Mechanism of inactivation of tobacco mosaic virus with ozone

Wat. Res. Vol. 22, No. 7, pp. 933-938, 1988 Printed in Great Britain.All fights reserved

0043-1354/88 $3.00+0.00 Copyright © 1988PergamonPress pie

MECHANISM OF INACTIVATION OF TOBACCO MOSAIC VIRUS WITH OZONE NARIKO SHINRIKI,I KOZO ISHIZAKI,l TOSHIMICHIYOSHIZAKI,2 KAZUNOBUMIURA3 and Tox-mu UEDA 3 'Government Industrial Development Laboratory, Hokkaido, Tsukisamu-Higashi 2-17, Toyohira-ku, Sapporo 004, 2Hokkaido University of Education, Kita-ku, Sapporo 002 and 3Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060, Japan (Received June 1987; accepted in revised form February 1988)

Abstract--The inactivation mechanism of tobacco mosaic virus (TMV) in a phosphate buffer (pH 6.9) by ozone was studied. We previously reported that the damage of naked TMV-RNA occurred at the guanine moiety of RNA (Shinriki et aL, Biochim. biophys. Acta 655, 323, 1981). In this paper, we clarified the mode of the inactivation of TMV by using tritium-labeled TMV (TMV*) prepared by the reconstitution of tritium-labeled TMV-RNA (TMV-RNA*) and coat protein of TMV. It was found that the amount of extracted TMV-RNA* from ozone-treated TMV* decreased with the advance of ozonization, and that there was good correlation between the loss of infectivity and the decrease of recovery of TMV-RNA*. When TMV lost its infectivity due to ozone, tryptophan and tyrosine of the coat protein were also degraded by ozone. Polyacrylamide gel electrophoretic analysis of the substance produced during ozonization showed that the coat protein subunits were aggregated with each other and cross-linked with TMV-RNA*. From these results, it was concluded that the inability of uncoating is the major cause of the TMV inactivation by ozone. Key words---ozone, ozonization, tobacco mosaic virus, RNA, infectivity, inactivation, degradation, protein coat, tryptophan, tyrosine

INTRODUCTION

The use of ozone for disinfection and inactivation of viruses is attracting considerable attention because of its superior oxidative power. Ozone has already been in use for water disinfection in certain countries in Europe for a long period. Recently, other countries are contemplating the use of ozone, since it has been clarified that the use of chlorine for water and wastewater treatment leads to the induction of harmful organohalides. With this background, numerous kinetic data on the inactivation of microorganisms by ozone have been accumulated. However, there is a paucity of information on the inactivation mechanism. It has been reported that some viruses resist chlorination (Durham and Wolf, 1973; Engelbrecht, 1976). This is considered to depend on the structural constituents of the capsid or envelope that protects respective RNA or DNA. Then, in what manner does ozone attack the viruses? The mechanism of virus inactivation can be divided int,~ ~wo classes: (a) the damage to the coat protein and (b) the direct damage to the nucleic acids. The former results were obtained by studies on bacteriophage $ x 174 (De Mik and De Groot, 1977), f2 (Kim et al., 1980; Sproul et al., 1982), T4 (Sproul et al., 1982) and poliovirus type 2 (Riesser et al., 1977). In the case of poliovirus type 1 inactivation, Roy et aL (1981) reported that ozone broke down the polypeptide chains, but the main cause for its inactivation was the damage of RNA by ozone. Information on the mechanism of ozone-

inactivation would be useful in developing the use of ozone for water and wastewater disinfection. In this paper, tobacco mosaic virus (TMV) is selected as the target because the structure of TMV is well characterized. The shape of TMV resembles a rod, 16 nm in dia and 300 nm in length, having a hollow center (8 nm in dia). The TMV consists of RNA (5 wt%) of 6400 ribonucleotides (2 x 106Da) and 2130 protein subunits (95 wt%). One subunit consists of 158 amino acids and its molecular weight is 17,500. Each trinucleotide is bound to one subunit by hydrogen bonds (Holmes et al., 1975), which results in the formation of spiral RNA having the protein coat outside of it with a hollow center (Bloomer et aL, 1978). MATERIALS AND METHODS

