VIROLOGY

75, 306-318 (1976)

Synthesis

of Virus-Specific Polypeptides Mutants of Herpes Simplex RICHARD

Department

of Virology

by Temperature-Sensitive Virus Type 1’

J. COURTNEY,2 PRISCILLA A. SCHAFFER, AND KENNETH L. POWELL and Epidemiology,

Baylor

College of Medicine,

Houston,

Texas 77030

Accepted June 28, 1976 The polypeptide phenotypes of 22 temperature-sensitive (ts) mutants of herpes simplex virus type 1 were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis of mutant-infected cells at permissive and nonpermissive temperatures. Following analysis of isotopically labeled polypeptides synthesized from 4-24 hr postinfection, the mutants were divided into four major phenotypic groups which include: (1) DNA- ts mutants which share several common polypeptide defects, (2) DNA’ ts mutants which exhibit polypeptide profiles resembling the DNA- ts mutants, (3) DNA+ ts mutants which exhibit polypeptide phenotypes differing only slightly from that observed in wild-type virus-infected cells grown at 39”, and (4) DNA+ ts mutants which exhibit no detectable alterations in their polypeptide profiles when compared with that of the wildtype virus. When the polypeptide phenotypes of the mutants were compared with previously determined mutant characteristics, including synthesis of viral DNA, thymidine kinase, DNA polymerase, and physical virus particles, a correlation was consistently observed between mutant polypeptide and viral DNA phenotypes. INTRODUCTION

The potential use of temperature-sensitive (ts) mutants for studies of the herpes simplex virus (HSV) replicative cycle and for defining specific functions of virus-induced macromolecules in this cycle has been reviewed previously @chaffer, 1975; Subak-Sharpe et al., 1975). Twenty-two ts mutants of HSV type 1 (HSV-1) isolated in this laboratory have been classified into 15 complementation groups (Schaffer et al., 1970, 1973). Mutants in several HSV-1 DNA-negative complementation groups have proved useful for examining specific aspects of HSV protein synthesis. These studies have included the synthesis of HSV-induced glycoproteins (Schaffer et 1 This work was supported by Research Contract No. NO1 CP 53526 within The Virus Cancer Program of the National Cancer Institute, and Research Grant No. CA 10,893 from the National Cancer Institute. * Author to whom requests for reprints should be addressed. 306 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

al., 1971), the major capsid polypeptide (Bone and Courtney, 1974), an early nonstructural, high-molecular-weight polypeptide (Courtney and Benyesh-Melnick, 1974), and the relative amounts of individual polypeptides synthesized in the absence of viral DNA synthesis (Powell et al., 1975). These mutants have also been used in studies of HSV-1 DNA replication (Schaffer et al., 1976) and viral DNA synthetic enzyme induction by HSV-1 (Aron et al., 1973, 1975). The present investigation was undertaken to examine the ability of mutants in the 15 complementation groups to synthesize virus-specific polypeptides at the nonpermissive temperature. Although the function of each of the detectable polypeptides is currently not known, high-resolution polyacrylamide slab gel electrophoresis offers the only available means for the simultaneous examination of the synthesis of a variety of virus-specific gene products. In this investigation a clear correlation was found to exist between the viral DNA

SYNTHESIS

OF VIRUS-SPECIFIC

phenotype of mutants at the nonpermissive temperature and their virus-specific polypeptide phenotypes. Some relationship between mutant polypeptide phenotype and the ability of mutants to induce the synthesis of physical particles was also observed. MATERIALS

