Proteins Specified by Herpes Simplex Virus - Journal of Virology

JOURNAL OF VIROLOGY, Nov. 1972, p. 1044-1052 Copyright @ 1972 American Society for Microbiology

Vol. 10, No. 5 Printed in U.S.A.

Proteins Specified by Herpes Simplex Virus VIII. Characterization and Composition of Multiple Capsid Forms of Subtypes 1 and 2 WADE GIBSON AND BERNARD ROIZMAN Department of Microbiology, The University of Chicago, Chicago, Illinois 60637

Received for publication 7 August 1972

Two classes of herpesvirus capsids, designated A and B, were isolated from the nuclei of human cells infected with herpes simplex virus (HSV). A and B capsids share in common four structural proteins, i.e., no. 5, 19, 23, and 24. B capsids contain 7.7 to 9.7 times more deoxyribonucleic acid than A capsids; moreover, they contain proteins no. 21 and 22a in addition. All of the proteins contained in the capsid except no. 22a are present in the enveloped nucleocapsids (virions) in approximately the same molar ratios. The capsid proteins of HSV-1 cannot be differentiated from their HSV-2 counterparts with respect to electrophoretic mobility. A third class of capsids, designated C capsids, was isolated from virions contained in the cytoplasm of infected cells by the same procedure used to obtain A and B capsids. The C capsids contain all of the proteins present in A capsids plus proteins 1 to 3 and 21. A preceding paper (25) in this series reported the composition, electrophoretic mobility, and molecular weight of proteins comprising the herpes simplex virus (HSV) virion. Subsequently (13) it was shown that a number of the glycosylated proteins present in the virion were also present in purified membranes of infected cells. The conclusion that these glycoproteins were constituents of the envelope of the virion was reinforced by the observation that NP40, a mild nonionic detergent, partially stripped them from the surface of the virion (25). The question still remained as to which proteins are the structural components of the viral capsid. Pertinent to the studies of the composition of viral nucleocapsids reported in this paper are the following observations and reports. (i) The nucleocapsid assembles in the nucleus, and in cells infected with most herpesviruses it acquires an envelope as it buds through the inner lamella of the nuclear membrane (3, 22). (ii) Electron microscopy studies (18-20, 23, 24) have shown that at least two types of capsids are present within the nucleus of HSV-infected cells, and that they appear to differ, at least with regard to staining properties, from the capsid contained within virions accumulating in the perinuclear space or cysternae of the endoplasmic reticulum. (iii) The three most current descriptions of herpesvirus nucleocapsids (1, 14, 21) have been based on structures obtained by widely differing techniques and, not surprisingly, are not in agreement on

about the exact protein composition. Furthertwo of the three descriptions (14, 21) are based on radioisotopic profiles obtained by sectioning the analytical acrylamide gels-a technique that does not afford as high a resolution as absorbance profiles from either stained gels or autoradiograms. We are presenting evidence here in support of the following: (i) two classes of capsids, differing with respect to both deoxyribonucleic acid (DNA) content and protein composition, are present in the nuclei of HSV-infected cells and (ii) the protein composition of capsids derived by stripping of the envelope from virions differs from that of either type of intranuclear capsid. more,

MATERIALS AND METHODS Virus and cells. The properties of HSV subtypes 1 and 2 used in these studies have been described elsewhere (7, 15, 25). Enveloped virions were produced in HEp-2 cells, and the intranuclear capsids were produced in either HEp-2 or Vero (simian) cells. Pertinent information about the growth, maintenance, infection, and radioisotopic labeling of cells has been presented in detail by Spear and Roizman (25). Deviations from these general techniques are indicated in the text. Isotopes and reagents. The isotopes and reagents were reconstituted 3H-protein hydrolysate (mixture no. 3130-08, 1 mCi/ml) and thymidine-2-14C (no. 2523-96, 10 /ACi/mi) from Schwarz/Mann, Orangeburg, N.Y.; uniformly labeled 14C-L-amino acid mixture (mixture no. NEC-445, 100 ,Ci/ml), and 1044

