VIROLOGY
72,&'-409
(1976)
The Expression
of the syn- Gene of Herpes Simplex
II. Requirements for Macromolecular
Virus Type 1
Synthesis
J. M. KELLER Department of Biochemistry, University of Washington, Seattle, Washington 98195 Accepted March 8,1976 The fusion of cells infected with a single-step syn- mutant of the Glasgow 17 strain of herpes simplex virus type 1 (HSV-11 is independent of the production of infectious virus. The fusion response of infected BHK cells occurs between 4 and 8 hr postinfection. Prior to this time, synthesis of both RNA and protein is required. The RNA required for the full expression of the syn- phenotype is synthesized by 4 hr postinfection, while the protein required for the full expression of the syn- phenotype is synthesized by 6 hr postinfection. In the presence of 2-deoxy-n-glucose (DOG), neither cell fusion nor the production of infectious virus occurs. As monitored by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, essentially all of the virus proteins are produced in the presence of DOG. However, the bands corresponding to glycoproteins are absent with a concomitant appearance of bands with faster mobilities. This observation suggests that the presence of DOG prevents the complete glycosylation of glycoproteins. These results support the idea that glycoproteins are required for cell fusion as well as for the production of infectious virus. INTRODUCTION
This work is directed towards elucidating the mechanism whereby strains of herpes simplex virus type 1 (HSV-11,’ which carry the syn- gene, cause fusion of infected cells, and the production of syncytia. The previous paper in this series described the morphological appearance of cells infected with either wild type, syn+, or single-step or deletion syn- mutants (Keller, 1976). An important result of that study was the demonstration that the syn+ gene is dominant in doubly infected cells unless the cells are preinfected with a synmutant. In the latter case, the syn- response, fusion, is the phenotype of the infected cells, although both virus genotypes are replicated. The basis for this observa1 Abbreviations used: HSV-1, herpes simplex virus type 1; CA, cytosine arabinoside, pi., postinfection; AD, actinomycin D; CH, cycloheximide; DOG, 2-deoxy-n-glucose; m.o.i., multiplicity of infection; TdR, thymidine; TCA, trichloroacetic acid; UdR, deoxyuridine; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide-gel electrophoresis. 402 Copyright All rights
0 1976 by Academic Presa, Inc. of reproduction in any form reserved.
tion remains unexplained and will necessitate more detailed information concerning the specific gene products synthesized in the infected cells. This paper describes experiments that identify the types of macromolecules and the time of their synthesis required for the expression of the syn- phenotype. My results support other data that implicate virus glycoproteins in both the fusion response and the production of infectious virus (Gallaher et al., 1973; Ludwig et al., 1974; Ludwig and R&t, 1975). MATERIALS
AND METHODS
Cells and viruses. The HSV-1 strains used in this study include syn+ Glasgow 17, a single-step syn- mutant of strain Glasgow 17, syn+ NT and a syn- deletion mutant of strain NT. BHK or HEp-2 cells were used in experiments as indicated. A detailed description of these cells and virus strains as well as the methods used in their propagation were described previously (Keller, 1976). Quantitation of fusion. The extent of
EXPRESSION
OF syn-
GENE
403
14C]glucosamine, 1 &i/ml, or a mixture of 14C-labeled L-amino acids, 1 &i/ml. The medium used to label cells with [14C]glucosamine contained one-tenth the amount of glucose present in the standard medium. The medium used to label cells with 14C-labeled amino acids contained one-twentieth the normal level of amino acids. The experiments with DOG were performed with medium containing onetenth the normal amount of glucose and Inhibition of macromolecular synthesis. one-twentieth the normal amount of The concentrations of various metabolic inhibitors used in these studies were se- amino acids. Control experiments showed lected on the basis of minimal concentra- that these modified media, in the absence tions required to inhibit synthetic proc- of added inhibitors, did not alter the esses. The extent of total DNA synthesis expression of the syn gene. Twenty hours in infected cells was determined by the after the start of infection, cell monolayers incorporation of [3H]TdR into cold 7.5% (60-n-m dishes were used) were washed TCA-insoluble material during a 2-hr pe- one time with phosphate-buffered saline riod at 6 hr p.i.’ Concentrations of cytosine (Keller, 1976), dissolved in 0.2 ml of the arabinoside (CA) > 10 pg/ml were found to solubilizing solution described by Spear block >85% of this incorporation. Synthe- and Roizman (1972) and then boiled for 5 sis of RNA was found to be >99% blocked min. Electrophoresis and autoradiography in the presence of 10 pg/ml actinomycin D of sodium dodecyl sulfate-polyacrylamide (AD), as monitored by the incorporation of slab gels (8.5%) were performed as de[14C]UdR into 7.5% TCA-insoluble mate- scribed by Honess et al. (1974). The prorial during a 2-hr incubation started at 4 teins used for molecular weight standards hr p.i.’ Protein synthesis during a 4-hr included bovine milk lactoperoxidase [MW period started at 2 hr p.i. was blocked by = 77,500 (Rombauts et al., 196711,bovine >98% in the presence of 100 pglml of cyclo- serine albumin [MW = 67,000 (Loeb and heximide (CH),’ as determined by the in- Scheraga, 1956)], chicken egg white ovalcorporation of f3H]amino acids into mate- bumin [MW = 45,000 (Fevold, 1951)], and rial precipitable with cold 7.5% TCA. The yeast glyceraldehyde-3-phosphate dehysynthesis of glycoproteins was altered (see drogenase [MW = 36,000 (Branden et al., Discussion) with 2-deoxyglucose (DOG).’ 197511,all obtained from Sigma and rabbit Studies with DOG were performed in me- muscle pyruvate kinase [MW = 57,000 dium containing 0.45 g of glucose/liter, (Darnall and Klotz, 1975)], obtained from which is one-tenth the concentration in the Calbiochem. normal virus growth medium. Low glucose Chemicals. Cytesine arabinoside, actimedium containing DOG was added to cul- nomycin D, cycloheximide, and deoxyglutures at the end of the adsorption period (2 case were obtained from Sigma. Radiohr p.i.). In the presence of the normal con- chemicals were obtained from New Engcentration of glucose (4.5 g/liter), the con- land Nuclear. centrations of DOG used in experiments RESULTS had no effect on either virus-induced cell Time Course of Fusion fusion or virus yield. Preparation and analysis of radioactive Infection of a monolayer culture of BHK cell extracts. The radioisotepically labeled cells with syn- Glasgow 17 at an m.o.i. of 5 infected-cell extracts were prepared from PFU/cell results in the fusion of all cells to cells grown in modified maintenance me- form a syncytium (Keller, 1976). The earlidium (see below) containing 1% dialyzed est macroscopic appearance of fusion occalf serum. The appropriate radioactive curs at 6 hr p.i., which is about the same precursor was added 4 hr p.i.; D-[ltime that infectious progeny begin to apfusion was judged by visual inspection of infected cultures stained with Giemsa and assigned values of 0 (no fusion), 1+ (ca. 25% of cells fused), 2+ (ca. 50% of cells fused), 3+ (ca. 75% of cells fused), or 4-t (full fusion), as described by Falke (1965). Although this method is only semiquantitative, it is fast and efficient for the purposes used in the experiments described in this paper.
