Solubilization of Trichoplusia ni granulosis virus proteinic crystal

JOVRSBL

OF

INVEIVl%BRATE

PATHOLOGY

Solubilization

19, 395-404

(1972)

of Trichoplusia ni Proteinic Crystal

Granulosis

Virus

I. Kinetics KOHJI The

Cell

Research

Institute

and

EGAWA~ The

AND

Department Austin, Receivecl

MAX

D.

of Botany, Texas 78712 January

SUMMXRS The

University

oj Texas

at Amtin,

4, iY71

Methods for the solubilization of an insect granulosis virus capsule from l’richopl~aia ni were studied using turbidity measurements. The solubilixation in the solvents employed was most strongly influenced by pH. Various alcohols and hydrogen bond cleaving agents also enhanced solubilization. The most effective solvent tested at pH’s close to neutrality was 607n n-propanol saturated with guanidine.HCl. The effectiveness of this solvent was minimal at pH 5.2. It was shown that the time courses of the solubilization were generally composed of three phases. A plot of the third phase levels versus pH indicated a normal distribution of those capsules or capsular fragments in resisting solubilization. Validity of the calculation of rate constants for the dissociation reaction in the second phase was discussed. Logarithm of the rate constant showed a linear relat,ionship with the reciprocal of absolute temperature. The activation energy for the dissoeiat,ion calculated from this relationship using the Arrhenius equation indicated that hydrophobic and hydrogen binding forces play the major roles for the stability of the crystalline structure of the capsular protein. Ionic binding forces were estimated to be of minor importance.

polyhedra have shown that they are cryetalline structures of protein subunits (Amott and Smith, 1965; Engstrom and Ii-ilkson, 1968) with lattice spacings of approximately 70-80 A and estimated lattice angles of 95”-120”. These cryst’als are very resistant to solubilization at pH values close to their isoelectric pointIs (Bergofd, 1959), resistant to proteolyt’ic action of enzymes, and usually soluble only in aqueous solutions of weak alkali or strong acids (Bergold, 1947, I%%).. In sodium hypoclorite solution, the soir;. bilizat,ion of a nuclear polyhedrosis v5us (KPV) of Trichoplusia ni has been shown to be a function of both reagent conceniratior: and time of exposure (Ignoffo and Dutky:, 1963). Polyhedra and capsules from diff ertm sources have shown variable resistance against alkali dissociation according to the species of the viruses (Day et al-, 1953), which perhaps suggest,s fundamental ehemi-

The intimate association of a prot’ein crystal and a rod-shaped Dn’A virus part,icle or a group of virus particles is a unique relationship found wit,h the occluded insect viruses commonly referred to as the granulosis viruses (or capsules) and nuclear polyhedrosis viruses. Alt’hough considered to be of virus origin, the source and/or biological role of this prot,einaceous crystal is not presemly known. One of the major difficult,ies previously encount’ered in the chemical characterization of the virus proteinaceous crystals has been their extreme st#ability and resistance to solubilization at neutral pH. Ultrastruct,ural studies of capsules and I Robert A. Fellow. Present Chemistry, The The University kyo, Japan. Copyright

Welch address: Institute of Tokyo,

0 1972 by hcadenGo

Foundation Research Department of Cell of Medical Science, P.O. Takanawa, To-

Press, Inc.

395

396

KOHJI

EGAWA

AND MAX D. SUMMERS

cal differences in the crystal structure of proteins of the different viruses. Some studies on the kinetics of the alkali solubilization of a NPV were attempted by Aizawa (1953), who demonstrated h,y turbidimetric measurement the presence of a lag phase initial to solubilization of the cryst’al struct,ure. This lag phase was attributed to t’he presence of a possible protective surface structure of the NPV. Physical and chemical studies for the crystal proteins are few and incomplete. Molecular weight estimates for alkali-dissociated proteins were reported to be 276,000, 336,000, and 378,000 for three different NPV and about 300,000 for a granulosis virus (Bergold, 1947; Bergold, 1948). These molecular aggregat’es could be dissociated into smaller subunits by stronger alkaline treatment. RNA (Aizawa and Iida, 1963; Faulkner, 1962; Himeno and Onodera, 1969) and silicate (Estes and Faust, 1966) have also been reported as structural component*s of occluded virus crystals. The results of certain RNA studies cited above have been somewhat obscure due to alkaline conditions used to isolate the nucleic acid. Although it has been suggested that the RKA may play a role in the initial infection process (Vago and Bergoin, 1968), the specific relationship of associated components to the structural and functional aspects of these virus crystals is not presently known. In this report, as the first step for the chemical characterization of the inclusion body protein and the granulosis virus of Trichoplusia ni, methods for solubilizing the inclusion body protein in neutral conditions are investigated. A proposal on the association and formation of t’he stable capsular protein struct’ure is also discussed in view of the results from the solubilization experiments presented in this study. MATERIALS

