VIROLOGY 48, 360-372 (1972)

Membrane-Bound Proteins of Jiapanese Encephalitis Virus-Infected

Chick Embryo Cells

DANIEL SHAPIRO, KATHLEEN KOS, WALTER E. BRANDT, AND PHILIP K. RUSSELL

Department of ‘VGXS Diseases, Walter Reed Army Institute of Research, Washington, D.C. dOOld

Accepted January 18, 197.2

The seven Japanese encephalitis virus specific polypeptides found in infected chick embryo cells were all bound to membranes. None were completely released from the membranes by treatment with neutral salt, alkaline salt, or dilute detergent, but two of them were partially released by both the neutral and alkaline salts. The poly- peptides were released or attacked by trypsin at unequal rates and in the sequence: NV-5 2 NV-4 > V-3. NV-5 was released as a relatively undegraded soluble poly- peptide, NV-4 was extensively degraded, and V-3 was degraded but part of its trypsin- derived fragment (TF-2) remained membrane bound. We suggest that the three largest viral polypeptides are bound in such a manner that the larger the polypeptide, the more exposed and superficial it is. Treatment of virions with trypsin produced low molecular weight material and three discrete polypeptide fragments, probably all derived from the large virion envelope protein V-3; two (TF-1 and TF-3) had electro- phoretic mobilities similar to the two naturally occurring nonvirion virus-specified polypeptides, NV-l and NV-3.

INTRODUCTION

Japanese encephalitis virus (JEV) is a group B arbovirus composed of three poly- peptides (Shapiro et al., 1971), the largest of which is a glycoprotein (Stellar, 1969; Shapiro et al., unpublished data). Electron microscopic studies of infected cells (Murphy et al., 1968; Ota, 1965) have indicated that viral morphogenesis occurs in intimate association with internal cellular mem- branes, neither “free” cytoplasmic struc- tures, such as viral cores, have been de- scribed, nor has viral “budding” through plasma membranes been observed as in group A arbovirus-infected cells (Acheson and Tamm, 1967, 1970). Instead, electron- dense, membrane-bound, round structures, about 27 nm in diameter, form on cyto- plasmic vacuoles; they penetrate the vacuoles and become enveloped to form apparently mature virions within the vacuoles.

JE-infected cells synthesize seven virus-

specified polypeptides (Shapiro et al., 1971). In this paper we have examined some aspects of the binding of these polypeptides to internal cellular membranes of infected chick embryo cells.

MATERIALS AND METHODS

Preparation of membranes. Methods for infection of chick embryo cells with JEV in the presence of actinomycin D, and for pulse inhibition with cycloheximide have been described (Shapiro et aZ., 1971). Briefly, infection of confluent monolayers in 32-0~ prescription bottles was at 0 hr, actinomycin D (1 pg/ml) was added at 9 hr, cyclohexi- mide (500 fig/ml) was added at 18 hr, medium was replaced with cycloheximide- free medium at 18.5 hr, and radioactive amino acids were added from 19 through 23 hr. The cells were scraped off with a rubber policeman into 0.25 M sucrose [unless other- wise indicated all sucrose solutions were made in RSB-0.01 M tris(hydroxymethyl)-