404
J. M. KELLER
pear. Fusion is generally complete (4+) by 6 to 8 hr p.i. (Fig. 1). Essentially identical results are obtained with syn- NT-infected BHK cells as well as syn- Glasgow 17- or syn- NT-infected HEp-2 cells. Macromolecular
Synthesis
The lack of a relationship between DNA synthesis and cell fusion in syn- Glasgow 17-infected BHK cells was shown with cytosine arabinoside, an inhibitor of DNA synthesis. Cultures infected in the presence of >lO pg/ml of CA exhibited full fusion (4+) although no infectious virus was produced. These results show that the inhibition of DNA synthesis does not effect the expression of the syn- gene response. The role of RNA synthesis in the fusion response of syn- Glasgow 17-infected BHK cells was studied with actinomycin D, an inhibitor of RNA synthesis. As shown in Fig. 2, the addition of AD (10 pg/ml) between 0 and 2 hr p.i. prevented virus-induced cell fusion. Addition of AD at or later than 4 hr p.i. had no effect on the appearance of cell fusion. However, the addition of AD at any time prior to about 7 hr p.i. prevented the production of infectious virus. These results demonstrate that RNA synthesis is required for the expression of fusion induced by syn- Glasgow 17 and, in addition, that the production of infectious progeny is not required, which is in agreement with the results obtained with CA. The relationship between protein synthesis and cell fusion was studied with cycloheximide, an inhibitor of protein synA
thesis. The addition of CH at any time between 0 and 3 hr p.i. of BHK cells with syn- Glasgow 17 completely prevented fusion (Fig. 3). If the inhibitor was added at 6 hr p.i., however, the fusion response was complete. These results indicate that protein synthesis is required for at least 5 hr p.i. for the full expression of the fusion response in BHK cells infected with synGlasgow 17. Essentially identical results were obtained with syn- NT-infected monolayers of HEp-2 cells.
‘+I r-----T’ I = J2
4
0
Time
of
Time
(hr)
IO4
PI)
0’ 2 0
4+ F $3+ B2+
06E B : 058
0 0 0 4
2 Time
1. The time course of fusion and virus production in BHK cells infected with syn- Glasgow 17 at an m.o.i. of 5. (GO), Extent of fusion; (A-A), total virus in cells and medium.
I2
IO
a Ihours
FIG. 2. The effect of actinomycin D (AD) on the expression of thesyn- Glasgow 17 phenotype and the production of infectious virus in BHK cells. The inhibitor (10 pg/ml) was added at the times indicated on the abscissa. The extent of fusion and the total virus yield were determined in cultures 24 hr p.i. (0). Yield of virus in the absence of inhibitor; (O-O), yield of virus when AD was added at the indicated time; (O-O), extent of fusion when AD was added at the indicated time.
$ ,+ I: 0 kf!, 0
FIG.
6 add,hon
of
addition
6 (hours
a
0’
PI1
FIG. 3. The effect of cycloheximide (CH) on the expression of thesyn- Glasgow 17 phenotype and the production of infectious virus in BHK cells. The inhibitor (100 pg/ml) was added at the time indicated on the abscissa. The extent of fusion and the total virus yield were determined in cultures 24 hr p.i. (A), Yield of virus in the absence of inhibitor; (O---O), yield of virus when CH was added at the indicated time; (O-O), extent of fusion when CH was added at the indicated time.
EXPRESSION
The possible involvement of glycoproteins in the syrz- HSV-induced cell fusion was studied with 2-deoxyglucose, a molecule which interferes with glycoprotein synthesis (see Discussion). The effect of various concentrations of DOG on the expression of the syn- phenotype and virus yield in syn- NT-infected HEp-2 cells is shown in Fig. 4. These data show that both the morphology of the sylz- response as well as the production of infectious virus are blocked when HEp-2 cells are infected in the presence of 10 mM DOG. Essentially identical results were obtained with synGlasgow 17 (data not shown). With the same medium (i.e., 0.45 g of glucose/liter), 10 mM DOG was only partially effective in blocking either fusion or virus production of syn- NT or syn- Glasgow 17 in BHK cells. The basis for this difference is unknown but presumably reflects metabolic differences between the two cell types. Proteins and Glycoproteins Infected Cells
Synthesized
in
The proteins and glycoproteins synthesized in virus-infected cells were examined by analysis with SDS-PAGE.’ As shown in Fig. 5, at least 20 protein bands ( amino acid label) and 6 glycoprotein bands (glucosamine label) can be distinguished. Both the protein and glycoprotein patterns are similar to those reported by Heine et al. (1974). Visual comparison of the protein and glycoprotein bands made in HEp-2 cells after infection with either the wild type or syn- mutant does not distinguish any major change common to the two fusion mutants (syn- NT and syn- Glasgow 17). However, there is at least one notable distinction between the glycoproteins from cells infected with syn+ NT compared to infection with syn- NT. In particular, there is an absence of glycoprotein bands in the 105,000 to 115,000 molecular weight region of the syn- NT gel [in the region indicated by the bracket, compare the glucosamine-labeled bands (+glcn) in the gels of syn+ NT and syn- NT]. A similar difference is not noted between glycoprotein profiles of the syn+ and syn- Glasgow 17infected-cell extracts. The proteins made in infected HEp-2
OF syn- GENE
[DOG]
mM
FIG. 4. The effect of various concentrations of DOG on the expression of the syn- NT phenotype and the production of infectious virus in HEp-2 cells. The DOG was added to cultures 2 hr pi. and the extent of fusion (0-O) and the total virus yield (O-0) were determined at 24 hr p.i. Note the scale change on the abscissa.