VOL. 10, 1972

HSV-SPECIFIED PROTEINS

D-glucosamine-1-14C (NEC-193X, 100 ,Ci/ml) from New England Nuclear Corp., Boston, Mass.; acrylamide from K & K Laboratories, Plainview, N.Y.; N,N'-methylenebisacrylamide and N,N,N' ,N'-tetramethylethylenediamine (TEMED) from Eastman Chemicals, Rochester, N.Y.; dextran 10 from Pharmacia, Uppsala, Sweden; Coomassie Brilliant Blue from Colab Laboratories, Inc., Glenwood, Ill.; ultrapure urea from Mann Research Laboratories, New York, N.Y.; Nonidet P-40, a gift from Shell Oil Co., New York, N.Y.; sodium desoxycholate from Schwarz/Mann, New York, N.Y.; BRIJ 58 (polyoxyethylene [20] cetyl ether) from Atlas Chemical Industries, Inc., Wilmington, Del.; herring sperm DNA, type IV, from Sigma Chemical Co., St. Louis, Mo.; bovine serum albumin (crystallized A-grade) from Calbiochem, Los Angeles, Calif.; and deoxyribonuclease I (electrophoretically purified) from Worthington Biochemical Corp., Freehold, N.J. Isolation of intranuclear virus particles. Cells were harvested between 18 and 24 hr after infection; collected by centrifugation at 1,500 X g for 10 min (low-speed centrifugation); and resuspended (5 X 107 cells/ml) in 0.15 M NaCl, 0.01 M tris(hydroxymethyl)aminomethane (Tris), pH 7.2, and 0.002 M MgCl2 containing 1%c NP40. After incubation for 30 min at 0 C, the nuclei were sedimented from the suspension by low-speed centrifugation and lysed by adding sodium deoxycholate (DOC) to a final concentration of 0.5%. The lysate was gently warmed in the presence of deoxyribonuclease (50 ,ug/ml) to decrease the viscosity. BRIJ-58 and urea were then added to final concentrations of 0.5% and 0.5 M, respectively, and the extract was clarified by lowspeed centrifugation. The supernatant fluid was layered on top of linear, 10 to 40% (w/w) sucrose density gradients, prepared in 0.15 M NaCl and 0.01 M sodium phosphate buffer (pH 7.2), and centrifuged for 60 min at 23,000 rev/min and 4 C in a Spinco SW27 rotor. Two light-scattering bands located near the middle of the tube were resolved during the course of centrifugation. Each was recovered mechanically from the top of the gradient and either analyzed immediately, or frozen and stored at -20 C. Isolation of enveloped virions. Enveloped virions were isolated essentially as described by Spear and Roizman (25). Briefly, infected cells were swollen in hypotonic buffer and disrupted by several strokes with a Dounce homogenizer, and the cytoplasmic fraction was layered onto 3 to 30% dextran-10 gradients and centrifuged at 23,000 rev/min for 60 min in a Spinco SW25.3 rotor at 4 C. Enveloped virions sedimented to a position just above the middle of the tube, and formed a light-scattering zone that was collected mechanically from the top of the tube. This material was not further purified. Preparation of detergent-treated cytoplasmic virions. (i) NP-40-extracted cytoplasmic virions were prepared by suspending approximately 108 infected cells in 1 ml of 0.15 M NaCl, 0.01 M Tris (pH 7.2), and 0.002 M MgCl containing 1% NP-40. After incubation for 20 min at 0 C the nuclei were removed from the lysate by low-speed centrifugation. The lysate was further clarified by a second low-speed centrifugation.

