Characterization of the effects of aryl-azido compounds and UVA irradiation on the viral proteins and infectivity of human immunodeficiency virus type 1

Article abstract of DOI:10.1111/j.1751-1097.2010.00780.x

Hydrophobic UV-activatable compounds have been shown to partition into the hydrophobic region of biological membranes to selectively label transmembrane proteins, and to inactivate enveloped viruses. Here, we analyze various UV-activatable azido- and iodo-based hydrophobic compounds for their ability to inactivate a model-enveloped virus, human immunodeficiency virus (HIV-1 MN). Treatment of HIV-1 with 1,5-diazidonapthalene (DAN), 1-iodo, 5-azidonaphthalene (INA), 1-azidonaphthalene (AzNAP) or 4,4′-diazidobiphenyl (DABIPH) followed by UVA irradiation for 2 min resulted in complete viral inactivation, whereas treatment using analogous non-azido-containing controls had no effect. Incorporation of an azido moiety within these hydrophobic compounds to promote photoinduced covalent reactions with proteins was found to be the primary mechanism of viral inactivation for this class of compounds. Prolonged UVA irradiation of the virus in the presence of these azido compounds resulted in further modifications of viral proteins, due to the generation of reactive oxygen species, leading to aggregation as visualized via Western blot analysis, providing additional viral modifications that may inhibit viral infectivity. Furthermore, inactivation using these compounds resulted in the preservation of surface antigenic structures (recognized by neutralizing antibodies b12, 2g12 and 4e10), which is favorable for the creation of vaccines from these inactivated virus preparations.

Full text of DOI:10.1111/j.1751-1097.2010.00780.x

Photochemistry and Photobiology, 2010, 86: 1099–1108
Characterization of the Effects of Aryl-azido Compounds and UVA
Irradiation on the Viral Proteins and Infectivity of Human
Immunodeficiency Virus Type 1
Julie M. Belanger1, Yossef Raviv2, Mathias Viard2, Michael Jason de la Cruz3†, Kunio Nagashima3
and Robert Blumenthal*1
1Center for Cancer Research Nanobiology Program, NCI-Frederick, Frederick, MD, USA
2Basic Research Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD, USA
3Advanced Technology Program, SAIC-Frederick Inc., NCI-Frederick, Frederick, MD, USA
Received 15 March 2010, accepted 27 May 2010, DOI: 10.1111/j.1751-1097.2010.00780.x
therefore contain viruses as close to their native configuration
as possible, while still being noninfectious. These inactivated
ABSTRACT
Hydrophobic UV-activatable compounds have been shown to
partition into the hydrophobic region of biological membranes to
selectively label transmembrane proteins, and to inactivate
enveloped viruses. Here, we analyze various UV-activatable
azido- and iodo-based hydrophobic compounds for their ability to
inactivate a model-enveloped virus, human immunodeficiency
virus (HIV-1 MN). Treatment of HIV-1 with 1,5-diazidonap-
thalene (DAN), 1-iodo, 5-azidonaphthalene (INA), 1-azidonaph-
thalene (AzNAP) or 4,4¢-diazidobiphenyl (DABIPH) followed
by UVA irradiation for 2 min resulted in complete viral
inactivation, whereas treatment using analogous non–azido-
containing controls had no effect. Incorporation of an azido
moiety within these hydrophobic compounds to promote photo-
induced covalent reactions with proteins was found to be the
primary mechanism of viral inactivation for this class of
compounds. Prolonged UVA irradiation of the virus in the
presence of these azido compounds resulted in further modifica-
tions of viral proteins, due to the generation of reactive oxygen
species, leading to aggregation as visualized via Western blot
analysis, providing additional viral modifications that may
inhibit viral infectivity. Furthermore, inactivation using these
compounds resulted in the preservation of surface antigenic
structures (recognized by neutralizing antibodies b12, 2g12 and
4e10), which is favorable for the creation of vaccines from these
inactivated virus preparations.
virus vaccines can, therefore, potentially elicit an immune
response comparable with that of the live virus. With the
appearance of new pandemic viruses, such as SARS and H1N1
influenza, the need for rapid, efficient and safe methods of
inactivation for the preparation of vaccines became essential.
The ideal inactivated virus vaccine should be free of residual
infectious material, while still maintaining the necessary anti-
gens and epitopes from the virion structure to produce an
effective immune response. The ideal method for this inactiva-
tion should not only be rapid, efficient and reproducible, but
should also be broadly applicable to a wide variety of viruses.
The most common approach for the preparation of inac-
tivated virus vaccines is to use chemical inactivation methods,
such as formaldehyde (formalin), glutaraldehyde and beta-
propiolactone treatment. Some of these methods were shown
to damage immunogenic epitopes, which could adversely effect
the efficacy of vaccines prepared using these methods (1–7).
There is also concern over the toxicity of residual chemical
inactivators, such as glutaraldehyde, formaldehyde and beta-
propiolactone because these are reactive until either allowed
enough time to fully react, removed from the preparation or
diluted to permissible levels. Photoactivatable compounds,
used for viral inactivation, have an advantage from this
perspective since their chemical reactivity can be controlled by
light. Psoralens, a group of UV-activatable compounds that
selectively bind and crosslink DNA were used for inactivation
of viruses with preservation of viral surface epitopes (8).
However, there were some concerns that repair and recombi-
nation of DNA could lead to the resurrection of infectious
virus (multiplicity reactivation) (9).