360 Copyright 0 1972 by Academic Press, Inc.

MEMBRANE-BOUND PROTEINS OF JEV-INFECTED CHICK CELLS 361

aminomethane (Tris), 0.01 M l\‘aCl, 0.0015 M ;\lgCl~, pW 7.41 at 4°C and broken with 25 strokes of a *i-ml tight-fitting Dounce homogenizer (Kontes Glass). The material was centrifuged at 2000 rpm (450 g) for 2 min in the Sorvall SS-34 rotor. The supernatant was then centrifuged through 0.3 M sucrose onto a 2.6 M sucrose cushion at either 65,000 rpm (300,000 g) for 45 min in the Beckman SW 65 rotor, at’ 50,000 rpm (203,000 y) for 1 hr in the SW 5OL rotor or at 40,000: rpm (106,000 9) in the type 40 rotor for 1.5 hr. The membranous material was then col- lected, resuspended with a Pasteur pipette and used dire6tly for experiments as “crude membranes.” When purified membrane bands were desired as starting material, “crude membranes” were made 1.33 M in sucrose and then placed on the discontinuous sucrose gradient shown in Fig. 1, which is a modification of previously described methods (Uhr and Schenkin, 1970; Bosmann et al., 1968; Caliguiri and Tamm, 1970; Spear et al, 1970). Centrifugation was a-b 65,000 rpm (300,000 9) for 1.5 hr in the SW 65 rotor; O.l-ml aliquots were collected by bottom puncture.

Treatment and analysis of mem,branes.

The various fractions were diluted with &her NaCl, KaHC03 + Na2C03, KC1 or NP-40 (Shell Chemical Co.) or else were incubated with t,rypsin :2X recrystallized, lyophilized, salt-free, Mann Research) as indicated in the text. Subsequently, the membranes were diluted at, least 25-fold and pelleted at 50,000 rpm for 60 min in the SW 5OL rotor or else were fractionated on the discontinuous sucrose gradient shown in Pig. 1.

Pelleted material was dissociat,ed with sodium lauryl sulfate (SLS) and 2-mercnpto- ethanol (2-ME) and electrophoresed on 8% po!yacrylamide gels (PAGE) as previously described (Shapiro et al., 1971). Determina- tion of radioactivity in the gel. slices was as described previously (Shapiro et al., 1971) except, that the NCS (Amersham Searle), LiquiAuor (Iiew England Nuclear), and t.oluene were added simultaneously to the vials, which were then incubated at 37” overnight and counted. All radioactive I- amino acids were obtained from New Eng-

land Nuclear and consisted of a synthetic mixture of 15 I-amino acids, either 311 or 14C~

P~roductim of radioactive JE vii-iom. JEV was propagated in either LLC-MI& or chick embryo cells, labeled with radioactive amino acids, and purified by r&e zona! centrifuga,- tion as previously described (Shapiro et a!., 1971).

RESULTS

SpeciJicity of membrane binding

Most of the virus-specified radioactive proteins in JEV-infected chick embryo cells were associated with sedimentable structures that could be solubilized by treatment with a detergent (1% -Uonidet P-40) (da$a not shown). Furthermore, when t!he nonsedi- mentable proteins of infected ceils (those proteins not sedimenting at 300,OO~ g for 60 min in the absence of detergent) were analyzed by polyacrylamide gel eiectro- phoresis (PAGE), much of the material did not coelectrophorese with marker “‘~+oie- cell extract" containing the seven previ- ously described virus-specified proteins (unpublished data, Shapiro et al., 1971). We therefore conciuded that, ihe greas, majority of virus-specific proteins rl-ere (II-, or enclosed by, detergent,-labile membmncs.

The infected-cell membranes ri-erc ob- tained from Dounce-homogenized ceils and were fra6tionated on t.hc discontinuorrs sucrose gradient illustrated in Fig. 1 t By tl-iis technique most of the “light” rne~~b~a,~~es were located in band 3, which was predomi- nantly smooth; the denser, “heavy” bands, 5, 6, and 7, were more heterogeneous, cork- sisting of rough membranes, cytopat~hic

vacuoles, and some cell organelles. We selected bands 3 and 5 for studying the binding of viral proteins to membranes. 1, the following, we present the data only for band 3, which is the most homogeneous membrane band isolated; however, similar results were obtained for band 5. The pobypeptide composition for UIL- treated band 3 is presented in Fig. 28. Ail seven of the virus-specified poigpeptides previously found within whole cell extracts ai JEV-infected chick ceils (Shapiro el al., 1971) were present, OT, the membranes. These

