Early functions of the genome of herpesvirus

VIROLOGY

65, 355-362

(1975)

Early Functions V. Serological TAMAR Department

BEN-PORAT, of Microbiology,

of the Genome

Analysis MARIJA Vanderbilt

of Herpesvirus

of “Immediate-early” KERVINA,

University

AND

Proteins’ ALBERT

School of Medicine,

Nashville,

S. KAPLAN Tennessee 37232

Accepted December 31, 1974 Immediate-early proteins synthesized by cells treated with cycloheximide during early stages of infection with pseudorabies virus do not react with serum prepared against mature virions. Antiserum against immediate-early proteins was prepared. These sera were used to follow the synthesis of immediate-early proteins during the normal course of infection. Proteins reacting with antiserum against immediate-early proteins are synthesized up to 3 hr postinfection only. Immediate-early proteins are stable and are present in the cells at later stages of infection but do not become part of virions. The polyacrylamide gel electrophoretic (PAGE) profile of the immediate-early proteins synthesized during the normal course of infection is similar to that of proteins synthesized by cells treated with cycloheximide during early stages of infection. Immediate-early proteins are less stable upon storage at 4” than are proteins synthesized at later stages of infection. This treatment alters the PAGE profile of the immediate-early proteins. INTRODUCTION

The RNA transcribed from the genome of pseudorabies (Pr) virus may be divided into three major classes which, to conform to the terminology used for RNA molecules synthesized in bacteriophage-infected cells, have been classified as follows: (1) immediate-early RNA-molecules that are transcribed in the infected cells in which protein synthesis has been inhibited from the time of infection; (2) early RNA-molecules transcribed before the onset of viral DNA synthesis; (3) late RNA-molecules not transcribed unless protein and DNA synthesis have occurred in the infected cells (Ben-Porat and Kaplan, 1973). We have focused on the immediate-early RNA and on the functions it specifies. This class of RNA molecules accumulates in greater abundance in cycloheximidetreated, infected cells than it does during the normal course of infection and is translated once the synthesis of protein is allowed to resume (Rakusanova et al., 1971; ‘This National

work was supported by a grant Institutes of Health (AI-10947).

from the 355

Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

Ben-Porat et al., 1974). The proteins translated from the immediate-early RNA (the “immediate-early” proteins) have a characteristic pattern of migration in polyacrylamide gels which differs from those of the proteins synthesized at any stage during the normal course of infection. Recently, Honess and Roizman (1974) have reported similar results in herpes simplex virus-infected cells. The only immediate-early functions that have been uncovered thus far are regulatory in nature; they are involved in the inhibition of cell-specific macromolecular synthesis and the modulation of the transcription of the viral genome (Jean et al., 1974; Ben-Porat et al., 1974). In this paper we present experiments which show the following: (1) immediateearly proteins differ antigenically from the proteins made at later times during the infective process; (2) antisera prepared against immediate-early proteins made by infected cells that have been treated with cycloheximide during early stages of infection can be used to identify the immediate-

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AND KAPLAN

early proteins synthesized in small amounts during the normal process of infection.

cated at maximum output for 2 min in a Raytheon sonic vibrator and centrifuged at 25,000 g for 1 hr. (Approximately 60% of the acid-precipitable counts in the samples MATERIALS AND METHODS were not sedimented under these condiVirus and cell culture. The properties of tions). The proteins remaining in the suPr virus and cultivation of rabbit kidney pernatant were used in the indirect im(RK) monolayer cultures were described munoprecipitation test. previously (Kaplan, 1969). Indirect immunoprecipitation test. This procedure has been described in detail Media (Hamada and Kaplan, 1965). In brief, EDS. Eagle’s synthetic medium (Eagle, radioactively labeled proteins are treated with rabbit antisera, prepared as described 1959) plus 5% dialyzed bovine serum. above, and the complex is precipitated EDSI2 and EDSIlO. EDS containing one-half, or one-tenth, respectively, the with antiserum against rabbit y-globulin. The amount of acid-precipitable radioacnormal amount of amino acids. tive material in the pellet is a measure of PBS. Phosphate-buffered saline (Dulthe amount of radioactive antigens present becco and Vogt, 1954). in the sample. Preparation