INTRODUCTION
There are a variety of strategies available for the preparation of
vaccines against a large number of viruses, such as virus-like
particles, live-attenuated virus, subunit, inactivated virus and
split virus vaccines. In particular, inactivated viruses have been
used successfully and are currently licensed in the USA in
vaccines against influenza, hepatitis A and poliovirus. Inacti-
vated virus vaccines are derived from infectious materials and
Hydrophobic membrane probes containing a UV-activat-
able labeling group (such as an aryl azide or aryl diazirine)
partition into the hydrophobic regions of biological membranes
and have been used to selectively label the hydrophobic
domains of transmembrane proteins (10,11). The UV-activat-
able groups produce either a nitrene or a carbene (respectively)
upon irradiation with UV light. When these compounds are
based on azidonaphthalene, they can be photoactivated to
generate the nitrene at wavelengths above 300 nm thus pre-
venting UV–irradiation-induced protein or nucleic acid damage
*Corresponding author email: blumenthalr@mail.nih.gov (Robert Blumenthal)
†Current address: Laboratory of Cell Biology, NCI Bethesda, Bethesda, MD,
USA.
ꢀ 2010 U.S. Government. Journal Compilation. The American Society of Photobiology 0031-8655/10
1099

1100 Julie M. Belanger et al.
1,5-Diazidonaphthalene (DAN) was synthesized according to the
general procedure above. The crude solid was dissolved in methylene
chloride, decolored by the addition of activated carbon and recrystal-
lized from methylene chloride, resulting in peach-colored needles. 1H
NMR (CDCl3, 300 MHz): 7.27–7.32 ppm (d, 1H), 7.45–7.52 ppm (t,
1H), 7.88–7.92 ppm (d, 1H).
4,4¢-Diazidobiphenyl (DABIPH) was synthesized according to the
general procedure above, with the exception that the reaction solution
was allowed to warm to room temperature overnight after the addition
of sodium azide. The product was extracted using ethyl acetate, dried
and purified by column chromatography using hexanes ⁄ dichlorome-
thane. Yellow solid. 1H NMR (CDCl3, 300 MHz): 7.07–7.12 ppm (m,
4H), 7.52–7.57 ppm (m, 4H).
(12). For example, 1-Iodo, 5-azidonaphthalene (INA) (12)
proved particularly useful for the study of membrane structure
and dynamics of enveloped viruses (13–15). We have recently
shown that INA effectively inactivated enveloped viruses when
photoactivated by UV light. We have demonstrated for a
variety of enveloped viruses that, by this approach, the
inactivation is complete with preservation of viral antigenicity
(16–19). This breadth of inactivation for a wide variety of
enveloped viruses makes this class of photoactivatable hydro-
phobic alkylating compounds ideal candidates for use in
chemical inactivation for whole virus vaccine preparations.
Herein, a study of various hydrophobic compounds with
azido functionality was carried out and analyzed for their
ability to inactivate HIV-1 (as a model enveloped virus) while
maintaining the integrity of hydrophilic surface antigens. The
mechanism of inactivation was studied, with particular atten-
tion to the formation of reactive oxygen species (ROS) with
prolonged UV irradiation times.
1,5-Diiodonaphthalene (DINAP) was synthesized according to
published procedures (23), extracted using dichloromethane, dried and
purified by column chromatography with hexanes ⁄ dichloromethane.
1
Off-white solid. H NMR (CDCl3, 300 MHz): 7.23–7.29 ppm (t, 2H),
8.11–8.17 ppm (m, 4H).
UV spectroscopy. Analysis of the UV absorbance spectra for the
compounds studied was performed using a SpectraMax M2 spectrom-
eter (Molecular Devices), with DMSO as a reference ⁄ blank.
Treatment of virus with hydrophobic naphthalene compounds. The
indicated HIV-1 MN isolate was diluted with phosphate-buffered
saline (PBS) to 0.5 mg mL)1 total protein. Stock solutions, 8 mM in
DMSO, of each naphthalene or biphenyl compound were added to the
diluted HIV-1 isolate with vortexing in a biosafety hood, for a final
concentration of 100 lM (unless otherwise noted). For samples that
contained glutathione (a subset of the UV = 15 min treated samples,
as noted), a stock solution of glutathione (1.0 M in PBS, pH 7.5) was
prepared and added to the viral suspension for a final glutathione
concentration of 20 mM. The viral suspension was incubated at room
temperature for 5 min then exposed to UV light for 2 or 15 min, as
specified. The light source was a 100-W ozone-free mercury arc lamp
placed in a lamp-house with collector lens (Olympus). Samples were
irradiated at a distance of 5 cm from the light source where the dose
was 10 mW cm)2, consistent with our previously published procedure
(18). A water filter (a polystyrene culture flask filled with water) was
placed between the sample and the lamp to prevent sample heating.
Sample volumes used for irradiation varied from 0.10 mL to 1.0 mL in
clear polypropylene microfuge tubes. The UV emission from the light
source was measured using a Stellarnet Spectroradiometer and was
found to have the mercury emission lines where k > 335 nm (data not
shown). The mercury emission lines below this wavelength were absent
due to the absorption by the lamp housing. Additionally, there was
negligible absorption by the water filter and polypropylene microfuge
tubes at these wavelengths.
Western blot analysis for protein aggregation ⁄ crosslinking. HIV-1
samples treated with the azido compounds plus UV irradiation, and
controls were lysed in SDS sample buffer for 15–20 min at 55–60ꢁC.
Aliquots of these were added to a 4–20% Tris-glycine gel. Gel
electrophoresis was done under reducing conditions. After electropho-
resis, the proteins were transferred to a nitrocellulose membrane. The
proteins were detected using various monoclonal antibodies to HIV-1
MN, anti-gp41 and anti-gp120. Secondary antibodies containing
AlexaFluor680 were used for readout on an Odyssey Infrared Imaging
System (LiCOR Biosciences). Blots were not reprobed; a new blot was
done for each antibody probe used.
