Studies on the structural proteins of vaccinia virus

VIROLOGY

(1967)

33, 717-725

Studies

on

the Structural

I. Structural

of Cell

Biology,

of Vaccinia

Virus

Proteins of Virions and Cores

J. A. HOLOWCZAK Department

Proteins

WOLFANG

AND

K. JOKLIK

Albert Einstein College of Medicine, Accepted September 11, 1967

Bronx, New York,

1046l

Radioactively labeled vaccinia virions grown in L cells were dissociated to yield the constituent polypeptide chains, which were then subjected to electrophoresis in polyacrylamide gels. Mechanical fractionation of these gels yielded complex profiles in which at least 17 components, some major, some minor, could be reproducibly identified. The relative mass in each component has been calculated from the amount of radioactivity incorporated. The principal component (VSP-4) accounts for about 2@ of the total viral protein mass. Cores derived from virions by chemical treatment contain the principal viral protein component, which is clearly multiple, as well as two minor ones (VSP-1 and VSP-2), which are probably single polypeptide species. These latter two components are the two slowest moving ones, and therefore most probably have the largest molecular weights. Treatment of virions with the nonionic detergent NP 40 liberates one major viral protein component (VSP-6). This component accounts for 18.7’3& of viral protein, the second highest amount, and is also multiple in nature. INTRODUCTION

The vaccinia virus particle has a highly complex structure. Extensive electron microscopic investigations by a number of investigators, in particular Peters (1960), Dales (1963), Westwood et al. (1964), and Easterbrook (1966) have led to the following model: an outer membrane surrounds a protein coat within which is situated the core which is itself surrounded by a membrane. Two structures, the lateral bodies, are recognizable within the protein coat, on opposite sides of the core. The function of the lateral bodies is unknown; the viral genome resides within the core. Such a complex structure is clearly composed of a large number of different protein species.Marquardt et al. (1965) attempted to disrupt vaccinia virus particles by various means and recognized seven different antigens by employing the Ouchterlony gel double-diffusion technique. However, the poxvirus particle is not readily dissociated into antigenically active subunits and there is little doubt that this number represents

only a fraction of the total number of protein species present. In extracts of cells within which a cycle of vaccinia virus multiplication has proceeded, approximately twenty components capable of reacting with antiviral antiserum can be detected (Appleyard et al., 1965; Rodriguez-Burgos et al., 1966). This work was undertaken in an attempt to define more accurately the number of protein speciesof which the vaccinia virus particle is composed. To this end vaccinia virions were dissociated with SDS’, urea and 2-mercaptoethanol, and the resulting mixture of polypeptides was subjected to electrophoresis in polyacrylamide gels, a technique which has recently been applied successfully to poliovirus (Summers et al., 1965) and adenovirus (Maizel, 1966). Further, it was our aim to define the location of some of the viral structural proteins within the vaccinia virion. For this purpose we 1 Abbreviations: SDS, sodium dodecyl sulfate; EB, elementary body; PFU, plaque-forming unit; VSP, vaccinia structural protein.

717

718

HOLOWCZAK

utilized the recent finding of Easterbrook (1966) that treatment of vaccinia virions with the nonionic detergent NP 40 plus 2mercaptoethanol leads to the dissociation of the outer membrane and protein coat and to the production of viral cores. Adaptation of this technique has allowed the identification of certain protein species that are located at the surface of the virion, and of the protein species of which the vaccinia core is composed. MATERIALS