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(ii) NP-40/DOC-extracted cytoplasmic virions were prepared by further processing a clarified NP-40 lysate, obtained as above, with DOC (0.5%), deoxyribonuclease (50 Mug/ml), BRIJ-58 (0.5%), and urea (0.5 M) in the same fashion as described above for the isolation of intranuclear virus particles. The lysate was clarified by low-speed centrifugation. Both NP40 and NP-40/DOC lysates were subjected to rate-velocity sedimentation on 10 to 40% (w/w), linear sucrose density gradients prepared in 0.15 M NaCl and 0.01 M sodium phosphate (pH 7.2) and centrifuged at 23,000 rev/min for 60 min at 4 C in a Spinco SW27 or SW25.3 rotor. One visible band was resolved from each lysate at about the mid-point of the tube; it was collected mechanically from the top of the gradient and stored at -20 C. Analytical gel procedures. The electrophoretic, staining, and autoradiographic techniques used in these studies have been described in detail by Spear and Roizman (25). The important features are as follows. (i) Samples to be analyzed were solubilized by heating at 100 C for 2 min in the presence of 2% sodium dodecyl sulfate (SDS), 5% beta-mercaptoethanol, and 50 mm Tris (pH 7.0). Solubilized preparations were applied to the gels in 50- to 150-Militer volumes generally containing between 50 and 100 ,ug of protein. (ii) Discontinuous SDS gels (4, 16) were formed in 12-cm glass tubes cleaned by soaking overnight in a solution of concentrated sulfuric aciddichromic acid and rinsed thoroughly with water, rather than siliconized as recommended in the original procedure. The main gel (8.5 cm in length) contained 8.5% acrylamide; the stacker gel (2.0 cm in length) contained 3% acrylamide. (iii) Proteins in the gel were fixed and stained with 0.03% Coomassie Brilliant Blue in 10% acetic acid and 25% 2-propanol according to the procedure of Fairbanks et al. (10). Destaining was in 10% acetic acid, 10% 2-propanol. (iv) Autoradiograms were made from gels sliced longitudinally and dried as described by Fairbanks (9). A mechanical press was used to hold the dried gel firmly against the X-ray film during exposure. (v) Absorbance profiles were made from stained gels and autoradiograms, both scanned at 550 nm, with the Gilford linear transport attachment and Gilford recording spectrophotometer. Protein and DNA analyses. Protein concentrations were determined by the Lowry procedure (17) as modified by Eggstein and Kreutz (6). Bovine serum albumin was used as the standard. DNA concentrations were determined by the diphenylamine technique described by Dische (5) as modified by Burton (2). Herring sperm DNA was used as the standard. Electron microscopy. Thin sections were prepared for electron microscopy examination from pellets of A and B capsids. Briefly, capsid suspensions recovered from nuclear lysates as described above were subjected to centrifugation at 25,000 rev/min for 2 hr at 4 C in a Spinco SW27 rotor. The pellets that were obtained were fixed in gluteraldehyde, postfixed in osmium tetroxide, embedded in Epon, and stained with uranyl acetate and lead citrate prior to examination in an AEI-EM6B microscope. A detailed description of this procedure and of the technique used to