MATERIALS AND METHODS
Safety. All handling of infectious HIV-1 isolates were done under
Biosafety Level 2 conditions, following Biosafety level 3 procedures,
with the proper personal protective equipment. Synthesis and handling
of the azido compounds was performed using the proper precautions
due to the potentially explosive nature of these compounds (including
handling in a chemical fume hood, no metal utensils, minimal use of
chlorinated solvents, small scale <1 g).
Reagents and cells. The chemical precursors to the azido compounds,
1-aminonaphthalene (98%; Aldrich), 1,5-diaminonaphthalene (97%;
Acros), 4,4¢-diaminoazobenzene (95%; Acros) and 4,4¢-diaminobiphe-
nyl (benzidine, 95%; Sigma), were purchased from Sigma-Aldrich.
1-Iodonaphthalene (INAP, 97%; Aldrich), dimethyl sulfoxide (DMSO)
(‡99.7%, Hybri-MaxTM; Sigma), sodium azide (SigmaUltra) and
sodium nitrite (ACS grade; Fisher) were used without further purifica-
tion. INA was custom-synthesized by Biotium (Hayward, CA). Glutar-
aldehyde (Tousimis, Rockville, MD), Sodium cacodylate, Osmium and
Embed-812 epoxy resin (Electron Microscope Sciences, Fort Washing-
ton, PA) were used in the transmission electron microscopy (TEM)
study. HIV-1 MN cl. 4 concentrated virus stock (lots #P3592 and
#P3602) were prepared by the AIDS Vaccine Program, SAIC.(20) The
following reagents were obtained through the AIDS Research and
Reference Reagent Program, Division of AIDS, NIAID, NIH: TZM-bl
indicator cell line from J.C. Kappes; anti–HIV-1 broadly neutralizing
antibodies 2G12 and 4E10 were from Herman Katinger; B12 antibody
was obtained from Dennis Burton and Carlos Barbas; anti-gp41
monoclonal antibody was purified from the hybridoma Chessie 8 from
Dr. George Lewis; anti-gp120 (ID6) monoclonal antibody used for
Western blots was obtained from Dr. Kenneth Ugen and Dr. David
Weiner; anti-p24 (183-H12-5C) monoclonal antibody from Dr. Bruce
Chesebro and Kathy Wehrly. Secondary antibody used for Western was
Alexafluor680 goat antimouse IgG (Invitrogen). Catalase and glucose
oxidase were purchased from Sigma-Aldrich.
Virus infectivity assay by luciferase reporter. The infectivity of HIV-1
MN samples were determined using the TZM-bl cell line as previously
reported (24). Cells were added (2 · 104 cells well)1) to a 96 well clear-
bottomed microtiter plate with 100 lL of complete Dulbecco’s modified
eagle medium (DMEM) per well. Plates were incubated to allow the cells
to adhere for 18–24 h at 37ꢁC. The media was aspirated from each well
and was replaced with 100 lL of fresh DMEM containing 40 lg mL)1
diethylaminoethyl-dextran (DEAE-dextran) per well. To each well was
added either the crosslinked or untreated HIV samples in serial
dilutions, diluted with DMEM ⁄ dextran. The plate was again incubated
at 37ꢁC for 18–24 h. Luciferase activity was analyzed using the Steady-
Glo Luciferase System (Promega). Using infectious controls of the same
viral lot in serial dilution, the sensitivity of this assay was determined to
be at least 2 logs of inactivation.
Synthesis of compounds. General procedure: The synthesis of the
diazido compounds from their diamine precursors was adapted from
previously published procedures (21,22). In brief, aqueous sodium
nitrite (5 mmol) was added to a cooled solution of the amino precursor
(2.4 mmol, for a 1.1:1 ratio of nitrite to amino group) in dilute
hydrochloric acid to create the diazonium salt. After stirring for 2 h, an
aqueous solution of sodium azide (5 mmol) was added dropwise, while
cooling in an ice bath, to the diazonium salt. The reaction was stirred for
another 15 min and the precipitate was recovered by filtration.
1-Azidonaphthalene (AzNAP) was synthesized according to the
general procedure above, with the exception of using half the amount
of sodium nitrate and sodium azide, and reaction was allowed to warm
to room temperature overnight after the addition of sodium azide.
Extracted with ethyl acetate, dried and purified by column chroma-
tography in hexanes. Yellow oil. 1H NMR (CDCl3, 300 MHz):
7.21–7.26 ppm (m, 1H), 7.40–7.53 ppm (m, 3H), 7.58–7.63 ppm (d,
1H), 7.76–7.83 ppm (m, 1H), 8.04–8.12 ppm (m, 1H).
HIV virus pellet preparation for electron microscope (EM)
analysis. HIV virus pellet preparation for the EM analysis was
described before (25). Briefly, HIV virus pellet was made by an

Photochemistry and Photobiology, 2010, 86 1101
ultracentrifuge at 100 000 g for 60 min with 2% glutaraldehyde (in
0.1 M cacodylate buffer, pH 7.2) solution. The pellet was post fixed in
1% osmium tetroxide in the same buffer for 1 h. The pellet was
dehydrated in ethanol (e.g. 35%, 50%, 75%, 95%, 100%) and 100%
propylene oxide. The pellet was infiltrated overnight in 1:1 mixture of
propylene oxide and epoxy resin, and embedded in a pure resin and
cured for 48 h in 55ꢁC. The 70 nm thin sections were made and
mounted on naked copper grids. The thin sections were double stained
(uranyl acetate and lead citrate), examined in a Hitachi (Tokyo, Japan)
H-7600 transmission electron microscope, and the images were taken
using an AMT (Danvers, MA) digital camera.