cells in the presence of 10 mM DOG are shown in Fig. 6. In the presence of DOG, two protein bands appear, which are not present or are present in very low amounts in cells infected in the absence of DOG (Fig. 6, bands b and c). These same bands appear in extracts of cells infected with either syn+ or syn- strains. Concurrent with the appearance of band b is the loss of band a. The appearance of band c (MW, 47,000) seems to correlate with a reduction in the bands of slightly higher molecular weight, but the results are not as clear. This may be due to the presence of a protein which migrates with the intact glycoprotein. There are additional changes that occur in the gel patterns of only one strain. For instance, there is a loss of a 30,000molecular-weight protein in the case of syn+ Glasgow 17 and the appearance of a 30,000-molecular-weight protein in the case of syn- Glasgow 17 grown in the presence of DOG. Similar changes were not observed with the NT strains. DISCUSSION
The results presented in this paper describe the requirements for macromolecular synthesis required for the induction of fusion by the syn- Glasgow 17 strain of HSV-1 in BHK cells. In a general sense, many of these results are similar to those reported by others with different strains of HSV-1 (see below). However, the HSV-1
406
J. M. KELLER
5
FIG. 5. An autoradiogram of an SDS-polyacrylamide slab-gel separation of the proteins and glycoproteins synthesized in HEp-2 cells following infection with various wild type and mutant strains of HSV-1. The vertical channels marked with +aa show the proteins labeled with “‘C-labeled amino acids, which were added to cultures 4 hr p.i. The channels marked with +glcn show the glycoproteins labeled with [14C]glucosamine, which was added to cultures 4 hr p.i.
strains used in these other studies are genetically poorly defined. The HSV-1 strain that I am using, namely, Glasgow 17, is being extensively studied by both genetic and biochemical techniques (Brown et al., 1973; Subak-Sharpe et al., 1975). In addition, the syn- Glasgow 17 mutant is a single-step mutant as judged by the fact that
revertants can be recovered (Brown et al., 1973; J. M. K., unpublished observations). Therefore, identification of a difference in the molecules produced in cells infected with the wild type or single-step syn- mutant of Glasgow 17 is very likely to be directly related to the fusion event. With the use of inhibitors of macromo-
EXPRESSION
lecular synthesis, 1 have shown that: (i) The production of infectious virus is not needed for the expression of the syn- phenotype, fusion. This conclusion is based upon the observation that inhibition of DNA, RNA, or protein synthesis at appropriate times after infection blocks the production of infectious virus, but allows vi-
OF syn-
GENE
407
rus-induced cell fusion to occur on schedule. This result is identical to those reported by others for HSV-l-induced cell fusion (e.g., Munk and Sauer, 1964; Falke, 1967). (ii) The expression of the syn- Glasgow 17 phenotype requires RNA synthesis for only 3 to 3.5 hr p.i., as reported for HSV-1 strain H-4 in primary rabbit kidney
PIG. 6. An autoradiogram of an SDS-polyacrylamide slab-gel separation of the proteins synthesized by infected cells in the presence of 10 n&f DOG. For comparative purposes, the data from Fig. 5 are reproduced. The vertical channels marked with +aa, DOG show the proteins synthesized in the presence of DOG. The proteins were labeled with W-labeled amino acids, which were added to cultures 4 hr p.i. See Fig. 5 for other designations.
408
J. M. KELLER
cells (Falke and Peterknecht, 1968). (iii) The synthesis of proteins is required for at least 5 hr p.i., again, similar to the results obtained with strain H-4 (Falke and Peterknecht, 1968). (iv) Glycoprotein synthesis is required for both fusion and the production of infectious virus. This glycoprotein requirement has previously been observed with other strains of HSV-1 (Gallaher et al., 1973) as well as for pseudorabies virus (Ludwig et al., 1974; Ludwig and Bott, 1975). The basis of the DOG effect on HSVinduced cell fusion is not fully understood. This drug is believed to act as an analog of both glucose and mannose (Schmidt et al., 1974). Since mannose is a component of HSV glycoproteins (unpublished observations), it is reasonable to believe that DOG is acting as a mannose analog in the infected cell. In both syn- Glasgow 17- or syn- NT-infected HEp-2 or BHK cells, the presence of DOG results in the appearance of new protein bands that have faster mobilities in the SDS-PAGE patterns than the glycoproteins that are missing. The chemical changes that are responsible for these changes in the SDS-PAGE patterns, however, have not been determined. An earlier investigation with a syn+ HSV-1 strain showed that DOG is incorporated into some virus proteins (Courtney et al., 1973). This presumably also occurs in the case of syn- strains. The observation that the glycoprotein bands are missing in cells infected in the presence of DOG but are replaced by protein bands with more rapid mobilities suggests that the molecules produced in the presence of DOG are smaller. This decrease in size is presumed to be the result of the absence or early termination of carbohydrate side chain synthesis. Structural studies are currently underway in order to resolve this issue. Despite evidence that links glycoproteins to HSV-l-induced cell fusion, the SDS-PAGE analysis of glycoproteins made by infected cells has not provided a direct relationship between fusion and the absence or presence of a specific glycoprotein. Although there is a broad glycoprotein band ( 105,000 to 115,000 MW) present in cells infected with syn+ NT that is absent
in cells infected with syn- NT, the same broad band is missing in cells infected with either syn+ or syn- Glasgow 17. In addition, all of the glycoproteins in this region disappear when cells are infected in the presence of DOG, and a single band with a lower molecular weight (93,000) appears. This result could arise from the presence of multiple species of proteins with the same molecular weight but which are glycosylated to different extents or to differences in extent of glycosylation of a single protein species. A second consideration regarding these glycoproteins is that their presence in infected cells is not necessarily correlated with their presence in either membranes or purified virus. Previous data have shown that purified smooth membranes (Keller et al., 19701, plasma membranes (Heine and R&man, cited by Roizman and Furlong (1974)) and virus (Honess and Boizman, 1973) contain a limited number of the total glycoproteins and/or proteins synthesized in infected cells. Therefore, my failure to observe differences between the glycoproteins synthesized in syn+ or syn- Glasgow 17-infected cells does not eliminate the possibility that there is a difference in the subcellular distribution of glycoproteins (e.g., only some are inserted into the membranes, a presumed requirement for fusion). A fusion-promoting glycoprotein of the single-step syn- Glasgow 17 mutant may differ by only a single amino acid change from the wild-type glycoprotein. Such a small change may cause a dramatic change in its interaction with the plasma membrane. Structural studies on purified glycoproteins are currently underway. ACKNOWLEDGMENTS This work is supported by NIH Grant No. CA16902. J. M. K. is an Established Investigator of the American Heart Association. I thank Ms. J. Lakner for technical assistance. REFERENCES BR&ND$N, C.-I., J~RNVALL, H., EKLUND, H., and FURUGREN, B. (1975). Alcohol dehydrogenases. In “The Enzymes” (P. D. Boyer, ed.), 3rd Ed., Vol. 11, Part A, pp. 103-190. Academic Press, New York.
