Pardaxin, a hydrophobic toxin of the Red Sea flatfish, disassembles the intact membrane of vesicular stomatitis virus.

Reaction of vesicular stomatitis virus with pardaxin, the hydrophobic toxin of the Red Sea flatfish, resulted in a profound morphological change of many virions and dissociation of their membrane and nucleocapsid into components readily separable by density gradient centrifugation. The basic matrix protein and acidic pardaxin segregated largely with the high density nucleocapsid. The dissociated virion membrane formed lipoprotein vesicles which retained glycoprotein spikes and a certain amount of N protein but no appreciable amounts of other nucleocapsid proteins and little if any RNA. Iodination of the tyrosine residue of the glycoprotein tail fragment provided supporting evidence that the COOH terminus of the glycoprotein extends beyond the inner layer of the membrane into the interior of the virion. These data indicate that pardaxin may serve as a probe for studying the organization of viral membranes, and, hopefully, other biological membranes.

This research was supported by Grants PCM77-00494 from the National Science Foundation, MV-9E from the American Cancer Society, AI-11112 and HL-17576 from the National Institutes of Health, and 2669/81 from the United States-Israel Binational Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. The abbreviations used are: VS, vesicular stomatitis; G protein, viral glycoprotein; M protein, viral matrix protein.
creted from the skin of the Red Sea flatfish Pardachirus marmoratus (Soleidae), is an acidic protein of M , = 17,000 composed of 162 amino acids (6, 7). It is rich in hydrophobic amino acids, including leucine (18 residues), phenylalanine (13 residues), isoleucine (11 residues), and valine (5 residues) but is devoid of arginine, histidine, and tryptophan (6). Pardaxin exhibits strong interaction with lipid bilayers and makes vesicle membranes permeable as evidenced by leakage of entrapped 6-carbo~yfluorescein.~ As previously described (8), plaque-purified VS virus of the Indiana serotype was used to infect baby hamster kidney-21 cells at a multiplicity of 0.1 plaque-forming units/cell. Homogeneous bullet-shaped virus harvested at 21 h postinfection was purified by differential, rate zonal, and equilibrium centrifugation in sucrose and tartrate gradients (5) Fig. 1 reveals that pardaxin profoundly alters the morphology of VS virions (50 pg) when incubated at 20 "C for 10 min with 20 pg of the purified toxin in 0.2 ml of phosphate-buffered saline, pH 7.4. Many of the typically bullet-shaped virions ( Fig. L 4 ) assumed a spherical shape and a large proportion of the internal nucleocapsids were extruded (Fig. 1B). Dissociated virion membranes assumed the shape of large lipid vesicles surrounded by glycoprotein spikes.
We next attempted to isolate and characterize the VS virion membrane vesicles from which the nucleocapsid cores had been extruded by the action of pardaxin. To this end, three suspensions of VS virions (300 pg/ml each) were treated for 10 min with 120 pg of pardaxin and then made 50% with respect to sucrose; this suspension was overlayered with a 10-50% continuous gradient of sucrose and centrifuged in the SW 27.1 rotor at 52,400 X g for 16 h. To identify by density flotation the location of virion RNA, proteins, and lipids, separate samples of VS virus had been biologically labeled with [3H]uridine, [3H]leucine, or [3H]palmitate. As shown in Fig. 2, most of the [3H]palmitate floated to the top of the gradient at p = 1.06 g/ml but a substantial amount of [3H] palmitate banded at p = 1.15 g/ml, presumably representing undisrupted virions which banded at this same density (data not shown). In contrast, only -20% of [3H]leucine-labeled viral protein floated to the top of the gradient, whereas the largest amount of protein banded at p = 1.2 g/ml, equivalent to that of delipidated nucleocapsids. The C3H]uridine label, representing viral RNA of pardaxin-extruded nucleocapsids, also banded at an equivalent density of 1.2 g/ml (data not shown). These experiments indicate that pardaxin dissociates VS virions into a lipoprotein fraction of low density and a ribonucleoprotein fraction of high density with a certain proportion of undisrupted virions of intermediate density.