Virus Capture Assay of treated virus. Neutralizing antibodies
against gp120 (b12 and 2g12) or gp41(4e10) were plated on Immulon
4HBX 96-well plates (Thermo Scientific) according to previously
published procedures (26). In brief, 100 lLof 10 lg mL)1 (b12 or
2g12) or 20 lg mL)1 (4e10) of each antibody in carbonate buffer was
added per well. Wells to assess background binding were also prepared
using only carbonate buffer with no antibody added. After incubation
at 4ꢁC overnight, the wells were washed with PBS and blocked with
3% BSA for 1 h. Wells were again washed with RPMI. Treated virus
samples were prepared (0.5 mg mL)1 total protein in PBS with 100 lM
of each hydrophobic compound, plus UV irradiation for 2 min) and
BSA was added to these samples for a final concentration of 1%.
Samples were diluted by an equal volume of RPMI containing 1%
BSA, and 100 lL of each was added to the specified wells. Incubation
was carried out at 37ꢁC for 1 h, after which wells were washed with
RPMI. To each well, 50 lL of Laemmli SDS reducing sample buffer
(1·) were added. Plate was sealed and incubated at 37ꢁC for 10 min.
Sample buffer containing the lysed sample was transferred to 200 lL
PCR tubes with lids for storage at )20ꢁC until analysis via SDS–
PAGE ⁄ Western blot.
Western blot analysis of virus captured on the virus capture
assay. Samples that were isolated via the virus capture assay were
stored at )20ꢁC in SDS Laemmli reducing sample buffer, and heated
at 60ꢁC for 15 min just prior to loading on the gel. Twenty microliters
of each sample was added to each lane of a Novex 4–20% Tris-glycine
15-well gel (Invitrogen). Proteins were transferred to nitrocellulose and
probed using MAb for p24. Secondary antibody, goat antimouse
Alexafluor680 was used and the resulting blot was imaged using an
Odyssey IR imaging system. Individual bands were integrated using
the Odyssey software to determine the overall raw integrated intensity.
Background samples were run as separate lanes on the same gel that
contained mock samples (that used no antibody in the virus capture
assay) to account for any signal due to nonspecific binding of the virus
to the well plate. Also run on the gel, in the remaining lanes, were
control samples of HIV-1 and INA-treated (UV = 2 min) HIV-1,
diluted from the treated suspensions and not subjected to the virus
capture assay. These were run at the same concentration on each gel to
normalize between gels. For the samples that were treated at prolonged
UV irradiation times (15 min), the entire lane of the gel was integrated
to account for the proteins that were aggregated, but were still
recognized by the antibody.
Oxygen depletion studies during viral inactivation. Samples of virus
were prepared that contained glucose oxidase, catalase and glucose to
remove oxygen from the system (27) prior to prolonged UV irradi-
ation. To HIV-1 in PBS (0.5 mg mL)1 total protein) was added: the
azido compound studied (from 8 mM stock in DMSO), glucose (1.0 M
stock in PBS), catalase (4 mg mL)1 in PBS) and glucose oxidase
(4 mg mL)1 in PBS) for final concentrations of 0.45 mg mL)1 total
viral protein, 100 lM azido compound, 50 mM glucose and
0.2 mg mL)1 of each enzyme in a final volume of 200 lL. Additional
mock-treated samples were prepared where the enzyme solutions were
replaced by PBS. Each mixture was allowed to sit at room temperature
for 1 h to facilitate the removal of oxygen from the solution. After 1 h,
each microfuge tube was carefully irradiated with UVA for 15 min.
Aliquots were either frozen at )80ꢁC for infectivity assays or placed
into sample buffer for Western analysis of viral proteins for the extent
of aggregation.
structure and function of an enveloped virus, HIV-1, following
photoactivation. Four of these compounds contained a pho-
toactivatable azido group, whereas the remainder (INAP and
DINAP) were used as controls. The compounds had absorp-
tion maxima ranging between ꢀ290 nm and 320 nm with
similar molar extinction coefficients (Fig. 1). The mercury light
source was found to emit at wavelengths above 335 nm,
therefore the activation of these naphthalenic compounds did
not occur at their kmax, but rather within the higher wavelength
tail in their absorption spectra. Three different parameters
were used to evaluate the effect of these compounds on HIV-1:
(1) electrophoretic mobility and state of aggregation of viral
proteins as revealed by SDS–PAGE and by Western blot
analysis of specific viral proteins of interest; (2) the infectivity
of the virus as measured by a luciferase reporter gene assay; (3)
the integrity of the immunogenic epitopes on the surface of the
virus as measured by a virus capture assay.
I
N3
N3
N3
N3
N3
AZNAP
INA
DAN
I
I
N3
I
DABIPH
INAP
Scheme 1. Various hydrophobic aromatic compounds studied. Azido-
DINAP
naphthalene (AzNAP), 1-iodo, 5-azidonaphthalene (INA), 1,5-diazi-
(DABIPH),
donaphthalene
(DAN),
4,4¢-diazidobiphenyl
1,5-diiodonaphthalene (DINAP), 1-iodonaphthalene (INAP). The
UV-activatable compounds contain azides, whereas DINAP and
INAP are the control compounds.
0.6
0.5
DINAP
AZNAP
0.4
0.3
0.2
0.1
0
DABIPH
INAP
DAN
INA
290
300
310
320
330
340
350
360
Wavelength (nm)
Abbreviation
AzNAP
INA
Wavelength (λmax), nm
300
315
320
300
305
290
DAN
DABIPH
DINAP
INAP
RESULTS
Several compounds, mostly naphthalene derivatives (see
Scheme 1), were analyzed in this study for their effect on the
Figure 1. UV absorbance of the various synthesized compounds from
Scheme 1.

