Identification of the spike proteins of Rous sarcoma virus

VIROLOGY

46, 185489

(1971)

Identification

of the Spike

Proteins

ROW sarcoma virus (RSV) is a spherical particle with a diameter of 80-120 nm (1). The virion is surrounded by a lipid-containing envelope derived from the plasma membrane of the host cell. The outer surface of the viral membrane contains knoblike projections 7.5 nm in diameter which extend radially 7.0 nm (9). The interior of the virus contains an electron dense core of 50 nm that, is thought to be composed of protein and nucleic acid (1, 3). The virion has been shown to contain at least eight proteins including three glycoprot’eins (4-8). Two of the proteins are type-specific antigens and are presumed to be located on the exterior of the virion (5, 6, Q-l l), while three other proteins show group-specific antigen activity and are believed to be internal (4, 7, 11). This assignment, of the viral proteins either to the outer surface or the interior rests almost enOirely upon immunological criteria. The present experiments localize the viral glycoprot,eins to t’he outer surface of the virion. The results show that the knoblike projections on the surface of the virion can be removed by controlled exposure to bromelain, and that their removal is correlated with the loss of infectivity. The Schmidt-Ruppin D strain of RSV was grown in chick embryo fibroblast cultures and purified as previously described (1~). E’or more efficient radioactive labeling of virus, the culture medium cont’ained only ${o of the usual concentration of amino acids and glucose. Glucosamine-3H was purchased from New England Nuclear Corporation, Boston, Jlassachusetts, and 14C- and 3H-lsbeled amino acid mixt’ures from Schwarz RioResearch, Orangeburg, New York. Purified radioact,ive RSV was incubated with bromelain (Sigma Chemical Co., St. Louis, Missouri) as described previously for influenza virus (13). After a 3-hr incubation period with the enzyme, the virus was repurified by isopycmc cent’rifugation using 485

of Rous Sarcoma

Virus

a 20-55 % (w/v) sucrose gradient, (1~?), dialyzed against 0.1 M Tris, 0.001 M EDTA, pH 7.1, and then pelleted at 50,000 rpm for 1 hr in an International Equipment Co. SB-405 rotor. Control preparations of virus were incubated at 37” for 3 hr and then repurified in the same manner as bromelaintreated virus. The focus-forming act,ivity of the virus was assayed by the method of Temin and Rubin (14). The preparation of specimens for electron microscopy and acrylamide gel electrophoresis was done as described previously (13, 15, 16’). lutoradiography of polyacrylamide gels was performed according t,o the method of I’airbanks et al. (17). Figure 1 shows negatively stained RSV particles from a control preparation. A layer of fine projections is present, on the outer surfaces of the particles, and in particles which have been penetrated by the stain, an elect’ron-lucent membrane is visible beneath the project’ions. Control preparations contain disrupted particles and droplets about 200 A in diameter int’erspersed among the virus particles. After exposure to bromehrirr for 3 hr the surface projections are absent from the particles (Fig. 2). The resulting smooth-surfaced particles are st:ble to purification in a sucrose density gradient, in which they have an equilibrium density of 1.165 gjml, compared wit.h 1.173 g/ ml for the original RSV preparation. The effect of bromelain on virus infect ivit’? is presented in Table 1. Compared n-it11 the control sample incubated without enzyme, the infectivity of bromelaintrent ed RSV’ (B-RSV) is reduced almost ten-tlrolls:tndfold. This indicates that t)he infectivity of RPV, like that of influenza (13, 18), Sindbis (/,I?), VSV (SO), and SV5 (N), is markedl~~ dccreased by removal of t.he surface spikt>s. The proteins of R.SV and H-NV were compared a,ftrr polyacrylamide gel t,lrctrophoresis by b& scintJillation counting and

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FIG. 1. Rous sarcoma virus particles negatively stained with sodium phosphotungstate. The surfaces of the particles are covered with a layer of fine projections 70 .& in length. Beneath the projections is an electron-lucent membrane (M) which varies from 70 t*o 150 B in thickness. One particle shows an internal strand (arrow) 80 1i in width. X 150,000. FIG. 2. Spikeless Rous sarcoma virus particles, after treatment with bromelain for 3 hr and repurification in a sucrose density gradient. X 150,000.