Materials

TMV, Japanese common strain OM, was infected on Nicotiana tabacum L. var White Burley for I0 days. Purified

TMV was collected from the infected leaves by the polyethylene glycol precipitation and differential centrifugation (Hebert, 1963; Yamamoto et aL, 1970; Otsuki et al., 1977). The coat protein was isolated by the acetic acid method (Fraenkel-Conrat, 1957). TMV-RNA was prepared by phenol-bentonite extraction (Fraenkel-Conrat et al., 1961) and TMV-RNA solution was finally subjected to ultracentrifugation to remove the fine bentonite particles. Tritium-labeled TMV-RNA (TMV-RNA*) was prepared by using the full length TMV-RNA by the methods of RajBhandary (1968) and Ohno et al. (1977). Both 5'- and 3'-ribosyl ends were oxidized by periodate, followed by the reduction with tritium-labeled sodium borohydride (New 933

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NARIKO SHINRIKI et al.

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Fig. 1. Agarose gel electrophoresis of purified TMV-RNA. Lanes: 1, markers of molecular weight, (a) 28s RNA (b) 18s RNA; 2, 3, TMV-RNA. England Nuclear, England) to give the tritiated RNA at the terminal triol-nucleosides. The molecular weight markers, 28s and 18s RNA were prepared from rat liver by phenol method. Gel electrophoresis To ascertain the full length of TMV-RNA, gel electrophoresis of TMV-RNA was carried out on a 1.2% agarose slab gel (Type II, Sigma Chemical Co.; 200 x 200 x I ram). Gel was prepared in 50raM sodium citrate-citric acid (pH3.5)--6M urea-10% glycerin, which is a modified method reported by Woo et al. (1975). It requires 2.5 h at 4°C to obtain perfect gel. A 6-7/~g sample of TMV-RNA was loaded onto a gel and electrophoresis was carried out in 50mM sodium citrate-citric acid buffer (pH 3.5) at 15mA for 4-5 h. After electrophoresis, the gels were stained with Stains-all (Eastman Kodak Co., U.S.A.) and photographed (Fig. 1). To ascertain the full length of TMV-RNA*, 1.2% agarose disk gel (~b4 × 70 mm) electrophoresis was performed for 5 h at 2 mA per disk. Gel constituents and running buffer used were the same as mentioned above. Following electrophoresis, the gels were sliced 2 mm thick by using a gel slicer and each gel slice was melted in 0.5 ml water. The radioactivity of the solutions with 5 ml of Aquasol-2 (New England Nuclear) was measured by a Beckman liquid scintillation counter model LS-230. For the analysis of precipitate on the boundary of phenolbuffer layer during the extraction of TMV-RNA* from tritium-labeled TMV (TMV*) by phenol, polyacrylamide disk gel electrophoresis was carried out on a 10% polyacrylamide gel (q~5 x 100mm) with a 3% polyacrylamide gel (5 ram) layered on top. The procedure was referred to the method by Laemmli (1970). Constant ampere was maintained at 5.0-5-5 mA for 4 disks until the Bromophenol Blue marker passed through 3 0 gel into 10% gel, after which it was kept at 7.5--8.0mA for 4 disks for 65rain. After electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250 and photographed. The 2 mm thick sliced gels were melted at 80°C for 2 h in a 0.6mi of 33% H~O 2 and the radioactivity was measured in the same way as mentioned above. Preparation o f tritium-labeled T M V The result of the gel electrophoresis showed that the full length TMV-RNA* was contaminated by shorter