Samples collected from the top three fractions of the sucrose gradient depicted in Fig. 2 were examined by negative stain electron microscopy.  density vesicles, varying in sue from large to very small resealed membrane fragments, all of which exhibit protruding spikes clearly analogous to those extending from the surface of intact virions (Fig. IA).
The protein composition of pardaxin-treated VS virus lipidrich fractions from the top of the gradient ( p = 1.06 g/ml) and the RNA-rich fractions from the bottom of the gradient ( p = 1.2 g/ml) was determined by electrophoresis on 12.5% polyacrylamide-sodium dodecyl sulfate gels. Gels stained with Coomassie blue were scanned by densitometry and the concentrations of each viral protein were determined by integration. Fig. 3 shows the electropherograms and Table I compares the relative concentrations of the five viral proteins in the low density and high density fractions with that of unfractionated virus. As noted, the high density nucleocapsid contains all five viral proteins but has a reduced concentration of G protein and was relatively enriched in N protein; as expected, this fraction was heavily contaminated with VS virions undisrupted by pardaxin, presumably accounting for the presence of G protein. By comparison, the low density lipid-rich fraction contained predominantly G protein. We are somewhat at a loss to explain why so much N protein was present in the low density vesicle fraction despite failure to detect appreciable amounts of nucleocapsid L and NS proteins. It is possible that pardaxin partitions into the hydrophobic regions of the nucleocapsid, thus dissociating some N protein to a monomeric form that can associate with membrane vesicles. We had previously shown that hydrophobic aryl azide probes covalently bind to N protein of intact VS virions (4). No intact nucleocapsids could be detected by electron microscopy of lipid-rich viral membrane vesicles dissociated with pardaxin. Most importantly, we could not detect any multilayer vesicles by electron microscopy which indicates that pardaxin does not release from the virion membrane appreciable amounts of free lipid that would form multilayer vesicles (bangasomes) under these conditions.
Of considerable interest is the observation that the M protein segregated with the nucleocapsids after pardaxin dissolution of virions and none could be detected in the low density virion vesicles (Table I). Moreover, most of the M , = 17,000 pardaxin which is readily detectable on polyacrylamide gels was found associated with the high density nucleocapsid and none was present in the vesicle fraction dissociated from the virions (Fig. 3). This finding can perhaps be attributed to charge interaction and association of the acidic pardaxin (6) with the basic M protein, which has a PI > 9.0 by isoelectric focusing (9). It should be noted that small amounts of M  (0). Purified VS virions (300 pg) were exposed for 10 min to pardaxin (120 pg) and made 50% with respect to sucrose. After overlayering with a 10-50% sucrose gradient containing 1 M NaCI, 50 m~ Tris-HCI, 1 mM EDTA (pH 7.5). the virion-pardaxin mixture was centrifuged at 52,400 X g for 16 h at 4 "C. Gradient fractions were analyzed for radioactivity by scintillation spectrometry and for density by refractometry. . 300 pg of purified VS virions labeled with ["HI palmitic acid or ["Hluridine were allowed to react with 120 pg of pardaxin or with buffer (intact virions) and subjected to equilibrium sedimentation by flotation through a 10-5056 sucrose gradient as described in Fig. 2. Three fractions containing ['Hluridine label were collected from the bottom of the gradient and pooled, whereas fractions 19, 20, and 21 containing [.'H]palmitate label were collected from the top of the other gradient and pooled. Intact virions were collected from fractions 9, 10, and 11 from a separate gradient. Densities of peak fractions were measured by refractometry. Fractions were dialyzed free of sucrose and lyophilized. Proteins from each sample were extracted by boiling in sodium dodecyl sulfate and were analyzed by electrophoresis on a Tris-glycine-buffered polyacrylamide slab gel consisting of a 2-cm stacking gel of 4% acrylamide and a resolving gel of 12.5% acrylamide. After fixation for 30 min in 50% methanol and 10% acetic acid, the gel was stained for 1 h with 0.1% in the nucleocapsid fraction ( N O as determined by electrophoresis of the purified protein on a separate gel (not shown).