AND

METHODS

Cells and virus. Mouse L cells were grown in spinner culture in Eagle’s medium (1959) supplemented with 10% fetal calf serum. HeLa S3 cells were grown similarly in Eagle’s medium supplemented with 5% fetal calf serum. A highly purified stock of the WR strain of vaccinia virus, propagated in mouse L cells, containing 2.4 X 10n EB/ml and assaying at 5 X lo9 P&U/ml when titrated on chick embryo fibroblasts, was used as the inoculum for these experiments. Cells were infected by procedure A as described by Becker and Joklik (1964). Purification of virus. Virus was purified according to the method of Joklik (1962a) with some minor modifications. After centrifuging the crude virus suspension through 36% sucrose, the virus was banded twice in 20-40% (w/w) sucrose density gradients (13,000 rpm for 1 hour in the SW-25 rotor of the Spinco Model L ultracentrifuge). After the second banding, the specific activity of radioactively labeled virus preparations became constant. Purified virus preparations were free of soluble antigens. The amount of virus was estimated by measuring the absorption at 260 rnp of briefly sonicated virus suspensions. One optical density unit is equivalent to 64 pg of viral protein and 1.2 X lOlo virus particles (Joklik, 1962b). Preparation of virus un
AND

JOKLIK

concentration was about 8 X 10” cells/ml. Immediately after dilution, 0.1 PC! of a complete mixture of 14C-labeled amino acids (U.L.) or 0.1 PC of reconstituted 3Hlabeled protein hydrolyzate was added per ml of culture. The cultures were then incubated for 24 hours and harvested by centrifugation; virus was isolated and purified. Isolation of material released by controlled degradation of vaccinia virions. Vaccinia virions labeled with 14C-amino acids or with 3H-amino acids were collected by centrifugation and resuspended in Tris buffer (1 mM, pH 8.0) at a density of 1 X 10” EB/ml. To the suspension one-tenth volume of a 5% solution (v/v in water) of the nonionic detergent NP40 was added. The mixture was incubated at 37” with continuous agitation for 1 hour. At 20-minute intervals small samples were removed, and the amount of solubilized material determined by removing particles by centrifugation (1 hour, 15,000 rpm, SS-34 rotor of the Servall ultracentrifuge) and precipitating the clear supernatant with 6 % trichloroacetic acid. When the reaction was judged to be complete, material released into the supernatant was collected for electrophoretic analysis. To analyze the next step of the degradation, the reaction mixture, incubated as just described, was transferred t’o room temperature, 2-mercaptoethanol was added to a final concentration of 0.12 M, and incubation was continued for 1 hour. Solubilized material was again assayed as above, and the released material was collected when the reaction was complete. 2,2’-Dithiodiethanol (final concentration 0.1 n/r) was sometimes used in place of 2-mercaptoethanol, as suggested by Smithies (1965). Isolation oj viral cores. Vaccinia virions were subjected to the two-step incubation just described. When 2-mercaptoethanol was employed, the reaction was terminated by the addition of 2 mmoles of a-iodoacetamide for each millimole of 2-mercaptoethanol used. If iodoacetamide was omitted, the cores aggregated badly. When 2,2’-dithiodiethanol was used, addition of iodoacetamide was not necessary. The reaction mixture was then layered onto 10 ml of a solution of 70 % sucrose and centrifuged (40 minutes, 15,000 rpm, SS-34 rotor of the Sarvall

PROTEINS

OF VACCINIA

ultracentrifuge). The particulate fraction was recovered from the top of the 70% sucrose cushion and diluted in Tris buffer (1 mM, pH 8.0) ; the centrifugation was repeated. The particulate fraction, again suspended in buffer, was then sonicated for 3(t60 seconds with an MSE 60-watt ultrasonic disintegrator and layered onto a 2(r 40% (w/w) sucrose density gradient and centrifuged for 1 hour at 13,000 rpm in the SW 25 rotor in the Spinco Model L ultracentrifuge. Fractions of 0.7-0.9 ml were collected after the optical density at 260 rnp was recorded by passagethrough a Gilford absorbance recorder. The banded material was sampled for electron microscopy and acid-precipitable radioactivity and the remainder was solubilized for analysis by electrophoresis. Dissolution of virus for gel electrophoretic analysis. Approximately 500-1000 pg of virus (7-15 optical density units at 260 rnp) was suspended in 1 ml of Tris buffer (1 mM, pH. 8.0) and gently sonicated as above. The suspensionwas made successively 2.5 % with respect to SDS, 0.7 1M with respect to urea, and 1.5 M withrespect to 2-mercaptoethanol, allowed to stand at 37” for at least 1 hour, and then dialyzed for 18-24 hours against 0.01 M phosphate buffer, pH 7.1, containing 0.1% SDS, 0.5 M urea, and 0.1% 2-mercaptoethanol. Material liberated from virions and viral cores was treated similarly. Techniques of gel electrophoresis and gel fractionation. For the system under study, gels composed of 8.5% acrylamide, 0.23% N ,N’-bis-methylene acrylamide, 0.1% SDS, 1.0 M urea and 0.1 M phosphate buffer, pH 7.2, produced the most satisfactory separations. Met,hods for preparing gels, the techniques for electrophoresis, mechanical fractionation of the gelsand gel staining have been published (Summers et al., 1965; Maizel, 1966). Counting of radioactivity. Gels were fractionated into approximately 160 fractions; the pumping rate of the diluent into the gel fractionator (Maizel, 1966) was adjusted so that each fraction was approximately 0.S ml. If the radioactive label was 14C, these fractions were collected on aluminum planchettes and counted in a Nuclear Chicago or Beckman Widebeta low back-

