192

Biochimica et Biophysica Acta, 4 9 6 ( 1 9 7 7 ) 1 9 2 - - 1 9 6 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 28122

INHIBITION OF VIRUS-INDUCED PLAQUE F O R M A T I O N BY ATOXIC D E R I V A T I V E S OF P U R I F I E D COBRA N E U R O T O X I N S

K E N T D. M I L L E R , G E O F F R E Y G. M I L L E R , M U R R A Y S A N D E R S a n d O L I V E R N. FELLOWES

Departments o f Medicine and Microbiology, University o f Miami School o f Medicine, Miami, Fla. 33152 and the Sanders Medical Research Foundation, Boca Raton, Fla. 33431 (U.S.A.) (Received J u n e 4 t h , 1 9 7 6 )

Summary Venom from Naja naja siamensis was resolved into 16 toxic and nontoxic fractions by chromatography on SP-Sephadex, C-25. The principal neurotoxin preparations were chromatographically and electrophoretically homogeneous. Of the purified constituents, only the principal neurotoxin and minor neurotoxins were precursors of inhibitors of plaque formation among baby hamster kidney fibroblasts infected with Semliki Forest Virus.

Introduction

Subspecies of cobra venom contain a variety of biologically active constituents among which are (a) p o t e n t curarimimetic neurotoxins [1--3], (b) a complement inactivating factor [4,5], (c) phospholipases [6,7], (d) a nerve growth stimulating factor [8,9], (e) a noncytotoxic, mast cell degranulating substance [ 1 0 ] , and (f) cardiotoxins [11]. Whole cobra venom, detoxified by oxidation, also inhibited plaque formation induced by Semliki Forest Virus (SF virus) added to tissue culture cells [ 1 2 ] . The oxidized venom, called modified neurotoxin, was investigated as a protective agent for animals with experimental virus infections [13,14] and as a therapeutic agent for patients with recurrent herpetic corneal lesions [15] and amyotropic lateral sclerosis [16]. The present studies were undertaken to determine which constituents of cobra venom are responsible for the antiviral activity of modified neurotoxin. Purification studies establish that the neurotoxins are the only precursors of inhibitors of SF virus-induced plaque formation in tissue culture cells. Abbreviations: SF virus, Semliki Forest Virus; p.f.u., plaque-forming unit; BHK-21, baby hamster kidney fibroblasts.

193 Materials and Methods

Venoms and chromatographic fractions: Lyophilized venoms from the cobra, Naja naja siamensis, and the krait, Bungarus multicinctus, were obtained from the Miami Serpentarium, {Miami, Fla.). The cobra venom was chromatographed on SP-Sephadex, C-25, according to Chatman and DiMari [17], but with linear salt gradients flattened by doubling the developing buffer volumes (Fig. 1). The principal neurotoxin (Peak VI, Fig. 1) occasionally displayed a small amount of impurity at the leading edge of its peak. Such fractions required rechromatography, employing linear salt gradients generated from equal volumes of 50 mM phosphate buffers (pH 6.0) containing, respectively, 0.115 M and 0.53 M Na + as regulated by addition of NaC1 {Fig. 2). Analytical: Neurotoxic fractions were identified by the characteristic respiratory paralysis induced in 20 g female mice receiving 0.5 ml intraperitoneal injections containing 5 pg of each pooled, chromatographic fraction. The LDs0 of the major neurotoxin {Fraction VIb), was calculated according to Reed and Muench [18]. Determination of purified neurotoxin concentrations utilized the coefficient, A~em (1.0 mg/ml = 1.06 [19]). Purity was monitored by the second ion 279nm exchange column described above and by disc electrophoresis at pH 4.0 [20]. Oxidative detoxifications: Aliquots of the neurotoxic, chromatographic fractions and of mixtures of whole cobra and krait venoms [16] were adjusted to pH 7.4 and oxidized in 0.17 M H20~ and 30 pM CuSO4. The purified neurotoxins were completely inactivated in 24 h while 6 days were required for

ABSORBANCE (279 nm)

~fL "I × 104

bO VI 5.0

I

4.0

30

v,,lll

2.0

_k,, xWx,

1.0

0

I

I

I

100

200

300

!