362 SHAPIRO ET AL.

VOLUME (ML IAND

0.2 0.3

0.6

I

2 1.0 M

-a--

1.3 M

---- 1.33 M

LOAD ZONE m-m-

1.35 M

- ---

1.55 M

3

0.7

0.7

0.7

0.7

0.7

0.4

0.3

4

5

the neutral and alkaline salt partially re- leased NV-5 and V-2 from the membranes. In order to examine this further, membranes were treated with 3 M KCl, a procedure which has been shown to extract membrane- bound transplantation antigens (Kahan and Reisfeld, 1971; Reisfeld et d., 1971). As a result of this salt treatment, NV-5 was par- tially extracted (Fig. 3) ; probably V-2 was also partially extracted. We therefore sug- gest that the binding of NV-S, and probably of V-2, to membranes is partially stabilized by polar or hydrogen bonds.

----

1.75 M

6

Trypsinixation of Virions 7

2.0 M

w 8

Two recent studies have indicated that the sensitivity of 30 S ribosomal subunit proteins to tryptic digestion reflects the topographic arrangement of the proteins: those that are digested first are assembled last on to the subunit and are the most superficial (Chang and Flaks, 1970; Craven and Gupta, 1970). When we initially trypsinized infected membranes in order to study the topog- raphy of the virus-specified proteins, we found a complex pattern; it was therefore necessary to first look at the sensitivity of virions to trypsin.

FIG. 1. Discontinuous sucrose gradient for membrane fractionation. The samples were made 1.33 M sucrose with the aid of a refractometer and loaded in the middle of the discontinuous gradient illustrated above. The volumes used are indicated on the left. Centrifugation was at 65,000 rpm (300,000 g) for 90 min, after which O.l-ml fractions were collected from the bottom. The various membrane-containing visible bands that formed at the interfaces are indicated on the right.

ranged from NV-5 (nonvirion, molecular weight about 94,000 daltons) down to NV-1 (molecular weight about 10,500 daltons), and included V-3 (the large virion envelope protein) and V-2 (virion core protein). Since it has recently been shown that nonspecific binding of proteins to membranes can be greatly reduced or eliminated by washing membranes successively in 0.15 M NaCl, 1.0 M NaCl, 0.1 M (NaHC& + Na2C03), and 0.075% deoxycholate (Hinman et al., 1970), we treated band 3 membranes simi- larly: aliquots were diluted 25-fold with (1) 0.25 M sucrose (untreated control) ; (2) 1.5 M NaCl; (3) a solution of equal parts of 0.2 M NaHC03 and 0.2 M Na2C03; or (4) 0.075 % sodium deoxycholate (DOC). After 20 min at 4”C, the material was pelleted and analyzed by PAGE (Fig. 2). None of these treatments eliminated any of the virus- specified polypeptides from the membranes, which suggests that the binding was not adventitious.

However, there was a suggestion that both

When JE virions were treated with 0.1% trypsin, three discrete polypeptide frag- ments were produced, along with low- molecular weight material (Fig. 4). The three discrete trypsin-derived fragments were designated as TF-1 (with a mobility similar to that of NV-l), TF-2 (with a mobility corresponding to an estimated molecular weight of 32,000 daltons), and TF-3 (with a mobility similar to that of NV-3). TF-3 and TF-2 were derived ulti- mately from V-3; TF-1 could have been derived from V-2 but, by Occam’s razor, was also probably derived from V-3. The facts that TF-1 and TF-3 were not readily distinguishable from NV-l and NV-3, and that a high background was present in the low molecular weight region of gels of trypsinized membrane samples, and that NV-5 and NV-4 did not appear to be trypsinized to small membrane-bound frag- ments (see Fig. 8) indicated that readily interpretable results could be obtained only by studying the disappearance from tryp-

MEMBRANE-BOUND PROTEINS OF %EV-INFECTED CIXICM CELLS

Y-3

2000 A CONTROL

C. (No, C03’NaHC0& 1200

1000

800

z 0 600

400

200

IO 20 30 40 50 60

2000 B. NaCI v-3

I 1600

1; ,

3600 v-3

FRACTION

FIG. 2. Polyacrylamide gel electrophoresis of band 3 membranes treated with various salts or de- tergent. After treatment with either 1.5 M NaCl, 0.2 144 (NaHCOs + Na&03) or 0.0750/, deoxycholate (DOG), the membranes were pelleted at 50,000 rpm for 60 min; dissociated with SLS and 2-X@ and eleetrophoresed.

sinized membranes of the relatively large peptides YV-5, NV-4 and V-3.