of Antisera

Indirect

RK cultures were infected with Pr virus in the presence of cycloheximide (100 &ml) and incubated for 5 hr. The cultures were then extensively washed (to remove the cycloheximide) and further incubated for 3 hr. The cells were scraped into PBS and precipitated with 3.5 vol of cold acetone ( - 20’). The precipitate was resuspended in PBS and dialyzed overnight against the same buffer. Four rabbits were injected at weekly intervals both intramuscularly and interperitoneally four times with 5 mg of protein mixed with Freund’s adjuvant. The animals were bled and the serum was absorbed with acetone powder made from uninfected RK cells. Against Pr uirions. Partially purified virions (lo9 PFU/ml) were injected into roosters at weekly intervals. The roosters were bled and the serum was absorbed with acetone powder made from uninfected RK cells. Against

immediate-early

proteins.

Preparation of proteins for indirect immunoprecipitation test. Cultures treated

as indicated in Results were incubated for 2 hr in EDS/lO containing either [l’C]leutine (2 &i/ml) or t3H]leucine (20 &i/ml). They were washed once with PBS, scraped into PBS at a concentration of 2 x lo6 cells per ml, and stored at - 20” until used. Prior to immunoprecipitation the cells were soni-

immunofluorescence

test. Sec-

ondary RK cells grown on coverslips were infected and treated as described in Results. The coverslips were washed twice in cold (4”) PBS and fixed in cold (-20”) acetone-methanol (1: 1) for 5 hr. The coverslips were exposed to the indicated serum at 37” for 30 min, washed, and again exposed for 30 min to fluorescein-conjugated anti-y-globulin. After a final wash in PBS, the cells were mounted in 90% glycerol-10% PBS and were examined in an ultraviolet microscope for fluorescence. Purification of uirions. Mature Pr virions were purified from the extracellular fluids as described previously (Kaplan and BenPorat, 1970). Polyacrylamide gel electrophoresis (PAGE). This was performed essentially

according to the method of Laemmli (1970) and has been described in detail (Kaplan et al., 1975). Reagents and radiochemicals. L- [“H]Leutine (sp act, 46 Ci/mmole) and L-[“Clleutine (sp act, 312 mCi/mmole) were purchased from Schwarz/Mann. Fluoresceinconjugated and unconjugated sheep antirabbit and rabbit anti-chicken y-globulin were purchased from Grand Island Biological Company. Assay of radioactivity. This was determined as described previously (Kaplan et al., 1970).

“IMMEDIATE-EARLY”

HERPESVIRUS

RESULTS

Antigenic Characterization Early Proteins

of Immediate-

Cells infected with Pr virus and incubated with cycloheximide during the early stages of infection synthesize proteins with a characteristic migration pattern (the immediate-early proteins), once the cycloheximide is removed. Although some of the proteins synthesized at early stages of the normal infective process migrate in gels to the same positions as those of some of the immediate-early proteins, their identity cannot be established since cellular proteins synthesis is still going on at this time. To determine the time sequence of synthesis of immediate-early proteins during the normal course of infection, antisera were prepared against these proteins, as well as against mature viral particles, and the reactivity of these antisera with the proteins synthesized at various stages of the infective process was tested by indirect immunofluorescence and the indirect precipitation test. No reaction of this serum with cells at any stage of the normal infective process could be detected by immunofluorescence. Thus, during the normal course of infection, immediate-early proteins are synthesized, if at all, in amounts too small to be detected by this technique. On the other hand, cells which had been treated with cycloheximide during early stages of infection showed brilliant nuclear fluorescence, indicating that the immediate-early proteins were localized in the nucleus. Serum against mature virions reacted with in. fected cells as early as 3 hr postinfection and at 5 hr bright nuclear fluorescence was evident. Thus, proteins reactive with serum against mature virions are synthesized relatively early during the course of normal infection. However, cells which had been treated with cycloheximide and which synthesize immediate-early proteins did not react with the serum prepared against mature virions. The reactivity of sera prepared against immediate-early proteins and against mature Pr virions with proteins synthesized

