Proc. Nati. Acad. Sci.
USA
Vol. 90, pp. 158-162, January 1993
Medical Sciences
Photodynamic inactivation of infectivity of human immunodeficiency
virus and other enveloped viruses using hypericin and rose bengal:
Inhibition of fusion and syncytia formation
(vesicular stomatitis virus/influenza virus/Sendai virus/hemolysis)
JOHN LENARD*, ARNOLD RABSONt, AND ROGER VANDEROEF*
*Department of Physiology and Biophysics, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 675 Hoes Lane,
Piscataway, NJ 08854-5635; and tDepartment of Molecular Genetics and Microbiology, Center for Advanced Biotechnology and Medicine, Piscataway,
NJ 08854
Communicated by Aaron J. Shatkin, September 29, 1992 (received for review May 15, 1992)
ABSTRACT The mechanism of the antiviral activity of
hypericin was characterized and compared with that of rose
bengal. Both compounds inactivate enveloped (but not unen-
veloped) viruses upon illumination by visible light. Human
immunodeficiency and vesicular stomatitis viruses were pho-
todynamically inactivated by both dyes at nanomolar concen-
trations. Photodynamic inactivation of fusion (hemolysis) by
vesicular stomatitis, influenza, and Sendai viruses was induced
by both dyes under similar conditions (e.g., I50 = 20-50 nM for
vesicular stomatitis virus), suggesting that loss of infectivity
resulted from inactivation of fusion. Syncytium formation,
between cells activated to express human immunodeficiency
virus gpl20 on their surfaces and CD4+ cells, was inhibited by
illumination in the presence of 1 ,uM hypericin. Hypericin and
rose bengal thus exert similar virucidal effects. Both presum-
ably act by the same mechanism-namely, the inactivation of
the viral fusion function by singlet oxygen produced upon
illumination. The implications of this photodynamic antiviral
action for the potential therapeutic usefulness of both hypericin
and rose bengal are discussed.
As the worldwide epidemic of human immunodeficiency
virus (HIV) infection and AIDS grows, the rapid develop-
ment of effective, affordable agents with anti-HIV activity is
an urgent need. To date, the most effective anti-HIV agents
have been nucleoside analogs that inhibit the HIV reverse
transcriptase (RT). Recently, much attention has been given
to classes of potential antiviral agents that target other
aspects of the viral life cycle. In this regard, hypericin has
been regarded as a promising potential anti-AIDS drug since
1988, when it was reported that a single injection of this
compound prevented splenomegaly and death in mice in-
fected with Friend leukemia virus. Hypericin was "coadmin-
istered" with the virus; 50% survival was conferred by 10 jig
of hypericin, whereas 150 Ag was needed for complete
protection (1, 2). In addition, biweekly injections of hypericin
into mice greatly reduced the viremia resulting from infection
with the slower acting LP-BM5 murine leukemia virus (2).
Others, however, could not replicate some of these results
(ref. 3, note added in proof; N. R. Stevenson and J.L.,
unpublished results).
Subsequent reports rapidly established two important
facts: (i) hypericin inactivated a wide variety of lipid-
containing (i.e., enveloped) viruses, but was ineffective
against viruses lacking membranes (3-7); and (ii) light in-
creased the virucidal potency of hypericin against several
different enveloped viruses by at least 100-fold (6, 7).
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158
Microscopic observations of hypericin's intrinsic red flu-
orescence in cultured cells showed it to be localized in plasma
membranes (2). This suggests that hypericin acts in a manner
quite different from the well-known nucleoside analogs.
Observations of hypericin's activities in vitro have led to
suggestions that it may act on viral assembly (1, 2) or by
inhibiting RT (4). Since these experiments did not control
light as a variable, their interpretation is difficult.
Hypericin has a long and well-documented history as a
photodynamic compound. It is the active principle contained
in plants of the Hypericum genus; ingestion by grazing