Trypsinixation 0s Membranes

When crude membranes of JEV-infected chick cells were treated with trypsin and then fractionated, there were two changes (Fig. 5); some of the radioactivity was solubilized (the radioactivity remained in the load zone) and some was converted to membranous material of low density (the radioactivity floated to the top of the gradient). The various fractions were

analyzed by PAGE (Fig. 6). zation, V-3, NV-5, and XV-4 were t,he most. prominent polypeptide peaks (Fig, 6.A) ~ After trypsinization, before membrane fine-- tionation, the major peak present was a broadened NV-5 peak, indicating some degradation; NV-4 disappeared (or was not resolved within the broadened NV-5 peak), and V-3 was reduced.

By contrast,, the low densit,y membranes contained a reduced amount of NV-5 rela- tive to V-3; NV-4 was not present (Fig. CC). Since NV-5 was present in large amounts in

364

6 V-3 I

A. UNTREATED 8 3M KCI

SHAPIRO ET AL.

IO 20 30 40 50 60

FRACTION

FIG. 3. Polyacrylamide gel electrophoresis of crude membranes extracted with 3 M KCl. Infected cells were disrupted in RSB and crude membranes were obtained by sedimenting the 2000 rpm super- natant onto a 2.0 M sucrose cushion. The membranes were divided into two 0.5-ml aliquots; each was diluted to 5.1 ml with either RSB or 3 M KC1 (containing 0.004 M Tris, pH 7.4) and incubated at 4” for 18 hr. Each was sedimented at 50,000 rpm for 50 min in the SW 50L rotor. The pellets were both re- suspended in 5.1 ml RSB, resedimented, dissociated with SLS and 2-ME, and electrophoresed.

A. UNTREATED JE VIRIONS B. TRYPSINIZEO JE VIRIONS

240 r

IO 20 30 40 50 60 IO 20 30 40 50 60

FRACTION

FIG. 4. Polyacrylamide gel electrophoresis of trypsinized JEV virions. (A) 1GAmino acid labeled sucrose-density purified JEV virions grown in LLC-MKz cells were dissociated with SLS and 2-ME and electrophoresed on an 8% gel. (B) After addition of 2 ~1 of 0.1% trypsin to 0.25 ml of lGamino acid labeled JEV grown in LLC-MK2 cells, the mixture was incubated at 38” for 1 hr, then diluted to 5 ml with 10% sucrose in RSB and pelleted at 50,000 rpm for 1 hr. The pellet was dissociated with SLS and 2-m and electrophoresed on an 8% gel.

MEMBRANE-BOUND PROTEINS OF JEV-INFECTED CHICK @ELLS

A. UNTREATED MEMBRANES 6. TRYPSiNlZED MEMBRANES t I

IO 20 30 40 50 IO 20 30 40 50

FRACTION

FIG. 5. Fractionation of trypsinized JEV-infected chick embryo cell membranes. Grade membranes were collected from previously frozen pulse-inhibited infected chick cells at the interface between 0.3 TV sllcrose and 2.6 X sucrose as described in Materials and Methods. The material was divided into two 0.5-ml aliguots and 20 ~1 of 1% trypsin was added to one aliq;uot. Incubation was at 37” for 4 hr, nfte~. which 0.1 m! of 0.2T0 of STI was added. The membranes were then fractionated on the discontinuous sucrose gradient as in Materials and Methods.