length TMV-RNA*. This preparation was used for the reconstitution with TMV-protein in 0.1 M phosphate buffer (pH 7.0) at 25°C for 20 h (Otsuki et al., 1977). The collected TMV* containing shorter rods was then subjected to 8.8-23.8% (w/v) linear sucrose density gradient ultlacentrifugation. An 8 mg sample of the crude TMV* was loaded onto 36.7 ml of the gradient and the centrifugation was carried out at 2°C at 24,000rpm for 2.5h. Each gradient was fractionated into 40 fractions. The suspensions of the full length TMV* which was confirmed by examining by an electromicroscope (Hitachi HV-12A, Japan), were dialyzed against water for 16 h at 4°C. Then the full length TMV* was collected by polyethylene glycol precipitation and was stocked in 10mM phosphate buffer (pH 7.4) at - 30°C. The count of the radioactivity was approx. 24 cpm per 1/zg of TMV*. Ozone treatment Ozone was generated from pure oxygen using a Model 0-1 ozonizer (Nippon Ozone Co., Tokyo, Japan). Ozone-oxygen gas (ozone, 40 + 1 mg 1-t; flow rate, 330 ml min-t) was blown onto the surface of 100/~1. of 40 mM phosphate buffer (pH 6.9) containing I mg of TMV or TMV* in a glass tube (~b15 mm) at 2°C. Ozone-treated TMV was subjected to infectivity assay and ozone-treated TMV* was subjected for analysis of the damage. In the case of the amino acid analysis of coat protein, 7ml suspension of TMV (2,04mgml -~) was blown by ozone-oxygen gas (ozone, 20 + 0.5 mg l-t; flow rate, 670 ml min -t) under vigorous stirring, and 0.1 and 0.5 ml of suspension were withdrawn at appropriate intervals for bioassay and analysis of amino acids of coat protein, respectively. Infectivity assay o f T M V Ozone-treated TMV suspension was diluted by a 0.1 M phosphate buffer (pH 7.0) and the final concentration of TMV was adjusted to l-6/~gml -t. Each sample was subjected to bioassay by the half-leaf method using about 20 leaves of Nicotiana glutinosa L. (Holmes, 1929). Amino acid analysis o f coat protein o f T M V A 1 mg sample of TMV, obtained by drying 0.5 ml of ozone-treated sample suspension/n vacuo, was hydrolyzed by 4 N methanesulfonic acid containing 0.2% 3-(2-aminoethyl)indole (Simpson et al., 1976). The estimated value of the amount of tryptophan (Trp) by this method is often affected by the presence of carbohydrates of more than 5%. Accordingly, this effect was examined, in advance, using an authentic mixture of amino acids and ribonucleoside 5'-monophosphates in the constitution ratio of TMV. There was no effect found on the determination of Trp in the presence of 5% mononucleotides. A neutralized hydrolysate was subjected to a Hitachi model KLA-3B amino acid analyser. Extraction o f T M V - R N A * from ozone-treated T M V * The method was the same as that used in the extraction of TMV-RNA from TMV except for the minimized scale of extraction without bentonite. Ozone-treated TMV* (1 mg) was transferred into an Eppendorf tube with buffer used for rinsing the reaction tube to come to 100pl and phenol extraction was carried out. The distribution of the radioactivity, i.e. the radioactivity of pure TMV-RNA* and of all other residual solution, was determined. Precipitates on the boundary of phenol-buffer layer produced in the ozonetreatment for (a) 5, (b) 10 and (c) 30 rain, were subjected to gel el~trophoresis after dissolving them into 25/~1 of I% SDS [20 mM Tris--HC1 (pH 7.0)--1 mM EDTA] solution at 60°C for 30 min. The precipitates of (a) and (b) were perfectly dissolved and half of each solution was loaded on the gel. In the case of (c), two-thirds of pricipitate was kept in 1% SDS solution but almost half failed to dissolve. The SDS solution was also subjected to gel electrophoresis.

Ozone-inactivation mechanism of TMV

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Time (rain) Fig. 2. Inactivation and degradation of TMV-RNA. Starting cohen of TMV-RNA, 0.25 mg ml-[; ozone cohen in inlet gas, 0.1 mgl-~; flow rate, 70mlmin-~; temp., 2°C; pH6.9; O, AMP, CMP; A, GMP; O, UMP; ~7, infectivity of ozone-treated TMV-RNA; Q, control. *Four ribonucleoside 5'-monophosphates were obtained after digestion of ozone-treated TMV-RNA by nuclease Pl.