animals causes photopoisoning (hypericism) leading to skin
irritation, fever, and death (8, 9). Hypericin's photodynamic
activity against membranes in vitro was also noted very early,
when it was found to be a promoter of light-induced hemol-
ysis (H. F. Blum, quoted in ref. 10). In this regard it resem-
bled rose bengal, another potent inducer of photodynamic
hemolysis (11-13).
ICH
CHb
Cl HO 0 OH
Rose Bengal Hypeiln
The similarities between hypericin and rose bengal extend
to their mechanism of action. Both compounds catalyze the
light-induced formation of singlet oxygen, a highly reactive
oxidizing species that is largely or completely responsible for
their membrane-disruptive effects (14-16). In a further par-
allel, rose bengal has been shown to inactivate enveloped
viruses upon illumination. The photodynamic inactivation of
vaccinia virus by rose bengal was attributed to alterations in
viral proteins, as opposed to nucleic acids (17). Rose bengal's
virucidal activity against herpes simplex virus was noted as
a complication attending its use in a common diagnostic
procedure for ocular disease (18). However, in contrast to
hypericin, rose bengal has not previously been evaluated as
a potentially useful antiviral agent.
In view of these very similar properties of hypericin and
rose bengal, a comparison of their effects on enveloped virus
Abbreviations: HIV, human immunodeficiency virus; VSV, vesic-
ular stomatitis virus; RT, reverse transcriptase; PMA, phorbol
12-myristate 13-acetate.
 Medical Sciences: Lenard et al.
infectivity and fusion was carried out. Fusion was chosen for
study because it is the membrane-specific process that all
enveloped viruses must perform. As reported below, both
compounds appear to act by the same mechanism. Both are
potent inactivators of fusion by all the enveloped viruses
studied. Loss of fusion activity appears to account for the
inactivation of infectivity of both vesicular stomatitis virus
(VSV) and HIV, induced in the presence of light, by either of
these photodynamic agents.
MATERIALS AND METHODS
Viruses. Sendai (Z strain) and influenza (APR/8/34 strain)
viruses were grown in 10- to 11-day-old embryonated eggs and
purified on 5-40% potassium tartrate gradients. VSV Indiana
(Birmingham strain) was grown in BHK-21F cells and purified
as described (19). HIV-1 (LAV strain) was prepared by
passage in phytohemagglutinin-stimulated human peripheral
blood lymphocytes and was a kind gift of Kathleen Clouse and
the Viral Biology Unit (Georgetown University).
Reagents. Rose bengal was obtained from Sigma, and
hypericin was from Sigma or Atomergic (Farmingdale, NY).
Stock solutions of rose bengal (100 AM) were made up in
water. Stock solutions of hypericin (400 AuM) were made up
by adding 1 vol of methanol or dimethyl sulfoxide to the solid
compound, heating gently as necessary (especially with
methanol) to dissolve, and then adding 9 vol of phosphate-
buffered saline at pH 7.2 (PBS) containing 0.1% Tween 80 (5).
Samples were prepared under conditions of reduced light;
that is, lights immediately overhead were turned off, but
incident light remained sufficient to carry out the required
operations (measured illumination, <1 footcandle; 1 footcan-
dle = 10.76 lx).
Illumination. The purified or stock viruses were suspended
in translucent plastic tubes or plate wells in 100 ul of PBS
containing the desired concentrations of hypericin or rose
bengal. Illumination was carried out for 1 h in ice by using a
standard fluorescent desk lamp (containing two Philips F15
T8/CW 15-W bulbs) placed 5 cm above the sample. Illumi-
nation was 8-900 footcandles, as measured with a Spectra
Candela light meter.
Hemolysis. After illumination of the virus sample, 1.4 ml of
buffer and 0.1 ml of a 10% suspension of fresh washed human
erythrocytes in PBS were added, and the sample was mixed.
PBS was used for Sendai virus-induced hemolysis. Citric
acid/phosphate buffer at pH 5.0 (20) was used for influenza
virus- and VSV-induced hemolysis; the buffer contained
DEAE-dextran (3 ,ug/ml) for the VSV experiments (21). The
suspension was incubated in the dark for 1 h on ice, followed
