Activation of cellular stress protein genes by herpes simplex virus temperature-sensitive mutants which overproduce immediate early polypeptides

VIROLOGY

123, 113-122 (1982)

Activation of Cellular Stress Protein Genes by Herpes Simplex Virus Temperature-Sensitive Mutants Which Overproduce Immediate Early Polypeptides ELENA M.R.C.

L. NOTARIANNI Virology

AND

CHRIS

M. PRESTON’

Unit, Church Street, Glasgow, Gll

5JR, Scotland

Received May 25, 1982; accepted July 22, 1982 This paper describes the activation of cellular genes after infection of chick embryo fibroblasts (CEF) with temperature-sensitive (ts) mutants of herpes simplex virus type 1. One mutant, tsK, has a mutation in the gene sequences which encode the immediateearly (IE) polypeptide Vmw175. After infection of CEF with tsK at the nonpermissive temperature (38.5’), IE polypeptides are overproduced but early and late viral gene products cannot be detected. In addition, the synthesis of three cellular polypeptides is stimulated in a manner which closely resembles the “stress” or “heat-shock” response. Peptide mapping confirmed that the tsK-induced polypeptides are stress proteins. No induction was observed when viral gene expression was prevented, leading to the conclusion that one or more IE polypeptides is responsible for the stress response. Two other mutants, tsD and tsT, which have mutations in different regions of the Vmw175 coding sequences, also induce stress proteins at 38.5’. INTRODUCTION

The interaction of herpes simplex virus (HSV) with eukaryotic cells can result in productive, abortive, transforming, persistent, or latent infections. Most extensively studied is the productive infection of tissue culture cells, which entails virus replication and cell death. Viral gene expression is characterised by the synthesis of different temporal classes of mRNAs and polypeptides which can broadly be classified as immediate-early (IE), early, and late (Honess and Roizman, 1974; Powell and Courtney, 1975; Marsden et al., 1976; Clements et al., 1977). IE polypeptides are normally synthesised very early after infection, and the amounts produced are barely detectable because transcription and translation of IE gene products is rapidly turned off. Nevertheless, it is thought that IE polypeptides have important roles in the progress of the viral replication cycle and that their interactions with cellular components might influence the infection process. ’ To whom reprint

requsts should be addressed.

Only two pieces of evidence relating to the functions of IE polypeptides exist at present. One is based on the properties of temperature-sensitive (ts) mutants whose mutations map in DNA sequences which encode the IE polypeptide Vmw175. These virus mutants, of which tsK (derived from the Glasgow HSV-1 strain 17) is an example, all overproduce IE polypeptides and synthesise reduced or undetectable amounts of early and late products at the nonpermissive temperature (Courtney and Powell, 1975; Marsden et al., 1976; Preston, 1979a; Dixon and Schaffer, 1980). The extent of the restriction in gene expression depends on the site of the amino acid substitution in Vmw175 (Marsden et al., 1976; Preston, 1981). Experiments with tsK have shown that its defect directly affects viral transcription and that a normal function of Vmw175 is to modulate the viral transcription pattern by suppressing the synthesis of IE mRNA and activating early and late genes (Preston, 1979a, b; Watson and Clements, 1980). In contrast to the vital role of Vmw175 in productive infection, deletion mutagenesis has shown that 113

0042-6822/82/150113-10$02.00/O Copyright All rights

Q 1982 by Academic Press, Inc. of reproduction in any form reserved.

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114

a complete copy of another IE polypeptide, Vmw68, is not essential for virus replication in tissue culture cells (Post and Roizman, 1981). We show here that infection of chick embryo fibroblasts (CEF) with tsK activates a specific set of cellular genes which encode polypeptides known as “stress” or “heat shock” proteins. These proteins are produced in response to increased temperature or alterations in the cellular environment which are considered to cause stress. The most detailed analyses of the stress response have used Drosophila melanogaster, but more recently an analogous system in eukaryotic cells, especially CEF, has been identified and studied (Hightower and Smith, 1978, Kelley and Schlesinger, 1978; Ashburner and Bonner, 1979; Johnston et al., 1980). Induction of stress proteins in uiuo has also been demonstrated (Currie and White, 1981). In all examples so far examined, activation of stress protein genes primarily involves a dramatic increase in the rate of RNA synthesis, accompanied by changes in chromatin structure (Ashburner and Bonner, 1979; Wu et al., 1979). Although the stress response clearly represents a major, physiologically important, alteration in cellular metabolic state, neither the intracellular signals for its induction nor the functions of the proteins themselves are understood at present. The experiments reported here provide information on a possible interaction of virus and cell at early stages of infection, since we show that HSV IE polypeptides are responsible for the induction of stress proteins in CEF. MATERIALS