VIRIONS

AND CORES

719

ground counter. If the label was 3H, or if a double-label analysis was being carried out, the fractions were collected in vials containing 10 ml Bray’s solution and counted in a Packard Tri-Carb scintillation spectrometer. Radioactivity in fractions derived from density gradients was determined by precipitation with trichloroacetic acid (after addition of carrier RNA and protein), filtration onto Millipore membranes, and counting either after mounting on planchettes (for ‘“C) or after addition to vials containing Bray’s solution or toluene plus scintillator as just described (for 3H). Materials. Eagle’s medium was purchased from the Grand Island Biological Company in the form of Joklik’s modification of minimum essential medium. The 14C-labeled amino acid mixture (U.L.) (0.67 mC/mg) was purchased from the New England Nuclear Corporation. The reconstituted 3Hprotein hydrolyzate (l-2 mC/pmole) was obtained from Schwarz BioResearch. Acrylamide and N ,N’-bis-methylene acrylamide were purchased from Eastman Organic Chemicals; Nonidet P40 (NP 40) was obtained form the Shell Chemical Company, Lt’d., England. RESULTS

The Proteins of Vaccinia Virus The first part of the investigation reported here involved an examination of the structural proteins of the vaccinia virus particle. For this purpose highly purified virus preparations were dissociated into the individual protein speciesand the resulting mixtures were separated by means of electrophoresis in polyacrylamide gels. A large number of different treatments were tested for ability to achieve optimal dissociation of vaccinia virus particles, which is readily observed macroscopically as the conversion of the milky virion suspension into a crystal-clear solution. The solvent which was finally adopted consisted of 2.5 % SDS, 0.7 M urea, and 1.5 M 2-mercaptoethanol (final concentration in all cases).The mixture was incubated at 37” for at least 1 hour and then dialyzed against 0.01 M phosphate buffer, pH 7.1, containing 0.1% SDS, 0.5 M urea, and 0.012 M 2-mer-

720

FIG. 1. trophoresis, shows the accurately trophoresis

HOLOWCZAK

AND JOKLIK

Polyacrylamide gel in which dissociated vaccinia virus strain WR was subjected to elecstained with Coumassie Blue (Maizel, 1966; Faeekas de St Groth et al., 1963). This figure relative positions of the major viral structural protein groups, but staining does not reflect their relative amounts. Length of gel: 27 cm; electrophoresis conditions as in Fig. 2. Elecwas from left to right.