1102 Julie M. Belanger et al.
Figure 3. Western blot analysis showing increased effects of com-
pounds on proteins within the virus with increased UV irradiation
(15 min). Blots were probed using (a) anti-gp41, (b) anti-p24, (c) anti-
gp120. Lane A is the HIV control (no UV), the remaining lanes are
HIV + compound listed + UV irradiation for 15 min. Lane B:
DMSO; lane C: INAP; lane D: DINAP; lane E: AzNAP; lane
F:DAN; lane G: INA; lane H: DABIPH.
Figure 2. Western blot analysis showing minimal effect of the com-
pounds on the electrophoretic migration of HIV-1 viral proteins after
treatment with compounds plus UV for 2 min. Blots were probed
using (a) anti-gp41, (b) anti-p24, (c) anti-gp120. Lane A is the HIV
control (no UV), the remaining lanes are HIV + compound
listed + UV irradiation for 2 min. Lane B: DMSO; lane C: INAP;
lane D: DINAP; lane E: AzNAP; lane F:DAN; lane G: INA; lane H:
DABIPH.
solution in DMSO, followed by irradiation with UV light
(k > 335 nm) for 2 min. Following this treatment, specific
viral proteins of interest were examined by electrophoretic
mobility using SDS–PAGE followed by Western blot analysis
(see Fig. 2). The envelope glycoprotein of the HIV-1 that
mediates fusion between viral and cell membranes consists of
two noncovalently associated subunits, gp120 and gp41. The
outer surface subunit, gp120, contains sites necessary for
specific binding to target cells whereas the transmembrane
gp41 is responsible for the fusion. Since we are interested in the
effects of our treatments on the structure and function of
Photoinduced aggregation of HIV-1 proteins by azide-containing
compounds
Each of the compounds was added to a purified HIV-1
suspension for a final concentration of 100 lM from a stock

Photochemistry and Photobiology, 2010, 86 1103
surface-exposed proteins, we examined those subunits. In
addition, we examined effects on the capsid protein, p24. We
found that the compounds had only a minimal effect on the
migration or aggregation of the probed proteins when 2 min of
UV irradiation was applied. Compounds that were expected to
behave as crosslinkers, DAN and DABIPH, also did not show
a pronounced increase in aggregation or oligomerization under
these conditions. However, when the HIV-1 samples were
irradiated for longer periods of time (15 min), the proteins that
were treated by the azido-functionalized naphthalene deriva-
tives exhibited a higher extent of molecular aggregation as
visualized by Western blot analysis for the specific proteins of
interest (Fig. 3). The appearance of high molecular weight
oligomers was evident for HIV-1 samples treated with the
azido-functionalized naphthalenes and diazidobiphenyl
(AzNAP, INA, DAN, DABIPH), but was not evident in
the controls (DINAP, INAP). The higher molecular weight
aggregates were more pronounced in the transmembrane
(gp41) and capsid (p24) proteins, but not within the external
surface proteins (gp120). Transmission electron microscopy
images of the treated HIV-1 virions and the untreated controls
show that the viral structure was preserved in all viruses
regardless of irradiation time (Fig. 4). The untreated HIV-1
shows intact virions with dark cores (capsid), as well as
microvesicles (28), and appears indistinguishable from the
HIV-1 preparation that was treated with an azido compound
followed by UV irradiation for 15 min (DAN was chosen as a
representative sample).
Treatment of HIV-1 with the azido-containing hydrophobic
compounds plus UV irradiation results in loss of infectivity
of the virus
When HIV-1 was treated with 100 lM of the azido-based
compounds, the virus was inactivated after 2 min of UV
irradiation when analyzed using a 24 h cell-based luciferase
reporter assay (see Fig. 5). Similar results were obtained for
concentrations down to 12.5 lM for the azido-containing
compounds when tested in the same assay (data not shown).
Treatment of HIV-1 with 100 lM of each of the control
compounds, without azido functionality, showed no change in
the infectivity of the virus when compared with the UV
irradiated control, with the exception of the DINAP. Although
DINAP shows a partial inactivation, the residual infectivity is
still significantly above the background limits.
Pretreatment of HIV-1 with glutathione or enzymatic depletion
of oxygen eliminates photoinduced aggregation while
maintaining viral inactivation
To elucidate the mechanism for the aggregation, samples were
tested with and without the addition of glutathione. Glutathi-
one is typically used as a radical scavenger and reacts with any
hydrophilic reactive radical intermediates that may be
1000000
100000
10000
1000
Figure 5. Hydrophobic azido-containing compounds and analogs
induce viral inactivation when added to HIV-1 followed by UV
irradiation for 2 min. The concentration of HIV-1 used in this assay
was 3.1 · 103 ng mL)1 of total protein added per well. Readout using
a TZM luciferase reporter assay. Data are the average of two separate
experiments and are normalized to 100 000 luminescence units. The
error bars represent the difference in the values obtained from two
independent experiments.
Figure 4. Treatment of HIV virus with hydrophobic compounds does
not disrupt viral integrity as assessed by TEM. (a) HIV-1 MN, (b)
HIV-1 MN treated with 100 lM DAN + UV irradiation for 15 min.
Vsc, microvesicles; V, HIV viron.

1104 Julie M. Belanger et al.
Figure 6. Aggregation of viral proteins is induced by reactive oxygen species. (a) The addition of glutathione (20 mM) prior to UV irradiation
significantly reduced the higher molecular weight aggregates, as can be seen in Western blots probed for p24 and gp41. (b) Oxygen depletion prior
to UV irradiation for 15 min in the presence of the azido compounds, significantly reduced the aggregation at higher molecular weights, while
maintaining viral inactivation. Lane A is the HIV control (no UV), the remaining lanes are HIV + compound listed + UV irradiation for 15 min.