AND

METHODS

Cells. Embryos from lo-day-old fertilised eggs were used to prepare CEF. These cells were grown in Eagles medium (Glasgow modification) containing 10% calf serum and used between the third and sixth passage. Virus stocks. Wild-type virus and temperature-sensitive mutants of HSV-1 strain 17 were grown as described previously (Brown et al., 1973; Marsden et al., 1976),

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PRESTON

using the nonsyncytial (syn’) plaque morphology phenotypes of tsB, tsD, and tsE. Mutants tsK and tsT were initially isolated in the syncytial (syn) plaque morphology phenotype (Marsden et al., 1976) and were converted to syn+ by backcrossing with strain 17 syn+ (V. G. Preston, personal communication). The experiments reported here were performed with tsK syn+ and tsT syn+. The mutant ts9 was derived from HSV-2 strain HG52 (Timbury, 1971). Radiolabelling of cells. Almost confluent monolayers were infected with 40 PFU/ cell of the appropriate virus preparation. After incubation at 38.5” for 1 hr, the inoculum was replaced by growth medium. At suitable times, the growth medium was removed, the cell monolayer was washed with phosphate-buffered saline (PBS), and PBS containing 700 pCi/ml of [35S]methionine (The Radiochemical Centre, Amersham, England) and 2 pg/ml actinomycin D was added. After incubation for 30 min at 38.5’, the labelling medium was removed, monolayers were washed with ice-cold PBS, and cells were disrupted in electrophoresis sample buffer as previously described (Marsden et al., 1976). Cycloheximide, where present, was added at 20 pg/ml and removed by thoroughly washing cells with prewarmed growth medium (Preston, 1979a). Polyacrylumide gel electrophoresis. Radiolabelled samples were analysed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis using gels of 9% polyacrylamide cross-linked with 10% diallytartardiamide (relative to acrylamide). Gels were dried and autoradiographs prepared. Polypeptide identification by partial proteolysis. Radiolabelled bands were excised from gels, electroeluted, and partially digested with Staphyloccus aureus V8 protease or chymotrypsin, following procedures described previously (Anderson et al., 1973; Cleveland et al., 1977; Cash et al., 1979). Inactivation of tsK by uu irradiation. Culture fluids from BHK cells infected with 0.01 PFU/cell of tsK at 31” were collected when most cells showed cytopathic

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effects, and virus was harvested by centrifugation at 20,000 g for 2 hr. The pellet was resuspended in growth medium lacking calf serum and phenol red and exposed to an Englehard-Hanovia bactericidal lamp with an output of 32 ergs/mm2/sec at a distance of 20 cm. Virus was irradiated for various times, and the inactivation of infectivity was monitored at 31’. Six minutes was found to be the minimum time necessary to prevent detectable production of IE polypeptides after infection of CEF at 38.5’, and this sample was used in the experiment described in Fig. 4A.

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MI

Dis

2

115

infection 4

6

10

RESULTS

Protein

Synthesis

in tsK-Infected

CEF

Figure 1 shows a time course of protein synthesis in CEF cells infected with the HSV-1 mutant tsK at the nonpermissive temperature (38.5’). By 4 hr postinfection, new polypeptide bands can be detected. Four of these, Vmw175, Vmw136, VmwllO, and Vmw63, are the virus-specified IE polypeptides which have previously been identified in BHK cells infected with this mutant (Preston, 1979a). Three others, labelled SPl, SP2, and SP4, have approximate molecular weights 90,000, 70,000, and 25,000 and comigrate with major polypeptides induced by incubation with the reagent disulfiram. As shown in previous studies, these are stress proteins which can also be induced by a variety of treatments, including incubation at supraoptimal temperatures (Levinson et al., 1980; E. Notarianni, unpublished results). We have routinely added 0.3 PM disulfiram or 50 pM sodium arsenite to cell growth medium to obtain radiolabelled stress protein markers, since these compounds are active at 38.5” and do not significantly inhibit cellular protein synthesis after incubation for 4 hr. Additional features of the stress response in CEF cells should be noted. Low levels of polypeptides which comigrate with SPl and SP2 are always present in untreated cells, whereas SP4 is never detected without induction. Furthermore, the degree of stimulation by infection with tsK differs for the three polypeptides.

