Photochemistry ond Phorobralogy. Vol. 29. pp. 7 x 5 to 790

0 Pcrg,,rnun Press Ltd. 1979. Printcd in Great Brltmn

INACTIVATION OF LIPID-CONTAINING VIRUSES BY HYDROPHOBIC PHOTOSENSITIZERS AND NEAR-ULTRAVIOLET RADIATION WALLACE

SNIPE$*

GREGOKY KELLEK,JOHN WOtK. TOMVICKROY. REGINALDDIXKINC~

and ALFC KHTH Biophysics Laboratory. Department of Biochemistry and Biophysics, Thc Pennsylvania State University, University Park, PA 16802, U.S.A.

Abstract--The hydrophobic photosensitizers acridine and phenothiazine inactivate the lipid-contnining viruses PM2, 46,and herpes simplex when samples are illuminated with near-1JV radiation. 423-1-0. which is insensitive to organic solvents and presumably contains n o lipids. is not inactivated under comparable conditions. For acridinc, the inactivation of virus requires that oxygen bc present and is inhibited by sodium azide, implicating the involvement of singlet oxygen. For phenothiazine, oxygen IS not required for photosensitized inactivation. Treatment of PM2 with acridine and near-UV light caused a complete disruption of the virion, as determined by sucrose gradient analysis of treated and untreated samples. These data and related observations suggest that lipid-containing viruses are inactivated through photosensitized membrane damage.

INTRODUCTION

Early studies by Clifton (1931) and Perdrau and Todd (1933)showed that viruses can be inactivated by photosensitizing dyes and light. More quantitative experiments followed with the work of Burnet (1933), Welsh and Adams (1954), Yamamoto (1958)and Helprin and Hiatt (1959)on bacterial viruses. Hiatt et al. (1060) reported the inactivation of several animal viruses by photosensitizers and visible light. The clinical potential of photodynamic therapy was recognized by Herzberg (1933) and advanced by Melnick and his colleagues whose first successful application of the technique in tliuo was the treatment of herpes keratitis in laboratory rabbits (Moore et a/., 1972). Subsequently, clinical investigations of the photodynamic treatment of herpes simplex virus (HSV) infections were carried out in a number of laboratories (Friedrich, 1973;Kaufman et a!., 1973; Roome et a/., 1975; Chang and Weinstein, 1975; Myers et d.,1975). The eficacy reported in these studies was somewhat variable, and the question of whether photodynamic therapy is effective is not yet satisfactorily resolved. Concern over the safety of photodynamic therapy was raised by the observation of Rapp et a/. (1973) and Li or LII. (1975)that cultures of HSV treated with dyes and light had the capacity to transform cultured cells. These transformed cells. when implanted in appropriate laboratory animnls, produced tumors. More recently, it was shown that cells treated with proflavine and light are more susceptible to transformation by subsequent infection with UV-irradiated HSV (Verwoerd and Rapp, 1978).Although no report of cancer resulting from thc treatrncnt of HSV lesions *To whom correspondence and reprint requests should

be addressed.

with dyes and light has appeared, thc work from Rapp’s laboratory has generated considerable caution amongst researchers and clinicians in developing the method for general use. The two photodynamic dyes that have been utilized to the greatest extent for treatment of HSV infections are neutral red and proflavine. These are both positively charged, water-soluble dyes that bind to DNA. Viruses that are grown in the presence of such dyes incorporate them into the virus structure and become irreversibly photosensitized (Crowther and Melnick, 1961;Schaffer, 1962;Wilson and Cooper, 1962, 1963; Schaffer and Hackett, 1963;Rapp et d.,1973). Exposure of such cultures to light then inactivates the infectious virus particles, presumably by damaging viral nucleic acid. Cultures treated in this manner, nevcrtheless, are capable of infecting cells and, with some small probability, causing transformation. This most likely is due to the incorporation of part or all of the viral genome into the cellular DNA. It is not known whether transformation is brought about by inactivated virus that were previously infectious, or by defective HSV particles present in the initial virus preparation. It may be that the transformation of cells by inactive HSV is a unique characteristic of virus that have been inactivated by DNA damage. Viruses that have been inactivated by other mechanisms. such as modifications of the viral membrane, may not give rise to comparable transforming events. We are exploring the possibility that hydrophobic photosensitizers can inactivate lipid-containing viruses via localized membrane damage. This paper describes our initial work with three lipid-containing viruses and two hydrophobic photosensitizers. The viruses that were used are 46, PM2, and HSV. For comparative purposes, some experiments were also