400

I

I

I

I

500

600

700

800

900

EFFLUENT (ml)

GRADIENT Fig. 1. Chromatographic separation o f constituents in 0.5 g N. siamensis v e n o m on 2.5 X 20 c m columns of SP-Sephadex, C-25, previously equilibrated with 50 m M sodium phosphate buffer ( p H 6.0). A linear elution gradient was generated from 500 m ! equilibration buffer, 1.0 M with respect to NaCl ( p H 6.0), added to an equal vol. of equilibration buffer.

194 ABSORBANCE (279 nm'.'

M~,z_l

501

" 1°4 VIb

3(? 2O 10 0

~ 1@0

I 200

I 300

I 40(~

I 500

I 600

I 700

0

EFFLUENT Imll Fig. 2. S e p ~ a t i o n o f t h e p~ineip~l n e u r o t o x i n o f N. siemensis v e n o m f r o m a c o n t a m i n a n t . T h e 8PS e p h a d e x , C-25, c o l u m n ( 2 . 5 X ~-0 era) w ~ e q u i l i b r a t e d w i t h 50 m M s o d i u m p h o s p h a t e b u f f e r ( p H 6 . 0 ) m a d e 0 . 1 1 5 M w i t h respect t o N a + b y a d d i t i o n o f N a C I . T h e p o o l o f t o x i n a n d c o n t a m i n a n t , f r o m a prior c h r o m a t o g r a p h i c separation, w a s d i l u t e d t w o - f o l d w i t h w a t e r a n d a p p l i e d t o t h e c o l u m n . Linear gradient elution involved 3 7 5 m l 5 0 m M s o d i u m p h o s p h a t e b u f f e r , 0 . 5 3 M w i t h r e s p e c t t o N a + b y a d d i t i o n o f NaC1 (pFI 6 . 0 ) , a d d e d t o a n e q u a l vol. o f e q u i l i b r a t i o n bt~ffer.

detoxification of the whole venoms. Catalase was added (final concentration 60 #g/ml) to stop each reaction. The oxidized fractions were then adjusted to pH 6.8, dialyzed against 15 mM phosphate buffer (pH 6.8) containing 0.12 M NaCl, and sterile filtered. Following initial observations that SF virus-induced plaque inhibition was unique to the neurotoxic fractions, aliquots of the nontoxic chromatographic fractions were also oxidized. They were then re-examined for SF virus-inhibition to ascertain whether antiviral activity might be induced nonspecifically among nontoxic as well as toxic venom constituents. Inhibition of SF virus-induced plaque formation: Aliquots (0.2 ml) of each sterile, purified venom fraction, oxidized neurotoxin, or oxidized whole venom mixture, were added to separate monolayers of BHK-21, clone 13 cells (Flow Laboratories) in 25 cm 2 flasks. The flasks were incubated 1 h at 37°C, and 0.2 ml of 10-fold dilutions (10-s--10 -~) of Semliki Forest virus {American Type Culture Collection), propagated on BHK-21 cells, were then added to the treated and untreated cells. One virus dilution was added per flask and each set of conditions was duplicated. After an additional 1 h at 37°C each flask received 8 ml of plaquing overlay medium. The inverted flasks were held 48 h at 37 ° C, and the plaques were visualized by treatment of the cells with 1o0 mg/ml neutral red (0.2 ml per flask) for 10 h at 37°C. Plaque-forming units (p.f.u.) were determined from the plaque count of each flask multiplied by the dilution of the stock virus added to that flask (in 0.2 ml). Results

In addition to the major neurotoxin (Peak VIb, Fig. 2), materials represented by chromatographic Peaks V, Via and VIII (Figs. 1 and 2) were also neurotoxic, perhaps confirming the report by Karlsson et al. of three minor neurotoxins in

