Photochemistry und Photobiology Vol. 5 3 , No. 2. pp. 169-174, 1991 Printed in Great Britain. All rights reserved

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RAPID COMMUNICATION

PHOTOSENSITIZATION IS REQUIRED FOR INACTIVATION OF EQUINE

INFECTIOUS ANEMIA VIRUS BY HYPERICIN

SUSAN CARPENTER1' AND GEORGE A. KRAUS2

(Received 24 September 1990; accepted 5 November 1990)

lDepartment of Veterinary Microbiology and Preventive Medicine, and 2Department of Chemistry, Iowa State University, Ames, IA 50011 U.S.A.

Abstract. Hypericin, a photoreactive polycyclic quinone, was found to dramatically reduce infectivity of cell-free stocks of equine infectious anemia virus. However, the antiviral activity of hypericin was completely dependent on the presence of light. Short periods of photosensitization resulted in a partial loss of reverse transcriptase activity and complete inhibition of viral infectivity. These results suggest that the photodynamic effect of hypericin interferes with more than one stage in the virus replication cycle.

INTRODUCTION Hypericin (11, a pigment found in certain plants and ciliates, exhibits

bactericidal activity and has been employed as an antidepressant (Daniel, 1949). In a seminal series of papers, Meruelo and co-workers have described the potent antiretroviral effect of hypericin and the structurally related quinone, pseudohypericin (2) (Meruelo et al., 1988; Lavie et al., 1989): Interestingly, both compounds were found to inhibit not only the production of infectious virus from chronically infected cells, but were also shown to inhibit reverse transcriptase activity of mature virions. We have previously demonstrated the effect of hypericin on cells chronically infected with equine infectious anemia virus (EIAV) (Kraus et al., 19901, a retrovirus genetically related to human immunodeficiency virus (Gonda et al., 1988). At concentrations of 10 &mL, hypericin was able to reduce production of EIAV by 99.99% without causing obvious cytopathic effects.

OH 0 OH

1 : X=H 2: X=OH

*To whom correspondence should be addressed. +Abbreviations: DMSO, dimethyl sulfoxide; EIAV, equine infectious anemia virus; FFU, focus forming unit; HPD, hematoporphyrin derivative; HSV, herpes simplex virus; RT, reverse transcriptase

169

170 SUSAN CARPENTER and GEORGE A. KRAUS

Hypericin is also responsible for hypericism, a disease characterized by sensitivity of the skin to visible light (Pace and Mackinney, 1941). Hypericin photosensitization requires molecular oxygen and involves the generation of singlet oxygen (Racinet et al., 1988). A timely review on the photodynamic activity of hypericin has been published by Duran and Song (1986). Recently, it was repo’rted that fluorescent light was able to enhance the antiviral activity of hypericin against murine cytomegalovirus and Sindbis virus (Hudson et al., 1990). These findings indicate that antiviral activity of hypericin may depend on the photoreactive properties of the compound. In the present study, we have examined the direct antiviral effect of hypericin on mature virions and have explored the importance of photosensitization for antiviral activity. Our results indicate that photosensitization is required for the antiretroviral activity of hypericin.

MATERIALS AND METHODS Photosensitization of hypericin and related quinones. Cell free stocks of the

MA-I strain of EIAV (Carpenter et al., 1987; Carpenter and Chesebro, 19891, containing approximately 105 infectious units of virus per mL, were serially diluted in Dulbecco minimum essential medium supplemented with 20% fetal bovine serum, 100 unitdml penicillin, 100 &mL streptomycin and 1% dimethyl sulfoxide. Virus was mixed with an equal volume of hypericin to final concentrations of 1 0 , l and 0.1 &mL hypericin. Samples were incubated for various times at room temperature in the presence or absence of fluorescent light. The light source consisted of 24 40W standard fluorescent bulbs. The light intensity at the experiment was 120 footcandles as measured by a Spectra Professional Model P-251 incident exposure meter. Following incubation, samples were returned to the dark and assayed for infectious virus and reverse transcriptase (reverse transcriptase) activity.

Focal immunofluorescence assay. Titers of infectious virus were determined using a focal immunofluorescence assay as previously described (Carpenter et al., 1987). Titers are expressed as focus forming units (FFU) per mL inoculum.

performed using methods similar to those previously described (Carpenter and Chesebro, 1989; Goff et al., 1980).

previously described (Kraus et al., 1990).

Reverse transcriptase assay. Assays for reverse transcriptase activity were

Synthesis of quinones. Hypericin and related quinones were synthesized as

RESULTS AND DISCUSSION Hypericin was able to inactivate infectivity of cell-free EIAV. Importantly,

however, our results indicated that the antiretroviral activity of hypericin was wholly dependent on the presence of light. No decrease in viral infectivity was observed when hypericin and virus were incubated in the dark (Table 1). Moreover, it appeared that light was an absolute requirement for the antiviral activity, as even high concentrations of hypericin (10 &mL) were unable to reduce infectivity of as few as 100 infectious virions. These findings contrast somewhat with the report that the antiviral activity of hypericin is enhanced by, but not dependent on, the presence of fluorescent light (Hudson et al., 1990). Rather, it appears that photoinactivation of retroviruses by hypericin may be more similar to the antiviral activity of hematoporphyrins which also require photosensitization for antiviral activity (Lewin et al., 1980; Matthews et al., 1988; Chanh et al., 1989).