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by cycloheximide-treated, infected cells after the removal of the drug, as well as with proteins synthesized by cells at various times during the normal course of infection, was also tested by indirect immunoprecipitation. The results are summarized in Table 1. Serum against mature virions did not react with the proteins synthesized by cells which had been treated with cycloheximide during early stages of infection; homologous precipitation of immediate-early proteins by serum against immediate-early proteins occurred, as expected. On the other hand, the serum against immediateearly proteins did not react significantly with the proteins synthesized by cells during a normal course of infection between 3 and 8 hr while the serum against mature virions did. However, approximately 5% of the proteins synthesized between 1 and 3 hr postinfection were precipitated by the serum against immediate-early proteins. Thus, although immediate-early proteins cannot be detected by immunofluorescence in normally infected cells, their synthesis at early stages of infection can be detected by an indirect immunoprecipitation test. These data may be summarized as follows: (1) Immediate-early proteins are synthesized in large amounts in cells which have been treated with cycloheximide during the early stages of infection. (2) These proteins are mainly associated with the nuclei of the cells. (3) Immediate-early proteins are synthesized during the normal course of infection in relatively small amounts only at early times after infection (l-3 hr). Stability of the Immediate-Early in the Infected Cells

Proteins

To test the stability of the immediateearly proteins synthesized during the normal course of infection, infected cells were labeled with [9H]leucine between 1 and 3 hr postinfection and were harvested either immediately after the labeling period or after an additional chase period (see legend to Table 2). The proteins were assayed in an indirect immunoprecipitation test. Table 2 shows that there was no decrease in

358

BEN-PORAT,

KERVINA, TABLE

REAIXIVIT~

AND KAPLAN 1

OF PROTEINS SYNTHESIZED BY INFECTED CELLS WITH SERA PREPARED AGAINST IMMEDIATE-EARLY PROTEINS AS DETECTED BY THE INDIRECT PRECIPITATION

Cells

Labeling period (hr after infection)

Input (cm)

Precipitated Anti-Pr

virus serum

VIRIONS TESTO

AND AGAINST

with Anti-IEP* (cpm)

serum

(cpm)

(%I

(S)

9,820 6,570 6,360 13,540

212 493 743 0

2.1 7.5 11.6

497 11 42 0

5.1 0.1 0.7

3,097 8,300

53 0

1.7

1,680 0

54.5

Untreated Infected Uninfected Cycloheximide-treated Infected Uninfected

1.0-3.0 2.5-4.5 5.5-7.5 5.5-7.5 O-5 hr 5.5-7.5 5.5-7.5

“The proteins synthesized by the virus-infected cells under the conditions indicated were labeled for 2 hr with [3H]leucine (20 &i/ml) and used in an indirect immunoprecipitation test, as described in Materials and Methods, with serum prepared either against mature Pr virus, against immediate-early viral proteins, or preimmune serum. The number of counts precipitated specifically (after subtraction of those precipitated by preimmune serum) are tabulated. b IEP, Immediate-early proteins. TABLE

2

STABILW OF IMMEDIATE-EARLY PROTEINS SYNTHESIZED BY INFECTED CELLS DURING ‘THE NORMAL COURSE OF INFECTION=

Input

Pulse Pulse and Chase

29206 2080

Precipitated by serum against IEP

Percent precipitated

103’ 95

3.5 4.5

(LCells were infected and incubated between 1 and 3 hr postinfection in EDWO with [3H]leucine (20 &i/ml) and were then washed with EDS. Part of the cultures was harvested immediately (pulse) and part incubated further in EDS containing 100 rglml unlabeled leucine for 4 hr and then harvested (pulsechase). The cellular proteins were solubilized and reacted with serum against immediate-early proteins in an indirect immunoprecipitation test, as described in Materials and Methods. b Counts per minute per sample. c Counts per minute precipitated after subtraction of the amount precipitated by preimmune rabbit serum.

the amount of labeled proteins reacting with serum against immediate-early proteins after the chase period, indicating that the immediate-early proteins were not degraded. Thus, while immediate-early pro-

teins of Pr virus are synthesized in the infected cells only at early stages, these proteins are stable and are present in the cells at later stages of infection. This has also been established by indirect anticomplementary fluorescence (G. Klein, personal communication). Classification

of Immediate-Early

Proteins

To ascertain whether the immediateearly proteins synthesized during the normal course of infection are structural viral proteins, i.e., whether they become integrated into virions, the following experiment was performed: One set of infected cultures was incubated with [3H]leucine between 1 and 3 hr postinfection, a time when immediate-early proteins are synthesized. These cultures were then incubated in medium containing an excess of unlabeled leucine. Another set of infected cultures was incubated with [‘%]leucine between 3 and 24 hr postinfection. The virions produced by both cultures were collected, mixed, purified, and the PAGE profiles of their proteins were analyzed (Fig. 1). Despite an effective chase in the presence of a large excess of unlabeled leucine