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GIBSON AND ROIZMAN

J. VIROL.

prepare whole mounts negatively stained with sodium phosphotungstic acid has been published elsewhere (25). RESULTS Centrifugation of nuclear lysates. Nuclear extracts prepared as described above, on centrifugation through a density gradient, formed two bands designated as top and bottom (Fig. 1). A third band occasionally appeared near the bottom of the tube; its occurrence generally indicated incomplete solubilization of cytoplasmic membranes with NP-40. The bands shown in Fig. 1 were obtained from approximately 109 cells infected with HSV-2. Some variability has been noted in the amount of material in the top and bottom bands recovered from different preparations; however, several lines of evidence indicate that it does not result from the extraction procedure per se. Specifically, (i) extending NP-40 extraction to 24 hr at 0 C or substituting Triton X-100 for NP-40, (ii) changing the NP-40 extraction to 15 min at 37 C, and (iii) allowing the final extract to stand for 24 hr at 0 C prior to centrifugation do not visibly affect the yield of the material in either the top or bottom bands. Electron microscopy. Electron microscopy studies revealed the presence of capsids in both FIG. 1. Top and bottom bands in a sucrose density top and bottom bands. Enveloped or partially obtained by centrifugation of a niuclear lysate gradienit the In general enveloped particles were absent. HEp-2 cells inzfected with HSV-2. The lysate was capsids in each band were uniform in size and from layered on top of a 10 to 40% (w/w) sucrose density appearance. In negatively stained (sodium phos- gradient prepared in 0.15 M NaCI and 0.01 .f sodium photungstic acid) preparations, the capsids in the phosphate (pH 7.2) and centrifuged at 23,000 rev/min at top band were filled with stain, whereas those in 4 Cfor 60 miii in a Spinico S W27 rotor. the bottom band were filled only partially, delineating an internal structure (Fig. 2A, C). In the second experiment, approximately 4 X Examination of thin sections of top- and bottomband material (Fig. 2B, D) stained with uranyl 108 cells were infected with hSV-1 and labeled acetate and lead citrate showed that top-band with 3H-amino acids (1 ,Ci/ml) and '4C-thymicapsids appear to be empty, whereas bottom-band dine (0.4 ,Ci/ml) from 1 to 24 hr pcstinfection. capsids contain a centrally located, densely stain- The nuclear lysate prepared from these cells was ing structure. Cross-contamination of the bands centrifuged on sucrose density gradients. After was estimated by particle counts to be less than centrifugation, the top and bottom bands were collected; an equal volume (50 Mliters) of each 10%. For the sake of brevity and uniformity, we shall was precipitated with cold, 7%sY- trichloroacetic designate the particles in the top and bottom acid, collected by vacuum filtration on 0.45-,m bands as A and B capsids, respectively, without HA nitrocellulose discs, dried, and counted in a differentiating between the terms capsid and Packard Tri-Carb spectrometer. The results summarized in Table 1 indicate that nucleocapsid. DNA-protein composition of top- and bottom- A capsids contain 7.7 to 9.7 times less DNA than band capsids. Two series of experiments were B capsids. Analyses of the size, structure, and done. In the first experiment DNA and protein density of the B capsid DNA by rate velocity composition were determined on an unlabeled sedimentation in neutral and alkaline sucrose preparation of A and B capsids obtained from density gradients, and by isopycnic centrifugacells infected with HSV-2. Following their isolation in CsCl solutions, respectively, indicate that tion as described above, the capsids were pelleted it has the same characteristics as that isolated by additional centrifugation. Sequential DNA and from mature virions reported previously from protein determinations were done on each pellet. our laboratory (11, 12, 15).

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VOL. 10, 1972

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niegatively stained with 2%7o sodium phosphotungstic acid containing 0.01% bovine serum albumin (A an2d C). Tkinsectionted preparationis were stained with uranyl acetate and lead citrate (B and D). The capsids are approximately 100 imn in diameter. TABLE 1. DNA and protein content of A and B capsids DNA/protein of

Material analyzed

DNA

Protein

DNA/protein

DNA/protein of A capsids

Expt 1 (HSV-2) A capsids B capsids

9.27,ug

671 ,/g 297 ,Ag

0.014 0.136

9.7

40.43 JAg

Expt 2 (HSV-1) A capsids B capsids

58 counts 'mina 282 counts/min

1,194 counts,/minb 766 counts/min

0.048 0.368

7.7

a

14C-thymidine was used to label HSV-DNA. bH-amino acid mixture was used to label HSV proteins.

Structural proteins of A and B capsids. The protein components of the A and B capsids were identified on the basis of three szries of experiments. The first series was a direct comparison

of A and B capsid proteins and an estimation of their molar proportionality. For the comparison, 3 x 109 cells were infected with HSV-2 and labeled with '4C-amino acids from 4 to 20 hr

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GIBSON AND ROIZMAN

postinfection. The nuclei were then isolated and the extract was centrifuged in sucrose density gradients. The top and bottom bands were collected, and the capsids were solubilized and subjected to electrophoresis in 8.5% polyacrylamide gels. Absorbance profiles of the stained gels and of their respective autoradiographic images are shown in Fig. 3. Estimations of the molar proportionality of the capsid proteins were based on staining analyses of three different preparations of B capsids, solubilized and separated electrophoretically in 8.5 % polyacrylamide gels as described above. As shown in Table 2, the molar proportions of B capsid proteins 5, 19, 21, 22a, 23, and 24 are approximately 12:4:1:16:8:1, respectively. The purpose of the second series was to establish the identity of the A and B capsid proteins. This was accomplished by subjecting HSV-1, B capsid proteins to electrophoresis alone and in an artificial mixture with partially purified, enveloped HSV-1 virion proteins. Absorbance profiles of the stained gels are shown in Fig. 4. The objective of the third series was to compare the proteins of HSV-1 and HSV-2 intranuclear capsids. Electropherograms of the HSV-1 and HSV-2, B capsid proteins are shown in Fig. 5. The results of these analyses can be summarized as follows. (i) B capsids contain four major proteins and several minoir components detectable by autoradiography (Fzig. 3, bottom band). On the basis of co-electrn ophoresis with virion proteins (Fig. 4), we have identified three of the major proteins as being no. 5, 19, and 23. The fourth major protein is not present in the virion and has been designated ats protein 22a.