AND

METHODS

Cells and cell cultures. Serially propagated human embryonic lung (HEL) fibroblasts and African green monkey kidney (Vero) cells were used in this study. Cells were grown in Eagle’s medium (Eagle, 1959) supplemented with 10% fetal bovine serum (FBS) and 0.075% NaHC03 for cells in stoppered bottles or 0.225% NaHCO, for cells in petri dishes. Virus growth and assay. The KOS strain of herpes simplex virus type 1 (HSV-1) was used as the wild-type (WT) virus. The isolation, complementation, and partial characterization of the 22 temperature-sensitive (ts) mutants used in this study have been described previously (Schaffer et al., 1970, 1973). Virus stocks were prepared in HEL cells and Vero cells were used for virus assay @chaffer et al ., 1973). Infection and labeling of cultures. Nearly confluent HEL cell monolayers in 8-0~ prescription bottles containing approximately 4 x lo6 cells were washed once with phosphate-buffered saline and infected at a multiplicity of infektion of lo20 PFU/cell of each mutant and the WT virus. After adsorption for 1 hr at 37”, monolayers were washed twice, Eagle’s medium containing 2% FBS and 0.075% NaHC03 was added to each culture, and cultures were incubated at either 34” (permissive temperature) or 39” (nonpermissive temperature). Cultures incubated at 34” were held in a water-jacketed incubator with a temperature variation of rtO.2”, and cultures incubated at 39” were held in a constant temperature water bath with a temperature variation of 0.1”. All cultures were isotopically labeled with either 5 &i/ml of ‘“C-amino acid mixture (specific activity, 57 mCi/mAtom) or 10 @Zi/ml of [s5S]methionine (specific activity, 40 Ci/ mmol). Isotopes were obtained from Amersham-Searle Inc., Arlington Heights, Ill.

POLYPEPTIDES

307

The labeling medium containing the 14Camino acid mixture consisted of Eagle’s medium with l/lo the prescribed concentration of unlabeled amino acids, normal amounts of glutamine and arginine, and 2% FBS. When labeling with [YS]methionine, the medium was identical to that containing 14C-labeled amino acids, however, it contained no unlabeled methionine. At 4 hr postinfection (p.i.), culture medium was removed and 5 ml of prewarmed labeling medium was added to each culture. At the end of the labeling period (24 hr p.i.), cells were harvested by scraping into ice-cold phosphate-buffered saline as described previously (Powell and Courtney, 1975). Samples were resuspended in water and frozen at -70”. For infectivity assays, samples were thawed, sonicated for 45 set (10 kc, Raytheon sonic oscillator), and assa’yed in Vero cell monolayers at 34 and 39” @chaffer et al., 1973). The protein concentration was determined by the method of Lowry et al. (1951) and the acid-insoluble radioactivity was measured as previously described (Powell and Courtney, 1975). The remaining suspension was used for the assay of virus-induced polypeptides on polyacrylamide gels. Polyacrylamide gel electrophoresis. All samples were analyzed by polyacrylamide slab gel electrophoresis as previously described (Powell and Courtney, 1975) using the discontinuous buffer system of Ornstein (1964) and Davis (1964), and the modification for the inclusion of sodium dodecyl sulfate (SDS) (Dimmock and Watson, 1969). Immediately prior to electrophoresis, samples were solubilized by boiling for 2 min in 0.05 M Tris/HCl, pH 6.7, 1% SDS, 1% mercaptoethanol, and 0.5 M urea. Individual polypeptide bands were visualized by autoradiography of dried gel slabs as previously described (Powell and Courtney, 1975). Evaluation of polypeptide synthesis was accomplished by visual examination of the X-ray films. Each of the samples (one set labeled with 14C-amino acids and one set with [35S]methionine), was analyzed by six or more separate electrophoretie runs. Our designation as to the increase or decrease in the synthesis of a particular polypeptide refers only to the

308

COURTNEY,

SCHAFFER

major differences observed and is not intended therefore to be strictly quantitative. Molecular weights were determined by electrophoresis of known polypeptide standards in parallel with samples of unknowns (Weber and Osborn, i969). The molecular weights of the standards used covered the entire range of unknown polypeptide molecular weights. RESULTS