AND METHODS

Isolation and puri$cation of the capsule. Occluded virus was purified by a combination of differential and sucrose density gra-

dient centrifugations as described previously (Summers and Paschke, 1970). Purified capsules were lyophilized and stored at 5°C under desiccated conditions. Incubation procedure and turbidity measurement. Buffers used were potassium chloridehydrochloric acid for pH 1.0-2.0, potassium phosphate-hydrochloric acid for pH 2.5, sodium phosphate-citric acid for pH 3.6-6.5, sodium phosphate for pH 7.0-7.5, sodium borate for pH 8.0-9.0, and sodium carbonate for pH 9.5-10.5. For the reaction, 0.75 ml of 0.2 M buffer (0.05 M as final concentration) and 2.15 ml of the designated solvents, such as aqueous alcohol, were placed in a 15 mm X 100 mm Pyrex test tube, st,oppered, and equilibrated t’o the desired temperature in a t’emperaturecontrolled water bath. To this mixture was added 0.1 ml of a water suspension containing occluded virus (0.8 mg/ml; also equilibrated to the desired temperature). The reaction mixture was immediately shaken with a mechanical agitator and the turbidity was measured at various time intervals after addition of the capsule suspension as optical density at 560 nm (Bausch & Lomb, spectronic 20). The turbidity at zero react,ion time was obtained by extrapolation from the OD values at 10, 20, and 30 see after init,iating the reactions. This value was within the range of a previously determined linear relationship of capsule concentrat’ion versus optical density. Calorimetric determinations. The amount of protein was determined by Lowry’s method (Lowry et. al; 1951) using bovine serum albumin as the standard. RESULTS

The time course of solubilization under a dilute alkaline condition is shown in Fig. 1. The decrease of turbidity resulted in a concomitant increase of t,he solubilized protein, showing that turbidimetric measurements directly reflect the loss of an organized cryst,al structure to that of a soluble form of

SOLUBILIZATION

Trichoplusia

OF

TIME

IN

307

ni Chndosis

MINUTES

1. The relat,ionship between turbidity and solubilization of capsular protein. (I) Time cowx of the change of the turbidity in 0.05 M Xa carbonate buffer (pH 10.0) shown as percent of the initial turbidity. (2) Time course of the amount of protein in the supernatant expressed as percent of the t,otai protein in the mixture. After incubation, the mixture was cooled to 0°C and centrifuged at 30,000 g Ear 20 min. FIG.

polymeric peptides. Figure 2 shows the effect of different’ solvents on t)he solubilizatjion of the proteinaceous crystal as determined by experiments of similar design. In most, cases there was a slight lag phase (phase I) followed by an exponentially decreasing phase (phase II) and finally a, phase of considerabl> slowe? decrease (phase III). The lag phase was most distinct during soluklization with urea, and least’ distinct at extreme pH’s. Results of a few-: the most significant, of the solubilixatiou experiments conducted in our investigations are summarized in Table 1 and Fig. 3. The efficacy of various solvents arc expressed as the reciprocal of the time in minutes necessary for a 50 % decrease in turbidity in Table 1 and as t,he logarithm of tlj2 in Fig. 3. In most cases there was a linear relationship between the logarit,hm of 11,2and pH of the solvent on both sides of the isoelect~rie point’, indicating a strict dependence of t,he solubilization on t#he hydrogen ion concentration regardless of solvent systems employed. At 25°C without additives, solubiiization is undetect)able or only parGal below pH 10.0. Limited solubility without’ additives is observed at pH’s around 1.0. Additions of alcohols also enhanced the rate of the solubilixation. The relative effectiveness of alcohols listed according to the size of the hydrocarbon moieties were as follows: met’hanol < ethanol < isopro-