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50

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FIG. 3. Electropherograms of the p)roteins contained in the top (A capsids) and bottom (EI capsids) banids isolated from HEp-2 cells infected uvith HSV-2 and labeled beginning 4 hr postinfkction with '4C-amino acids. Absorbance profiles of both the Coomassie Brilliant Blue-stained gels and of their. autoradiographic imnages are

show,z.

J. VIROL.

TABLE 2. Protein compositionz of HSV-2 B Capsids Protein no.

5 19 21 22a 23 24

Percent total protein in

Molecular

capsida ai SD

weightb

2.8 1.1 0.6 2.8 4 0.5 i 0.1

155 53 44 38.8 33 25

62.5 6.8 1.5 20.5 8.1 0.8

i i i i

(Xi10-3)

Molar ratio' 4- SD

12.59 4.00 1.06 16.50 7.66 1.00

i 0.56 i 0.66 i 0.44 4: 2.25 4 0.47 i 0.12

a Average + standard deviation of four determinations: two were made from gels stained with Coomassie Brilliant Blue stain and scanned at 550 nm, as described in Materials and Methods; the third was made from a gel fixed overnight in 25% isopropanol-10% acetic acid, stained with Fast Green stain (1% in water, pH 3.0) for 12 hr, destained overnight, and scanned at 620 nm; and a fourth was made from one of the Coomassie Brilliant Blue-stained gels scanned at 500 nm. I Determined as described in reference 25 on the basis of migration in 6, 7, 8.5, and 9% polyacrylamide gels by using proteins of known molecular weight as standards. c Calculated by dividing the percent of the total protein by the molecular weight and normalizing with respect to the protein present in lowest amount, no. 24.

Based on its electrophoretic mobility in the "mixture" gel (Fig. 4), we estimate that protein 22a has a molecular weight of 38,800. Only two of the minor B capsid proteins occur reproducibly in the same relative amounts from preparation to preparation; one of these corresponds to virion protein 24 (Fig. 4). The second has been tentatively identified as protein 21 because it is electrophoretically indistinguishable from protein 21 and the amounts of protein in band 24 and putative band 21 are about the same, as shown both by staining (Fig. 4 and 5) and autoradiography (Fig. 3). A similar proportionality was found between viral proteins 21 and 24 in highly purified preparations of virions (reference 25, Fig. 6, "4C-amino acid autoradiogram). (ii) A capsids lack protein 22a and the minor component tentatively identified above as protein 21, but are otherwise indistinguishable from B capsids in protein composition. (iii) Three principal lines of evidence indicate that the proteins listed above are structural components of the A and B capsids and are not impurities. Briefly, proteins 5, 19, 23, and 24 are present in approximately the same molar ratios in A and B capsids and in virions (Fig. 3 and 4) as those shown in Table 2 for B capsids. Secondly, in one experiment the bottom-band material was layered on top of a discontinuous density

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Coomassie Brilliant Blue stain

envelope with that of A and B capsids isolated from nuclear lysates. Approximately 4 x 108 HEp-2 cells were infected with HSV-1 and labeled beginning 2 hr after infection with '4C-glucosamine. Three types of particles were isolated from the cells 24 hr after infection, as described above: partially purified virions, NP-40-extracted cytoplasmic virions, and NP-40/DOC-extracted cytoplasmic virions. Briefly, the partially purified virions were isolated from half of the infected cells. The remaining cells were treated with 1 %c NP-40 in 0.15 M NaCl, 0.01 M Tris (pH 7.2), and 0.002 M MgCI2 for 20 min at 0 C. The nuclei which were not lysed by this procedure were pelleted by centrifugation at 1,500 x g for 10 min. Half of the clarified NP-40 lysate was not further processed; the other half was treated sequentially with DOC (0.5%c), deoxyribonuclease (50 ug/ml), BRIJ-58 (0.5%), and urea (0.5 M) in exactly the same fashion as nuclei were lysed for the preparation of A and B capsids. The NP-40 and the NP-40/DOCtreated lysates were then banded by centrifugation on sucrose density gradients as described Coomassie Brilliant Blue Stain