FIG. 1. Protein synthesis in tsK-infected CEF. Cells were infected with tsK and radiolabelled at various times after infection (tracks 3 to 6). Polypeptides synthesised by mock-infected cells (track l), or cells treated for 4 hr with 0.3 pM disulfiram (track 2) are also shown. Virus specified IE polypeptides are labelled on the right of track 6, and stress proteins (SP) on the right of track 1. A disulfiram-induced polypeptide which is seen only occasionally is also labelled (0).

Quantitation from numerous experiments shows that the increase in synthesis of SPl is not greater than 3-fold and sometimes is barely detectable. Synthesis of polypeptide SP2 increases approximately 20-fold, and the stimulation of SP4 is more than 50-fold. These values correlate well with the relative induction by disulfiram. Polypeptide SP3 (molecular weight approximately 35,000) has been detected by others (Levinson et al., 1978) but is not synthesised in significant amounts after tsK infection. Production of this polypeptide is inhibited by treatment of cells with cycloheximide, in contrast to the behaviour of SPl, SP2, and SP4 (Kelley and Schlesinger, 1978). Additional stress proteins of approximate molecular weights 100,000

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FIG. 2. Partial proteolysis of disulfiram and SP4 induced by 0.3 a disulfiram (D) or partial proteolysis. The peptide patterns (150 pg/ml for SPl, 50 pg/ml for SP2) or

PRESTON

and tsK-induced polypeptides. Polypeptides SPl, SP2, or tsK (K) were analysed after mock digestion (No enz) after treatment with Staphylococcus aureus V8 protease chymotrypsin (150 rg/ml for SP4) are shown.

(labelled “0” in Fig. 1) and 20,000-23,000 are sometimes observed (see Fig. 4). Further identification of the tsK-induced polypeptides as stress proteins was achieved by partial proteolysis mapping (Fig. 2). The peptide patterns of tsK- and disulfiram-induced SP2 and SP4 were indistinguishable. In the case of SPl, the patterns were sufficiently similar to confirm the identity of the tsK-induced polypeptide, but minor differences possibly due to heterogeneity or differential processing of SPl @later et al., 1981; Wang et al., 1981) were observed. Involvement of HSV IE Polypeptides Stress Response

AND

in the

Cells infected with tsK contain two types of virus-associated products, either or both of which might cause induction of stress proteins. Virion components or cell debris in the virus inoculum could be responsible or, alternatively, IE polypeptides synthesised after infection might mediate the response. To distinguish between these

possibilities, cells were infected with wildtype (wt) HSV-1 or tsK in the absence or presence of cycloheximide, and protein synthesis was analysed 4 hr later (after removal of cycloheximide where appropriate). As a control to determine whether these treatments impair the stress response, a duplicate set of cultures was treated with disulfiram at 4 hr after infection. The result of this experiment is shown in Fig. 3. Infection of cells with wt HSV-1 or tsK in the presence of cycloheximide did not induce stress proteins (tracks 3 and 5) even though the cells remained susceptible to induction by disulfiram (tracks 4 and 6). The combined treatments did, however, cause a reduction in overall protein synthesis. In contrast, infection for 4 hr with tsK (track 9) or treatment with disulfiram (track 2) gave a strong response. This experiment, therefore, shows that a polypeptide newly synthesised after infection with tsK causes the stress response. Figure 3 further shows that cells infected with wt HSV-1 do not produce