by 1 h at 37°C. The samples were then centrifuged at 1200 x
g for 10 min at 4°C, and absorbance of the supernatant was
measured at 590 nm. Values corresponding to complete
(100%) hemolysis were determined in each experiment from
samples lysed in distilled water. The amount of virus used in
each experiment (1-5 ,ug of virus protein) was controlled so
as to produce 30-80% hemolysis under these conditions.
Infectivity. Stock VSV was illuminated in the presence of
hypericin or rose bengal as described above and then used to
infect BHK-21F cells at a multiplicity of infection of 5. The
infection was allowed to proceed overnight. The cell medium
was then collected, serially diluted, and titrated for plaque-
forming units on BHK-21F cell monolayers. Similarly, HIV-1
was treated with hypericin or rose bengal, illuminated, and
then added to 4 x 105 MT-4 cells (22) at a multiplicity of
infection of 0.01 in 1.5 ml of RPMI 1640 with 10% (vol/vol)
fetal calf serum (GIBCO/BRL) in 24-well plates. Culture
medium was changed on days 4 and 6, and aliquots of culture
supernatant were harvested daily for RT assay. Cell viability
was determined by trypan blue dye exclusion.
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HIV RT Assay. HIV production in infected cultures was
determined by a 32P-based RT assay previously described
Proc. Natl. Acad. Sci. USA 90 (1993) 159
(23) in which samples were incubated with RT cocktail for 1.5
h and then spotted on DEAE paper and washed as described.
RT activity was determined by quantitation of 32P bound to
the filter by using a Molecular Dynamics phosphorimager
following a 3-h exposure to a phosphorimaging screen.
HIV gpl20-Induced Syncytium Formation. A culture of
ACH-2 cells was induced to active production of HIV by
incubation with phorbol 12-myristate 13-acetate (PMA) as
described (24, 25). After 24 h of induction, 4 x 103 ACH-2
cells were suspended in 100 gl of buffer in a microtiter dish,
treated with hypericin or rose bengal with or without illumi-
nation, then mixed with 4 x 105 Sup-T1 cells (a CD41 T-cell
line that forms large syncytia upon HIV infection; ref. 26),
and incubated overnight at 370C; "dark samples" were
wrapped in foil. Syncytia were identified by microscopic
examination, and those exceeding a minimum size offour cell
diameters were counted.
SDS/PAGE. SDS/PAGE was carried out by the method of
Laemmli (27) using 10% polyacrylamide gels with 6% stack-
ing gels.
RESULTS
Inactivation of Infectivity. Stock preparations of HIV or
VSV were mixed with various concentrations of hypericin or
rose bengal and illuminated prior to infecting their respective
host cells as described. Dark samples were prepared and
incubated identically, but were shielded from the light by
wrapping in aluminum foil. Results with VSV are shown in
Fig. 1, and those with HIV are shown in Fig. 2. Both viruses
lost infectivity upon illumination with either photosensitizer,
in a concentration-dependent manner.
The concentration of hypericin or rose bengal required to
cause 50% inactivation (150) under standard illumination
conditions is shown in Table 1. Values for VSV inactivation
were 20 nM hypericin and 50 nM rose bengal. The '50 value
for loss of HIV infectivity depended upon the time of assay.
Thus, on day 3 postinfection 150 for rose bengal was =8 nM,
while on day 5 postinfection it was 50 nM; a similar increase
occurred for hypericin between day 5 and day 7 postinfec-
tion. The higher I50 on later days resulted because the lowest
concentrations delayed, but did not completely prevent, new
virus production (Fig. 2). This delay was due to a 3 log
reduction in infective titer in the viral stock, induced by the
drug treatment and illumination (data not shown). Complete
inhibition of HIV infectivity was achieved by using 30 nM
C.
0
0 30 100 300 1000 300010,000 10 30 100 300 1000
Hypericin, nM Rose bengal, nM
FIG. 1. Photodynamic inactivation of infectivity of VSV by
hypericin (A) or rose bengal (B). pfu, Plaque-forming units.
 160 Medical Sciences: Lenard et al. Proc. Natl. Acad Sci. USA 90 (1993)