TIME

IN

MINUTES

FIG. 2. Representative time course solubilixation of the occluded virus solvent systems. Curves: (1) 0.05 M Na buffer (pH 10.0, 25°C); (2) 0.05 M Ka buffer (pH 10.5, 25°C); (3) 0.05 .\I Na buffer (pH 9.5) + 1% SLS (25°C); (4) carbonate buffer (pH 9.0) + 7 M urea, Initial turbidity; 8, turbidity after cubation periods.

stildies of varicius rarbonate carbonuw carbonate 0.K M Na (25’C); AO, various inin

pan01 < normal propanol < tert’iary butano1 < normal butanol. Kormal propanoi was the most effective among these, the optimal concentration of normal propanol being about, .iO%. The relative effwtivenws of organic acids were: formic acid > propionic acid > butyric acid > acetic acid. The lack of correlation bet,ween the cffecti.veness of the acid and the size of the hydrw carbon moiety is probably due to the difference in dissociation constants. The ionic st)rengtb of the solvent also affected thy

TABLE Solvent

0.05 M Bu$ers KC1 -HCl KCI-HCl Na borate Na borate Na carbonate Na carbonate Na carbonate Detergents Nonionogenic : 1% Tween 80 1% Tween 20 1% Triton X-100 Anionic : 1% Na deoxycholate 1% SLS 1% SLS 1% SLS Alcohols 30% Methanol 60% Methanol 30% Ethanol 607, Ethanol 3Oyo lsopropanol 309?o n-Propanol 60% n-Butanol + 12% 607, tButano1 60% Isopropanol 60% lsopropanol 60% lsopropanol 60% lsopropanol 60% lsopropanol 10% n-Propanol 10% n-Propanol 10% n-Propanol 2Ooj, n-Propanol 20% n-Propanol 20% n-Propanol 4Oyo n-Propanol 407, n-Propanol 4Oyo n-Propanol 5Oyo n-Propanol 50% n-Propanol 50y0 n-Propanol 60% n-Propanol 60% n-Propanol 60% n-Propanol 60% n-Propanol 607, n-Propanol 607. n-Propanol SOY0 n-Propanol 60% n-Propanol 60% n-Propanol

System

methanol

398

1

(l/h/2)

PHa

Temp. (“C)

1.0 1.5 8.5 9.0 9.5 10.0 10.5

25 25 25 25 25 25 25

0 0 0 0 0.4 8 570

9.5 9.5 9.5

25 25 25

7 9 I1

9.5 9.0 9.5 10.0

25 25 25 25

7 0 67 2000

9.5 9.5 9.5 9.5 9.5 9.5 8.0 8.0 1.0 1.5 8.5 9.0 9.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 1.5 2.0 7.5 8.0 8.5 7.5 7.5 7.5 2.0

25 25 25 25 25 25 25 25 25 25 2.5 25 25 70 80 89 60 77 85 40 60 77 45 60 70 25 25 25 25 25 40 60 60 60

2 4 200 500 400 1000 0 0 2000 50 6 400 1000 0 0 100 0 300 1700 0 59 1300 0 133 1250 3000 83 0 40 500 0 59 2000 500

x

103b

XC

x x X X

X

X X

2 X *d X

2 X X

x

X

X

X

TABLE Solvent

l--Continued

System

Temp. (“Cl

60% n-Propanol SOY0 n-Propanol 6OcI, n-Propanol

(l/h/n) x 103*

2.5 7.0 8.0

60

60

II 500

10.5 11.0

25 25 25

5 .iQO 500

23 25 25 25

6 23 140 670

3.0 3.5 7 ..5 8.0 8.5 9.0 9.5 10.0 10.3

25 25 25 25 25 25 25 25 25 25

2000 59 0

* + *

0 19 4,5 83 71 290 3000

z 2 Lr ;P

7 .K 7.5 7.5

17 25 30

180 460 590

7.5 7.5 7.5

25 4Q 60

100 260 1300

3.5 4.0 4 .5 5.0 5.5 6.0 B..? 7.0 7.3 8.0

60 60 60 60 60 60 SO 60 2j 25

1000 330 63 16 30 105 2.X 1250 11 230

25 25 25 2.5 25 25

0 0 0

*1 * *

12 670 1000

2”