EXPRESSION BROWN, S, 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. Viral. 18, 329-346. COURTNEY, R. J., STEINER, S. M., and BENYESHMELNICK, M. (1973). Effects of 2-deoxy-n-glucose on herpes simplex virus replication. Virology 52, 447-455. DARNALL, D. W., and KLOTZ, I. M. (1975). Subunit constitution of proteins: A table. Arch. Biochem. Biophys. 166, 651-682. FALKE, D. (1965). Untersuchungen iiber die Beziehungen zwischen Riesenzellbildung und Infektiositat von Herpes-simplex Virus. Arch. VirusfOFSCh. 15, 387-401. FALKE, D. (1967). Ca++, Histidin und Zn++ als Faktoren bei der Riesenzellbildung durch das HerpesVirus hominis. Z. Med. Mikrobiol. Immunol. 153, 179-189. FALKE, D., and PETERKNECHT,W. (1968). DNS-, RNS- und Proteinsynthese und ihre Relation zur Riesenzellbildung in vitro nach Infektion mit Herpesvirus hominis. Arch. Virusforsch. 24, 267287. FEVOLD, H. L. (1951). Egg proteins. Advan. Protein Chem. 6, 187-252. GALLAHER,W. R., LEVITAN, D. B., and BLOUGH,H. A. (1973). Effect of 2-deoxy-n-glucose on cell fusion induced by Newcastle disease and herpes simplex viruses. Virology 55, 193-201. HEINE, J. W., HONESS, R. W., CASSAI, E., and Ron+ MAN, B. (1974). Proteins specified by herpes simplex virus. XII. The virion polypeptides of type 1 strains. J. Viral. 14, 640-651. HONESS, R. W., and ROIZMAN, B. (1973). Proteins specified by herpes simplex virus. XI. Identification and relative molar rates of synthesis of structural and nonstructural herpes virus polypeptides in the infected cell. J. Virol. 12, 1347-1365. KELLER, J. M. (1976). The expression of the syngene of herpes simplex virus type 1. I. Morphology
409
OF s.yn- GENE of infected cells. virology 69, 490-499. KELLER, J. M., SPEAR, P. G., and ROIZMAN,
B.
(1970). Proteins specified by herpes simplex virus. III. Viruses differing in their effects on the social behavior of infected cells specify different membrane glycoproteins. PFOC. Nat. Acad. Sci. USA 65, 865-871. LOEB, G. I., and SCHERAGA,H. A. (1956). Hydrodynamic and thermodynamic properties of bovine serum albumin at low pH. J. Phys. Chem. 60, 1633-1644. LUDWIG, H., BECHT, H., and Roar, T. (1974). Inhibition of herpes virus-induced cell fusion by concanavalin A, antisera, and 2-deoxy-n-glucose. J. Virol. 14, 307-314. LUDWIG, H., and ROTT,R. (1975). Effect of 2-deoxy-nglucose on herpesvirus-induced inhibition of cellular DNA synthesis. J. Viral. 16, 217-221. MUNK, K., and SAUER, G. (1964). Relationship between cell DNA metabolism and nucleocytoplasmic alterations in herpes virus-infected cells. Virology 22, 153-154. ROIZMAN, B., and FURLONG,D. (1974). The replication of herpesviruses. In “Comprehensive Virology” (R. R. Wagner, ed.), Vol. 3, pp. 229-403. Plenum Press, New York. ROMBAUTS,W. A., SCHROEDER,W. A., and MORRE SON, M. (1967). Bovine lactoperoxidase. Partial characterization of the further purified protein. Biochemistry
6, 2965-2977.
SCHMIDT,M. F. G., SCHWARTZ,R. T., and SCHOLTIS SEK, C. (1974). Nucleoside-diphosphate derivatives of 2-deoxy-n-glucose in animal cells. Eur. J. Biochem.
49, 237-247.
SPEAR,P. G., and ROIZMAN,B. (1972). Proteins specified by herpes simplex virus. V. Purification and structural proteins of the herpes virion. J. Viral 9, 143-159.
SUBAK-SHARPE,J. H., BROWN, S. M., RITCHIE, D. A., TIMBURY, M. C., MACNAB, J. C. M., MARBDEN, H. S., and HAY, J. (1975). Genetic and biochemical studies with herpesvirus. Cold Spring Harbor Symp. Quant. Biol.