Coomassie
protein and pardaxin could have been present in other regions of the gradient that were not examined by polyacrylamide gel electrophoresis. These studies support the hypothesis (2,4,5, 10) that the M protein of both rhabdoviruses and paramyxoviruses serves as the "glue" that binds the nucleocapsid to the lipoprotein membrane of VS virus during the process of assembly and budding from the infected cell surface membrane.
The M protein may have a greater affinity for the nucleocapsid than for the membrane as judged by its segregation during pardaxin-mediated fractionation of the two components of VS virions. A similar preferential association of M protein with nucleocapsids was found after VS virions were exposed to octylglucoside or Triton X-100 in the absence of salt (11).
An important question about the VS virion membrane is whether the intrinsic G protein traverses the bilayer and, if so, does the COOH terminus protrude from the inner surface? The capacity of pardaxin to dissociate the VS virion membrane, thus exposing the inner surface, seemed to provide a means to determine the interior location of the G protein. The primary amino acid sequence of the VS viral G protein is known from the studies of Rose et al. (12) who determined the nucleotide sequence of the cDNA copied from the G protein mRNA. According to their model, there is a hydrophobic domain of 20 amino acids extending from position 30 to 49 from the COOH terminus, ending in a COOH-terminal sequence of 29 hydrophilic amino acids. There is one tyrosine residue at position 11 from the COOH terminus; the next tyrosine is a t position 111 (12). Therefore, it seems feasible by proteolytic digestion of the major external segment of the G protein, followed by selective iodination of hydrophobic tail segment, to determine whether the COOH-distal segment does indeed protrude beyond the inner membrane surface. For this purpose, VS virions were labeled with by the chloramine T oxidation method as well as by the lactoperoxidase method (13) after digesting away the outer 90% of the G protein with thermolysin (3). Table I1 shows that the 66amino acid G protein tail fragment generated by exposure of virions to thermolysin was specifically and heavily labeled with ' "I in vesicles prepared from pardaxin-treated virions as determined by electrophoresis on a 17.5% sodium dodecyl sulfate-polyacrylamide gel (14). The G protein tail fragment of untreated virions was also labeled to some extent probably due to permeabilization and mechanical disruption during protease treatment of the virions. However, iodination of the tail fragment was 2-3-fold higher in the presence of pardaxin compared to untreated controls. These data suggest that the COOH terminus of the G protein protrudes from the inner surface of the VS virion bilayer a t least as far as the tyrosine residue a t position 11. It should be noted that there are also two histidine residues at positions 18 and 25 from the COOH terminus, both of which presumably protrude inward from the bilayer and could also be iodinated, but at lower efficiency.
Although detergents have served as useful tools for studying membrane dissolution and in vitro transcription of negative strand viruses, it is unlikely that this reaction mimics the

Comparative protein composition of high density nucleocapsids and low density membrane vesicles prepared from pardaxintreated VS virions
The percentage of each viral protein (L, G , N, NS and M) present in the intact virions, nucleocapsids, and vesicles of the electropherograms shown in Fig. 3 was determined by densitometry and integration.  viral protein) for 30 min at 37 "C and then purified by centrifugation in a 10-40470 sucrose density gradient. Control and pardaxin-treated spikeless virus was iodinated by chloramine T or by lactoperoxidase as described by Moore et al. (13). Iodinated samples were subsequently dialyzed overnight against phosphate-buffered saline (pH 7.4) and then subjected to 17,5% polyacrylamide gel electrophoresis to separate the hydrophobic piece of G protein (13). The gel fraction containing the stained G protein tail fragment was analyzed for radioactivity by scintillation spectrometrv. -" _I.

Mode of iodination Thermolysin
Thermolysin + alone pardaxin cpm x 10' cpm X 10' events that lead to dissociation of virion membrane and nucleocapsid when virus penetrates the host cell as infection is initiated. We have recently demonstrated that pardaxin at low doses can induce in vitro transcription of VS virions in the absence of detergents and at somewhat greater efficiency (15). The studies presented here suggest that pardaxin may provide a more physiological means for studying in vitro the dissociation of virion membrane and nucleocapsid that more closely resembles the intracellular events leading to initiation of infection.