Structural proteins of adenoviruses

VIROLOGY

61, 252-256 (1973)

Letter Structural IX. Molecular

Weight

to the

Proteins of Adenoviruses

and Subunit

Composition

The fiber is a capsid protein which extends from each of the 12 vertices of the adenovirus icosahedron (1, 2). Electron microscopy has revealed that fibers consist of a thin rod-like structure with a knob at the end (1, 3). Fibers from different adenovirus serotypes differ in length but serotypes belonging to the same immunological subgroup (4) have fibers of identical length (5). Fibers from serotype 2 are 20 nm in length and have a terminal knob approximately 4 nm in diameter (1, 3). Although several methods have been described for purification of this protein, there is disagreement on the molecular weight of the fiber, its subunit composition, and amino acid composition (1, 5, 6-11). The reported values for the molecular weight of fibers from subgroup III range between 60,000 and 500,000 daltons (3, 7-11). We have, therefore, purified fibers from adenovirus type 2, by two different methods, and determined the molecular weight under both native and denaturing conditions. Adenovirus type 2 was propagated in suspension cultures of KB-cells as previously described (3). The soluble antigen fraction, consisting of capsid proteins produced in excess during infection was used as the source of fibers. Two methods were used for purification both giving preparations which were homogeneous by polyacrylamide gel electrophoresis (Fig. 1). Fibers were either purified by DEAE-chromatography, isoelectric focusing (ld), and agarose chromaessentially as described by togwhy, Pettersson et al. (3) (Method A), or by a procedure where the isoelectric focusing was replaced by chromatography on CM-cellulose (Method B). The fractions containing fiber after DEAE-cellulose chromatography @ 1973 by Academic Press, of reproduction in any form

Inc. reserved.

of Adenovirus

Type

2 Fiber

were dialyzed against 0.1 M ammonium acetate buffer, pH 5.5, and chromatographed on CM-cellulose (Whatman CM32) equilibrated with the same buffer. After thorough washing the columns were eluted with a linear gradient of 0.1-0.4 ammonium acetate, pH 5.5. The final step of purification in Met.hod B was also exclusion chromatography on 6 % agarose in 0.05 M Tris-acetate pH 8.0 with 0.5 M NaCl (3). Molecular weight of native fibers. The molecular weight was determined in a Spinco model E analytical ultracentrifuge by the met.hod of high-speed sedimentation equilibrium as described by Yphantis (1s) as modified by Chervenka (14). Three independently purified samples were analyzed in buffers of different ionic strength (Table 1). An average molecular weight of 207,000 daltons was calculated using a partial specific volume of 0.725, as estimated from the amino acid composition (Table 2) as previously described (22). All preparations were homogeneous as judged by the concentration distribution after obtaining equilibrium (Fig. 2). The high axis ratio of the fiber which can be calculated from the molecular weight and the S value 6.1 (5) suggest a highly asymmetric molecule, which has been confirmed by electron microscopy (1, 3). Determination of the molecular weight of the subunits 0s the fiber. Fibers were denatured in 6 M guanidine-HCl and analyzed by analytical ultracentrifugation as described by Chervenka (14). A molecular weight of 61,000 was calculated at equilibrium (Fig. 2b, Table 1) assuming a partial specific volume of 0.725. Fibers were also denatured in 2 % sodium dodecyl sulfate (SDS) and 1% mercaptoethanol a,t 100°C for 2 min and 252

Copyright All rights

Editor

LETTER

TO THE

253

EDITOR TABLE

1

MOLECULAR WEIGHT OF PURIFIED SEROTYPK 2

-3

.A g

2

z:g a, OG z

L

B

c

B

d

B

FIRERS FROM

Buffer for ultracentrifugation

Molecular weight”

0.01 M phosphate, pH 8.0 phosphate, pH 0.15 M NaCl 0.05 M phosphate, pH 6.8 0.1 M NaCl 6 M guanidine-HCl 0.1 M mercaptoethanol, pH 6.1

200,000 224,000 199,000 61,000

= A partial specific volume of 0.725 was used for all calculations. TABLE 2 AMINO ACID COMPOSITION SEROTYPE

OF

FIBERS FROM

‘2

Residues per lo@

FIG. 1 A. Analytical polyacrylamide gel electrophoresis of fibers purified according to method “A” (1) and “B” (2). The gels contain 8% acrylanude and 0.05% N,N’-bismethylene acrylamide in 0.05 &l Tris-acetic acid, pH 8.0. Electrophoresis was performed for 2 hr at, 20 V/cm with the anode toward the bottom. B. The same preparations analyzed on gels containing 13% acrylamide, 0.34ye cross-linker in 0.1% SDS according t,o Ref. 15.

subsequently analyzed on polyacrylamide gel elcctrophoresis in 0.1% SDS essentially as described by Maize1 (15). Gels containing 13% acrylamide were used for molecular weight determination. Only one sharp band was observed in the purified preparations (Fig. 1). The molecular weight of this polypeptide was estimated to be 60,00065,000 in gels which were calibrated with markers of known molecular weight (t,ransferrin, bovine serum albumin, heavy chains of IgG, and egg albumin (Fig. 3)). The size of the subunit was furthermore determined by gel chromatography 011 4% agarose (Sepharose 4B, Pharmacia Fine Chemicals, Uppsala, Sweden) in the presence of .5 M