RESULTS

Ozone damage o f T M V - R N A and coat protein The degradation of T M V - R N A and the loss of its infectivity by ozone, have already been reported by the present authors (Shinriki et al., 1981). These results are reproduced in Fig. 2. Each amount of mononucleotides was determined by HPLC after the ozone-treated T M V - R N A was digested with nuclease PI to four ribonucleoside 5'-monophosphates. The degradation rate of guanosine 5'-monophosphate (GMP) was the highest among these ribonucleotides, which means that the guanine moiety of T M V - R N A was attacked rapidly by ozone (Ishizaki et al., 1981; Shinriki et al., 1983). To estimate the damage of protein coat of TMV, ozone-treated TMV was hydrolyzed and analyzed by an amino acid analyzer. The time courses of inactivation of TMV and of degradation of amino acids in the coat protein? are shown in Fig. 3. It was found that only Trio and Tyr were degraded while all other amino acids were not changed, when TMV lost its infectivity after 20 min-ozonization,

84.4%), and that of the ozone-treated TMV* decreased with the time of ozonization. These results are summarized in Fig. 4. There seems to be a good correlation between the loss of infectivity and the decrease of recovery of TMV-RNA*. A

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Recovery of T M V - R N A * from ozone-treated TMV* While ozone-treated TMV was subjected to the infectivity assay, ozone-treated TMV* was treated by phenol and the extraction rate of TMV-RNA* was examined. The recovery of TMV-RNA* from intact TMV* was found in the range 79.4-90.1% (average tA subunit of coat protein of TMV is constructed of 158 amino acids: Alanine (Ala) 14, Arginine (Arg) 11, Aspartic acid (Asp) 18, Cysteine (Cys) 1, Giutamic acid (Glu) 16, Glycine (Gly) 6, Isoleucine (lie) 9, Leucine (Leu) 12, Lysine (Lys) 2, Phenylalanine (Phe) 8, Proline (Pro) 8, Serine (Ser) 16, Threonine 0%0 16, Tryptophan (Trp) 3, Tyrosine (Tyr) 4, Valine (Val) 14.

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Fig. 4. Inactivation of TMV and change in recovery yield of TMV-RNA by ozone treatment. TMV or TMV* (reconstituted by using tritium-labeled TMV-RNA), l mg in 100#1 buffer; ozone concn, 40mgl '; flow rate, 330 ml min '; temp., 2°C; pH 6.9; O, tritium-labeled RNA extracted; O, infectivity of ozone-treated TMV.

Analysis

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As shown in Fig. 4, the radioactivity of the pure T M V - R N A * decreased with the time of ozonization, while the radioactivity of the other residues increases. This means that some T M V - R N A * migrates into the extraction residues. To clarify the mode of this migration, ozonization of TMV* was carried out during 5, 10 and 30 min, respectively. Each 1% SDS solution of these three kinds of precipitates on the boundary of phenol-buffer layer was subjected to the polyacrylamide disk gel electrophoresis. The stained gel pictures are shown in Fig. 5. They show several protein bands at the positions of T M V coat protein subunit and of its aggregates. Some of these protein bands showed radioactivity as shown in Fig. 6, which means that T M V - R N A * and coat protein were crosslinked together. The occurrence of the cross-linkings and the aggregations was also supported by the fact that almost half of the precipitate produced by 30 min-ozone-treatment did not dissolve in 1% SDS solution. DISCUSSION

In our previous papers, it has already been clarified that the ozone degradation of T M V - R N A and tRNAs occurred most rapidly at the guanine moiety and that the strand scission of R N A did not occur in the initial stage of ozonization (Shinriki et al., 1981, 1983). In addition, it was confirmed that the amino acid acceptor activity of phenylalanine t R N A was reduced to 45% when only 1.1 guanine moieties were

Fig. 5. Polyacrylamide disk gel electrophoresis of precipitates obtained by phenol-extraction of ozone-treated TMV*. TMV*, ling in 100#1 buffer; ozone concn, 40mgl ~; flow rate, 330mlmin-t; temp., 2°C; pH6.9. Lanes: 1, 5 min; 2, 10 min; 3, 30 min; 4, markers of molecular weight, (a) 28s RNA (b) 18s RNA (c) TMV-protein subunit. degraded (Shinriki et al., 1981). Thus, we conclude that the loss of the infectivity of T M V - R N A is attributed to the damage of the guanine moieties. In the present paper, we attempted to clarify the