the unfractionated trypsinized mixture (Fig. 6B) and was present in only small amountIs in the low density membrane frac- t,ion (E’ig. GC), then it should have been present in large amounts in the solubilized material, which was confirmed (Fig. 6D). NoJT.ever, by this reasoning, the recovery of the other polypeptides was not quantitative. One can conclude that NV-5 was readily released from the membranes (relative to V-3) in a relatively undegrnded soluble form. By contrast, NV-4 appeared to be released and degraded. Finally, V-3 was degraded but not readily released (relative to NV-5) ; at least some of TF-2 (a derivative of V-3) remained membrane-bound (Figs. 6D, 7G, 7D). These fact’s suggest that the sensi- tivity of a membrane-bound polypeptide to trypsin Liattacki’ can be expressed in at least three ways: (I) It can be released in a

relatively undegraded form (KV-5). (2) It can be released and degraded (ST-4). (3) It can be degraded but still diEcult to release (V-3). The initial stimuhE for release under the aegis of trypsin is unelea,r; whether covalent bond breakage is necessary is not known, We therefore use the term “release” only to indicate disappearance of a poiy-- peptide from trypsinized membranes re- gardless of the exact mechanism involved, under the assumption that the topographic arrangement of the polypeptide on the membrane determines its sensitivity Chile its intrinsic na.ture determines its response.

Ki&?h In order to obtain a clearer understanding

of trypsin mediated release, kin& studies mere done (Figs. 7 and 8). With relatively high concentrations of t’rypsin (0,1 tic and

SHAPIRO ET AL.

A UNTREATED CRUDE MEMBRANES 0.

260

240 -

160 -

C TRYPSINIZED MEMBRANES: “LIGHT” MEMBRANES

TRYPSINIZEO CRUDE MEMBRANES

D. TRYPSINIZED MEMBRANES: SOLUBLE MATERIAL

I IO 20 30 40 50 60

FRACTION

FIG. 6. Polyacrylamide gel electrophoresis of trypsinised membrane fractions. Fractions from the experiment in Fig. 6 were dissociated with SLS and 2-ME and electrophoresed on 8% gels, including: (A) crude membranes, control; (B) crude membranes, trypsinized; (C) solubilized trypsinized mem- branes (fractions 36-38 pooled) ; (I)) low density trypsinized membranes (fraction 53).

1.0 %) the order of release was NV-5 M leased without the production of small NV-4 > V-3 (Fig. 7). In order to determine membrane-bound trypsin-derived fragments the release sequence of NV-5 relative to other than the trypsin-derived fragments of NV-4, lower concentrations of trypsin were V-3, indicating the reliability of determining used (0.001 %a.Ol%) (Fig. 8); NV-5 was the respective release rates of NV-5, NV-4, released at a slightly faster rate than NV-4. and V-3. Moreover, both NV-5 and NV-4 were re- The order of trypsin-mediated release is

A. UNTREAiED MEMBRANES

900

800

a00

coo

5 B 500 u

400

300

200

100

2000

leoc

16CC

i 400

IZOC

IOOC

8OC

6OC

4oc

MEMBRANES+O,% TRYPSIN- 2 HOURS

v-3

D. MEMBRAMES+IC % TRYPSlM - 4 )-1RS

4

I

IO 20 30 40 50 60 IO 2” 30 40 50 6C

FRACTlOhi

FIG. 7. Polyacrylamide gel electrophoresis of pelleted, trypsinizcd membranes. (A, B, C) Crude membranes, 0.15 ml samples, were treated as follows: (A) no addition; (B) 2 ~1 of 0.1% trypsin for 20 min; (C) 2 ~1 of O.lya trypsin for 2 hr at 35”, after which 10 pl of 0.2y0 STI was added. The mixturee were diiut,ed with 30 ml of RSB and centrifuged at 30,000 rpm for 15 hr at 4’ in a Beckman 3Q rotar, The pellets were dissociated with SLS and 2-ME and electrophoresed on 8y’n gels. (D) Crude membranes, 0.2-ml samples, were treated with 5 ~1 of 1% trypsin for 4 hr, after which 10 ~1 of 1% STI was added and the membranes were diluted 25-fold, pelleted at 65,000 rpm for I hr, dissociated with SLS and 2- ME, and electrophnresed on 8% gels.