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stress proteins (track 7) and have a greatly reduced response to disulfiram (track 8). This result may be due to inhibition of cellular RNA synthesis after infection (Pizer and Beard, 1976; Wagner and Roizman, 1969), although interference could also occur at many other stages in the induction process. Further experiments have shown that inhibition of the stress response occurs at early times, between 1 and 2 hr postinfection (E. Notarianni, unpublished results). The results shown in Fig. 3 demonstrate that protein synthesis after infection is necessary for induction of stress proteins. The major new proteins synthesised in tsK-infected cells are the virus-specified IE polypeptides, but it is conceivable that a component of the input virus particles stimulates the transcription of a cellular gene whose polypeptide product mediates the stress response. Although the latter mechanism seems unlikely, since agents which induce SPl, SP2, and SP4 do not require cellular protein synthesis (unless they are amino acid analogues), it was tested by two approaches. Virus transcription can be inactivated by irradiation of virions with uv light without seriously affecting absorption, penetration, or uncoating (Eglin et al., 1980). Protein synthesis was, therefore, examined in cells “infected” with uv irradiated tsK (Fig. 4A). The results presented show the effects of virus treated with the minimum uv dose needed to prevent detectable IE protein synthesis. Under these conditions, the virus titre at 31” was reduced from 3.0 X 10’ to 1.2 X lo5 PFU/ml, a degree of inhibition which reflects inactivation of the genome rather than any interference with absorption, penetration or uncoating (Eglin et al., 1980). It can be seen that no stimulation of stress protein synthesis occurred when cells were infected with uv-inactivated virus, strongly suggesting that IE polypeptides are responsible for the stress response. The use of the HSV-2 mutant ts9 provided a further test for any effects of virion components on the stress response. This mutant, which adsorbs to cells, does not produce any detectable viral polypeptides

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MI -D

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I InfOCtia, +CH I Infection -CH I WT trK WT tsK +D -D +D -D +D -D +D -D +D

FIG. 3. Involvement of IE polypeptides in induction of stress proteins. Cells were infected with wt HSV1 (wt) or tsK in the presence (tracks 3 to 6) or absence (tracks 7 to 10) of cycloheximide. After incubation for 4 hr at 38.5’, monolayers were either washed and radiolabelled (tracks 3, 5, 7, and 9) or treated with 0.3 NM disulfiram and incubated a further 3 hr at 38.5” before being washed and radiolabelled (tracks 4, 6, 8, and 10). Polypeptides synthesised by mock-infected cells (track 1) or mock-infected cells incubated for 3 hr with 0.3 j&f disulfiram (track 2) are also shown. Virus specified polypeptides are labelled on the right of track 10, and stress proteins are labelled between tracks 1 and 2.

after infection of BHK cells for 8 hr at 38.5” and may have a defect in penetration or uncoating (H. S. Marsden, personal communication). A similar HSV-1 mutant, tsB7, which maps at a comparable genome location to HSV-2 ts9, has recently been described in detail (Knipe et al., 1981). Figure 4B shows that CEF cells infected with ts9 at 38.5” produced no detectable virus-induced polypeptides and did not induce stress proteins. When ts9infected cells were incubated at 31”, after adsorption at 38.5”, typical viral polypeptides were synthesised.

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U.V. tsK tsK 1

2

MI 3

AND

PRESTON

As

tsK

4

39S’ 1

ts9 As 2

MI 3

39.5’ 4

ts9 31’ 5

FIG. 4. Failure of uv-inactivated tsK or HSV-2 ts9 to induce stress proteins. (A) Cells were infected with uv-irradiated tsK (track 1) or untreated tsK (track 2) and radiolabelled at 6 hr postinfection. Polypeptides synthesised by mock-infected cells (track 3) or mock-infected cells treated for 3 hr with 50 pM sodium arsenite (track 4) are also shown. (B) Cells were infected at 38.5” with tsK or HSV-2 ts9. After absorption, incubation was continued for 5 hr at 38.5’ (tracks 1 and 4) or 31’ (track 5) before radiolabelling. Polypeptides synthesised by mock-infected cells (track 3) or mockinfected cells treated for 3 hr with 50 pM sodium arsenite (track 2) are also shown. “Late” HSV2 polypeptides are labelled on the right of track 5.

Taken together, the data presented in Figs. 3 and 4 show that one or more IE polypeptides, rather than any direct or indirect effect of input virion components, are responsible for induction of stress proteins in tsK-infected cells. Induction

of Stress Proteins by Other HS V-

1 ts Mutants

Compared with tsK, the HSV-1 mutants tsD and tsT have mutations which result in amino acid substitutions closer to the

carboxy terminus of Vmw175 (Preston, 1981). Phenotypically, in BHK cells, tsD and tsT resemble tsK in the overproduction of IE polypeptides but differ in permitting synthesis of some early and late polypeptides at the nonpermissive temperature (Marsden et al., 1976; Preston, 1981). When CEF cells were infected with these mutants at 38.5’, stress proteins SP2 and SP4 were produced as in tsK-infected cells (Fig. 5, tracks 5 and 6). It should be noted that tsD and tsT produced apparently normal amounts of many early and