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Ozone-inactivation mechanism of TMV mode of the inactivation of TMV. Thus, our object is to clarify whether the first reason of the inactivation of TMV is (a) the damage of coat protein or (b) the direct damage of TMV-RNA. The results of the examination of ozone-treated TMV* showed that there was a good correlation between the loss of the infectivity and the decrease of the recovery of TMV-RNA*. Furthermore, the results of gel electrophoresis of precipitates formed during the ozonization for 5, 10 and 30 min showed that some protein bands had radioactivity due to TMV-RNA*. This means that TMV-RNA*, especially the degraded quanine moieties and coat protein are cross-linked together, resulting in the decrease of the extracted TMV-RNA*. On the other hand, when TMV lost its infectivity, only 90% of 3 Trps and 50% of 2 Tyrs of 158 amino acids were degraded by ozone. According to the side view of a sector through the coat protein disk which was investigated using X-ray diffraction of 2.8 A, resolution (Bloomer et al., 1978), 3 Trps, 4 Tyrs and 1 Cys are assumed to be located between the outer side of protein coat to the central portion of the coat, far from the RNA. We have previously reported that ozone attacked preferentially the ozone-reactive portion in the outside of the higher-order structure and ozone did not penetrate into the inner higher-order structure as long as highly ozone-reactive moieties remained intact (Shinriki et al., 1983; Sawadaishi et al., 1985, 1986). Thus, since highly reactive Tyrs (50% of Tyr, i.e. 2 Tyrs) remain intact, ozone seems to still react with them when the infectivity is almost lost. However, since Trps and Tyrs were degraded by ozone, they could become more reactive and could link with the other moieties, resulting in the formation of aggregates of protein subunits (Kuroda et al., 1975; Matus et al., 1980; Dooley et al., 1982). We did not provide the details of these aggregates, but it appears to be an attractive speculation. In conclusion, the mode of the inactivation of TMV may be summarized as follows. Ozone attacks both protein coat and RNA. The RNA degraded at the guanine moieties cross-links with amino acids of the coat protein subunits, which were also aggregated by the damaged Trps and Tyrs. Thus, it is considered that TMV loses the infectivity because of its inability of uncoating. Acknowledgements--The authors wish to express their

sincere gratitude to Professor Yoshimi Okada of Tokyo University for his kind advice and encouragement. They also wish to thank Dr A. Ikehata for his encourgement and Ms Yoshie Watab¢ and Mr Kazuyuki Sawadaishi for their valuable assistance in the experimental work. REFERENCES

Bloomer A. C., Champness J. N., Bricogne G., Staden R. and Klug A. (1978) Protein disk of tobacco mosaic virus at 2.8 A resolution showing the interactions within and between subunits. Nature 276, 362-368.