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carried out with 423, a bacterial virus that is insensitive t o organic solvents and presumably contains no lipids. The photosensitizers that were used are acridine (ACR) and phenothiazine (PTZ), whose structures are shown below. In unsubstituted form, these molecules are quite hydrophobic and are expected t o partition into membrane hydrocarbon zones. Data presented here characterize the inactivation of viruses by photosensitized membrane damage and form the basis for further experiments on the potential use of hydrophobic photosensitizers for the treatment of HSV infections. Acridine

Phenothiazine

MATERIALS AND METHODS

Orynnistns. 46 and $23-1 -a were kindly supplied by Prof. A. K. Vidaver, Department of Plant Pathology, University of Nebraska. The host for both 46 and 423 is the bacterium Pseudornonus phaseoiicolu, strain HB 1OY. PM2 and its host, Pseudomonus BAL-31, were obtained from Prof. Eugene Cota-Robles, University of California, Santa Cruz. From BAL-31 we isolated strain TT167, which requires tryptophan and thymidine for growth. Herpes simplex virus, type 2, was supplied by Prof. John Docherty, Department of Microbiology and Cell Biology, The Pennsylvania State University. Human embryonic lung (HEL) cells, used in monolayer culture for plaque assays of HSV-2, were obtained from Prof. Stanley Person, Department of Biochemistry and Biophysics, The Pennsylvania State University. Techniques for the culture of 4 6 and 423 (Wanda et ul., 1976; Snipes et al., I977), PM2 (Snipes et a/., 1974; Cupp et al., 1975), and HSV (Person et al., 1976; Snipes ef nl., 1977) have been reported. Near-U V liyht exposure'. Samples were exposed to near-lJV radiation from Westinghouse F15T8/BLB (blacklight blue) bulbs at a distance of 6.5 cm. Four bulbs were arranged vertically with the sample at their geometrical center. For all experiments except those with HSV, all four bulbs were used. Due to the greater sensitivity of HSV to photosensitized inactivation. only two bulbs were used in experiments with this virus. Measurements of sample temperature before and after exposure showed less than 2 C increase for the exposure times used. Sucrose yrudient analysis. Sucrose gradients (2&30:/,, w/v) were prepared in a solution containing 50% Medium 25 (Snipes et a/., 1974) and 507; distilled water. Samples were layered on top of the gradients and centrifuged for 130min at 32,000rprn in a SW41 rotor. The temperature was 4°C. Afterward, the tubes were punctured and 0.5 m/fractions were collected for analysis. Addition of phorosensirizers. Stock solutions of acridine and phenothiazine were prepared in 95% ethanol at 100 times the final concentration desired for the experiment. Just prior to near UV exposure, 0.1 m / of the stock was added to ]Om/ of diluted virus and vortexed. Control samples were treated in a similar manner with 95% ethanol lacking photosensiti7,er.