The finding that the photodynamic properties of hypericin were required for inactivation of EIAV suggests that other photodynamic compounds may also display antiretroviral activity. In fact, it has recently been found that human immunodeficiency virus and simian immunodeficiency virus are inactivated in the

Rapid Communication 171

Table 1. Photoinactivation of EIAV by hypericin

FFUs EIAV per mL*

104 103 102

pg/mL Hypericin Dark Light Dark Light Dark Light

10.0 4.13+ 0.0 2.84 0.0 2.11 0.0

1.0 3.98 0.7 3.15 0.0 2.08 0.0

0.1 3.87 3.77 2.98 2.34 1.90 1.65

0.0 3.84 3.99 3.11 3.04 1.98 1.89

*Titer of EIAV prior to treatment with hypericin.

+Titer of EIAV, expressed as loglo FFU/mL, following treatment with hypericin.

presence of dihematoporphyrin ether and light (Matthews et al., 1988; Chanh et al., 1989). In our studies of the structural requirements for antiviral activity of quinones (Kraus et al., 19901, we synthesized a series of basic analogs structurally related to hypericin. Although no antiviral activity was observed among these analogs, it was reasoned that prolonged exposure to fluorescent light may result in photoactivation and increased antiviral activity. Thus, 1000 FFUs of virus were mixed with each quinone and exposed to light for 0,10, or 60 min. Hypericin was the only compound which was found to reduce viral infectivity (Table 2). As little as 10 min of light were required for hypericin to inactivate infectious virus. In contrast, none of the analogs were able to effect significant reduction in viral infectivity, even after 60 min of light. The structure of quinone 6 contains the aromatic ring system of calphostin C, a quinone that exhibits photodynamic activity; however, 6 was unable to inactivate EIAV. Thus, photoreactivity alone may not be sufficient for antiretroviral activity.

Table 2. Effect of Photosensitization on the Antiviral Activity of Hypericin and Related Analogs

Minutes of Fluorescent Light ExDosure

Compound+ 0 10 60

1% DMSO 3.45* 3.28 3.28

Hypericin 3.14 0.00 0.00

4 3.34 3.25 3.15

3 3.52 3.40 3.41

5 3.36 3.45 3.36

6 3.40 3.36 3.46

172 SUSAN CARPENTER and GEORGE A. KRAUS

*Loglo FFU/mL virus following treatment of approximately 3 loglo EIAV with 10 g/mL of quinone. -

+ OH 0 0

R

3: R=OH, R'=Me

4 : R=R'=H HO 0 0 5 6

To ascertain the possible mechanism by which hypericin is able to inactivate EIAV, virus samples treated with hypericin in the presence or absence of light were assayed for the presence of viral RT. Care was taken to keep all samples in the dark prior to, and during, the RT assay. RT activity was completely inhibited by hypericin and'6O min of fluorescent light (Fig. 1). Interestingly, 10 min of light exposure reduced, but did not abolish, RT activity. Because the same length of light exposure was able to completely eliminate viral infectivity (Table 21, these results suggest that inhibition of RT activity is not the sole mechanism of viral inactivation. Studies with herpes simplex virus (HSV) demonstrated that photoinactivation by hematoporphyrins prevented viral adsorption and penetration into susceptible cells (Lewin et al., 1980). These and other studies (Skiles et al., 1985) suggest that photoinactivation involves the viral envelope.

0 10 60 Minutes in Light

Figure 1. Photodynamic effect of hypericin on EIAV RT activity. loK FFUs EIAV were diluted 1:2 in tissue culture media containing 0 or 10 p g / d hypericin. Samples were ex- posed to fluorescent light 0,lO or 60 min and assayed for RT activity. Results are expressed as % RT activity in hypericin treated samples as compared to controls.

Rapid Communication 173

Photodynamically active porphyrins have been shown to effect cell membrane damage by crosslinking proteins (Moan, 1986). A similar effect on viral membranes may interfere with receptor binding and entry into susceptible cells, such as was observed with HSV (Lewin et al., 1980). The mechanism by which hypericin is able to photoinactivate RT activity in mature virions is less clear. Photosensitized hematoporphyrin derivative has been shown to produce DNA damage in cultured cells (Blazek and Hariharan, 1984) and to damage viral DNA (Schnipper et al., 1980). Effects of HPD include protein-DNA crosslinks as well as protein-RNA crosslinks (Boegheim et al., 1985) and strand breaks (Blazek and Hariharan, 1984). Additional experiments are needed to elucidate the interactions between hypericin and retroviral proteins and nucleic acid. An understanding of the mechanisms of photoinactivation may aid in development of photodynamic therapies for treatment of retroviral diseases, including acquired immunodeficiency syndrome.

Acknowledgements. The authors gratefully acknowledge Therese Cotton for helpful discussions and thank Yvonne Wannemuehler and Nancy Qvale for technical assistance. The work was supported in part by a grant from the Iowa State University Agricultural Biotechnology Council to S. C.

Blazek, E. R. and P. V. Hariharan (1984) Alkaline elution studies of hematoporphyrin-derivative photosensitized DNA damage and repair in Chinese hamster ovary cells. Photochem. Photobiol. 40,5-13.

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