39, 717-730.
72,&'-409
(1976)
The Expression
of the syn- Gene of Herpes Simplex
II. Requirements for Macromolecular
Virus Type 1
Synthesis
J. M. KELLER Department of Biochemistry, University of Washington, Seattle, Washington 98195 Accepted March 8,1976 The fusion of cells infected with a single-step syn- mutant of the Glasgow 17 strain of herpes simplex virus type 1 (HSV-11 is independent of the production of infectious virus. The fusion response of infected BHK cells occurs between 4 and 8 hr postinfection. Prior to this time, synthesis of both RNA and protein is required. The RNA required for the full expression of the syn- phenotype is synthesized by 4 hr postinfection, while the protein required for the full expression of the syn- phenotype is synthesized by 6 hr postinfection. In the presence of 2-deoxy-n-glucose (DOG), neither cell fusion nor the production of infectious virus occurs. As monitored by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, essentially all of the virus proteins are produced in the presence of DOG. However, the bands corresponding to glycoproteins are absent with a concomitant appearance of bands with faster mobilities. This observation suggests that the presence of DOG prevents the complete glycosylation of glycoproteins. These results support the idea that glycoproteins are required for cell fusion as well as for the production of infectious virus. INTRODUCTION
This work is directed towards elucidating the mechanism whereby strains of herpes simplex virus type 1 (HSV-11,’ which carry the syn- gene, cause fusion of infected cells, and the production of syncytia. The previous paper in this series described the morphological appearance of cells infected with either wild type, syn+, or single-step or deletion syn- mutants (Keller, 1976). An important result of that study was the demonstration that the syn+ gene is dominant in doubly infected cells unless the cells are preinfected with a synmutant. In the latter case, the syn- response, fusion, is the phenotype of the infected cells, although both virus genotypes are replicated. The basis for this observa1 Abbreviations used: HSV-1, herpes simplex virus type 1; CA, cytosine arabinoside, pi., postinfection; AD, actinomycin D; CH, cycloheximide; DOG, 2-deoxy-n-glucose; m.o.i., multiplicity of infection; TdR, thymidine; TCA, trichloroacetic acid; UdR, deoxyuridine; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide-gel electrophoresis. 402 Copyright All rights
0 1976 by Academic Presa, Inc. of reproduction in any form reserved.
tion remains unexplained and will necessitate more detailed information concerning the specific gene products synthesized in the infected cells. This paper describes experiments that identify the types of macromolecules and the time of their synthesis required for the expression of the syn- phenotype. My results support other data that implicate virus glycoproteins in both the fusion response and the production of infectious virus (Gallaher et al., 1973; Ludwig et al., 1974; Ludwig and R&t, 1975). MATERIALS
AND METHODS
Cells and viruses. The HSV-1 strains used in this study include syn+ Glasgow 17, a single-step syn- mutant of strain Glasgow 17, syn+ NT and a syn- deletion mutant of strain NT. BHK or HEp-2 cells were used in experiments as indicated. A detailed description of these cells and virus strains as well as the methods used in their propagation were described previously (Keller, 1976). Quantitation of fusion. The extent of
EXPRESSION
OF syn-
GENE
403
14C]glucosamine, 1 &i/ml, or a mixture of 14C-labeled L-amino acids, 1 &i/ml. The medium used to label cells with [14C]glucosamine contained one-tenth the amount of glucose present in the standard medium. The medium used to label cells with 14C-labeled amino acids contained one-twentieth the normal level of amino acids. The experiments with DOG were performed with medium containing onetenth the normal amount of glucose and Inhibition of macromolecular synthesis. one-twentieth the normal amount of The concentrations of various metabolic inhibitors used in these studies were se- amino acids. Control experiments showed lected on the basis of minimal concentra- that these modified media, in the absence tions required to inhibit synthetic proc- of added inhibitors, did not alter the esses. The extent of total DNA synthesis expression of the syn gene. Twenty hours in infected cells was determined by the after the start of infection, cell monolayers incorporation of [3H]TdR into cold 7.5% (60-n-m dishes were used) were washed TCA-insoluble material during a 2-hr pe- one time with phosphate-buffered saline riod at 6 hr p.i.’ Concentrations of cytosine (Keller, 1976), dissolved in 0.2 ml of the arabinoside (CA) > 10 pg/ml were found to solubilizing solution described by Spear block >85% of this incorporation. Synthe- and Roizman (1972) and then boiled for 5 sis of RNA was found to be >99% blocked min. Electrophoresis and autoradiography in the presence of 10 pg/ml actinomycin D of sodium dodecyl sulfate-polyacrylamide (AD), as monitored by the incorporation of slab gels (8.5%) were performed as de[14C]UdR into 7.5% TCA-insoluble mate- scribed by Honess et al. (1974). The prorial during a 2-hr incubation started at 4 teins used for molecular weight standards hr p.i.’ Protein synthesis during a 4-hr included bovine milk lactoperoxidase [MW period started at 2 hr p.i. was blocked by = 77,500 (Rombauts et al., 196711,bovine >98% in the presence of 100 pglml of cyclo- serine albumin [MW = 67,000 (Loeb and heximide (CH),’ as determined by the in- Scheraga, 1956)], chicken egg white ovalcorporation of f3H]amino acids into mate- bumin [MW = 45,000 (Fevold, 1951)], and rial precipitable with cold 7.5% TCA. The yeast glyceraldehyde-3-phosphate dehysynthesis of glycoproteins was altered (see drogenase [MW = 36,000 (Branden et al., Discussion) with 2-deoxyglucose (DOG).’ 197511,all obtained from Sigma and rabbit Studies with DOG were performed in me- muscle pyruvate kinase [MW = 57,000 dium containing 0.45 g of glucose/liter, (Darnall and Klotz, 1975)], obtained from which is one-tenth the concentration in the Calbiochem. normal virus growth medium. Low glucose Chemicals. Cytesine arabinoside, actimedium containing DOG was added to cul- nomycin D, cycloheximide, and deoxyglutures at the end of the adsorption period (2 case were obtained from Sigma. Radiohr p.i.). In the presence of the normal con- chemicals were obtained from New Engcentration of glucose (4.5 g/liter), the con- land Nuclear. centrations of DOG used in experiments RESULTS had no effect on either virus-induced cell Time Course of Fusion fusion or virus yield. Preparation and analysis of radioactive Infection of a monolayer culture of BHK cell extracts. The radioisotepically labeled cells with syn- Glasgow 17 at an m.o.i. of 5 infected-cell extracts were prepared from PFU/cell results in the fusion of all cells to cells grown in modified maintenance me- form a syncytium (Keller, 1976). The earlidium (see below) containing 1% dialyzed est macroscopic appearance of fusion occalf serum. The appropriate radioactive curs at 6 hr p.i., which is about the same precursor was added 4 hr p.i.; D-[ltime that infectious progeny begin to apfusion was judged by visual inspection of infected cultures stained with Giemsa and assigned values of 0 (no fusion), 1+ (ca. 25% of cells fused), 2+ (ca. 50% of cells fused), 3+ (ca. 75% of cells fused), or 4-t (full fusion), as described by Falke (1965). Although this method is only semiquantitative, it is fast and efficient for the purposes used in the experiments described in this paper.