90 70 50 Distance (mm) FIG. 4. Electropherograms of B capsid and virion proteins alone an1d in an artificial mixture. Three companion gels were subjected to electrophoresis, onze coiztaimzing partially purified virions (HSV-J virionzs), another containing HSV-1 B capsid proteins, and a third containing approximately equal amounts of virion anld B-capsid proteins (mixture). Absorbance profiles of the Coomassie Brilliant Blue-stained gels are shown. 10

30

gradient, formed by layering equal volumes of CsCl solutions prepared in 0.005 M sodium phosphate (pH 7.1) with densities of 1.25, 1.35, and 1.9 g/cm3, respectively. The gradient was then centrifuged for 14 hr at 30,000 rev/min and 20 C in a Spinco SW41 rotor. A discrete band formed below the initial 1.25 to 1.35 g/cm3 interphase and contained all of the proteins in the relative amounts present in the sucrose density gradient. Thirdly, the electrophoretic mobility of the proteins is independent of the host, HEp-2 or Vero cells, in which they are produced. (iv) The electrophoretic mobilities of HSV-1 and HSV-2, B capsid proteins are the same (Fig. 5). Analysis of the capsids produced by stripping virons of their envelope. These experiments were designed to compare the protein composition of capsids produced by stripping virions of their

9

223HSV-1

_Oj

MIXTURE

10

30

50

70

90

Distance (mm) FIG. 5. Electropherograms of HS V-J and HS V-2 B capsid proteins. Companion gels con2taining, respectively, HSV-J, B capsid proteins (HS V-i); HSV-2, B capsid proteins (HS V-2); and a mixture of approximately equal amounts of HSV-i and HSV-2, B capsid proteins (mixture) were subjected to electrophoresis. Shown here are absorbaiice profiles of the Coomassie Brilliant Bluestained gels.

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J. VIROL.

5 and 19. Parenthetically, increasing the detergent concentrations from 0.5% to 1 % had little noticeable effect on the protein composition of the C capsid. DISCUSSION In this paper we are reporting the isolation and composition of three classes of HSV capsids. 1-3 The salient features of the data, particularly as they relate to the structure of the virion and its assembly, may be summarized as follows. (i) Nucleocapsids class A and B are derived from the nucleus of the infected cells. As discussed below, they had not been enveloped at the time of isolation. The A and B capsids differ in four C -Capsids respects. First, they differ in hydrodynamic properties in that A capsids sediment more slowly ~~~~~22a than B capsids. Second, they differ morphologically in that A capsids lack the core associated with B- Capsids viral DNA (reference 8, Fig. 2B, D). Third, as would be predicted from the electron micrographs, they differ in DNA content. B capsid 30 50 70 10 90 contains 10 times more DNA than A capsids; this DNA is hydrodynamically and structurally Distance (mm) the same as that isolated from virions and previFIG. 6. Electropherograms of B and C capsids iso- ously described (11, 12, 15). Lastly, A and B lated from HSV-J-infected HEp-2 cells. Absorbance profiles of the Coomassie Brilliant Blue-stained gels are capsids differ in protein composition. Although A and B capsids of HSV-1 cannot be differentiated shown. from their HSV-2 counterparts, with respect to the proteins present or their electrophoretic above. One band formed in each gradient and was mobilities, the B capsids of both subtypes have a major protein (no. 22a) not present in the A collected and stored at -20 C. Analyses of these three types of particles re- capsids. It is pertinent that protein 22a is not a vealed the following. (i) NP-40 treatment alone contaminant of the B capsid but rather a tenareduced the specific activity (counts per minute ciously bound, structural component of that of "C-glucosamine per microgram of protein) of particle. This conclusion is based on two findings. the NP-40 particle by 90% as compared with the First, it is associated in the sucrose density gradipartially purified virions. The electrophoretic ents only with the bottom band; it is absent both profile of the particles obtained after treatment from the gradient fractions above and below the of the cytoplasm with NP-40 alone was essentially bottom band. Moreover, it sediments through a the same as that of particles produced by NP-40 2 M urea-20% sucrose cushion and bands in CsCl treatment of highly purified virions previously solutions in association with the B capsid. Second, described (25). (ii) NP-40 treatment followed by protein 22a is synthesized and becomes a strucDOC extraction further reduced the specific tural component of B capsids in both HEp-2 activity to less than 2% that of the virion. Fig. 6 and Vero cells long after host synthesis is shut off. (ii) The C capsids have been derived by extracshows the electrophoretic profile of the proteins present in these particles, designated as C capsids. tion of virions with the same reagents as those The data are noteworthy from two points of used to lyse the nuclei of infected cells. As eviview. First, the C capsids contain all of the B denced by a 98% decrease in the specific activity capsid proteins except protein 22a. Secondly, of "IC-glucosamine, this procedure removes they contain several proteins present in the virion essentially all of the glycoproteins associated with (proteins 1 to 3) absent from the A and B capsids the virions. The C capsids differ substantially and purified membranes. They lack, however, from both A and B capsids with respect to prothe nonglycosylated proteins 4 and 22, as well as tein composition. This, basically, is the datum all of the glycoproteins present in purified virions which led us to conclude that A and B capsids and in the purified membranes. We should add do not represent degradation products of enthat the electropherograms of C capsid proteins veloped cytoplasmic virions. The most interesting exhibited a number of less well-defined and less feature of the C capsid is its retention of specific consistently reproducible bands between proteins nonglycosylated proteins throughout the rigorous 5