Infectivity

and Protein Content of Samples

Monolayers of HEL cells were infected with each of the mutants and the WT virus and labeled with either 14C-amino acids or [35S]methionine from 4-24 hr p.i. The yield of infectious virus at both 34 and 39, the protein concentration, and TCA-precipitable counts were determined for each sample (Table 1). With respect to the efficiency of replication of 21 of the 22 mutants, the yield of infectious virus from mutant-infected cultures at 34” ranged from 1.3 x lo8 to 5.9 x 10s PFU/ml, with an average yield of 1.2 x lo9 PFU/ml. The yield of infectious virus at 39” ranged from 2.0 x lo2 to 2.6 x lo6 PFU/ml, with an average yield of 4.2 x 105. The yield of one mutant, tsG3, was lower at 34” (i.e., 4 x 107), while at 39” it was 2.0 x 103. The replication efficiency of mutants ranged from 3.1 x 10m7(tsL14) to 1.0 x 10e3 (tsG8). Virus yields at 39” were produced primarily by small quantities (52 PFU/cell) of ts virus (leak); revertants comprised ~1% of the virus yield in most cases at this temperature. Thus, the polypeptide profiles discussed below reflect the synthesis of proteins in cells infected by ts rather than ts+ revertant virus. The efficiency of incorporation of 14Clabeled amino acids and [“Slmethionine was determined for each sample. The values presented in Table 1 represent the ratio of 14C-labeled amino acid counts per minute per milligram (cpm/mg) of protein incorporated at 39” to the cpm/mg of protein incorporated in cultures incubated at 34”. The average cpm/mg of protein at both temperatures for the l”C-amino acid-labeled samples was 7.1 x lo6 and for the [35S]methionine-labeled samples, 13.6 x 106. In most cases the incorporation of iso-

AND

POWELL TABLE

1

COMPARISON OF THE YIELD OF INFECTIOUS VIRUS AND INCORPORATION OF ISOTOPE FROM HSV-lINFECTED CULTURES INCUBATED AT 34 OR 39”

r

Mutant

i

3 9”134”” “C-labeled amino acid cpmimg X 10-qd

1,ype at 39””

34 Wild-type tsAlb tsA15g tsA16g tsB2b tsB2lu tsC4b tsC7b tsD9b tsE5b tsE6b tsFl7g tsF18g tsG3b tsG8b tsHlOb tsIllb tsJ12g tsKl3g tsL14g tsM19u tsN2Ou ts022u

+ + + + + *c kc + + + kc kc TC + fC

4.4 2.1 3.1 5.0 6.8 2.2 2.0 6.8 5.6 4.6 1.3 1.0 5.9 1.0 2.8 7.7 1.3 1.8 3.1 1.6 1.6 8.1

726 0.3 x 10-4 170 x 10-S 143 x 10-e 156 x 10-S 147 x 10-S 129 x 10-d 907 x 10-S 381 x 10-4 134 x lo+ 808 x 10-G 720 x 10-j 862 x 10-d 775 x 10-S 645 x 10-S 707 x IO-* 975 x 10-e 1467 x 10-S 674 x lo+ 666 x 10-7 1012 x 1O-5 832 x 1O-4 851 x lo+ 1414

39” 840 198 196 179 150 173 913 496 116 1217 938 1060 640 1159 687 1064 889 657 606 1338 942 1170 1011

1.16 1.16 1.37 1.15 1.02 1.34 1.01 1.30 0.87 1.51 1.30 1.23 0.83 1.80 0.97 1.09 0.61 0.97 0.91 1.32 1.13 1.37 0.71

a Viral DNA phenotypes at 39” from Schaffer et al. (1973). * EOR (efficiency of replication) = yield of infectious virus (PFU/ml) 39”/34”. Yields were assayed at 34 and 39”. c These mutants have been shown to synthesize markedly reduced quantities of viral DNA as measured by incorporation of [3Hlthymidine (Aronet al., 1975, and unpublished observations). d Acid-precipitable ‘%-labeled amino acid counts per minute (cpm) per milligram of protein. e Ratio of ‘%-labeled amino acid cpm/mg of protein incorporated at 39” as compared to the cpm/mg ofproteinincorporated at34”.

tope into protein was equal or higher at 39” than it was at 34” (Table 1). Virus-Specific Polypeptides in Mutant-Znfected Cultures

WT and

The electropherograms of virus-induced polypeptides of the 22 ts mutants and the WT virus maintained at both 34 and 39” are presented in Figs. 1 and 2; the polypep-