25 25 2,’ 2.5

93 200 670 300

60

4

Salt 0 .,5 rs NaCl 0.5 M NaCl 0.2 M NaCl 7 36 Urea

10.5 8.0 8.5 9.0 9 .c5

z

i; ,M Guanidine~HCl 2 5

Alcohol + salt 605& n-Propanol 60% n-Propanol 60% n-Propanol Alcohol + Urea 60% n-Propanol SO70 12.Propanol 607, n-Propanol AlcohoE + guanidine, 60% n-Propanol 60% n-Propanol 60yG n-Propanol 60% n-Propanol 60% n-Propanol 60% n-Propanol 60% n-Propanol SOY0 n-Propanol 600/, n-Propanol 605% n-Propanol C:dnuffered systems HCl 1.0 N Hcl o.ri N HCI 0.2 N XaOH

0.005

+ + +

2 x KI 2 Y KI 2 M KI

+ + +

4 x urea 4 M urea 4 M urea HCl saturated with guanidine.HCle sat,urat,ed with guanidine.HCl saturated with guanidine.HCl saturated with guanidine.HCl saturated with guanidine.HCl saturated with guanidine.HCl saturated with guanidine.HCl sat)urated with guanidine.HCl saturat’ed with guanidine.HCl saturated with guanidine.HCl

s

Xa,QH 0.010 N NaOH 0.015 N Organic Acids 3 s Formic acid 5 s Acetic acid 5 x Propionic acid 5 x Butylis acid 399

X

400

KOHJI

EGAWA

AND

MAX

TABLE-l Solvent

Other

3077, 30yo 3077, 3Oy,

D.

SUMMERS

Continued

System

fiH&

Temp. (“C)

9.5 9.5 9.5 9.5

25 25 25 25

(l/h/2) x 1035

Solvents

Phenol-30yo methanol m-cresol-30~o methanol Benzyl alcohol-30% methanol DMSO

1300 430 10,000 0

a pH values indicate the pH of the buffers used in the incubation mixture. b tl/z: Time in minutes necessary for 50% decrease of the turbidity. 0 x : Decrease of turbidity after overnight incubation was less than 90%. d *: Precipitation occurred after some period of incubation. e 607, n-propanol was saturated with guanidine.HCl at 25°C. To 2.9 ml of this water suspension of occluded virus was added.

solution,

X X X

0.1 ml of

1

0

? 5

-1

m 0

-2

-3

I

I

1

I

I

I,

!

123456789

,

I

I

I

10

11

12

PH

FIG. 3. pH dependence of solubilization of the occluded virus in various solvent systems. (1) 60% n-propanol saturated with guanidine-HCl (60°C) ; (2) 60yo n-propanol (60°C) ; (3) SOrr/, n-propanol (25%) ; (4) 60% isopropanol (25°C) ; (5) 7 M urea (25%); (6) 5 M guanidine.HCl (25°C) ; (7) 0.05 M buffer (25°C); (8) 0.05 l\n buffer + 0.5 M NaCl (25%). pH values indicate the pH’s of the buffer systems used in the reaction mixtures. tl/s: Time in minutes necessary for 50% decrease of the turbidity.

SOLUBILIZATION

OF

Trichoplusia

(a)

ni Grunzllosis

(b)

FIG. 4. Temperat’ure dependence of solubilization of the occluded virus in solvent systems containing0.05 M Na phosphate buffer (pH 7.5). (a) 60% n-propanol. (5) 60yC n-propanol + 4 M urea. (c) SOoi;, n-propanol + 2 M KI. A,, initial turbidity. A, turbidity after various incubat,ion periods. Temperature of the reaction mixtures are indicated in the graphs.

solubilization. High concentration of salt inhibitled the solubilizat8ion at, extreme pH’s, while it enhanced the solubilizat,ion at pH’s close to the isoelectric point,. Hydrogen bond cleaving agents such as urea and guanidine. Cl also increased the rat,e of the solubilizaion. In all cases studied, the rate of solubilization was temperature dependent. The most effective condition for solubilization of the capsule at a pH close to neutrality was 60% normal propanol saturated with guanidine.HCl a.t an elevated temperature. The rate of solubilization under these conditions was minimal at pH 5.2, a value similar to the reported isoelectric point#s of insect inclusion body prot’eins (Bergold, 1959). Figure 4 shows the temperature dependency of the time course of solubilization in solvents containing normal propanol and a buffer system of sodium phosphate (pH 7.5). Note that the steeper the curve during phase If, t(he lower the t’urbidity at the end of this phase. In a given solvent neither curve nor the final level of phase III after a prolonged incubation period were dependent on the capsule concentration. Plotting the final level against, the pH of the solvent resulted in a sigmoid curve with an inflection point at pH 9.5 (Fig. 5).