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De Mik G. and De Groot I. (1977) Mechanism of inactivation of bacterophage ~ x 174 and its D N A in aerosols by ozone and ozonized cyclohexene.J. Hyg. 78, 199-211. Dooley M. M. and Mudd J. B. (1982) Reaction of ozone with lysozymeunder different exposure conditions. Archs Biochem. Biophys. 218, 459--471. Durham D. and Wolf H. W. (1973) Wastewater chlorination: panacea or placebo? Wat. Sewage Wk 120, 67-70. Engelbrecht R. S. (1976) Removal and inactivation of enteric viruses by wastewater and water treatment processes. In Advanced Wastewater Treatment (Edited by Japan Research Group of Water Pollution, Tokyo), pp. 109-143. Fraenkel-Conrat H. (1957) Degradation of tobacco mosaic virus with acetic acid. Virology 4, 1-4. Fraenkel-Conrat H., Singer B. and Tsugita A. (1961) Purification of viral RNA by means of bentonite. Virology 14, 54-58. Hebert T. T. (1963) Precipitation of plant viruses by polyethylene glycol. Phytopathology 53, 362. Holmes F. O. (1929) Local lesions in tobacco mosaic. Bot. Gaz. 87, 39-55. Holmes K. C., Stubbs G. J., Mandelkow E. and Gallwitz U. (1975) Structure of tobacco mosaic virus at 6.7 A resolution. Nature 254, 192-196. Ishizaki K., Shrinriki N., Ikehata A. and Ueda T. (1981) Degradation of nucleic acids with ozone. I. Degradation of nucleobases, ribonucleosides and ribonucleoside 5'-monophosphates. Chem. Pharmac. Bull. 29, 868-872. Kim C. K., Gentile D. M. and Sproul O. J. (1980) Mechanism of ozone inactivation of bacterophage f2. Appl. envir. Microbiol. 39, 210-218. Kuroda M., Sakiyama F. and Narita K. (1975) Oxidation of tryptophan in lysozyme by ozone in aqueous solution. J. Biochem. (Tokyo) 78, 641-651. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Matus V. K., Mernikova A. M., Okun I. M., Slobozhanina I. M. and Konev S. V. (1980) Structure-modifying action of ozone on plasmic membranes. Vestsi Akad. Navuk BSSR, Ser. Biyal. Navuk 1980, 125-128. Ohno T., Sumita M. and Okada Y. (1977) Location of the initiation site on Tobacco Mosaic virus RNA involved in assembly of the virus in vitro. Virology 78, 407-414. Otsuki Y., Takabe I., Ohno T., Fukuda M..and Okada Y. (1977) Reconstitution of tobacco mosaic virus rods occurs bidirectionally from an internal initiation region: demonstration by electron microscopic serology. Proc. natn. Acad. Sci. U.S.A. 74, 13-19. RajBhandary U. L. (1968) Studies on polynucleotides. LXXVII. The labeling of end groups in polynucleotide chains: the selectivemodified cation of diol end groups in ribonucleic acids. J. biol. Chem. 243, 556-564. Riesser V. W., Perrich J. R., Silver B. B. and McCammon J. R. (1977) Possible mechanisms of poliovirus inactivation by ozone. In Forum on Ozone Disinfection (Edited by Fochtman E. G., Rice R. G. and Bruwing M. E. ), pp. 186-192. International Ozone Institute, New York. Roy D., Wong P. K. Y., Engelbrecht R. S. and Chian E. S. K. (1981) Mechanism of enteroviral inactivation by ozone. Appl. envir. Microbiol. 41, 718-723. Sawadaishi K., Miura K., Ohtsuka E., Ueda T., Ishizaki K. and Shinriki N. (I 985) Ozonolysis of supercoiled pBR322 DNA resulting in strand scission to open circular DNA. Nucleic Acids Res. 13, 7183-7194. Sawadaishi K., Miura K., Ohtsuka E., Ueda T., Ishizaki K. and Shinriki N. (1986) Structure- and sequence-specificity of ozone degradation of supercoiled plasmid DNA. Nucleic Acids Res. 14, 1159-1169. Shinriki N., Ishizaki K., Miura K., Ueda T. and Harada F. (1983) Degradation of nucleic acids with ozone. III. Mode of ozone-degradation of mouse proline transfer

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NARIKO SHINRIKIel al.

ribonucleic acid (tRNA) and isoleucinc tRNA. Chem. Pharmac. Bull 31, 3601-3608.

Shinriki N., IshizakiK., Ikehata A., Yoshizaki T., Nomura A., Miura K. and Mizuno Y. (1981) Degradation of nucleic acids with ozone. II. Degradation of ycast RNA, phenylalanine t R N A and tobacco mosaic virus RNA. Biochim. biophys. Acta 655, 323-328. Simpson R. J., Neubcrger M. R. and Liu T. Y. (1976) Complete amino acid analysis of proteins from a single hydrolysate. J. biol. Chem. 251, 1936-1940. Sproul O. J., Pfister R. M. and Kim C. K. (1982) The

mechanism of ozone inactivition of water borne viruses. Wat. Sci. Teehnol. 14, 303-314. Woo S. L. C., Rosen J. M., Liarakos C. D., Choi Y. C., Busch H., Means A. R. and O'Malley B. W. (1975) Physical and chemical characterization of purified ovatbumin messenger RNA. J. biol. Chem. 250, 7027-7039. Yamamoto K. R., Albcrts B. M., Benzinger R., Lawhorne L. and Treiber G. (1970) Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology 40, 734-744.