SYNTHESIS

OF VIRUS-SPECIFIC

tide phenotypes of the mutants are summarized in Fig. 3. Only the profiles of the LQC-amino acid-labeled polypeptides are shown. However, as described above, the mutants and WT were also labeled with [35Slmethionine in the same manner. Analysis of the [“5S]methionine-labeled polypeptides by polyacrylamide gel electrophoresis confirmed results obtained with the 14C-amino acid-labeled polypeptides. However, as expected, several of the HSV-induced polypeptides were relatively inefficiently labeled with [3S]methionine, suggesting that these polypeptides contain only small amounts of this amino acid. Examination of the mutant polypeptide profiles at 34” reveals that mutants in complementation groups C, E, G, and L differ from that of the WT virus at this temperature. Several explanations for these differences exist: (1) Since ts mutants rarely function as efficiently as the WT virus at the permissive temperature, the altered polypeptide profiles of the mutants at 34” may reflect partial expression of the ts lesion at this temperature. Each mutant has a distinct cut-off temperature at which the ts defect is expressed. (2) The presence of a second, nonlethal lesion in a mutant could result in an altered polypeptide profile at 34”. Unfortunately, currently available genetic and phenotypic data do not permit us to distinguish between these two possibilities. The results presented here were obtained from a single polyacrylamide gel electrophoresis experiment under conditions in which all samples were prepared and run in an identical manner. It should be emphasized that each sample was run a minimum of six times and that the electrophoretic profiles varied little from run to run. Based upon their polypeptide phenotypes at 39” and the DNA phenotypes which have been previously determined (Aron et al., 1974), the mutants can be separated into four major groups which include: (1) DNA- ts mutants which share certain common polypeptide defects, (2) DNA’ ts mutants which exhibit polypeptide profiles resembling the DNA- ts mutants, (3) DNA+ ts mutants which exhibit polypeptide phenotypes which differ only

POLYPEPTIDES

309

slightly from that observed in WT virusinfected cells grown at 39”, and (4) DNA+ ts mutants which exhibit no detectable alterations in the polypeptide profile when compared with WT virus-infected cells. The DNA- ts mutants exhibited several common characteristics with respect to their polypeptide phenotypes (Figs. la and 3). The most unique feature of DNA- mutant profiles was the reduced synthesis of three major HSV-induced polypeptides: VP154 (the major capsid polypeptide), VP82, and VP67. These three polypeptides, which are structural components of the virion (Powell and Watson, 1975; unpublished observations), were reproducibly found to be present in reduced amounts in all of the DNA- ts mutants studied. In a previous study of the effects of viral DNA synthesis on HSV-induced polypeptide synthesis, the reduction of VP154 in tsAl-, tsB2-, tsC4-, and tsD9infected cells at the nonpermissive temperature was found to be similar to the reduction in VP154 synthesis observed in WT virus-infected cultures grown in the presence of Ara-C (Powell et al., 1975). In addition to the reduction in VP154, VP82, and VP67, polypeptide defects unique to each of the four DNA- complementation groups were also observed. Furthermore, in several instances differences were observed among mutants within a complementation group. With regard to the three mutants in group A, their polypeptide profiles appeared to differ significantly. This was especially evident in the profile of tsA16 which was markedly defective in the synthesis of most of the detectable HSV-induced polypeptides. This observation was made in cultures labeled with both 14C-amino acids and [35S]-methionine. The yield of infectious tsA16 virus in the 34” culture and the polypeptide phenotype at this temperature were similar to the other mutants tested and to the WT virus. Therefore, the reduced synthesis of viral polypeptides was not a consequence of aberrant infection procedures. With regard to tsA1, the synthesis of VP123, the broad diffuse region which represents the major envelope glycoprotein components, was reduced at 39”. This ob-

310

COURTNEY,

SCHAFFER

AND

POWELL

FIG. 1. Slab gel electropherograms of the polypeptides synthesized by ts mutants of HSV-1 belonging to complementation groups A through E. HEL cells were infected with ts mutants or WT virus and incubated at 34 or 39”. At 4 hr p.i., the cultures were labeled with %-amino acids. Whole-cell fractions were harvested at 24 pi. and prepared for analysis. (A) Polypeptpde profiles from infected cultures incubated at 39”. (B) Polypeptide profiles from infected cultures incubated at 34”.

servation is in agreement with a previous study in which we reported that tsAl was defective in the normal glycosylation of certain virus-induced proteins (Schaffer et al., 1971). Even though VP123 is reduced at 39” in tsAl-infected cells, no precursor polypeptides were detectable. In other studies in which the glycosylation event was altered by the addition of either deoxyglucose (Courtney, 1976) or cytochalasin