FRACTION

FIG. 2. Electropherogram of proteins in vaccinia virus strain WR propagated in mouse L fibroblasts in the presence of a mixture of 14C-L-amino acids (U.L.). Purified virus wa,s solubilized as described in Materials and Methods. A sample of the solubilized virus containing 830 pg of protein (specific activity 85 cpm per microgram of protein) was mixed with 50 ~1 of 60% sucrose (in HZO) and analyzed by gel electrophoresis (36 hours, 2.6 V/cm). The direction of electrophoresis was from left to right. captoethanol. This treatment most probably completely dissociates all viral protein into constituent polypeptide chains; certainly more drastic treatment failed to reveal additional components. Figure 1 shows a pattern of dissociated vaccinia virus stained with Coumassie Blue. Figure 2 shows the radioactivity

profile obtained by applying gel 830 pg of vaccinia virus with a complete mixture amino acids. The pattern complex,

but highly

to a 27 cm-long protein labeled of 14C-labeled is clearly very

reproducible.

There

is

no material which does not migrate into the gel; that is, the virions appear to be completely dissociated. We have divided the pattern into 17 regions which can be clearly discerned on every profile we have examined. VSP-1

and VSP-2

are minor

components

which are always well separated from the

rest. VSP-4 and 6 are major components which are clearly multiple and can be resolved on more prolonged electrophoresis. VSP-8 is well separated. VSP-10, 11, and 12, and VSP-14, 15, and 16 are two families of proteins the components of which are not well separated from each other. VSP-3, 5, 7, 9, 13, and 17 are minor components; it is difficult to quantitate these, as they are in close proximity to major components. Other minor components may be present, Table 1 lists the approximate relative amount of label in each of these 17 regions; since the virions are labeled with a complete mixture of amino acids, this probably provides a reasonable estimate of the relative mass in each of these regions. Several experiments were carried out in which virus was labeled with a pair of amino acids, one labeled with 14C,the other

PROTEINS

OF

VACCINIA

with 3H. A typical coelectropherogram is shown in Fig. 3. Clearly some of the peaks contain different relative amounts of tryptophan and lysine. Thus VSP-5 is rich in tryptophan and poor in lysine, as is VSP-7; on the other hand, VSP-8 and the VSP-1416 region is rich in lysine, but poor in tryptophan. TABLE

RELATIVE Protein VSP

1

AMOUNTS OF STRUCTURAL PROTEIN GROUPS IN VACCINIA VIRLW

group

Relative

amount

(70)

4.7 3.5 3.4 28.6 2.6 18.7 1.8 7.8 0.8 14.0 2.3 11.1 0.8

1 2 3 4 5 6 7 8 9 l&12 13 14-16 17

a The areas corresponding to proteins or protein groups in the electropherogram depicted in Fig. 2 were computed, and the relative amount of each calculated therefrom.

0

20

40

60

80

VIRIONS

AND

CORES

721

Vaccinia virus grown in HeLa and L cells and labeled with the complete mixture of amino acids, was subjected to coelectrophoresis. The profiles obtained coincided exactly. Identification of the Proteins in Vaccinia Virus Cores It is of primary interest to determine the location within the vaccinia virion of the various structural proteins revealed by electrophoresis. There is general agreement that the major morphological components of the vaccinia virion are an outer membrane, an outer region surrounding the core, two lateral bodies in this outer region, and the core which is itself surrounded by a membrane (Westwood et al., 1964). We are attempting to determine which of the protein components are associatedwith each of these morphological units. Definitive evidence has so far been obtained concerning the cores. In addition, one of the viral structural proteins has been identified as one which is readily dissociated from virions. Several techniques were tested in efforts to prepare and purify vaccinia virus cores. As described previously (Joklik, 1964), cores may be isolated from cells infected in the presence of actinomycin D or puromycin, in

100

120

140

160

FRACTION

FIG. 3. Relative amounts in L cells in the presence (4C/mmole) was purified activity 150 cpm/pg with phoresis as described in

of tryptophan and lysine in the polypeptides of vaccinia virus. Virus grown of 160 & of L-tryptophan-3-i% (21.8 mC/mmole) and 200 PC nn-lysine-4,5-*H and solubilized as described. A sample containing 525 pg protein (specific respect to 1% and 30 cpm/pg with respect to 3H) was analyzed by gel electrothe legend to Fig. 2.