TABLE COMPARISON INTACT

1

OF THE

AND

FOCUS-FORMING ACTIVITY BROMELAIN-TREATED SARCOMA VIRUS

Virus preparation Controlb*c Bromelain-treated

OF

Rous

FFU/mla RSV

2.5 X lo6 6.7 x lo2

0 The focus-forming units are presented as focus-forming units per 1 ml of the initial harvest fluid. b Purified RSV was incubated at 37” for 3 hr and repurified in the same manner as hromelaintreated virus. c The original virus preparation had a titer of 1.5 X lo7 FFU/ml. autoradiography. Figure 3A illustrates the protein pattern of intact virus, which resembles that reported by other investigators (4, 5, 8). Four glycoprotein components,

1, 2, 3, and 4, can be identified by glucosamine labeling, whereas proteins 5, 6, 7, and 8 are not labeled v&h glucosamine. Hung (4) also identified 8 proteins and co-workers

in the Bryan strain of RSV; however, they did not identify a component corresponding to glycoprotein 3, but identified an additional nonglycoprotein in the region of protein 6 by isoelectric focusing. A densitometry scan of an autoradiograph of amino acid-Wlabeled RSV proteins is shown in Fig. 4A. Glycoprotein 3 is more prominent, in the autoradiograph than in the radioactivity profile in Fig. 3A. The amount, of material in peak 1 was variable; it is not a significant, peak in the autoradiograph, and it may be an aggregate of other proteins. The glycoprotein 2 is seen in the autoradiograph to be composedof more than one species,and may represent several proteins of similar size, or a single polypeptide with different amounts

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20

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FIG. 3. (A) Electrophoresis of the proteins of RSV grown in chick embryo fibroblasts labeled with glucosamineJH and amino acid-i% mixture. The arrows indicate the positions of the major viral polypeptides. (B) Electrophoresis of the proteins of bromelain-treated RSV grown in chick embryo fibroblasts labeled with glucosamine-JH and amino acid-14C mixture, showing that the glycoprot,eins have been removed.

FIG. 4. (A) A densitometry scan of an autoradiograph of an acrylamide gel of the proteins of RSV grown in chick embryo fibroblasts labeled with amino acid-W mixture. (B) A densitometry scan of an autoradiograph of an ncrylamide gel of the proteins of bromelain-treated RSV grown in the presence of amino acid-14C mixture, showing that proteins 1, 2,3. and S have been removc~cl.

Incubation of KSV with bromelain completely removes all t’he glucosamine-cont,aining proteins (Figs. 23, 4R) but produces of carbohydrate. Alternat’ively, it ma’y be a no changes in t.he remaining four proteins single species which migrat,es as a split of low molecular weight. Thus, the proteins peak, as has been described with the glyco- in peaks 14 are on the surface of the virus protein of Sindbis virus under similar condiand are probably associated with the spikes, tions (dd). The remaining peaks in the autoradiograph correspond well with those in while the proteins in peaks 5-8 are resistant, Fig. 3R. Thus, these two methods used t,o the action of bromelain and are prrtogether indicate that t,he virion contains at sumably internal component,s of the virion. To ensure t,hat the proteins remaining least eight different, protein components of after digest,ion were not) degradation prodwhich four contain carbohydrate label.

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zyme treatment contain all of the nonglycoproteins, and attempts are now in progress to determine the location of these proteins in either the nucleoid or the membrane.

18 14 IO 6

2

Froctlon

number

FIG. 5. Coelectrophoresis of the proteins of RSV labeled with amino acid-l% mixture and bromelain-treated RSV labeled with amino acid-3H mixture, indicating that the remaining proteins of bromelain-treated virus correspond to those of the control virus, both in relative amounts and in distance migrated.