Lys His Arg Asp Thr* Serb Glu Pro Gly Ala Val Ile Leu Tyr Phe Met Trpc Half-cysd Total 127 residues

6.0 f 0.4 0.8 I-t 0.2 1.5 f 0.3 13.1 * 0.4 11.1 f 0.6 11.2 f 0.6 6.4 + 0.5 5.2 f 1.3 8.5 f 0.6 6.5 f 0.6 5.3 + 0.2 5.0 f 0.1 9.9 ztz 0.5 2.8 f 0.3 2.9 + 0.1 1.5 + 0.4 1.6 0.7 f 0.1 or 15,900 daltons

Residues per 1 His 8 1 2 16 14 14 8 7 11 8 7 6 12 4 4 2 2 1

(LAverage of five determinations. b Extrapolated to zero-time hydrolysis. c Det,ermined spectrophotometrically as described by Bencze and Schmid (21). d Determined as cystic acid after performic acid oxidation (20) ; two samples were analyzed.

guanidine-HCl as described by Davison (18). Proteins for analysis were reduced with 0.1 M dithiothreitol in the presence of 6 M guanidineeHC1 (“Ultrapure” Mann,

254

LETTER

I 50.00

I 51.00 r2 lcm2)

TO THE

J 52 00

FIG. 2. Sedimentation equilibrium of fibers in 0.05 M phosphate buffer, pH 6.8, and 0.1 M NaCl, T = radius. The concentration distribution was monitored by interference optics after centrifugation at 11,066 rpm for 21 hr at 20°C O---O. Sedimentation equilibrium of fibers in 6 M gusnidineHCl and 0.1 M p-mercaptoethanol, pH 6.1. Centrifugation was performed at 26,000 rpm for 25 hr at 20°C before the concentration distribution was measured with interference optics a--@.

Research Laboratories, New York) and 0.4 M Tris-chloride pH 8.6 in a final volume of 0.5 ml. After 60 min 0.05 ml of 2 M TrisHCI, pH 8.6, and solid iodoacetamide to a final concentration of 0.25 M was added. After an additional 30 min at room temperature the samples were added directly to the columns. Purified fibers, labeled with a mixture of l*C-labeled amino acids were cochromatographed with bovine serum albumin or egg albumin. The markers were obtained from Mann Research Lab (N.Y.). Fibers eluted between the two markers, corresponding to a K,, of 0.29 (17) (Fig. 4). A molecular weight around 60,000 was calculated for the fiber polypeptide by the method of Fish et al. (18). Amino acid composition of jibers from serotype 6. The amino acid composition of purified fibers was det.ermined as described

EDITOR

by Pettersson et al. (19) and is shown in Table 2. Analysis of five different batches showed only minor variations and preparations purified by method “A” and “B” showed no significant differences. In order to establish the presence of half-cystine in the fiber, two batches were oxidized by performic acid and analyzed as described by Hirs (SO). An average of 0.72 % half-cystine was detected in two separately analyzed preparations (0.63-0.80 %). Histidine and half-cystine are the amino acids present, in lowest amounts. A molecular weight of 16,000 daltons was calculated per residue of histidine. From the estimates on the size of the subunit described above, it appears likely that each repetitious unit in the fiber contains four histidines corresponding to a molecular weight of 64,000 daltons, assuming that all subunits have identical primary structure. In conclusion, although most previous reports (1,3, 7,9) have suggested that fibers from adenoviruses belonging to subgroup III have a molecular weight in the range

g---L 1 2 3 I: MOBILITY IN CM

4

FIG. 3. The relative electrophoretic migration rate of fibers in gels containing 13% polyacrylamide, 0.34% cross linker, 0.1% SDS, according to Ref. 16. Electrophoresis at 10 V/cm for 4 hr. The molecular weights of markers were plotted against the distance of migration on a semilogarithmic scale. The arrow indicates the position of the fiber. T = transferrin (76,000 daltons); BSA = bovine serum albumin (68,000 daltons); HC = heavy chains of IgG (50,060 daltons); EA = egg albumin (46,000 daltons).

LETTER

cpm

TO THE

255

EDITOR

E450

“t 4

40

50 60 FRACTION

70 NUMBER

80

go

100

110

FIG. 4. Exclusion chromatography of W-labeled fibers (O---a) and bovine serum albumin (0 -0) on 4% agarose in 5 M guanidine-HCl, 0.05 M Tris-acetate, pH 8.0, 0.01 M EDTA, and 0.02 M LiCl. The peak of the bovine serum albumin wa8 monitored t,urbidimetrically (16) after precipitation with 8% trichloracetic acid. Blue dextran 2000 (Pharmacia Fine Chemicals, Uppsala, Sweden) was used to determine the void volume and DNP-alanine to monitor the total volume of the column. The arrow indicates the position of egg albumin as determined in a separate experiment.