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GENES

late proteins in CEF, although overproduction of IE polypeptides still occurred. Two other mutants, tsB and tsE, which fail to synthesise viral DNA and are blocked at early (but later than IE) stages in the infection cycle, did not induce stress proteins (Fig. 5, tracks 7 and 8). A tsK stock of syncytial plaque morphology phenotype induced stress proteins (Fig. 5, track 3), but none of the mutants did so at the permissive temperature, 31’ (results not shown), nor did a revertant of tsK at 38.5” or 31” (results not shown). The phenomenon is therefore characteristic of infections in which a ts lesion is Vmw175 prevents repression of IE transcription. DISCUSSION

This paper reports a new aspect of virus/ cell interaction which can occur at the early stages of infection with HSV-1. The activation of cellular stress protein genes in tsK-infected cells is due to the presence of one or more IE polypeptides and, by analogy with previous studies (Ashburner and Bonner, 1979; Johnston et al., 1980), MI

Dis KsynK.syn’

D

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presumably involves dramatic transcriptional changes. Other examples of similar phenomena are the synthesis of cellular “non-viral T antigen” after transformation or infection with various viruses (Lane and Crawford, 1979; Linzer et al., 1979; Luka et al., 1980; Rotter et al., 1980), the production of interferon after virus infection (Stewart, 1979), and the stimulation of glucose-regulated protein synthesis after infection with paramyxoviruses (Peluso et al., 1978). In all cases so far examined, activation of stress protein genes occurs at the transcriptional level. Since many IE polypeptides are associated with chromatin and bind to DNA in vitro, a direct action on gene expression is possible. In most examples, however, the signal for transcription is thought to originate in the cytoplasm, and recently a protein which activates stress protein genes in vitro has been isolated from D. melunogaster cell cytoplasm (Craine and Kornberg, 1981). In situations where IE polypeptides are overproduced, such as infection with tsK at 38.5”, all IE polypeptides eventually acT

8

E WT

175 136 110

63

FIG. 5. Induction of stress proteins by HSV-1 ts mutants. Cells were infected at 38.5’ with tsK, tsD, tsT, tsB, tsE, or wt HSV-1, all of the syn+ morphology phenotypes, or with tsK syn. Cells were radiolabelled at 6 hr postinfection. Polypeptides synthesized by mock-infected (MI) cells or cells treated with 0.3 pM disulfiram for 4 hr (Dis) are also shown. IE polypeptides (,) and “late” polypeptides (a) are labelled.

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cumulate in the cytoplasm (as judged by cell fractionation in aqueous media), and, therefore, an effect on cytoplasmic components may also activate stress protein genes in this case. It may be necessary to reach a clearer understanding of the cellular changes leading to synthesis of stress proteins before the IE polypeptide(s) involved and its mode of action can be determined. Chick embryo fibroblasts were used in these studies because they are very sensitive to inducers of stress proteins. Infection of other cell types, such as mouse 3T3 cells, with tsK also results in a stimulation of stress protein synthesis, especially the putative analog of SP2 (E. Notarianni and C. Preston, unpublished results). No mammalian cell type with a response as dramatic as CEF has been found, and in some cases, for example BHK cells, the degree of response depends on aspects of the cellular growth state which have not been characterised at present. We envisage two general hypotheses to cover the possible relevance of cellular gene activation to infection under physiological conditions. One is that IE polypeptides of wt HSV-1 normally cause many of the changes associated with the stress response, but that stress protein synthesis is not detected because other viral proteins, probably including Vmw175, prevent their transcription. In support of this idea is the observation from Fig. 1 that induction in tsK-infected cells occurs soon after infection when only limited amounts of IE polypeptides are present. There is also some evidence that Vmw175, rather than an early or late polypeptide, is normally involved in preventing transcription of stress protein genes, based on the following points: (1) Induction by disulfiram is inhibited early after infection with wt HSV-1 (Fig. 3 and E. Notarianni, unpublished results). (2) Mutants tsD and tsT produce many early and late proteins in CEF at 38.5” but have a defect in Vmw175 which causes overproduction of IE polypeptides and induction of stress proteins. In these cases, activation of stress protein genes occurs even though cells contain many late gene products (Fig. 5). (3) Tem-