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Source of materiais. Acridine (Lot No. 112267) and phenothiazine (Lot No. 050347) were purchased from Aldrich Chemical Co., Inc., Milwaukee, WI. Sodium azide (Lot No. 721953) was supplied by Fisher Scientific Co., Fairlawn, NJ. "P as inorganic phosphate camc from New England Nuclear Corp., Boston, MA. RESULTS

Properties of photosensitizers Figure 1 shows the spectra of acridine and phenothiazine in aqueous solution, along with the emission spectrum of the BLB black light (Jagger, 1967). Acridine has extremely good overlap with the BLB emission over the entire range from 300 to 400 nm. Phenothiazine has its absorption maximum at about 320nm, and overlaps well with the BLB emission over a much narrower range of wavelengths. In organic solvents (ethanol, diethyl ether, octanol), the absorption maximum of phenothiazine is shifted only slightly to longer wavelengths (< 10 nm), so the spectrum of Fig. 1 should be a reasonable representation of that for molecules in a membrane. By integrating the product of photosensitizer absorption and BLB emission, normalized to photosensitizer concentration, we estimate that acridine absorbs approximately 3.5 times as much light over the BLB output range as does phenothiazine. We measured the partition coefficient of acridine between water and several organic solvents in order t o obtain a n estimate of the degree to which this molecule is expected to partition into membrane hydrocarbon zones. The data of Table 1 indicate that acridine partitions very strongly into hydrophobic environments, with coefficients ranging from 200 for hexane t o 2700 for decanol.

Treatment of 46 and 423 with ucridine We have found 46 and 423 to be very convenient for initial studies on the antiviral activity of agents I

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Figure I . Absorption spectra of acridine (12.5 pg/mt') and phenothiazine ( 1 1 &n/) and the emission spectrum of BLB bulbs (Jagger. 1967). The absorption spectra were recorded on a Bcckman Model 25 spectrophotometcr.

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Virus inactivation by hydrophobic photosensitizers

Table 1. Partition coefficient of acridine between several organic solvents and tris dilution buffer (Snipes et al., 1977) Solvent

Partition coefficient*

Hexanc Diethyl ether Toluene Chloroform Decanol

200 250 2000 2500 2700

* Dctcrmined by measuring the U V absorption of acridinc i n the aqueous and organic phases after partition equilibrium at room temperature. mM Sodium Azide

whose mode of action is through membrane damage (Snipes et al., 1975, 1977). 46, which is composed of an inner nucleocapsid surrounded by a rather loose membrane envelope (Vidaver et al., 1973), has many structural similarities to enveloped animal viruses. It is sensitive to organic solvents and to hydrophobic membrane perturbers (Vidaver et al., 1973; Snipes et d., 1975; Wanda et al., 1976). 623, which infects the same host cell as does 46, is generally not susceptible to agents that act on membranes and therefore provides a convenient, preliminary distinction between inactivation through membrane damage and through other mechanisms. Figure 2 shows data for the treatment of these two viruses with acridine and near-UV radiation. At 25 pg/m/, acridine alone has no effect on 46 or 423, and thc exposure of the viruses to the radiation in the absence of acridine is likewise without effect. If samples containing acridine are exposed to near UV I

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Figure 3. Effect or sodium azide on the inactivation of 46 by acridine and near-UV radiation. Samples were exposed for 6 min at room temperature to four BLB bulbs. radiation, 46, but not 423, is inactivated. The survival curve for 46 has a very pronounced shoulder, suggesting that considerable damage must be accumulated in the virion before it is inactivated. Control experiments (data not shown) gave identical survival curves for a sample irradiated immediately after addition of acridine and a sample stored in the dark for 25min after addition of acridine but prior to irradiation. These data suggest, but do not prove, that the inactivation of 46 is through photosensitized membrane damage. Experiments were carried out in the presence and absence of oxygen to investigate the mechanism of action for acridine. It was found that 4 6 is not inactivated by this treatment if the sample is bubbled with nitrogen prior to and during exposure (data not shown). Next, the involvement of singlet oxygen was investigated by the use of azide, an agent that is known to quench this species (Foote et a/., 1972; Hasty et al., 1972). The data of Fig. 3 show that the

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Figure 2. Treatment or 46 (A) and 423 (B) with acridine and ncar-UV radiation. Samples were exposed at room temperature to four BLB bulbs, as described in Materials and Methods. The cultures were bubbled continuously with air during exposure. Acridine concentration was 25 ,ug/m/.