60,000~80,000 daltons, the present results indicate that the molecular weight of t’he native fiber of serotype 2 is 200,000 daltons, as was first suggested (11) from measurcmcnts of St,okes’ radius according to the method of Laurent and Killander (17). The inconsist’ency of previous data may be due to the fact, that fiber is present in minute quantities in adenovirus-infected cells, and it is difficult to obt,ain enough material to make accurate physical studies. Two previous studies, including our own, using the analytical ultracent’rifuge have indicated a molecular weight of 70,000-80,000 daltons (3, ‘7). The amounts of fiber used for determination of the diffusion constant were probably too small to allow a correct interprctat’ion of the results. Valentine and l’clreira estimated the molecular weight of type .5 fiber to 70,000 from size measurements by electron microscopy (1). Molecular weights calculat’ed in this way may give erroneous results because of shrinkage of protein

molecules

after

negative

staining.

Maize1 and co-workers estimated the molecular weight’ of type 2 fibers to around 500,000 dultons (8). Their estimate was based on calculations of the proportion of radioactivity recovered as fiber polypeptidc when 3H-amino acid-labeled virions were analyzed iJy SIX-polyacrylamide gel electrophoresis.

This overestimate may have been caused by comigration of another polypeptide with the fiber polypeptide IV in the SDS gels (8, 15). The molecular weight of the subunit of the fiber appears to be 60,000-65,000 determined by three independent methods, suggesting the presence of three polypeptide chains in each native fiber molecule. It is not’ possible to establish whether the three polypeptides are identical. The electropherograms in SDS show one band which appears very homogenous in size, favoring the notion that all subunit’s are identical. On the other hand, it is entirely possible that a molecule with such complex morphology would be composed of different subunits. Fingerprinting of an enzymatic digest of the fiber is necessary to establish the answer unambigiously. The fiber has been found t)o cont’ain an average of four half-cystine per subunit., a fact’ which has been overlooked in previous amino acid determinations (3, 6). The amino acid composition reported here is in good agreement with that which we published previously (3), and it has recent’ly been found that fibers from serotype 5 have an amino acid composition similar to that shown in Table 2 (H. Ginsberg: personal communication).

LETTER

256

TO THE EDITOR

ACKNOWLEDGMENT The skilful technical assistance of Mrs. E. Hjertson and Miss S. Rosen is greatfully acknowledged. This work was supported by grants from the Swedish Cancer Society and the Swedish Delegation for Applied Medical Defense Research. One of us (U. P.) is presently a recipient of a fellowship from the International Agency for Research on Cancer. REFERENCES

1. VALENTINE, R. C., and PEREIRA, H. G., J. hfoZ. Biol. 13, 13-20 (1965). 2. NORRBY, E., Virology 28,236-243 (1966). and PHILIPSON, L., U., 3. PETTERSSON, HBGLUND, S., Virology

35, 204-215 (1968).

4. ROSEN, L., Amer. J. Hyg. 71, 120-128 (1960). 6. NORRBY, E., J. Gen. Viral. 5,221-236 (1969). 6. BOULANGER, P. A., FLAMENCOURT, P., and BISERTE, G., Eur. J. Biochem. 10, 116-131 (1969).

7. K~HLER, K. Z., Naturforschung 8. 9. 10. Il. 12.

20b, 747-752 (1965). MAIZEL, J. V., WHITE, I). O., and SCHARFF, M. O., Virology 36, 126-136 (1968). HOLLINSHEAD, A. C., and HUEBNER, R.. J., Nature London 211, 890-891 (1966). DORSETT, P. H., and GINSBERG, H., Bact. Proc. p. 223 (1971). thesis. WADELL, G., Habilitation I)avid Broberg’s printing Stockholm, Sweden, 1970. SVENSSON, H., Arch. Biochem. Biophys. Suppl. I. 132-138 (1962).

1s. YPHANTIS, D. A., Biochemistry 3, 297-317 (1964). 14. CHERVENKA, C. H., Anal. Biochem. 34, 24-29 (1970). 15. MAIZEL, J. V., Jlt., in “Methods in Virology” (K. Maramorosch and H. Koprowski, Eds.), Vol. V, 179-246, 1971. 16. DAVISON, P. F., Science 161,906-908 (1968). 17. LAURENT, T. C., and KILLANDER, J., .I. Chromatoo. 14.317-330 (1967). 18. FISH, W. WY, MXXN, K. &., add TANFORD, C., J. Biol. Chem. 244, 4989-4994 (1969). 19. PETTERSSON, U., PHILIPSON, L., and Hiic,LUND, S. Virology 33,575-590 (1967). 20. HIRS, C. II. W., J. Biol. Chem 219, 614-621 (1956). $1. BENCZE, W. L., and SCHMID, K., Anal. Chem. 8, 1193-1196 (1957). 22. SCHACHMAN, H. K., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, Eds.), Vol. 4, p. 32, New York, A(*ad. Press (1957). Bo SUNDQUIST ULF PETTERSSON LARS THELANDER LENNART PHILIPSON Department of Microbiology The Wallenberg Laboratory Uppsala University Uppsala, Sweden and Department of Biochemistry Medical Nobel Institute Karolinska Znstitutet Stockholm, Sweden Accepted October 5, lQY2