B (Dix and Courtney, 1976), a unique polypeptide which may represent a nonglycosylated precursor was detected. Other aspects of the tsA1 polypeptide profile include reduced synthesis of VP44, VP35, and VP25, and increased synthesis of VP47, VP34, and VP24. The significance of the altered expression of VP44 by group A and other mutants will be discussed below. With the exception of VP35, VP25, and

SYNTHESIS

OF VIRUS-SPECIFIC

VP24, the differences in the polypeptide profile of tsA15 were similar to tsA1. Of the group A mutants, tsA15 appeared to be the least defective with regard to its polypeptide phenotype. Mutants in group B (tsB2 and tsB21) exhibited polypeptide profiles uniquely different from those observed in other DNA- complementation groups. Group B mutants characteristically induced the extensive overproduction of VP175 as previously described (Courtney and BenyeshMelnick, 1974). Recent studies have indi-

POLYPEPTIDES

cated that VP175 is overproduced by all DNA- ts mutants at 39” and by the WT virus grown in the presence of Ara-C (Powell et al., 1975). The overproduction of VP175 by DNA- mutants not in group B and by the WT virus in the presence of Ara-C represented an approximate twofold increase as compared with the eight- to tenfold increase observed in profiles of the two mutants in complementation group B. It is of interest that one DNA’ mutant (tsL14) synthesized VP175 in amounts similar to those observed in tsB2- and tsB21-

312

COURTNEY.

SCHAFFER

AND

POWELL

FIG. 2. Slab gel electropherograms of the polypeptides synthesized by ts mutants of HSV-1 belonging to complementation groups F through 0. The procedures for infection and harvesting are the same as described in Fig. 1. (A) Polypeptide profiles from infected cultures incubated at 39”. (B) Polypeptide profiles from infected cultures incubated at 34”.

infected cells (Fig. 2a). Recent genetic analysis of this mutant has indicated that tsL14 maps close to the B group mutants (J. Jofre, personal communication). Other common characteristics of the group B mutants include reduced synthesis of VP125, VP69, and VP25, and accumulations of VP148, VP64, VP47, and VP45. As observed with tsA1, VP24 was present in increased amounts in tsB2.

The group C ts mutants include tsC4 and tsC7, which we have previously reported to be defective in the synthesis of the major capsid polypeptide VP154 (Bone and Courtney, 1974). Although mutants in all four DNA- complementation groups are defective in VP154 synthesis, the group C mutants appear to be the most defective (Fig. la). Additional characteristics of the group C mutants include the accumulation

SYNTHESIS

OF VIRUS-SPECIFIC

FIGURE

of VP134 (unique to the group C mutants as compared to the other DNA- mutants), VP64 (as observed in group 3) , and VP38 (in tsC4 only). In addition, only in tsC4 was the appearance of increased amounts of polypeptides VP77 and VP75 detected. The accumulation of VP77 and VP75 was also observed with one other mutant, tsL14, a DNA’ mutant which will be discussed below. The group D mutant (tsD9) appeared to

POLYPEPTIDES

313

2B

be the least defective of the DNA- mutants with regard to polypeptide synthesis. This mutant exhibited the typical reduction in the synthesis of the three polypeptides (VP154, VP82, and VP67) described for the other DNA- mutants. In addition, it exhibited reduced synthesis of VP69 and VP35 (as in tsA1). No other major alterations were noted. The second major group of ts mutants consists of DNA’ mutants which exhibited

+ + + c

;*+ * A

KD * ‘UD

ND ND B

++ ND + B

+ ND c

i + ND c

* * ND F!

* + ND B

+ + + c

* + * c

FIG. 3. Summary of the major alterations in polypeptide synthesis of the 22 ts mutants of HSV-1. The increased (A) or decreased (v) synthesis of each polypeptide was determined by comparing the mutant polypeptide present at 39” with the corresponding WT polypeptide synthesized at 39”. u Molecular weight of each polypeptide x 103. * A Effect also observed at 34”. c Viral DNA phenotype of ts mutants at 39” (Aron et kinase phenotype of ts mutants at 39” (Aron et nl., al., 1975; Schaffer, unpublished results). d Thymidine 1973). e DNA polymerase phenotype of ts mutants at 39” (Aron et al., 1975). f Electron microscopy study group; group A is most defective, B is intermediate, and C is least defective in viral morphogenesis. g ND = not done.