9.0

95

PH

10.0 105

in

5. pH dependence of the level of phase III after prolonged incubation. Solid Iine: A/A,. Dotted line: d(A/Ao)/d(pH). Ao, initial turbidity. A, turbidity aft,er incubation for 40 hr at’ 25°C. FIG.

IscussIoN

A t,heoretical interpretation of turbidimetric measurement,s is complicated. However, the validity of t,he method as used in this investigation is shown by the direct relationship between decrease in turbidity and protein solubilization. Capsular and polyhedral proteinareoue crystals are construct!ed of repeating units protein polymers, which, when solubilized, have been shown to have molecular weights in the range of 2 to 4 X 1Qj daltons, Summers and Egawa (unpubl.). The further

402

KOHJI

EGAWA

AND

dissociation of such polymers to smaller subunits appears not to be detectable by optical density measurements as used in this study. The change in turbidity of a capsule suspension, therefore, represents the solubilization of the crystal to the protein polymers. Curves relating the change in turbidity with time exhibit three phases as shown in the results. The shape of the curve and the level of phase III after prolonged incubation in a given solvent are independent of the concentration of the capsule: This indicates that phase III is due to the heterogeneity of capsules or capsule fragments in resisting solubilizat.ion. This fact was demonstrated by the sigmoid curve relating pH to the level of phase III after extended incubation. The differential of the curve indicates a normal distribution of this attribute. The level of phase III is thus not due to saturation of

MAX

D.

SUMMERS

the solvent with capsular protein. Consequently, the rate of disolution in phase II is also independent of solvent saturation, and this makes the calculation of a valid rate constant possible. Capsular protein contains an extremely high percentage of amino acids with hydrocarbon side groups (Summers and Egawa, unpubl.). Thus hydrophobic binding forces may be expected t’o play a considerable role in the stability of the proteinaceous crystals. In phase II, solubilization appears to follow first order reaction kinetics. The rate constants under various conditions of solubilization during this phase were calculated. As shown in Fig. 6, the logarithm of the rate constants (log k) shows a linear relationship with the reciprocal of absolute temperature (l/T). From the linear relationship a mean acbivation energy of the dissociation reaction

lr

FIG. 6. Temperature dependence of rate constants of solubiliaation of the occluded solvent systems containing 0.05 M Na phosphate buffer (pH 7.5). (1) 10% n-propanol; nol; (3) 4O’j& n-propanol; (4) 50% n-propanol; (5) 60% n-propanol; (6) 60% n-propanol 60% n-propanol + 2 M KI. k, Rate constants for the solubilization during phase II. perature.

virus in various (2) 200% n-propa-I- 4 M urea; (7) T, absolute tem-

SOLUBILIZATION

OF

Tkhoplusia

(kcal/mole of the soluble protein polymers) was calculated using the Arrhenius (1889) equation. From t,he values obtained using various concentrations of normal propanol in buffer (pH 7.5), the activation energy in pure buffer ($2 7.5) was estimated by extrapolation to be about 55 kcal/mole (Fig. 7). The value in 60 70 normal propanol (pH 7.5) decreased from 37 kcal/mole to 13 and 17 keal/mole with the addit’ion of concentrated salt (2 N potassium iodide) and hydrogen bond cleaving agent (4 M urea), respectively. The values should be considered only as est,imates because the precision of these calculat)ions is as yet unresolved. It can be concluded, however, that hydrophobic and hydrogen binding forces play the major roles in the formation and stabilization of t’he crystalline structure. Ionic binding forces contribute comparatively lit’tle to the total energy. In an alkaline environment, all t,he charged groups, including hydrogen-bonded groups, become negatively charged. The electrostatic repulsion between these groups then exceeds the hydrophobic binding forces. The addition of a monovalent salt under this condition neutralizes the repulsive charge to a significant extent and results in an inhibition of the solubilizat8ion. At pH values close to the isoelectric point, positively and negatively eharged groups will attract each other to give maximum stability t’o the crystal structure. Addition of a salt under these conditions neutralizes t’he electrostatic charges to enhance the solubilizat’ion. Combination of guanidine.IICl, a salt which has hydrogen bond cleaving activity, and a hydrophobic bond cleaving agent such as normal propanol results in the most effective solubilizing agent at neutral pH. Urea is more effective than guanidine.HCl at pH’s higher than 9.0, but is less effective at pH’s lower than 8.5 (Fig. 3). Contrary t)o a previous study (Estes and Faust, 1966), our investigations do not, indicate a dependency upon carbonate ions for solubilization of capsules.