ACKNOWLEDGMENTS The expert technical assistance of Mrs. Deborah Fowler and Mrs. Ann Erickson is gratefully acknowledged. This research was supported by Grant E-478 B from the American Cancer Society and Contract AT (30-l)-3983 from the U.S. Atomic Energy Commission. DANIEL B. RIFKIN RICHARD W. COMPANS The Rockefeller University New York, New York 10021 Accepted July 28, 1971

REFERENCES 1. VOGT, P. K., Advan. Virus Res. 11, 293 (1965). 1. DOURMASHKIN, R. R., and SIMONS, P. J., J* Ultrastruct. Res. 5, 505 (1961). 3. COFFIN, J. M., and TEMIN, H. M., J. Viral. 7,

ucts, and correspond to the original molecules, amino acid-3H-labeled B-RSV was 625 (1971). mixed with control ‘*C-labeled RSV, and 4. HUNG, P. P., ROBINSON, H. L., and ROBINSON, the mixture was analyzed by gel electroW. S., Virology 43, 251 (1971). phoresis. Both the positions of the “H-labeled 5. DUESBERG, P. H., MARTIN, S., and VOGT, P., proteins remaining after bromelain treatVirology 41, 631 (1970). ment and their relative amounts were equiva6. ROBINSON, W. S., HUNG, P., ROBINSON, H. L., lent to proteins from control 14C-labeled and RALPH, D. D., J. Viral. 6, 695 (1970). RSV (Fig. 5) indicating that they were not 7. DUESBERG, P. H., ROBINSON, H. L., ROBINSON, W. S., HUEBNER, R. J., and TURNER, H. C., generated by the enzyme. Virology 34, 73 (1968). All t’he enveloped viruses we have studied 8. BOLOGNESI, D. P., and BAUER, H., Virology 42, to date, including influenza (13, 18), Sindbis 1097 (1970). (19), SV5 (21), and vesicular stomatitis -~37, 157 virus (20), have spikes that are composed 9. BAUER, H., and GRSF, T., Virology (1969). of glycoprot’eins and are sensitive to digesH., BAUER, H., GRAF, T., and tion by proteolytic enzymes. In each case, 10. TOZAWA, GELDERBLOM, H., Virology 40, 530 (1970). the removal of the spikes renders the virus ~~BIUER, H., and BOLOGNESI, D. P.: Virology 42: noninfectious. Our data show that all the 1113 (1970). glycoproteins of RSV are susceptible to 12. RIFKIN, D., and REICH, E., Virology 45, 172 enzymatic digestion. These proteins must (1971). be on the surface of the virus, since their IS. COMPANS, R. W., KLENK, H. D., CALIGUIRI, disappearance coincides with that of the L. A., and CHOPPIN, P. W., Virology 42, 880 spikes. There is no evidence that all four (1970). - ~glycoprot,eins found by acrylamide gel elec- 14. TEMIN, HrM.,a%d RUBIN, HT V&oZo&6,-669 trophoresis represent unique species, since (1958). proteins of this type tend to form aggregates. 16. CALIGUIRI, L. A., KLENK, H. D., and CHOPPIN, Nor has the possibility been eliminated that P. W., Virology 39,460 (1969). the smaller glycoproteins are cleavage prod- 16. KLENK, H. D., CALIGUIRI, L. A., and CHOPPIN, ucts of the larger glycoproteins. The smoothP. W., Virology 42,473 (1970). G., LEVINTHAL, C., and REEDER, surfaced particles which remain after en- ~‘7. FAIRHANKS,

SH0R.T

-tS!b

COMiVUNICATIONS

R. II., Biochent. Biophys. Res. Commun. 393 (1965). 18. SCHULZE, 1. T., Virology 42, 890 (1970). 1.9. COMPANS, 11. W., -~atwe (Lo&o?L) 229, (1970). 20. M&HARRY, J. J., COMPANS, R. W.,

20,

114 and

P. iv., Bucferiol. Pro,.. 11. ?I,> (1971). 21. CHEN, C., COMPANS, R. W., and (‘I~oPI~~s, I’. W., ./. Gen. Viral. 11, 53 (1971). 22. STRAUSS, J. H., BLJRGE, B. W., and I).\ts~~m, J. IL, Virology 37, 367 (1969). (:HOWIN,