“IMMEDIATE-EARLY”

HERPESVIRUS

PROTEINS

359

of the virion (as such, or after alteration of their molecular weight) the ratio of 3H/1’C in some of the protein peaks in the gel should increase. Such a change in ratio should be readily detectable since immediate-early proteins are synthesized only up to 3 hr postinfection and these proteins will, therefore, not be labeled with “C!. The ratio SH/“C was, however, constant throughout the gel and it is clear, therefore, that the immediate-early proteins do not become part of mature virions and that they are nonstructural viral proteins.

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FIG. 1. PAGE profiles of virions obtained from cells labeled at early and late stages of infection. Cells (2 x 107) were infected and incubated in EDS/lO containing [3H]leucine (30 pCi/mll between 1 and 3 hr, washed extensively, and incubated further for 21 hr in EDS containing unlabeled leucine (100 pg/ml). Another set of infected cultures was incubated between 3 and 24 hr postinfection in EDSilO containing [“Clleucine (0.2 &i/ml). The virions produced by both sets of cultures were collected, purified, and the proteins subjected to PAGE, as described’in Materials and Methods. Open circles, ‘H; closed circles, “C.

(approximately 20% of the label incorporated in acid-precipitable form was lost during the chase period), some [SH]leucine was incorporated into the virions. This could be due, in part, to protein turnover and the reincorporation of 3H-labeled leutine into newly synthesized proteins, but could also be due to the synthesis of a small amount of structural proteins during the last part of the labeling period. Whatever the reason, the amount of aH-labeled leutine incorporated into virions was small and only represents approximately 7% of the label that would have been incorporated into virions, had the cultures been exposed to the label throughout the infective process. The specific activity of the 3H late proteins will, therefore, only be a fraction of the specific activity of the immediate-early proteins. Therefore, if the immediate-early proteins had become part

PAGE Profiles of Immediate-Early Proteins Synthesized During Early Stages of Infection To establish further the identity between the proteins synthesized by cells during early stages of infection that react with the sera against immediate-early proteins and those synthesized by infected cells which had been treated with cycloheximide (see Table l), we analyzed the PAGE profiles of these proteins. In the course of these experiments we found that during manipulations of the immediate-early proteins which are required to prepare the samples for the indirect immunoprecipitation test, there is a change in the PAGE profiles of these proteins, as illustrated in Fig. 2. Cells treated with SDS soon after harvest (Fig. 2A) show the characteristic profile of the immediate-early proteins that we have reported previously (Rakusanova et al., 1971; Jean et al., 1974). However, if the cells are first frozen, thawed, sonicated, and then stored at 4’ for several hours prior to treatment with SDS, the PAGE profile of the immediate-early proteins changes (Fig, 2B). Upon similar treatment, proteins synthesized during the normal course of infection and mixed with the immediate-early proteins show no significant change in their PAGE profile, indicating that immediateearly proteins are especially sensitive to storage at 4”. Figure 3 shows the PAGE profiles of the solubilized proteins synthesized by cells treated with cycloheximide during early stages of infection before and after precipitation by serum against immediate-early

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KERVINA.

AND KAPLAN

stages of infection (see Fig. 3). These results show that the proteins which are synthesized during the normal course of infection that react with serum against immediate-early proteins are the same as the proteins synthesized by the cycloheximide-treated, infected cells. DISCUSSION

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FIG. 2. PAGE profiles of immediate-early proteins before and after storage at 4”. RK cultures were infected in the presence of cycloheximide (50 rg/ml) and incubated in EDS containing cycloheximide up to 5 hr postinfection. The cultures were then washed extensively and further incubated in EDS/lO containing [‘Hlleucine (20 &i/ml) for 2 hr. The cells were scraped in PBS and sonicated. Panel (A) was treated with DNAse and SDS immediately thereafter; the remainder (Panel B) was stored for 12 hr at 4’ prior to this treatment. The samples were mixed with [“C]leucine-purified virions and electrophoresed as described in Materials and Methods. The arrow indicates the position of the major viral capsid protein (peak 2, Ben-Porat and Kaplan, 1970).