404
J. M. KELLER
pear. Fusion is generally complete (4+) by 6 to 8 hr p.i. (Fig. 1). Essentially identical results are obtained with syn- NT-infected BHK cells as well as syn- Glasgow 17- or syn- NT-infected HEp-2 cells. Macromolecular
Synthesis
The lack of a relationship between DNA synthesis and cell fusion in syn- Glasgow 17-infected BHK cells was shown with cytosine arabinoside, an inhibitor of DNA synthesis. Cultures infected in the presence of >lO pg/ml of CA exhibited full fusion (4+) although no infectious virus was produced. These results show that the inhibition of DNA synthesis does not effect the expression of the syn- gene response. The role of RNA synthesis in the fusion response of syn- Glasgow 17-infected BHK cells was studied with actinomycin D, an inhibitor of RNA synthesis. As shown in Fig. 2, the addition of AD (10 pg/ml) between 0 and 2 hr p.i. prevented virus-induced cell fusion. Addition of AD at or later than 4 hr p.i. had no effect on the appearance of cell fusion. However, the addition of AD at any time prior to about 7 hr p.i. prevented the production of infectious virus. These results demonstrate that RNA synthesis is required for the expression of fusion induced by syn- Glasgow 17 and, in addition, that the production of infectious progeny is not required, which is in agreement with the results obtained with CA. The relationship between protein synthesis and cell fusion was studied with cycloheximide, an inhibitor of protein synA
thesis. The addition of CH at any time between 0 and 3 hr p.i. of BHK cells with syn- Glasgow 17 completely prevented fusion (Fig. 3). If the inhibitor was added at 6 hr p.i., however, the fusion response was complete. These results indicate that protein synthesis is required for at least 5 hr p.i. for the full expression of the fusion response in BHK cells infected with synGlasgow 17. Essentially identical results were obtained with syn- NT-infected monolayers of HEp-2 cells.
‘+I r-----T’ I = J2
4
0
Time
of
Time
(hr)
IO4
PI)
0’ 2 0
4+ F $3+ B2+
06E B : 058
0 0 0 4
2 Time
1. The time course of fusion and virus production in BHK cells infected with syn- Glasgow 17 at an m.o.i. of 5. (GO), Extent of fusion; (A-A), total virus in cells and medium.
I2
IO
a Ihours
FIG. 2. The effect of actinomycin D (AD) on the expression of thesyn- Glasgow 17 phenotype and the production of infectious virus in BHK cells. The inhibitor (10 pg/ml) was added at the times indicated on the abscissa. The extent of fusion and the total virus yield were determined in cultures 24 hr p.i. (0). Yield of virus in the absence of inhibitor; (O-O), yield of virus when AD was added at the indicated time; (O-O), extent of fusion when AD was added at the indicated time.
$ ,+ I: 0 kf!, 0
FIG.
6 add,hon
of
addition
6 (hours
a
0’
PI1
FIG. 3. The effect of cycloheximide (CH) on the expression of thesyn- Glasgow 17 phenotype and the production of infectious virus in BHK cells. The inhibitor (100 pg/ml) was added at the time indicated on the abscissa. The extent of fusion and the total virus yield were determined in cultures 24 hr p.i. (A), Yield of virus in the absence of inhibitor; (O---O), yield of virus when CH was added at the indicated time; (O-O), extent of fusion when CH was added at the indicated time.
EXPRESSION
The possible involvement of glycoproteins in the syrz- HSV-induced cell fusion was studied with 2-deoxyglucose, a molecule which interferes with glycoprotein synthesis (see Discussion). The effect of various concentrations of DOG on the expression of the syn- phenotype and virus yield in syn- NT-infected HEp-2 cells is shown in Fig. 4. These data show that both the morphology of the sylz- response as well as the production of infectious virus are blocked when HEp-2 cells are infected in the presence of 10 mM DOG. Essentially identical results were obtained with synGlasgow 17 (data not shown). With the same medium (i.e., 0.45 g of glucose/liter), 10 mM DOG was only partially effective in blocking either fusion or virus production of syn- NT or syn- Glasgow 17 in BHK cells. The basis for this difference is unknown but presumably reflects metabolic differences between the two cell types. Proteins and Glycoproteins Infected Cells
Synthesized
in
The proteins and glycoproteins synthesized in virus-infected cells were examined by analysis with SDS-PAGE.’ As shown in Fig. 5, at least 20 protein bands ( amino acid label) and 6 glycoprotein bands (glucosamine label) can be distinguished. Both the protein and glycoprotein patterns are similar to those reported by Heine et al. (1974). Visual comparison of the protein and glycoprotein bands made in HEp-2 cells after infection with either the wild type or syn- mutant does not distinguish any major change common to the two fusion mutants (syn- NT and syn- Glasgow 17). However, there is at least one notable distinction between the glycoproteins from cells infected with syn+ NT compared to infection with syn- NT. In particular, there is an absence of glycoprotein bands in the 105,000 to 115,000 molecular weight region of the syn- NT gel [in the region indicated by the bracket, compare the glucosamine-labeled bands (+glcn) in the gels of syn+ NT and syn- NT]. A similar difference is not noted between glycoprotein profiles of the syn+ and syn- Glasgow 17infected-cell extracts. The proteins made in infected HEp-2
OF syn- GENE
[DOG]
mM
FIG. 4. The effect of various concentrations of DOG on the expression of the syn- NT phenotype and the production of infectious virus in HEp-2 cells. The DOG was added to cultures 2 hr pi. and the extent of fusion (0-O) and the total virus yield (O-0) were determined at 24 hr p.i. Note the scale change on the abscissa.