367

A UNTREATED MEMBRANES B MEMBRANES + 0.001 X TRYPSIN

40

36

32

28

24 VI b - x P 20 a. 0

16

12

8

4

C. MEMBRANES +O.Ol% TRYPSIN D. MEMBRANES + 0.1% TRYPSIN

32-

28 -

24 -

rQ 20 .

b -

-3

TF-3 /

l2C NY-4

T

8- NV-5

IO 20 30 40 50 60 IO 20 30 40 50 60

FRACTION

FIG. 8. Relative rates of disappearance of NV-5 and NV-4 from trypsinized membranes. Crude membranes were divided into four 0.2-ml aliquots treated as follows: (A) no addition. (B-D) 2 ~1 each of (B) O.OOl’%, (C) O.Ol%, and (D) 0.1% trypsin. After incubation at 37” for 30 min, 2 ~1 of 1% ST1 was added. The mixtures were diluted 25-fold, pelleted, dissociated with SLS and 2-ME, and electrophoresed on 8% gels.

368

MEMBRANE-BOUND PROTEINS OF JEV-IKFECTED CHICK CXXJ~S 369

370 SHAPIRO ET AL.

therefore NV-5 2 NV-4 > V-3. Despite the great sensitivity of NV-5 to trypsin, a small residual peak of radioactivity remained in the region of the gel even after extensive trypsinization (Figs. 7 and 8). This suggests that either (1) the release of NV-5 plateaus before total release is achieved, or (2) a resistant subpopulation of NV-5 exists, or (3) the residual radioactive peak represents an aggregate which coincidentally migrated similarly to NV-5, similar to what we have previously observed when electrophoresing dissociated virions (Shapiro et al., 1971).

No definite statements can be made about the release sequence of polypeptides smaller than V-3, especially NV-3 and INV-1. One puzzling feature is the apparent re- versal of the order of release of V-2 with respect to NV-2 (Figs. 7B, C); it is possible that a trypsin-fragment migrates similarly to one of them or that a resistant subpopula- tion of one of them exists.

SpeciJicity of Release of NV-5 and NV-4

We have assumed that NV-5 and NV-4 were rapidly released because of their specific arrangement on membranes. However, it is possible that any other polypeptide of similar molecular weight would be similarly released. To examine this, uninfected cells were labeled with Wamino acids and mixed with ‘%-amino acid labeled infected cells. Membrane band 3 was isolated, trypsinized, pelleted, and analyzed by PAGE (Fig. 9) ; the specific loss of NV-5 and NV-4 from viral infected membranes exceeded the loss from normal membranes of host polypep- tides of molecular weight exactly equal to those of NV-5 and NV-4. Similar results were also obtained when crude membranes were obtained from cells labeled with 3H- amino acids prior to infection, then infected and labeled with 14C-amino acids (data not shown).

DISCUSSION

We have shown that all seven JE-specified intracellular polypeptides (both virion and nonvirion) bind to membranes. Two of them, NV-5 and probably V-2, are partially re- leased by salt and are therefore presumably stabilized by polar or hydrogen bonds. The sequence of trypsin-mediated release from

membranes is NV-5 2 NV-4 > V-3. There- fore, our results suggest that the large viral proteins bind to membranes in an orderly fashion, such that the larger the polypeptide, the more superficial and “exposed” it is. Furthermore, since unequal rates of loss of radioactivity were observed for the polypep- tides, mediated by either trypsin or salt, it seems likely that they are bound as mono- molecular polypeptides rather than as qua- ternary complexes of multiple species of polypeptides, for the latter would imply simultaneous loss of radioactivity for every component. However, we cannot completely exclude the possibility that NV-5 and NV-4 are two polypeptide subunits of one mem- brane-bound protein.