a 300-
cc

4 5 6 -7 8 1

Dav at.

300 -

200- 400~

100A 2001

01 01
mock 0 10 30 100 300 1000 1090 mock 0 10 30 100 300 10001000
dark arK
Hypeicin. nM Rose bergalI,
FIG. 2. Photodynamic inactivation of HIV-1 infectivity by hypericin (A and C) or rose bengal (B and D). (A and B) RT activity present in
the culture supernatant at the indicated times was measured as described in Materials and Methods. Numbers refer to concentrations (nM) of
the appropriate drug. (C and D) RT activity values determined 5 days postinfection as a function of drug concentration. RT activity was recorded
as phosphorimager pixel units x 104. The values shown are the average of three independent wells at each point. Error bars indicate standard
deviations. HIV, untreated control.
hypericin or 100 nM rose bengal. No inactivation was seen in tration of either dye (data not shown). This indicates that
the presence of 1 AM hypericin or rose bengal in the absence fusion rather than binding was inactivated by the photody-
of light. Further, growth of MT-4 cells was not inhibited by namic action of the dyes.
the illuminated HIV stocks containing 1 ,AM hypericin or rose The susceptibility of erythrocytes to direct photodynamic
bengal, showing that inhibition of HIV replication was due to hemolysis (i.e., in the absence of virions) by hypericin and
effects on the virus, not on the cells. rose bengal were also measured. Both reagents induced
Inactivation of VSV, Influenza Virus, and Sendai Virus- direct photodynamic hemolysis (data not shown), confirming
Induced Hemolysis. In view of the broad antiviral activity previous reports (10-13). 150 values were much higher than
demonstrated by hypericin and by rose bengal against envel- those for viral inactivation of hemolysis, however (Table 1).
oped but not nonenveloped viruses, it was of interest to It should be noted that the drug concentrations shown in Fig.
determine the effects of these agents on viral fusion. This is 3 were those that were present during illumination of the
conveniently measured for many viruses by virus-induced
hemolysis (21, 28, 29), since fusion of the viral envelope with
the erythrocyte membrane is an essential prerequisite to Table 1. 15o values for photodynamic effects of rose bengal or
leakage of hemoglobin. To test for photodynamic inactivation hypericin on hemolysis, infectivity, and protein cross-linking
of viral fusion, therefore, hypericin or rose bengal was added I5o (±50%o), nM
to purified virus preparations and illuminated as described;
hemolytic activity was then assayed in the dark. As shown in Virus Function Hypericin Rose bengal
Fig. 3, the fusion activity of VSV and influenza and Sendai None Hemolysis at pH 7.2 14,000 350
viruses was inactivated by nanomolar concentrations of both Hemolysis at pH 5 6700
drugs. Inactivation was absolutely dependent upon light (Fig. VSV Inactivation of hemolysis 20 50
3) and increased with increasing time of illumination (data not Inactivation of infectivity 25 20
shown). I5o values differed for each virus/photosensitizer Cross-linking of G 1,000 500
combination, but most fell in the 20-80 nM range at the virus Cross-linking of M 5,000 1000
concentration used (Table 1). HIV-1 Inactivation of infectivity <10 (5)* 8 (3)*
It was possible that the inactivation of fusion shown in Fig. 20 (7)* 50 (5)*
3 might have resulted from an impaired ability of the virus to Inf Inactivation of hemolysis 80 50
bind to the erythrocyte cell surface. To test for this, hemag- Sendai Inactivation of hemolysis 220 35
glutination titers were determined for aliquots of influenza Inf, influenza.
and Sendai viruses that had been photodynamically treated as *The numbers in parentheses are the days at which the assay was
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in Fig. 3. Hemagglutination was unaffected by any concen- performed.

 Medical Sciences: Lenard et al. Proc. Natl. Acad. Sci. USA 90 (1993) 161
Table 2. Effect of hypericin or rose bengal and light on
syncytium formation by HIV-producing ACH-2 cells
with SUP-T1 cells
Treatment of ACH-2 cells* Syncytia per platet
None 155 ± 34
None (no PMA) 3 ± 3
1 AM hypericin + light 4± 4
1 AM hypericin (dark) 227 ± 64
1AM rose bengal + light 38 ± 3
1AM rose bengal (dark 205 ± 17
*Treatment 24 h after induction of HIV with PMA, except as
E 0.8eniSendai Sendai indicated.
0.8 -

tAverage ± SD of three plates.

0

0.6T
esters (24, 25). Induced ACH-2 cells efficiently form syncytia
~0.4- when incubated with Sup-T1 cells (26). ACH-2 cells were
treated with hypericin or rose bengal, with or without light,
d: 0.2-
24 h after induction of HIV protein expression by PMA, in
order to study the effects of these treatments on HIV-induced
cell fusion. Syncytium formation between these cells and
Sup-T1 cells was completely abolished by 1 1LM hypericin,
but illumination was required; syncytium formation was
unaffected by hypericin in the dark (Table 2). Treatment with
0.6
O~ I
F.m
1 gM rose bengal decreased syncytium production by >80P/o
0.4-
and was similarly light dependent (Table 2).
Cross-L of Viral Proteins. Treatment of membranes
0.2- with rose bengal has been reported to result in the cross-
linking of membrane proteins, evidenced by their disappear-
C30 100 30010003000 3 10 30 1003001000 ance from their usual positions in SDS/PAGE and their
Hypericin, nM Rose bengal, nM accumulation at the top of the gel (13, 15). We therefore
examined the effects of hypericin and rose bengal on cross-
FIG. 3. Photodynamic inactivation of hemolysis of VSV (A and
B), Sendai virus (C and D), or influenza virus (E and F) by hypericin
linking of VSV proteins. As shown in Fig. 4, both compounds
caused a.similar pattern of protein cross-linking. Of the three
(A, C, and E) or rose bengal (B, D, and F). A relative hemolysis value major VSV proteins, the integral membrane protein G was
of 1.0 equals 30-80o hemolysis. most readily cross-linked, followed by M, a peripheral mem-
brane protein that partially penetrates the viral bilayer (30).
virus; as described in Materials and Methods, they were The nucleocapsid protein, N, which binds the genomic RNA
diluted 15-fold prior to the hemolysis assay. but is not membrane-associated, was not significantly cross-
Inactivation of 1IV gp120-Induced Syncytium Formation. linked by either com ound. These findings provide an addi-
Viral fusion activity may also be assayed by the formation of tional indication that the predominant action of both hyper-
cell syncytia mediated by cells expressing the viral fusion icin and rose bengal is at the viral membrane and that both
protein on their plasma membranes. For HIV, syncytia dyes are exerting similar effects.
formation involves the fusion of uninfected CD4+ cells with
cells expressing cell surface HIV gpl20. ACH-2 cells are a DISCUSSION
chronically infected T-cell line in which high levels of HIV In this report we have provided evidence that the photody-
protein expression can be induced by treatment with phorbol namic inactivation of enveloped viruses by hypericin may
A
L ig h t ---- '7 B
Hypericin, uM 0 0.41 1
10 10 Light
...- --- W--- -
Rose bengal, nM 0 3 " CD 3 01 00 300' 1 000 1
--. - - M.W
"-
w -..