polypeptide profiles resembling those of the DNA- ts mutants discussed above. These mutants include tsK13, tsL14, tsM19, and ts022 (Figs. 2a and 3). As with the DNA- mutants, polypeptides VP154, VP82, and VP67 were found in reduced amounts in cells infected at 39” with these four mutants. Polypeptide VP69 was also detected in reduced quantities in all four mutants; this defect was characteristic of five of the eight DNA- ts mutants. Additional unique properties of DNA’ mutants also observed with the DNA- mutants include (1) the marked overproduction of VP175 by tsL14 as observed with the group B mutants; (2) the increased production of VP148 in tsK13-infected cells as observed

with the group B mutants; (3) the altered synthesis of VP148, VP134, VP125, and VP123 by tsKl3 as seen in several of the DNA- mutants; (4) the increased synthesis of VP77 and VP75 by tsL14 which was only seen in tsC4-infected cells; (5) the increased synthesis of VP64 in tsK13- and tsLlCinfected cells which was also seen with the group B and C mutants; and (6) the increased synthesis of VP60 by tsL14 and tsM19 which was not seen with any other mutant. Two additional DNA’ mutants (tsG3 and tsG8) exhibited certain polypeptide defects which resembled those of other DNA’ mutants. These include the reduction in VP154 synthesis by both mutants and the reduced synthesis of VP82 by

SYNTHESIS

OF VIRUS-SPECIFIC

tsG3. A unique feature of these mutants was that the defect in VP154 synthesis was also observed at 34” (Figs. 2a, b and 3). The third major group of mutants includes 5 DNA+ mutants (tsE5, tsE6, tsF17, tsH10, and tsI11) which exhibit polypeptide phenotypes with only minor alterations when compared to the WT virus grown at 39” (Figs. la, 2, and 3). The mutants included in this group are tsE5 and tsE6 which showed increased synthesis in VP64 (a characteristic common to the DNA- group B and C mutants) and tsF17 with increased synthesis of VP38 (also seen in tsD9). The other mutants, tsH10, and tsI11, as well as some of the abovementioned mutants in this group, showed either reduced or elevated levels of VP45 and VP44 which will be discussed below. A fourth group of mutants includes three DNA+ mutants in which no detectable differences in the polypeptide profile were observed when compared with the polypeptide profile of WT virus grown at 39”. The mutants which belonged to this group include tsF18, tsJ12, and tsN20 (Figs. 2a and 3). DISCUSSION

The purpose of this report is to describe the polypeptide phenotypes of HSV-1 ts mutants with defects in 15 cistrons. Comparison of the general polypeptide phenotypes of all 22 mutants examined in this study with the phenotypic information previously obtained should provide a clearer understanding of the nature of the ts defects of these mutants. Hopefully, this comparison will facilitate the identification of the groups of mutants which would be most useful (1) for defining specific events within the HSV replicative cycle and (2) for examining HSV-induced macromolecular synthesis in greater detail. Since the gene products encoded by ts mutant genes are synthesized but function abnormally at the nonpermissive temperature, one would not expect to observe defects in individual ts gene products. Rather, we would expect to observe the effects of the altered function of the ts protein as it relates to the control of later events in the replication cycle. Such con-

POLYPEPTIDES

315

trol could affect the synthesis and/or modification of other virus-specific polypeptides such as glycosylation, phosphorylation, and/or post-translational cleavage. If a ts mutant were defective in a gene with a control function, one might expect to see multiple polypeptide defects based on the work of Honess and Roizman (1974) who demonstrated interlinked “cascade” regulation of HSV protein synthesis. Indeed, multiple defects were most prominent among the DNA- and DNA’ mutants in which (presumably) a single ts defect resulted in altered synthesis of several polypeptides. When the DNA and polypeptide phenotypes of the ts mutants at 39” were compared, certain features were evident (Fig. 3). All the DNA- mutants exhibited reduced synthesis of three polypeptides, VP154, VP82, and VP67. In addition, six DNA’ mutants (tsG3, tsG8, tsK13, tsL14, tsM19, and ts022) also exhibited these defects. When the latter four mutants were analyzed for the production of viral DNA at 39”, ts022 synthesized only 2% of the amount of viral DNA synthesized by WT virus-infected cells at 39” (Aron et al., 1975). Mutants tsK13, tsL14, and tsM19 also synthesized