722

HOLOWCZAK

AND JOKLIK

;riIfYjJ

fj----j

20

30

FRACTIONO FIG. 4. Sedimentationof the corefraction of vaccinia virus in 2040% (w/w, lO+M phosphatebuffer) sucrosedensity gradientsafter centrifugation for 60 minutes at 13,000rpm in the SW 25 rotor of the SpincoModel L Ultracentrifuge at 4’. The coreswere collectedon sucrosecushionsas described,then diluted to a concentrationof 3.5 X lO~o/ml(referred to the starting concentrationof wholevirus particles which was1 to 2 X 10” EB/ml), sonicatedand l-2 ml layered onto the gradient. Panel A: Cores preparedasdescribedin Materials and Methods usingNP 40, 2-mercaptoethanol,and alkylation with ol-iodoacetamide. Panel B: CorespreparedusingNP 40 and 2,2’-dithiodiethanol.

which the first, but not the second, stage of uncoating proceeds. Treatment with sodium deoxycholate also yields structures which sediment less rapidly than intact virions: however there is always also formed much aggregated material. The best method for dissociating vaccinia virions was one based on that of Easterbrook (1966) and consisted of the addition of the nonionic detergent NP 40, followed either by 2-mercaptoethanol and subsequent acetylation with iodoacetamide, or by 2,2’-dithiodiethanol. Various combinations of NP 40 and urea were also tried, but were lesssuccessful.The method finally adopted is described in the section on Materials and Methods. Figure 4 shows the sedimentation characteristics of vaccinia cores as released by NP 40, Z-mercaptoethanol, and iodoacetamide (panel A) and NP 40 plus 2,2’dithiodiethanol (panel B), in relation to that of vaccinia virions. The former treatment released two bands (designated H and L); the latter treatment released one band only, which sedimented with the characteristics of band H. All bands could be rebanded without change in sedimentation characteristics. Electron micrographs of both H and L bands, as well as of the particles prepared with 2,2’-dithiodiethanol, showed typical cores, identical with those observed by Easterbrook (1966) using a very similar method. While there is no doubt that both H and L bands consist predominantly of viral cores, a conclusion borne out by the electrophoretic pattern (seebelow), we cannot offer

an explanation at this time as to why cores should form two discrete bands. Possible reasonsare discussedlater. Figure 5 shows a polyacrylamide gel electropherogram of the L band shown in Fig. 4A, derived from virions labeled with the complete mixture of 14C-labeledamino acids. Intact virus labeled with a complete mixture of 3H-labeled amino acids was coelectrophoresed. It is seenthat cores consist of VSP-1, VSP-2, and the VSP-4 complex. The H band (Fig. 4A) gave an identical pattern, as did the cores prepared with 2,2’-dithiodiethanol (Fig. 4B). Demonstration qf the Sequential Release of Protein from Virions When vaccinia virions are treated with NP 40 alone (for conditions see Materials and Methods), protein material is released from them. Figure 6 showsthe profile of this material. Over 90% of it is VSP-6. When virions are treated first with NP 40, and then with 2-mercaptoethanol, cores are produced. The released material should therefore contain all the viral structural proteins except VSP-1, VSP-2, and VSP-4, which constitute the core. Figure 7 showsthe profile of the total released material. It is seen that it does indeed contain all the expected protein species. In particular, it clearly contains the minor component VSP3, the presence of which could not be definitely established in cores, since it is situated in electropherograms in the ascending slope of VSP-4.

PROTEINS

OF VACCINIA

VTRIONS

AND

CORES

723

FRACTION FIG. 5. Proteins associated with vaccinia virus cores. Purified vaccinia virus labeled with W-labeled amino acids was degraded with NP 40 and 2,2’-dithiodiethanol as described in Materials and Methods. The core fraction was banded in sucrose density gradients, recovered, resuspended, and solubilized as described for whole virus. A sample of this dissolved core fraction was mixed with proteins from purified vaecinia virus IabeIed with 3H-labeled amino acids to serve as markers. Electrophoresis was performed as described in the legend to Fig. 2.