195

N. siamensis venom [19]. However, Peak Via was not observed consistently

nor was toxic activity always present in Peak V. Also, fractions from the ascending limb of Peak VII were nontoxic, whereas pooled Peak VII contained toxin, presumably overlapping activity from invariably toxic Peak VIII. Therefore in this laboratory, Fractions VIb and VIII are considered, respectively, the invariant principal and minor neurotoxins. Fraction VIb preparations were homogeneous by disc electrophoresis and yielded LDs0 values of 1.1--1.5 #g per 20 g mouse. The SP-Sephadex chromatographic systems described herein result in concentration as well as separation of venom constituents. Since (a) the final concentration of cobra venom present in the clinical toxoid, modified neurotoxin, is 1% [16] and (b) 0.5 g of whole venom samples were applied to the first chromatographic columns employed in the present studies, each pooled, chromatographically purified constituent would be present at the same concentration as it exists in modified neurotoxin if it is diluted to 50 ml. Under the conditions described, each pooled chromatographic fraction was in the range 30--60 ml. Therefore, no dilution or concentration procedures were necessary. The only chromatographically purified venom fractions that inhibit SF virusinduced plaque formation are the detoxified neurotoxins (Table I). For example, the derivative of the principal neurotoxin (Fraction VIb) blocked formation of more than 99% (106 p.f.u.) of plaques formed by the uninhibited virus (108~ p.f.u.). Derivatives of the minor neurotoxins (Peaks V and VIII) gave similar results (Table I}. The rather weak plaque inhibition obtained with oxidized Fraction VII was probably due to a small a m o u n t of overlapping neurotoxin from Fraction VIII (Fig. 1).

TABLE I EFFECTS OF COBRA VENOM FRACTIONS ON SF VIRUS-INDUCED PLAQUE FORMATION Substance addcd

p.f.u. *

Buffer

108.3

Fraction I (native)

108.3

F r a c t i o n II ( n a t i v e )

108.4

F r a c t i o n III ( n a t i v e )

108.3

F r a c t i o n IV ( n a t i v e )

108.7

Fraction V (oxidized)

106.5

Fraction VIb (oxidized)

106.0

F r a c t i o n VII ( o x i d i z e d )

107.9

F ~ a c t i o n VIII ( o x i d i z e d )

106.0

F r a c t i o n s IX, X ( n a t i v e )

108.2

F r a c t i o n s XI, XII, XIII ( n a t i v e )

108"1

Fractions XIV, XV (native)

108.3

M o d i f i e d n e u r o t o x i n ( L o t 1 5 2 ) **

106.4

Modified neurotoxin (Lot 153)

107.0

* P l a q u e - f o r m i n g u n i t s i n d u c e d b y 0 . 2 m l S e m l i k i F o r e s t V i r u s o n B H K - 2 1 cells. ** O x i d i z e d w h o l e v e n o m .

196

Discussion The ability of oxidatively detoxified cobra neurotoxins to inhibit SF virusinduced plaque formation in tissue culture cells appears specific, based on failure of all other venom constituents, whether oxidized or not, to block cell injury. This suggests that the neurotoxin molecules are precursors of the virusinhibiting material also present in oxidized whole venom (modified neurotoxin). The stronger virus inhibition by purified toxin derivatives compared to the modified neurotoxin (Table I) may be due to the long oxidation times (6 days) required for inactivation of the toxic activity in whole venom [16] compared to those for inactivation of the purified toxins (24 h}. Yet to be resolved is the relationship between the virus-inhibiting function of the detoxified, purified neurotoxins, described herein, and evidence that oxidized whole venom (modified neurotoxin) prevents some neurotropic virus infections in experimental animals [13,14], and, possibly, alters the progress of amyotropic lateral sclerosis [16]. The latter studies may now be reevaluated employing purified toxin derivatives. Continued correlations between clinical and laboratory phenomena should lead to the biochemical mechanisms of action based on the substantial information extant concerning animal toxins. For example, the 71-residue amino acid sequence of the principal neurotoxin ofNaja naja siamensis (Fraction VIb) is known [2]. Finally, SF virus-induced plaque formation offers a useful end-point for chemical delineation of the active sites on the neurotoxins responsible for virus inhibition and for clarification of the relationship between those sites and the toxic sites. Several microbial and plant toxins give evidence that distinct sites may be responsible for toxicity and cell membrane-binding functions [21--23]. Acknowledgements Supported by the John A. Hartford Foundation. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

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