Lane B: DMSO; lane E: AzNAP; lane F: DAN; lane G: INA; lane H: DABIPH.
produced as a result of the UV irradiation. When glutathione
was added (20 mM) prior to irradiation with UV light, the
previous aggregation that was seen significantly reduced (see
Figs. 3 and 6a). Additionally, enzymatic depletion of oxygen
was studied, using a catalase ⁄ glucose ⁄ glucose oxidase system
for the removal of oxygen prior to UV irradiation of virus in
the presence of the azido compounds. When oxygen was
removed from the system, the photoinduced aggregation of
viral proteins was significantly reduced (for both p24 and gp41;
see Fig. 6b). Although the aggregation of the proteins was
reduced under these conditions, the viral preparations were
still inactivated, and the controls were still infectious, as seen
using a 24-h cell-based luciferase reporter assay (data not
shown).
neutralizing antibody is one that when added to the virus,
causes the virus to be noninfectious by binding to specific
epitopes on the viral surface. Three known neutralizing
antibodies 4E10 (anti-gp41), 2g12 (anti-gp120) and b12 (anti-
gp120) were studied for their ability to recognize and capture
the treated HIV-1. A virus capture assay, using these antibod-
ies to capture the whole inactivated virus, was performed in a
96 well format. The readout for the assay was done using
quantitative Western analysis for p24 (capsid protein) with
Alexafluor IR dyes. Detection and quantitation of the p24
bands provided an indication of how much HIV was recog-
nized by the neutralizing antibodies. The resulting bands were
integrated and compared with mock samples run at the same
time (same protocol without capturing antibodies) to assess
any nonspecific background binding of HIV to the plate (see
Fig. 7a). Control lanes comprising of lysed HIV-1 were also
run for normalization between gels for the various antibodies
studied. A working range of the assay was established (see
Fig. 7b) in which the capture of the HIV-1 did not saturate the
plate and resulted in a linear response with decreasing HIV-1
added for binding. For the three antibodies tested (b12, 2g12
and 4E10), the various treatments do not have a significant
Structure of surface viral immunogens are preserved after
treatment with the azide-containing compounds
After treatment of HIV-1 with the azido-containing hydro-
phobic compounds and UV irradiation for 2 min, the resulting
noninfectious virus preparation was studied for the preserva-
tion of known neutralizing epitopes on the viral surface. A

Photochemistry and Photobiology, 2010, 86 1105
Figure 7. Virus capture assay (VCA) using neutralization epitopes indicates preservation of surface epitopes on HIV-1 MN after treatment with
compounds plus UV for 2 min. (a) Representative blot showing p24 amounts after capture with b12 (2g12 and 4e10 not shown), where labe A:
DAN, lane B: INA, lane C: AzNAP, lane D: DABIPH, lane E: UV, lane F: control captured by b12, and A¢–F¢ are mock samples added to the
virus capture assay without b12, lanes G and H are controls not subjected to the virus capture assay run on the gel to normalize between gels; lane
G: INA control, lane H: HIV control. (b) Capture of untreated virus with varying concentrations showing functional range of assay (data not
normalized between data sets); (c) 4e10 capture of 0.25 mg mL)1 virus after UV for 2 min; (d) b12 capture of 0.25 mg mL)1 virus after UV for
2 min; (e) 2g12 capture of 0.25 mg mL)1 virus after UV for 2 min, (f) b12 capture of 0.25 mg mL)1 virus after UV for 15 min; (g) 2g12 capture of
0.25 mg mL)1 virus after UV for 15 min. Readout using quantitative Western analysis for p24, where raw integrated intensity is the integration of
the main p24 band (for UV 2 min) or integration of entire lane (for UV 15 min, to include protein aggregates that contain the desired protein in the
integration). Error bars for (c)–(g) are the difference in the values obtained between two independent experiments which were normalized to an HIV
control lane, containing HIV not subjected to the virus capture assay, run on each gel.
effect on the recognition of these viruses by these antibodies
(see Fig. 7c–e), and the variations in intensity shown are all
within the deviation between the separate experiments (as
shown by the error bars). There is minimal apparent damage
to the epitope for b12 when the virus is treated with INA plus
UV irradiation for 2 min, although the majority of the b12
binding sites appear unaffected (Fig. 7d) when the overall
intensity is compared with that of the control versus the
background. Similar results were obtained upon treatment
with UV for 15 min, via integration of the main band plus
aggregated area, indicating the majority of the epitopes 2g12
and b12 were maintained (Fig. 7f–g). The one exception was
INA-treated HIV-1 with UV for 15 min, which lost b12
recognition. This, however, was regained after the addition of
glutathione, indicating the loss of b12 recognition was due to
secondary side reactions and not the reaction of the INA itself
(data not shown).