In addition to being the two largest poly- peptides, NV-5 and NV-4 are also nonvirion polypeptides. The release sequence there- fore also indicates that two nonvirion poly- peptides are more superficial than one virion polypeptide (V-3) ; whether or not this is a general pattern of organization of nonvirion versus virion polypeptides on cellular mem- branes is not known. Finally, we have ig- nored the possible contributions to the ob- served release sequence of factors related to target theory.

Our results may be limited by technical factors, which include (1) nonspecific binding and (2) inadequate fractionation of mem- branes.

1. Nonspecific binding. This would in- clude adventitious binding and the trapping of proteins within vesicular structures rather than actual binding to membranes. The washing experiments with detergent, salt, and alkali were attempts to minimize or evaluate these problems. In connection with this is a recent report demonstrating the binding of two or three vesicular stomatitis virion polypeptides to uninfected plasma membranes (Cohen et al., 1971). The authors regarded this as evidence for “nonspecific” binding and suggested that such controls are necessary for interpretation of membrane binding. However, the polypeptides that did bind in this manner were probably originally membrane bound and were solubilized by sonication (Wagner et al., 1970). Further- more, a polypeptide that was present intra-

MEMBRANE-BOUND PROTEINS OF JEV-INFECTED CHICK CELLS 371

cellularly in a soluble form did not bind to uninfected (or infected) membranes. There- fore, the problem of nonspecific binding can be a moot one.

2. Inadequate fractionation of mem- branes. Our data may represent averages of different populations of morphologically sim- ilar membranes which have different poly- peptide compositions. One would have to de- vise other methods for membrane fract’iona- Lion in order to eliminate this possibility. Related to this problem is the fact that an unknown, presumably large, proportion of our membranes are probably “inside-out.” Since membrane surfaces have asymmetric compositions and st’ructures (Bretscher, 1971), this may complicate, but should not invalidat,e, the interpretation of our data. Finally, band 3 has been variably contami- nated with rough membranes (or membranes lined with the 27 nm particles). We do not feel this to be significant because normal smooth and rough mkxosomes have similar polypeptide compositions (Schnaitman, 1969; Kiehn and Holland, 1970; Fleischer and Fleischer, 1970; Hinman and Phillip, 19’70; Ernster et csl., 1962). Furthermore, similar results mere obtained with band 5 and even with total crude membranes, both of which are morphologically quite hetero- geneous, indicating that the varioils mem- branes behaved similarly regardless of their heterogeneity. In support of the last, it has been shown in influenza-infected cells that al! the various membranous fractions had very similar polypeptide compositions (Hol- land and Kiehn, 1970). However, we cannot totally exclude the possibility t’hat, NV-5 and NV-4 are predominantJy components of the 27-nm particles,

Finally, and quite apart from considera- tions about membrane-binding, it should be noted that txo trypsin-derived poiypeptide fragmenk have molecular weights similar to two naturally occurring polypeptides. The relationships, if any, between the t,u-o groups of polypeptides remain to be deter- mined..

ACKNOWLEDGMENT

We thank Timothy Van Duser for his excellent

technical assistance.

REFERENCES

ACHESQN, N. II., and TXM~M, I. (1967). Replieatiol; of Semliki Forest virus: ,4n electron microscopic study. Virology 32, 128-143.

A~HEsoN,N.H.,~~~:TA~~~M,I. (1970). Pur~katioii

and propert’ies of Semliki Forest virus nucleo- capsids. Virology 41, 306-320.

BOSM.INS, H. B., HAGOPIAN, A., and EYLAK, E. H. (1968). Cellular membranes: The isolation and characterization of the plasma and smooth membranes of HeLa cells. Arch. Bioch~~z. Bio-. phys. 128, 51-69.

BRETSCHER, M. S. (1971). Major human erythro- cyte glycoproiein spans the cell membrane. Nature New Bid. 231, 229-232.

CALIGUIRI,E. A..: and TRMM,~. (1970).The role of cytoplasmic membranes in poliovirus bio- synthesis. Virology 42, 100-111.

