).
487
FUNDING INFORMATION
488
489
490
This work was supported by Grant Number 5 SC1 AI098238-02 from the National Insti-
tute of General Medical Sciences (NIGMS), National Institutes of Health (NIH), (to ML).
LE thanks the US National Science Foundation, grant CHE-1408865, and the Robert A.
491 Welch Foundation for an endowed chair, grant # AH-0033, for generous support. The
492
493
494
authors also thank the College of Science at University of Texas at El Paso for providing
seed funds for this work through the grant 5G12MD007592 from the National Institutes
on Minority Health and Health Disparities (NIMHD-NIH). The University of Texas at El
495 Paso core facilities are funded by the BBRC grant 5G12RR008124.
496
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497
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