Coomassie Brilliant Blue stain

19

I I

23

~~~

VOL. 10, 1972

1051


detergent-isolation procedure. Thus, whereas proteins 4 and 22 were completely removed, protein band 1 to 3 was nearly quantitatively retained, None of these are found as structural components of A or B capsids. Preliminary studies based on pulse-chase experiments and some properties of the proteins suggest that protein 22a is a precursor to protein 22. The nature and function of proteins 1 to 3 are not certain. As previously reported (N. Frenkel and B. Roizman, Proc. Nat. Acad. Sci. U.S.A., in press; Proc. 25th Annu. Symp. Fund. Cancer Res. M. D. Anderson Hosp. Tumor Inst., in press), these proteins are synthesized later than the remaining proteins of the virion and correspond, in time of synthesis and amount of DNA template required for their synthesis, to species of ribonucleic acid transcribed from the same amount of DNA late in infection. One hypothesis, which reconciles the data reported in this paper, the transcriptional program of the virus (N. Frenkel and B. Roizman, Proc. Nat. Acad. Sci. U.S.A., in press; Proc. 25th Annu. Symp. Fund. Cancer Res. M. D. Anderson Hosp. Tumor Inst., in press), the time of synthesis of viral proteins (Gibson and Roizman, manuscript in preparation), and the observation that in HSV-infected cells empty capsids do not, as a rule, become enveloped (22), is as follows. Protein 22a is found in association only with capsids containing DNA; B capsids, containing this protein, are then able to bind proteins 1 to 3 and subsequently become enveloped. In the process protein 22a is modified, perhaps cleaved, such that it is subsequently removable from the enveloped particles by detergent treatment. According to this scheme, protein 22a is on or exposed to the surface of the capsid and, as previously suggested (N. Frenkel and B. Roizman, see in press references cited above), proteins 1 to 3 regulate and enable envelopment. (iii) We do not know at present whether A capsids are by-products or the precursors to B capsids. The major differences between A and B capsids are the presence in B capsids of a "core," more DNA, and two additional proteins, no. 21 and 22a. If protein 22a, present in higher abundance than protein 21, is indeed on the surface of the capsid, as we suspected based on its disappearance from virions, it would follow that the only protein which conceivably might be associated with the DNA core of the capsid is protein 21. (iv) The data presented in this paper permit us to order the virion proteins reported in preceding papers of this series into four groups as shown in Table 3. They are as follows: group 1

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