proteins. Figure 4 shows profiles of proteins treated identically but synthesized by cells at early stages of a normal infection (1-3 hr). Figure 3 shows that serum against immediate-early proteins precipitated proteins with a PAGE profile similar to that of the total solubilized proteins, On the other hand, the serum against immediate-early proteins reacted only with specific proteins synthesized at early stages of infection. The total solubilized proteins have a complex profile (Fig. 4A). Figure 4B shows that serum against the immediate-early proteins precipitated only proteins with migration characteristics similar to the immediate-early proteins synthesized by cells treated with cycloheximide during early

Infected cells synthesize at early stages of infection both cellular and viral proteins (Kaplan et al., 1970; Ben-Porat et al., 1971). Because of the complex PAGE profiles of the cellular proteins, it is difficult to identify unequivocally the viral proteins synthesized at early stages of infection. The immediate-early proteins synthesized in relatively large amounts by cells treated with cycloheximide during early stages of infection are immunogenic and can be used to produce potent specific antisera. These specific serological reagents have enabled us to detect a small amount of immediateearly proteins synthesized in infected cells soon after initiation of a normal infective process. The immediate-early proteins are unstable at 4” and their PAGE profiles change considerably upon storage at this temperature in the absence of SDS. This is characteristic for immediate-early proteins, since proteins synthesized by infected cells at later stages of infection do not undergo such a change when mixed with immediate-early proteins prior to storage at 4”. The antisera produced against immediateearly proteins were made with the altered proteins, because in the course of the preparation of the material to be injected into the animals the samples are kept at 4” for at least 48 hr. It is doubtful, however, that the alterations of the PAGE profiles that result from storing the samples at 4” are accompanied by a complete change in antigenicity, since serum against immediate-early proteins which had been stored at 4” reacts in an indirect fluorescence test with cells which had been fixed immediately after harvest. It is possible that the change in the electrophoretic profile of the immediate-early proteins is due to their susceptibility to proteolytic enzymes. If so,

“IMMEDIATE-EARLY”

HERPESVIRUS

this breakdown could be avoided by treatment of the samples with inhibitors of proteolytic enzymes. This has as yet not been attempted. However, the breakdown of these proteins does not alter the basis for our conclusions that sera against immediate-early proteins are useful probes for the detection of these proteins in samples in which they are present in only small amounts. 16

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NUMBER

FIG. 3. PAGE profiles of the proteins synthesized by cycloheximide-treated infected cells which are precipitated by serum against immediate-early proteins. RK cultures were infected in the presence of cycloheximide (50 pg/ml) and incubated in EDS containing cycloheximide up to 5 hr postinfection. They were washed extensively and further incubated for 2 hr in EDS/lO containing [‘Hlleucine (20 rCi/ ml). The cells were scraped into PBS and the proteins solubilized for indirect immunoprecipitation, as described in Materials and Methods. A. Solubilized proteins. B. Solubilized proteins precipitated by serum against immediate-early proteins. The samples were mixed with [‘Elleucine-purified virions and electrophoresed as described in Materials and Methods. The arrow indicates the position of the major viral capsid protein (peak 2, Ben-Porat and Kaplan, 1970).

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FIG. 4. PAGE profiles of the proteins synthesized at early stages of infection which are precipitated by serum against immediate-early proteins. RK cultures were infected and incubated in EDS/lO containing [aH]leucine (20 &i/ml) between 1 and 3 hr postinfection. The cells were scraped in PBS and the proteins solubilized for indirect immunoprecipitation, as described in Materials and Methods. A. Solubilized proteins. B. Solubilized proteins precipitated by serum against immediate-early proteins. The samples were mixed with [“Clleucine-purified virions and electrophoresed as described in Materials and Methods. The arrow indicates the position of the major viral capsid protein (peak 2, Ben-Porat and Kaplan, 1970).