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PRESTON

perature upshift experiments show that inactivation of Vmw175 in tsK-infected cells at late times after infection can resensitise cells to induction by disulfiram (E. Notarianni, unpublished results). An alternative hypothesis is that overproduction, rather than normal levels, of IE polypeptides causes induction of stress proteins. This possibility may have biological relevance, since a major class of HSV defectives consists of tandem repeats of DNA sequences which include two complete IE genes. It has been shown that infection with HSV-1 stocks containing high levels of defective particles results in overproduction of Vmw175 and might, therefore, mimic some aspects of infection with tsK (Frenkel et al., 1975; Murray et al., 1975). Furthermore, it has been claimed that natural isolates of HSV-1 frequently have ts mutations in the DNA sequences encoding Vmw175 which result in overproduction of IE polypeptides at 39“ (Knipe et al., 1981). Most work with tissue culture cell systems has concentrated on events in productive infection by HSV-1, which leads to cell death. It is clear, however, that in natural infections some cells, especially neuronal cells, can survive infection and maintain HSV in a latent state (Stevens and Cook, 1971; Lofgren et al., 1977; McLennan and Darby, 1980). In this case, as well as in in vitro latency systems, some aspects of virus growth and cytopathic effects must be suppressed to prevent cell death. One mechanism for such suppression could be an inefficient transition from IE to early and late transcription, and under these circumstances activation of cellular stress protein genes might be expected. It is relevant to note that a successful way of maintaining infections in vitro is by incubation of cell cultures at supraoptimal temperatures (O’Neill, 1977; Levine et al., 1980; Wigdahl et al., 1981). In these cases, the restriction to virus growth may be due to a cellular response, since reactivated virus can productively infect cells at supraoptimal temperatures. Herpesvirus IE polypeptides should be included in the list of “natural” inducers of stress proteins, along with hyperther-

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mia, recovery from anoxia (Guttman et al., 1980), tissue damage (Hightower and White, 1981), and treatment with hormones (Ireland and Berger, 1982). It will be important to determine which IE polypeptides are responsible for the effect, whether other aspects of the stress response occur in tsK or wt HSV-1 infected cells, and if the response or the stress proteins themselves can modify the infection process. Answers to these questions may clarify some functions of IE polypeptides in HSV-infected cells. ACKNOWLEDGMENTS We thank Professor J. H. Subak-Sharpe for his interest and encouragement. E. L. Notarianni was a recipient of a Medical Research Council Research Training Award. REFERENCES ANDERSON, C. W., BAUM, P. R., and GESTELAND, R. F. (1973). Processing of adenovirus 2-induced proteins. J. Viral. 12, 241-252. ASHBURNER, M., and BONNER, J. J. (1979). The induction of gene activity in Drosophila by heat shock.Cell 1'7, 241-254. BROWN,~. M., RITCHIE, D. A.,and SUBAK-SHARPE, J. H. (1973). Genetic studies with herpes simplex virus type 1. The isolation of temperature-sensitive mutants, their arrangement into complementation groups and recombination analysis leading to a linkage map. J. Gen. Virol. 18, 329-346. CASH, P., PRINGLE, C. R., and PRESTON, C. M. (1979). The polypeptides of respiratory syncytial virus: Products of cell-free protein synthesis and posttranslational modifications. Virology 92, 375-384. CLEMENTS, J. B., WATSON, R. J., and WILKIE, N. M. (1977). Temporal regulation of herpes simplex virus type 1 transcription: location of transcripts on the viral genome. Cell 12, 275-285. CLEVELAND, D. W., FISCHER, S. G., KIRSCHNER, M. W., and LAEMMLI, U. K. (1977). Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Bid. Chem. 252, 1102-1106. COURTNEY, R. J., and POWELL, K. L. (1975). Immunological and biochemical characterisation of polypeptides induced by herpes simplex virus types 1 and 2. In “Oncogenesis and Herpesviruses” (G. de The, M. A. Epstein, and H. xur Hausen, eds.), Vol. 2, pp. 63-73. IARC, Lyon. CRAINE, B. L., and KORNBERG, T. (1981). Activation of the major drosophila heat-shock genes in uitro. Cell 25, 671-681. CURRIE, R. W., and WHITE, F. P. (1981). Trauma-

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