cells in the presence of 10 mM DOG are shown in Fig. 6. In the presence of DOG, two protein bands appear, which are not present or are present in very low amounts in cells infected in the absence of DOG (Fig. 6, bands b and c). These same bands appear in extracts of cells infected with either syn+ or syn- strains. Concurrent with the appearance of band b is the loss of band a. The appearance of band c (MW, 47,000) seems to correlate with a reduction in the bands of slightly higher molecular weight, but the results are not as clear. This may be due to the presence of a protein which migrates with the intact glycoprotein. There are additional changes that occur in the gel patterns of only one strain. For instance, there is a loss of a 30,000molecular-weight protein in the case of syn+ Glasgow 17 and the appearance of a 30,000-molecular-weight protein in the case of syn- Glasgow 17 grown in the presence of DOG. Similar changes were not observed with the NT strains. DISCUSSION
The results presented in this paper describe the requirements for macromolecular synthesis required for the induction of fusion by the syn- Glasgow 17 strain of HSV-1 in BHK cells. In a general sense, many of these results are similar to those reported by others with different strains of HSV-1 (see below). However, the HSV-1
406
J. M. KELLER
5
FIG. 5. An autoradiogram of an SDS-polyacrylamide slab-gel separation of the proteins and glycoproteins synthesized in HEp-2 cells following infection with various wild type and mutant strains of HSV-1. The vertical channels marked with +aa show the proteins labeled with “‘C-labeled amino acids, which were added to cultures 4 hr p.i. The channels marked with +glcn show the glycoproteins labeled with [14C]glucosamine, which was added to cultures 4 hr p.i.
strains used in these other studies are genetically poorly defined. The HSV-1 strain that I am using, namely, Glasgow 17, is being extensively studied by both genetic and biochemical techniques (Brown et al., 1973; Subak-Sharpe et al., 1975). In addition, the syn- Glasgow 17 mutant is a single-step mutant as judged by the fact that
revertants can be recovered (Brown et al., 1973; J. M. K., unpublished observations). Therefore, identification of a difference in the molecules produced in cells infected with the wild type or single-step syn- mutant of Glasgow 17 is very likely to be directly related to the fusion event. With the use of inhibitors of macromo-
EXPRESSION
lecular synthesis, 1 have shown that: (i) The production of infectious virus is not needed for the expression of the syn- phenotype, fusion. This conclusion is based upon the observation that inhibition of DNA, RNA, or protein synthesis at appropriate times after infection blocks the production of infectious virus, but allows vi-
OF syn-
GENE
407
rus-induced cell fusion to occur on schedule. This result is identical to those reported by others for HSV-l-induced cell fusion (e.g., Munk and Sauer, 1964; Falke, 1967). (ii) The expression of the syn- Glasgow 17 phenotype requires RNA synthesis for only 3 to 3.5 hr p.i., as reported for HSV-1 strain H-4 in primary rabbit kidney
PIG. 6. An autoradiogram of an SDS-polyacrylamide slab-gel separation of the proteins synthesized by infected cells in the presence of 10 n&f DOG. For comparative purposes, the data from Fig. 5 are reproduced. The vertical channels marked with +aa, DOG show the proteins synthesized in the presence of DOG. The proteins were labeled with W-labeled amino acids, which were added to cultures 4 hr p.i. See Fig. 5 for other designations.
408
J. M. KELLER
cells (Falke and Peterknecht, 1968). (iii) The synthesis of proteins is required for at least 5 hr p.i., again, similar to the results obtained with strain H-4 (Falke and Peterknecht, 1968). (iv) Glycoprotein synthesis is required for both fusion and the production of infectious virus. This glycoprotein requirement has previously been observed with other strains of HSV-1 (Gallaher et al., 1973) as well as for pseudorabies virus (Ludwig et al., 1974; Ludwig and Bott, 1975). The basis of the DOG effect on HSVinduced cell fusion is not fully understood. This drug is believed to act as an analog of both glucose and mannose (Schmidt et al., 1974). Since mannose is a component of HSV glycoproteins (unpublished observations), it is reasonable to believe that DOG is acting as a mannose analog in the infected cell. In both syn- Glasgow 17- or syn- NT-infected HEp-2 or BHK cells, the presence of DOG results in the appearance of new protein bands that have faster mobilities in the SDS-PAGE patterns than the glycoproteins that are missing. The chemical changes that are responsible for these changes in the SDS-PAGE patterns, however, have not been determined. An earlier investigation with a syn+ HSV-1 strain showed that DOG is incorporated into some virus proteins (Courtney et al., 1973). This presumably also occurs in the case of syn- strains. The observation that the glycoprotein bands are missing in cells infected in the presence of DOG but are replaced by protein bands with more rapid mobilities suggests that the molecules produced in the presence of DOG are smaller. This decrease in size is presumed to be the result of the absence or early termination of carbohydrate side chain synthesis. Structural studies are currently underway in order to resolve this issue. Despite evidence that links glycoproteins to HSV-l-induced cell fusion, the SDS-PAGE analysis of glycoproteins made by infected cells has not provided a direct relationship between fusion and the absence or presence of a specific glycoprotein. Although there is a broad glycoprotein band ( 105,000 to 115,000 MW) present in cells infected with syn+ NT that is absent