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Figure 4. Treatment of 46 (A) and 423 (B) with phenothiazine and near-UV radiation. Conditions were as described i i i Fig. 2. The phenothiazine concentration was 1 pg/m/.

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inactivation of 46 is greatly diminished in the presence of sodium azide, suggesting that inactivation by acridine and near UV light is a classical photodynamic effect mediated through singlet oxygen. Treatment of 46 und 423 with phenothiazine In similar experiments, 46, but not 423, was found to be sensitive to treatment with phenothiazine and near UV light. Phenothiazine is less soluble in water than is acridine, and lower concentrations were used to avoid precipitation and direct inactivation of the virus in the absence of light. Figure 4 gives the results of an experiment in which 1 pg/m/ phenothiazine was used. Somewhat longer exposure times are required than for comparable inactivation with acridine, as might be expected for a lower concentration of photosensitizer and a less optimal match of its absorption spectrum to the emission spectrum of the light source. In the absence of specific information about the comparative partitioning of acridine and phenothiazine into the 46 membrane, further comparisons of the photosensitizing efficiency of these two molecules cannot be made. In contrast to acridine, photosensitization by phenothiazine does not require oxygen (data not shown). The pronounced shoulder on the survival curve is lacking with phenothiazine, suggesting that less cumulative damage is required for inactivation with this compound. Solutions of phenothiazine turn pink upon exposure to near-UV radiation, indicating that the molecule is being chemically modified in I

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2 5 rg/rnl ACR t Ligh 25pg/rnl ACR -Ligh -ACR t Llght -ACR- Llght

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Figure 6. Sucrose gradient analysis of PM2 treated with acridine and near-UV radiation. A lysate made in the prcsence of 32P0, was centrifuged at low speed to remove cell debris. One portion was treated with acridinc (25 pg/m() and near U V radiation (6 min exposure to four BLB bulbs) at room temperature. Another portion was maintained as control. Samples were centrifuged at high speed to pellet any virus-like particles and analyzed on sucrose gradients as described in Materials and Methods. The direction of sedimentation is from right to left. some way. No further experiments were carried out with this compound.

Effects of ucridine and neur U V radiution v n PM2 The structure of PM2 has been well characterized by Franklin and his co-workers (Harrison et al., 1971; Hinnen et al., 1974; Franklin, 1974). The DNA is located in an icosahedral core surrounded by a phospholipid bilayer. An external icosahedral protein coat encloses the bilayer, making PM2 somewhat atypical with regard to structure. Figure 5 shows data for thc effects of acridine and black lights on PM2. The upper portion (A) shows the inactivation in the presence of 25 pg/mf acridine. As with 46, there is a pronounced shoulder on the survival curve followed by a precipitous drop in stirvival. In another experiment, a PM2 lysate was prepared in the presence of 25 pg/m/ acridine in the dark. and the virus were exposed to near UV radiation immediately after being diluted 100-fold in medium lacking acridine. In this case, no inactivation was observed for comparable exposure times (Fig. 5B). This result indicates that insufficient acridine becomes irreversibly bound to PM2 during growth to cause significant photosensitization in the absence of exogenous acridine. Additional experiments were carried out with PM2 to investigate the mechanism of photosensitized inactivation. A portion of a lysate prepared in the presence of 32P0, was treated with acridinc and near-UV radiation and then analyzed on sucrose gradients, to determine the degree to which the PM2 virion is disrupted. An untreated sample was maintained as a control. Comparison of the two samples (Fig. 6) clearly indicates that the PM2 virion is completely

Virus inactivation by hydi.ophobic photosensitizers

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other interpretations of these data are clearly possible, rthe collective data with this system strongly support

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Figure 7 . Treatment of type 2 HSV with acridine and near UV radiation. Samples were exposed to two BLB bulbs at room temperature. Acridine concentration was 25 pgJmY.