CARDIFF, R. D., BRANDI', 'Mi. E., bf&LOUu;

T. G., &M~IRO,~)., and RCISSELL, P.ki. (1971).

Immunological and biophysical separation of dengue-2 antigens. J. Virot. 7, 15-23.

CH.~NG, F. N., and FLAI<S, 9. 2;. (1970). Topog- raphy of t,he E. coli 30s ribosomal subunit and streptomycin binding. Proc. ATut. Acad. Sci. LT,S. 47, 1321-1328.

COHEN, G. N., ATICIXSON, P. II., and SUMMEXS,

D. F. (1971). Interactions of vesicular stomatitis virus proteins with HeLa plasma membranes. Mature Near Biol. 231, 121-123.

CRSVEN, G. R., and GUPTA, V. (1970). Three- dimensional organization of the 305 ribosomal proteins from E. co&, I. Preliminary classifica- tion of the proteins. Proc. Arat. Acad. Sci. U.S. 47, 1329-1336.

ERMTER, L., SIEKEVITZ, I?.; and PAL~DE, G. E.

(1962). Enzyme structure relattonships in the endoplasmic reticulum of rat iiver, J. CelE Bioi. 15, 541-562.

FLBXSCHER, B., ~~~FLEISC~ER, S. (1970). Prepara- t,ion and characterization of Golgi membranes from rat liver. Biochim. Biophys. Aeta. 219, 301-319.

HISXYX, N. D.? and PHILLIPS, A. I-1. (197Oj. Similarity and limited mult,iplicity of membrane proteins from roagh and smooth endoplasmi&: reticulum. Science 170, 1222-1223.

HOLL.~SD, J. J., and KIEHN, E. D. (1970). I&c- enza virus effects on cell membrane prot,eins Science 147, 202-205.

KAHAN, B. D., and REISBELD, R. 9. (1972). Chemical markers of transplantation tndi-. viduality solubilized with sonic energy. Rac- teriol. Rev. 35, 59-85.

KIEHN, IS. D., and HOLL~KD, J. J. (1970), Mem- brane and nonmembrane proteins of mam-

malian cells. Organ species and tumor specffici- ties. Biochemistry 9, 1729-1738.

372 SHAPIRO ET AL.

MURPHY, F. A., HARRISON, A. K., GARY, G. W., WHITFIELD, S.G.,and FORRESTER, F.T. (1968). St. Louis encephalitis virus infection of mice. Electron microscopic studies of central nervous system. Lab. Invest. 19, 652-662.

OTA, Z. (1965). Electron microscopic study of the development of Japanese B encephalitis virus in PS cells. ‘virology 25, 372-378.

REISFELD, R.A., PELLEGRINO, M.A.,and KAHAN, B. D. (1971). Salt extraction of soluble HL-A antigens. Science 172, 1134-1136.

SCHNAITMAN, C. A. (1969). Comparison of rat liver mitochondrial and microsomal membrane pro- teins. Proc. Nat. Acad. Sci. U.S. 63,412419.

SHAPIRO, D., BRANDT, W.E., CARDIFF, R.D.,and RUSSELL, P. K. (1971). The proteins of Japanese encephalitis virus. Virology 44, 108-124.

SPEAR, P. G., KELLER, J. M. and ROIZMAN, B. (1970). Proteins specified by herpes simplex virus. II. Viral glycoproteins associated with cellular membranes. J. liirol. 5, 123-131.

STOLLAR, V. (1969). Studies of the nature of dengue viruses. IV. The structural proteins of type 2 dengue virus. Virology 39,426-438.

UHR, J. W., and SCHENICIN, I. (1970). Immuno- globulin synthesis and secretion. IV. Sites of incorporation of sugars as determined by sub- cellular fractionation. Proc. Nat. Acad. Sci. U.S. 66, 952-958.

WAGNER, R.R., SNYDER, R.M., and YAIVSZBRT, S. (1970). Proteins of vesicular stomatitis virus: Kinetics and cellular sites of synthesis, J. Viral. 5, 54&558.