FRACTION FIG. 6. Proteins released from vaccinia virus labeled with W-labeled amino acids by treatment with NP 40. After incubation with NP 46 at 37O for 2 hours as detailed in Materials and Methods, the particulate fraction was separated from the suspension by centrifugation at 15,600 rpm for 1 hour in the 85-34 rotor of a Servall ultracentrifuge. The clear supernatant liquid was dialyzed for 48 hours against 0.1% SDS at room temperature. The solution was concentrated 16-15 times by packing the dialysis bag in Sephadex G-75. The concentrated extract was made 0.7 M with respect to urea and 1.5 M with respect to 2-mercaptoethanol and dialyzed as described. A sample was mixed with proteins from purified virus labeled with 3H-labeled amino acids and subjected to analysis by electrophoresis as described in the legend to Fig. 2.

724

HOLOWCZAK

AND JOKLIK

FRACTION FIG. 7. Proteins released from vaccinia virus labeled with “C-labeled amino acids upon treatment with NP 40 and 2,2’-dithiodiethanol. The solubilized fraction was separated from the particulate fraction and treated and analyzed as described in Fig. 6.

Thus, to summarize: cores are composedof VSP-1, 2, and 4; NP 40 alone releasespredominantly VSP-6; and on addition of 2mercaptoethanol to an NP 40-treated preparation, all other viral proteins except VSP-1, 2, and 4 are solubilized and clearly recognizable in gel profiles. DISCUSSION

As expected, the structural protein complement of vaccinia virus particles turns out to be very complex. Polyacrylamide gel electrophoresis of dissociated virus reveals at least 17 components. Many of these are undoubtedly multiple; in fact, results so far do not allow the placing of an upper limit on the total number of polypeptide chain speciesin vaccinia virus structural protein. The major components are the regions designated VSP-4, VSPS, VSP-8, VSP-10-12 and VSP-14-16. In addition, VSP-1, 2, and 3 can be unequivocally identified, and these are possibly single protein species. The individual components comprising the families of polypeptide chains designated VSP-4, VSP-6, VSP-l(tl2, and VSP-14-16 display someremarkable relationships. First, they are very similar in size and charge since they are imperfectly resolved on gels as long as 27 cm. Second, they are closely

associatedwithin the virus particle. Thus all the components of the VSP-6 region are released by treatment of virions with NP 40, and are therefore most probably situated near the surface of the virion; and all the components of the VSP-4 region reside in the core. Finally, as will be shown in the succeeding paper (Holowczak and Joklik, 1967), the individual components of these families behave as units as regards the time in the viral growth cycle when they are formed. Vaccinia virus cores consist of VSP-1, VSP-2, and VSP-4 which is the major component. As prepared from virions with NP 40 and 2-mercaptoethanol followed by acetylation, viral cores yielded two bands in sucrose density gradients which have the sameprotein complement. As prepared with NP 40 and 2,2’-dithiodiethanol, the cores form onlv one band, which coincides with the heavier of the two bands just described, and again has exactly the same protein complement. The reason for this sedimentation behavior is not clear. Electron micrographs of all these bands show particles very similar to the cores described by Easterbrook (1966). Band H and the band from the virions treated with 2,2’-dithiodiethanol reveal rather more doublets than band L. It may be recalled that viral cores aggregate