The immediate-early RNA represents the transcript of approximately 15% of the genome normally transcribed throughout the infective process (Rakusanova et al., 1971, Jean and Kaplan, unpublished results). In principle, this RNA can specify at least 10 polypeptides. Few of the functions specified by this RNA are known at present. However, it is clear from the experiments presented in this paper that the immediate-early proteins do not include structural proteins. Some members of the herpesviruses, which normally undergo cytocidal interac-

362

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tions with cells in culture, have been implicated in oncogenic transformation; the putative oncogenic potential of these viruses is expressed only when the viruses have been extensively inactivated (Rapp and Jerkofsky, 1973). The expression of herpesvirus oncogenic functions in the transformed cells has been difficult to detect because of the lack of a specific probe. Since the viral functions that are expressed in most cells transformed with oncogenic DNA viruses are also those which are expressed transiently at early stages of productive infection, it is possible that one of the functions of the immediate-early proteins of the herpesviruses may include a putative oncogenic function. Antisera against immediate-early proteins may be useful as a probe to determine whether this is the case. REFERENCES BEN-P• RAT, T., JEAN, J.-H., and KAPLAN, A. S. (1974). Early functions of the genome of herpesvirus. IV. Fate and translation of immediate-early viral RNA. Virology 59, 524-531. BEN-P• RAT, T., and KAPLAN, A. S. (1970). Synthesis of proteins in cells infected with herpesvirus. V. Viral glycoproteins. Virology 41, 265-273. BEN-P• RAT, T., and KAPLAN, A. S. (1973). Replication-biochemical aspects. In “The Herpesviruses,” (A. S. Kaplan, ed.), pp. 163-220. Academic Press, New York. BEN-P• RAT, T., RAKUSANOVA, T., and KAPLAN, A. S. (1971). Early functions of the genome of herpesvirus. II. Inhibition of the formation of cell-specific polysomes. Virology 46, 890-899. DULBECCO,R., and VOGT, M. (1954). Plaque formation and isolation of pure lines of poliomyelitis viruses. J. Exp. Med. 99, 183-189. EAGLE, H. (1959). Amino acid metabolism in mamma-

AND KAPLAN

lian cell cultures. Science 130, 432-437. HAMADA, C., and KAPLAN, A. S. (1965). Kinetics of synthesis of various types of antigenic proteins in cells infected with pseudorabies virus. J. Bacterial. 89, 1328-1334. HONESS, R. W., and ROIZMAN, B. (1974). Regulation of herpesvirus macromolecular synthesis. I. Cascade regulation of the synthesis of three groups of viral proteins. J. Viral. 14, S-19. JEAN, J.-H., BEN-P• RAT, T., and KAPLAN, A. S. (1974). Early functions of the genome of herpesvirus. III. Inhibition of the transcription of the viral genome in cells treated with cycloheximide early during the infective process. Virology 59, 516-523. KAPLAN, A. S. (1969). Herpes simplex and pseudorabies virus. In “Virology Monographs” (S. Gard, C. Hallauer, and K. F. Meyer, eds.), Vol. 5, Springer, New York. KAPLAN, A. S., and BEN-P• RAT, T. (1970). Synthesis of proteins in cells infected with herpesvirus. VI. Characterization of the proteins of the viral membrane. Proc. Nat. Acad. Sci. USA. 66, 799-806. KAPLAN, A. S., ERICKSON, J. S., and BEN-P• RAT, T. (1975). Synthesis of proteins in cells infected with herpesvirus. X. Proteins excreted by cells infected with herpes simplex virus, types 1 and 2. Virology, in press. KAPLAN, A. S., SHIMONO, H., and BEN-P• RAT, T. (1970). Synthesis of proteins in cells infected with herpesvirus. III. Relative amino acid content of various proteins formed after infection. Virology 40, 90-101. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 277, 680-685. RAKUSANOVA, T., BEN-P• RAT, T., HIMENO, M., and KAPLAN, A. S. (1971). Early functions of the genome of herpesvirus. I. Characterization of the RNA synthesized in cycloheximide-treated, infected cells. Virology 46, 877-889. RAPP, F., and JERKOFSKY,M. A. (1973). Persistent and latent infection. In “The Herpesviruses” (A. S. Kaplan, ed.), pp. 271-289. Academic Press, New York.