disrupted by the treatment and that no virus-like particles remain. Inactivation of H S V

We have found that HSV is particularly sensitive to inactivation in uitro by acridine and near-UV radiation. Figure 7 shows data for HSV-2 from an experiment in which only two black light bulbs were used. Appropriate controls established that acridine alone (25pgimf) and near-UV alone do not inactivate the virus under the conditions of this experiment. DISCUSSION

In several recent publications, evidence has been presented that microorganisms can be inactivated by photodynamic action on the membrane of the cells (Jacob and Hamann, 1975; Ito, 1977; Ito and Kobayashi, 1977; Cohn and Tseng, 1977). These studies were carried out with photodynamic dyes that could not cross the plasma membrane and enter the cell. Thus, singlet oxygen produced outside the cell was presumed to diffuse to and damage the cell envelope. The most extensive work to appear has been that of Ito and his colleagues on the photodynamic inactivation of yeast by toluidine blue. In contrast to acridine orange (Kobayashi and Ito, 1976), the treatment of yeast with toluidine blue and visible light produced no mutations, suggesting to the authors that DNA is not the site of damage produced by this phbtosensitizer (Ito, 1977). Additional experiments showed that treatment of yeast cells with toluidine blue rendered them more susceptible to the subsequent action of acridine orange. From this result the authors concluded that toluidine blue treatment damaged the cell envelope, allowing more rapid entry of acridine orange to the cell and enhancing its DNAmediated effects (Ito and Kobayashi, 1977). Although

the notion that inactivation is through membrane damage. Similar conclusions have been reached by Cohn and Tseng (1977), who studied the photodynamic effects of eosin Y and light on yeast cells. Jacob and Hamann (1975) have presented evidence both for photosensitized membrane damage and its repair in Proteus mirahilis. These workers observed that cells treated with methylene blue and light were more susceptible to osmotic lysis, as detected by release of DNA, than were untreated controls. Furthermore, incubation of the damaged cells for a short period of time between photodynamic treatment and osmotic shock reduced the extent of lysis. It was concluded that the cells possess a repair system capable of reversing photosensitized membrane damage. Hydrophobic photosensitizers, in principle, localize damage to membranes in a different manner than that for the dyes previously used. Rather than being excluded from the organism, a molecule such as acridine is most likely concentrated in the membrane hydrocarbon zone. Thus, the region of maximum generation of reactive species should be nearest the membrane. For acridine, the concentration of photosensitizer may be several hundred times greater in the membrane than in the surrounding aqueous zones. In the case of viruses, hydrophobic photosensitizers may be much more effective in damaging membranes in preference to DNA than molecules such as toluidine blue, since the virus membrane may not exclude these latter charged species the way a cell plasma membrane does. The sucrose gradient analysis of PM2 treated with acridine and near UV radiation suggests that inactivation is caused by structural damage to the virion which results in its disassembly. This is most likely damage to the PM2 membrane. Previous experiments in our laboratory have shown that PM2 is readily inactivated by butylated hydroxytoluene (RHT), a hydrophobic membrane perturber, and in that case also the PM2 virion was completely disrupted with the viral DNA being released to the medium (Cupp et al., 1975). Research in our laboratory along several other lines also indicates that acridine and near UV radiation damage membranes. Bacterial cells treated with acridine, but not acridine orange, undergo lysis. The phospholipid species extracted from membranes treated with acridine migrate differently on thin-layer chromatograms than do those of untreated controls. And finally, the fluidity properties of phospholipid vesicles, as measured by spin labeling and electron spin resonance, are significantly modified by treatment with acridine and near UV light. These studies of photosensitized membrane damage are actively being pursued. Ackriowlrdllrrnerits-This research was supported by the U.S. Department of Energy. Excellent technical assistance was provided by Margaret Murrer.

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WALLACE SNIPESet a1 REFERENCES

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