DISCUSSION
We have recently described the use of the hydrophobic
photoactivatable compound INA as a general approach for
the inactivation of enveloped viruses for vaccine application
(18,19). In this work, we were interested in systematically

1106 Julie M. Belanger et al.
evaluating the structural features of hydrophobic aromatic
derivatives, based off naphthyl or biphenyl that could make
them efficient agents for the preparation of whole virus
vaccines. The various azido-based compounds studied herein
(see Scheme 1) all showed the ability to inactivate a model
enveloped virus, HIV-1, when used in conjunction with UVA
irradiation at short irradiation times (2 min). Similar non–
azido-based controls that absorb at similar wavelengths
(Fig. 1) did not show this ability indicating that the inactiva-
tion of the HIV-1 was due to the covalent binding of the azido
moiety upon UV irradiation. Previous work using radiolabeled
INA had shown that the INA forms a covalent bond with
HIV-1 proteins through the reaction of the nitrene formed
through photoactivation.(15,18) We surmised that viral inac-
tivation is due to this covalent reaction. Indeed, several studies
have shown that the transmembrane segment of fusion
proteins was involved in the fusion process. Mutational studies
of the membrane spanning domain of HIV have indicated that
its amino acid composition influenced the ability of gp41 to
undergo proper fusion (29). In the case of influenza virus, the
replacement of the transmembrane segment of HA2 by a gpi
anchor yielded a protein that was able to induce hemifusion
but no longer full fusion of the bilayers (30). Due to their
hydrophobicity, the azido compounds we use will primarily
target the transmembrane portion of fusion proteins thereby
presumably preventing them from performing their critical
role in the overall fusion process.
Analysis of various HIV-1 proteins via Western blot
indicated that these compounds, along with their nonazido
analogs, did not effect the electrophoretic migration of these
proteins after treatment with UV for 2 min, which is consistent
for modifications of proteins with small molecules. However,
for the diazido compounds (DAN and DABIPH), it may be
expected that covalent crosslinking could occur if both azides
were activated. For short UV irradiation times, this was not
evident for these compounds, as can be seen in Fig. 2, where
there was no significant aggregation at higher molecular
weights. This is most likely due to the short length of these
molecules and the potential side reactions of azides (31,32).
Once covalent binding of one azide has occurred, it is likely
that the second azide would undergo another reaction that
does not result in a crosslink, such as proton abstraction or
ring expansion, should another protein not be in close-enough
proximity for a crosslinking reaction to occur. Clustering of
proteins by photoactivation of DAN and DABIPH had been
previously shown in erythrocyte membranes although in that
study it could not be conclusively established whether it was a
result of aggregation or crosslinking (33).
glycoprotein, (gp120) indicating a preference for the aggrega-
tion on internal and hydrophobic proteins (see Fig. 3a,b). This
mechanism appears to be due to secondary reactions that are
promoted by the azide-containing compounds, since the UV
control and non–azido-containing compounds do not indicate
such an increase in aggregation. These secondary reactions
may involve the formation of chromophores created by
prolonged irradiation of naphthyl-azides, such as from olig-
omerization of the reactive naphthyl species, or from coupling
to form diazo compounds. The products of these secondary
reactions or the covalently bound compounds themselves can
then act as photosensitizers to produce ROS. ROS have been
previously shown to promote aggregation of proteins due to
the formation of disulfide links from cysteine oxidation as well
as other covalent modifications by oxygen radicals (34).
Support for this hypothesis comes from the finding that the
addition of glutathione or the enzymatic depletion of oxygen
completely blocked the formation of protein aggregates at
prolonged irradiation times (Fig. 6). The addition of glutathi-
one results in the scavenging of radicals as they are formed
therefore preventing the viral protein aggregation seen for long
UV irradiation times. The glutathione does not inhibit the
covalent binding of the azido compounds and therefore, the
virus is still inactivated even in the presence of glutathione (see
Fig. 6a). Additionally, the removal of oxygen from the system
removed the source of the ROS thereby preventing aggregate
formation, but still resulted in viral inactivation when the
compounds were used with UV light (see Fig. 6b). The
observation that the DINAP derivative can also induce protein
aggregates, although to a lesser extent than the azido deriv-
atives, is also consistent with the generation of ROS
(Fig. 3a,b). The two iodines in this molecule can induce, upon
irradiation, a high yield of triplet exited states with lower
excitation energy more suitable for triplet sensitization of
molecular oxygen. Previous studies with INA used for hydro-
phobic protein labeling and viral inactivation were not done
for such long irradiation times, therefore this phenomenon was
not observed. These aggregates are seen for internal viral
proteins such as capsid (p24) and matrix protein (p17, data not
shown), as well as the hydrophobic transmembrane protein
(gp41). Aggregates are not seen for the hydrophilic external
glycoprotein (gp120) which is heavily glycosylated as the
sugars that hinder the amino acid side chains do not readily
react with ROS. Remarkably, this longer UV irradiation time
(15 min) does not effect the overall structure of the virus, as
can be seen by TEM, where the virions are still intact and are
indistinguishable from the untreated control (see Fig. 4).
Further studies will be necessary to evaluate the inactivated
virus after prolonged irradiation as an effective vaccine.
Although treatment of HIV-1 with the azido-based com-
pounds at low irradiation times (UV for 2 min) did not affect
the mobility of HIV-1 proteins in SDS–PAGE, it did result in
the complete loss of infectivity of the virus when monitored by
a cell-based luciferase reporter assay (see Fig. 5). The non–
azido-containing controls did not have an effect on the
infectivity of the virus, with the exception of DINAP that
had a small effect, indicating that the azide is needed for this
efficient and rapid inactivation. For the samples that were
treated with UV for 15 min, it is important to note that the
higher molecular weight aggregates are not needed for viral
inactivation, since inactivation occurs after 2 min of irradia-
In an effort to assess the effect of prolonged UV irradiation
on this system, and to see if longer UV irradiation times would
promote crosslinking of proteins using the diazido com-
pounds, the UV irradiation times were increased to 15 min.