."mm - - -

M -_AOb _m -Ws
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FIG. 4. Photodynamically induced cross-linking of VSV proteins by hypericin (A) or rose bengal (B). L, G, M, and N are the major VSV
proteins.
 162 Medical Sciences: Lenard et al.
arise chiefly from the destruction of its fusion function. The
fusion activity of three different enveloped viruses was
photoinactivated by concentrations of hypericin ranging from
20 to 220 nM. HIV gpl20-mediated cell fusion, as measured
by syncytium formation, was photoinactivated completely by
1 gM hypericin and >80% by 1 ,uM rose bengal. The higher
concentrations used for inhibition of syncytium formation
were likely necessitated by the larger amounts of membrane
present in the cell samples as compared to viral stocks.
Similar concentrations of hypericin have previously been
reported to inactivate Sindbis virus and murine cytomegalo-
virus in a light-dependent fashion (7). Higher concentrations
were required for photodynamic inactivation of equine in-
fectious anemia virus (6), and of other lipid-containing vi-
ruses in experiments in which light was not controlled (1-5).
Comparison of the effects of hypericin and rose bengal
permitted the following observations. (i) Both dyes act
similarly, inactivating infectivity and/or fusion in all four
enveloped viruses tested and promoting similar cross-linking
of viral membrane proteins (Table 1). Both dyes are efficient
producers of singlet oxygen upon illumination (14-16), and
both are known to associate with biological membranes.
Reactions of singlet oxygen induced by either compound thus
presumably cause most or all of the observed effects. (ii)
Loss of fusion function may be the general reason enveloped,
but not nonenveloped, viruses are inactivated by hypericin
and light. In support of this suggestion, the 150 for VSV
inactivation of infectivity and that for inactivation of hemno-
lysis were very similar, using either hypericin or rose bengal
as photosensitizer (Table 1).
As clinical trials of hypericin are currently under develop-
ment,t it is critical to understand the mechanism by which
this drug exerts its antiretroviral effects. Most importantly,
we have demonstrated that light is essential for all the
antiviral activities of hypericin and rose bengal that were
studied. In this connection, it should be noted that the action
of hypericin and rose bengal resembles quite closely that of
several other photodynamic antiviral compounds: merocya-
nine-540, certain porphyrin derivatives, and certain phthalo-
cyanine derivatives (31-33). All of these associate with
membranes and are thought to work at least in part by
generation of singlet oxygen (34). In common with these
agents, hypericin and rose bengal might prove to be suitable
agents for photodynamic inactivation of enveloped viruses
present in blood or blood products, although lack of speci-
ficity might be a problem with any of these agents. The
possibility of illuminating the blood of HIV patients after
administration of a photodynamic dye might also be inves-
tigated, as has been reported for psoralen (35).
The close similarity of effects seen with hypericin and rose
bengal and their similarity to effects seen with other photo-
dynamic compounds suggests that several of these should be
considered together and compared as potential anti-HIV
agents, both in vivo and in vitro.
tGulick, R., Lui, H., Anderson, R., Kollias, N., Hussey, S. &
Crumpacker, C., 8th International Conference on AIDS, July
19-24, 1992, Amsterdam, abstr, PoB3D18.
This study was supported in part by National Institutes of Health
Grant AI-13002 to J.L.
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