in cells infected with syn- NT, the same broad band is missing in cells infected with either syn+ or syn- Glasgow 17. In addition, all of the glycoproteins in this region disappear when cells are infected in the presence of DOG, and a single band with a lower molecular weight (93,000) appears. This result could arise from the presence of multiple species of proteins with the same molecular weight but which are glycosylated to different extents or to differences in extent of glycosylation of a single protein species. A second consideration regarding these glycoproteins is that their presence in infected cells is not necessarily correlated with their presence in either membranes or purified virus. Previous data have shown that purified smooth membranes (Keller et al., 19701, plasma membranes (Heine and R&man, cited by Roizman and Furlong (1974)) and virus (Honess and Boizman, 1973) contain a limited number of the total glycoproteins and/or proteins synthesized in infected cells. Therefore, my failure to observe differences between the glycoproteins synthesized in syn+ or syn- Glasgow 17-infected cells does not eliminate the possibility that there is a difference in the subcellular distribution of glycoproteins (e.g., only some are inserted into the membranes, a presumed requirement for fusion). A fusion-promoting glycoprotein of the single-step syn- Glasgow 17 mutant may differ by only a single amino acid change from the wild-type glycoprotein. Such a small change may cause a dramatic change in its interaction with the plasma membrane. Structural studies on purified glycoproteins are currently underway. ACKNOWLEDGMENTS This work is supported by NIH Grant No. CA16902. J. M. K. is an Established Investigator of the American Heart Association. I thank Ms. J. Lakner for technical assistance. REFERENCES BR&ND$N, C.-I., J~RNVALL, H., EKLUND, H., and FURUGREN, B. (1975). Alcohol dehydrogenases. In “The Enzymes” (P. D. Boyer, ed.), 3rd Ed., Vol. 11, Part A, pp. 103-190. Academic Press, New York.
EXPRESSION BROWN, S, 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. Viral. 18, 329-346. COURTNEY, R. J., STEINER, S. M., and BENYESHMELNICK, M. (1973). Effects of 2-deoxy-n-glucose on herpes simplex virus replication. Virology 52, 447-455. DARNALL, D. W., and KLOTZ, I. M. (1975). Subunit constitution of proteins: A table. Arch. Biochem. Biophys. 166, 651-682. FALKE, D. (1965). Untersuchungen iiber die Beziehungen zwischen Riesenzellbildung und Infektiositat von Herpes-simplex Virus. Arch. VirusfOFSCh. 15, 387-401. FALKE, D. (1967). Ca++, Histidin und Zn++ als Faktoren bei der Riesenzellbildung durch das HerpesVirus hominis. Z. Med. Mikrobiol. Immunol. 153, 179-189. FALKE, D., and PETERKNECHT,W. (1968). DNS-, RNS- und Proteinsynthese und ihre Relation zur Riesenzellbildung in vitro nach Infektion mit Herpesvirus hominis. Arch. Virusforsch. 24, 267287. FEVOLD, H. L. (1951). Egg proteins. Advan. Protein Chem. 6, 187-252. GALLAHER,W. R., LEVITAN, D. B., and BLOUGH,H. A. (1973). Effect of 2-deoxy-n-glucose on cell fusion induced by Newcastle disease and herpes simplex viruses. Virology 55, 193-201. HEINE, J. W., HONESS, R. W., CASSAI, E., and Ron+ MAN, B. (1974). Proteins specified by herpes simplex virus. XII. The virion polypeptides of type 1 strains. J. Viral. 14, 640-651. HONESS, R. W., and ROIZMAN, B. (1973). Proteins specified by herpes simplex virus. XI. Identification and relative molar rates of synthesis of structural and nonstructural herpes virus polypeptides in the infected cell. J. Virol. 12, 1347-1365. KELLER, J. M. (1976). The expression of the syngene of herpes simplex virus type 1. I. Morphology
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B.
(1970). Proteins specified by herpes simplex virus. III. Viruses differing in their effects on the social behavior of infected cells specify different membrane glycoproteins. PFOC. Nat. Acad. Sci. USA 65, 865-871. LOEB, G. I., and SCHERAGA,H. A. (1956). Hydrodynamic and thermodynamic properties of bovine serum albumin at low pH. J. Phys. Chem. 60, 1633-1644. LUDWIG, H., BECHT, H., and Roar, T. (1974). Inhibition of herpes virus-induced cell fusion by concanavalin A, antisera, and 2-deoxy-n-glucose. J. Virol. 14, 307-314. LUDWIG, H., and ROTT,R. (1975). Effect of 2-deoxy-nglucose on herpesvirus-induced inhibition of cellular DNA synthesis. J. Viral. 16, 217-221. MUNK, K., and SAUER, G. (1964). Relationship between cell DNA metabolism and nucleocytoplasmic alterations in herpes virus-infected cells. Virology 22, 153-154. ROIZMAN, B., and FURLONG,D. (1974). The replication of herpesviruses. In “Comprehensive Virology” (R. R. Wagner, ed.), Vol. 3, pp. 229-403. Plenum Press, New York. ROMBAUTS,W. A., SCHROEDER,W. A., and MORRE SON, M. (1967). Bovine lactoperoxidase. Partial characterization of the further purified protein. Biochemistry
6, 2965-2977.
SCHMIDT,M. F. G., SCHWARTZ,R. T., and SCHOLTIS SEK, C. (1974). Nucleoside-diphosphate derivatives of 2-deoxy-n-glucose in animal cells. Eur. J. Biochem.
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SPEAR,P. G., and ROIZMAN,B. (1972). Proteins specified by herpes simplex virus. V. Purification and structural proteins of the herpes virion. J. Viral 9, 143-159.
SUBAK-SHARPE,J. H., BROWN, S. M., RITCHIE, D. A., TIMBURY, M. C., MACNAB, J. C. M., MARBDEN, H. S., and HAY, J. (1975). Genetic and biochemical studies with herpesvirus. Cold Spring Harbor Symp. Quant. Biol.
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