PROTEINS

OF

VACCINIA

badly if not acetylated (see section on Materials and Methods). It is conceivable that the H band contains core doublets, and the L band singlets; and that treatment with 2,2’-dithiodiethanol also leads to the formation of core doublets. A more remote possibility is that particles in H and L bands differ in the extent to which lateral body material is still attached to the cores. Easterbrook (1966) had indeed noted that structures consisting of cores still combined with lateral bodies are intermediates, under certain conditions, in the formation of ‘(clean” cores. However, this alternative is unlikely, since the protein complement of H and L bands does not differ detectably. The nonionic detergent NP 40 removes just) one major protein component from virions. The whole VSP-6 family of proteins is thus presumably situated near the surface of the virion. VSP-6 acquires added interest from the finding that it is an “early” protein, that is, it is translated from messenger RNA t,ranscribed from parental viral genomes, as is VSP-1 and VSP-2. By contrast, the other core proteins, those of the VSP-4 complex, are late proteins. Experiments establishing the time when the various protein molecules destined to be incorporated into mature progeny are synthesized, and presenting evidence as to which of the structural viral proteins are early and late proteins, are described in the succeeding paper (Holowczak and Joklik, 1967). ACKNOWLEDGMENTS We are grateful to Dr. J. V. Maize1 of this Department for advice concerning the technique of polyacrylamide gel electrophoresis; we also wish to thank Dr. E. Robbins of this Department for taking some electron micrographs. This investigation was supported by grants number AI-04913 and AI-04153 and GM-876 from the National Institutes of Health. One of us (W. K. J.) is the recipient of a USPHS Research Career Award (No. I-K6-AI-22. 554). REFERENCES APPLEYARD, G., HUME, V. B. M., and WESTWOOD, J. C. N. (1965). The effect of thiosemicarbaeones on the growth of rabbitpox virus in tissue culture. Ann. N.Y. Acad. Sci. 130, 92-104.

VIRIONS

AND

CORES

725

BECKER, Y., and JOKLIK, W. K. (1964). Messenger RNA in cells infected with vaccinia virus. Proc. Natl. Acad. Sci. U.S. 51, 577-585. DALES, S. (1963). The uptake and development of vaccinia virus in strain L cells followed with labeled viral deoxyribonucleic acid. J. Cell Biol. 18, 51-72. EAGLE, H. (1959). Amino acid metabolism in mammalian cell cultures. Science 130, 432-437. EASTERBROOK, K. B. (1966). Controlled degradation of vaccinia virions in vitro: an electron microscopy study. J. Ultrastruct. Res. 14, 484496. FAZEKAS DE ST. GROTH, S., WEBSTER, R. G., and DATYNER, A. (1963). Two new staining procedures for quantitative estimation of proteins on electrophoretic strips. Biochim. Biophys. Acta 71, 377-391. HOLO~CZAK, J. A., and JOKLIH, W. K. (1967). Studies on the structural proteins of vaccinia virus. II. Kinetics of synthesis of individual groups of structural proteins. virology 33, 726-739. JOKLIK, W. K. (1962a). The preparation and characteristics of highly purified radioactively labeled poxvirus. Biochim. Biophys. Acta 61, 290-301. JOKLIK, W. K. (196213). The purification of four strains of poxvirus. viTiro2ogy 18, 9-18. JOKLIK, W. K. (1964). The intracellular uncoating of poxvirus DNA. II. The molecular basis of the uncoating process. J. Mol. Biol. 8, 277-288. MAIZEL, J. V. (1966). Acrylamide-gel electropherograms by mechanical fractionation: radioactive adenovirus proteins. Science 151,988-990. MARQUARDT, J., HOLM, S. E., and LYCKE, E. (1965). Immunoprecipitating factors of vaccinia virus. Virology 27, 170-178. PETERS, D. (1960). Struktur und Entwicklung der Pockenviren. Proc. .Qh Intern. Congr. Eteetron Microscopy, Berlin, 1968 pp. 552-573. Springer, Berlin. RODRIGUEZ-BURGOS, A., CHORDI, A., DIAZ, R., and TORMO, J. (1966). Immunoelectrophoretic analysis of vaccinia virus. Virology 30, 569-572. SMITHIES, 0. (1965). Disulfide bond cleavage and formation in proteins. Science 150, 1595-1598. SUMMERS, D. F., MAIZEL, J. V., and DARNELL, J. E. (1965). Evidence for virus-specific noncapsid proteins in poliovirus-infected HeLa cells. Proc. Natl. Acad. Sci. U.S. 54, 505513. WEST~OOD, J. C. N., HARRIS, W. J., ZWARTOUW, H. T., TITMUSS, D. H. J., and APPLEYARD, G. (1964). Studies on the structure of vaccinia virus. J. Gen. Microbial. 34, 67-78.