ni

Ql

403

i?ranulosis

0 TO 20 30 40 50 00 % OF

n-PROPANOL

FIG. 7. Activation energy the occluded virus in solvent 0.05 M Na phosphate buffer concentration, of n-propanol. ergy in kcal per mole of the calculated using the Arrhenius

for solubilization of systems containing (pH 7.5) and various The activation enprotein particle were equation

In addition, if silicate or ISA have a structural significance in the crystal, the contribution of ionic bonds to the tot)al binding energy would be expected t’o be greater. Aside from having extablished met,hods for dissociation of capsules at or near neutrality and elucidating some of the forces responsible for proteinaceous crystal structure, this study has provided a basis upon which inferences can be made concerning the process of crystal removal and release of infectious viruses in the gut lumen of lepidopterous insect’s. More significantly, ! his and further studies on the mechanism of assembly of monomeric polypeptides int,o the prot’ein polymers will be of general interest as a model of self-assembly of hydrophobic biopolymers. ACKNOWLEDGMEXT We would like to acknowledge the helpful suggestions given by Dr. J. Brown, Dr. P. Munk, and Dr. C. Y. Kawanishi. This study was supported by IX. A. Welch Foundation Grant No F-412 and PHS Grant AI 09765.

Amawa, K. 1953. 17, 145-154. (In mary.)

Jap. J. Appl. Japanese

with

Edomol. English

Zooi.,

su.m-

404

KOHJI

EGAWA

AND

AIZAWA, K., AND IIDA, S. 1963. Nucleic acid extracted from the virus polyhedra of the silkworm, Bombyx mori (Linnaeus). J. Insect Pathol., 5, 344-348. ARNOTT, H. J., AND SMITH, K. M. 1968. An ultrastructural study of the development of a granulosis virus of the moth Plodia interpunctella (Hbn.). J. Ultrastruct. Res., 21, 251-268. ARRHENIUS, S. 1889. Ueber die Reactionsgeschwindichkeit bei der Inversion von Rohrzucker durch SSiuren. Hoppe-Seyler’s 2. Physiol. Chem., 4, 226-248. 1947. Die Isolierung des BERGOLD, VON G. Polyeder-Virus und die Natur der Polyeder. 2. Naturforsch. B, 2, 122-143. BERGOLD, VON G. 1948. Ueber die KapselvirusKrankheit. Z. Naturforsch. B, 3, 338-342. BERGOLD, G. H. 1959. Biochemistry of insect viruses. In The Viruses. (Burnet, F. M., and Stanley, W. M., eds.) Vol. I, pp. 5055523. Academic Press, New York. DAY, M. F., COMMON, I. F. B., FARRANT, J. L., and Potter, C. 1953. A polyhedral virus disease of a pasture caterpillar, Pterolocera amplicornis Walker (Anthelidae). Austral. J. Biol., 6, 574-579. ENGSTROM, A., and KILKSON, R. 1968. Molecular organization in the polyhedra of Porthetria dispar nuclear polyhedrosis. Exp. Cell Res., 53, 305-310.

MAX

D.

SUMMERS

ESTES, Z. E., and FAUST, R. M. 1966. Silicon content of insect nuclear polyhedra from the corn earworm, Heliothis zea. J. Invertebr. Pathol., 8, 145-149. FAULI~NER, P. 1962. Isolation and analysis of ribonucleic acid from inclusion bodies of the nuclear polyhedrosis of the silkworm. Virology, 16, 479-484. HIMENO, M., and ONODERA, K. 1969. RNA isolated from polyhedra of a nuclear polyhedrosis of the silkworm. J. Invertebr. Pathol., 13, 87-90. IGNOFFO, C. M., and DUTICY, S. R. 1963. The effect of sodium hypochlorite on the viability and infectivity of Bacillus and Beauveria spores and cabbage looper nuclear polyhedrosis virus. J. Insect Pathol., 5, 422-426. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. 1951. Protein measuurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-275. SUMMERS, M. D., and PASCHKE, J. D. 1970. Alkali-liberated granulosis virus of Tricoplusia ni. I. Density gradient purification of virus components and some of their in vitro chemical and physical properties. d. Invertebr. Pathol., 16, 227-240. VAGO, C., and BERGOIN, M. 1968. Viruses in invertebrates. In Advan. Virus Res., 13, 247303.