At this longer irradiation time, there was an increase in the
amount of higher molecular weight aggregates observed by
Western blot (see Fig. 3). However, this aggregation was seen
for all the azido-containing compounds with prolonged UV
irradiation time, including those that would not be expected to
form crosslinks, specifically, those with only one azido
functionality. These higher molecular weight aggregates are
only seen for the transmembrane (gp41) and capsid (p24)
proteins in HIV-1 and not for the more hydrophilic external

Photochemistry and Photobiology, 2010, 86 1107
tion. Also, when the samples are treated with glutathione or
enzymatically depleted of oxygen prior to UV irradiation for
15 min, the aggregation is eliminated, but the viral inactivation
remains (Fig. 6, inactivation data not shown). This supports
the hypothesis that the mechanism for viral inactivation is due
to the covalent binding of the azido molecules to the viral
proteins and not the protein aggregation from the formation of
ROS. It is important to note, as well, for these preparations
that the virus was shown to be inactivated by 2 logs using a
24 h cell-based luciferase reporter assay, which is less than the
10 logs of inactivation that has been proposed as acceptable
for use as a killed virus vaccine (35). Our previous studies using
solely 1-iodo, 5-azidonaphthalene have shown 6 logs of
inactivation in a more involved 24 day assay (18). For the
studies within this manuscript, the cell-based assay was
sufficient to compare and contrast the inactivation of the
HIV under various conditions. More stringent, involved
assays, however, would need to be tested on the conditions
as outlined herein to be certain of greater than 2 logs of viral
inactivation, especially if these conditions were used to make a
killed virus vaccine.
In addition to viral inactivation, the preservation of viral
antigens and epitopes is necessary for efficient vaccine candi-
dates. After treatment of HIV-1 with the azido compounds
plus UV irradiation for 2 min, the epitopes for b12, 2g12 and
4e10 were all preserved (see Fig. 7). Similar results were
obtained with irradiation for 15 min except for the b12 epitope
on viruses treated with INA, which showed some damage
(Fig. 7f–g). The loss of b12 recognition with INA treatment
was recovered when glutathione was added to the suspension,
indicating that the epitope damage was due to secondary
reactions and not from the primary reaction of the azide with
the viral proteins (data not shown). Therefore, the treatment of
HIV-1 with the azido-based compounds, and UV irradiated
either for 2 or 15 min produced an inactivated virus that
remained intact with preservation of key immunogenic epi-
topes.
The increased aggregation of viral proteins as seen for
prolonged irradiation times, although not necessary for viral
inactivation, allows for the additional modification of proteins
within the virus similar to what is seen for treatments using
hydrophilic chemicals such as formalin. Formalin creates
covalent modifications and crosslinks proteins throughout the
virus, including the viral DNA (36). Thus, treatment with UV
for 15 min with the hydrophobic compounds allow for a
concurrent method of viral modification ⁄ inactivation that can
be applied to vaccines resulting in additional levels of vaccine
safety. However, unlike formalin treatment, when lethal
amounts of formalin are used (1,3–7), surface epitopes are
maintained, even with the ROS-induced modifications
throughout the virus. Additionally, treatment of live virus
with the UV-activatable hydrophobic compounds allows for
precise control over the chemical species that is inactivating
(i.e. the formation of nitrenes), and the time for viral
inactivation. Chemical inactivating agents such as formalde-
hyde can take hours or days, requiring extensive optimization
for inactivation conditions. For the hydrophobic compounds
herein, when the UV irradiation is stopped, any remaining
hydrophobic compound is non-toxic, unlike formalin, where
any remaining formaldehyde remains toxic and needs to be
removed prior to vaccine administration. Thus, the use of the
azido compounds, as described, provides a rapid, effective and
reproducible general approach for inactivation of enveloped
viruses with the preservation of viral epitopes for vaccine
applications.
CONCLUSION
Azido-containing hydrophobic compounds based on naphtha-
lenic or biphenyl structures can be used to rapidly and
efficiently inactivate HIV-1 when irradiated by UVA light with
short irradiation times. Longer UV irradiation times resulted
in further protein modifications, due to the generation of ROS
that could allow for concurrent inactivation methods for safer
inactivated vaccines. These further modifications via ROS may
provide an additional inactivation step for the safety of vaccine
preparations created using these compounds since the produc-
tion of the ROS modifies proteins throughout the virus and is
not limited to the viral membrane. The cell-based luciferase
reporter assay used herein has shown reproducibly 2 logs of
inactivation for the viral lot tested under these various
conditions. Further studies are needed and are ongoing on a
selected subset of these conditions using more sensitive,
involved tests to illustrate additional logs of inactivation
before this technique can be used to safely generate killed virus
vaccines. For the majority of the conditions tested, there was
no significant damage to the epitopes that are recognized by
the neutralizing antibodies 2g12 (gp120), b12 (gp120) and 4e10
(gp41) indicating that epitopes known to be targets for
neutralizing antibodies were still intact even under conditions
where ROS were created. These results indicate that all of the
azido-containing compounds studied herein, could be poten-
tially used for the generation of inactivated virus vaccines, with
minimal differences in the chemical structures (mono- or
di-azido) having insignificant effects on either changes in viral
inactivation, or epitope preservation.
Acknowledgements—The authors would like to thank Julian W. Bess,
Jr. of the AIDS and Cancer Virus Program, SAIC-Frederick, for many
helpful conversations and his critical reading of this manuscript. We
thank ARRRP for reagents, and Jeff Lifson and Julian W. Bess, Jr. for
generously providing purified virus. This research was supported (in
part) by federal funds from the Intramural AIDS Targeted Antiviral
Program and the Intramural Research Program of the NIH, National
Cancer Institute, Center for Cancer Research. This project has been
funded in whole or in part with federal funds from the National
Cancer Institute, National Institutes of Health, under contract
HHSN26120080001E. The content of this publication does not
necessarily reflect the views or policies of the Department of Health
and Human Services, nor does mention of trade names, commercial
products or organizations imply endorsement by the U.S. Govern-
ment.
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