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Quantitative studies on the maturation process of herpes simplex virus type 1 in vero cells
Virus Research, Elsevier
281
10 (1988) 281-286
VRR 00408
Short
Communication
Quantitative studies on the maturation process of herpes simplex virus type 1 in Vero cells A. Hajime Department
Koyama
of Vwology, School of Medicine, (Accepted
and Takahiro
Uchida
The Vnioersity of Tokushima,
for publication
12 January
Tokushima,
770 Japan
1988)
Summary We examined the time courses of viral DNA synthesis, the formation and envelopment of viral nucleocapsids, and the formation of infectious progeny virus in Vero cells infected with herpes simplex virus type 1 (HSV-1). The results showed that the formation of nucleocapsids coincided with the appearance of enveloped particles as well as of infectious progeny virus, although the synthesis of viral DNA took place approximately 2 h prior to the beginning of nucleocapsid formation. These results indicate that the rate-limiting step in the virogenesis of HSV-l-infected cells is the encapsidation of viral DNA and that the enveloped virus is formed immediately after the formation of nucleocapsids and is infectious without any further processing of virion constituents. Herpes simplex virus; Viral DNA of viral DNA; Envelopment
replication;
Maturation
of virus;
Encapsidation
The morphogenesis of herpes virus has been studied extensively (Roizman and Furlong, 1974; Watson, 1973). However, the chronological relationships between DNA synthesis, formation and envelopment of nucleocapsids, and the appearance of infectious progeny virus have not been conclusively determined. All we know is the sequence of the reactions, i.e., viral DNA is synthesized and encapsidated to form nucleocapsids in the nuclei of infected cells. These nucleocapsids are then enveloped by budding through the inner nuclear membrane into the perinuclear space to form an enveloped virus (Roizman and Furlong, 1974; Roizman, 1980).
Correspondence to: A.H. Koyama, Department of Virology, Tokushima Kuramotocho-3, Tokushima, 770 Japan.
016%1702/88/$03.50
0 1988 Elsevier Science Publishers
School
of Medicine.
B.V. (Biomedical
Division)
The
University
of
282 To understand the manner of the sequential events in the middle stage of HSV-1 multiplication, we examined the intracellular events in virus-infected cells chronologically and quantitatively. Vero cells grown in Eagle’s minimum essential medium (MEM) containing 10% calf serum were used. Herpes simplex virus type 1 (HSV-l), strain HF, was propagated in Vero cells and infectivity was measured by a plaque assay as described in previous papers (Koyama and Uchida, 1984, 1987). Infection was carried out by the following procedures. Monolayer cells in 35-mm dishes were washed with phosphate-buffered saline (PBS), and received 0.3 ml of the virus preparations in PBS containing 1% calf serum. The cells were incubated with the virus for 30 min at room temperature with continuous rocking. After removal of the inocula, the infection was started by incubating the infected monolayers at 37°C in an appropriate medium. We repeated the experiments several times with different multiplicities of infection (MOI). The difference in the MO1 between 10 and 20 PFU/cell did not affect the kinetics shown below. Determination of the amount of all nucleocapsids in the virus-infected cells was carried out as follows: virus-infected monolayer cells in 35-mm dishes were incubated at 37” C in 1 ml of MEM containing 2.5 pCi of [ 3H]thymidine (2 mCi/mmol). At intervals, the monolayer cells along with the culture fluid were subjected to two or three cycles of freezing and thawing, followed by an incubation with 40 pg of DNase I (Sigma) at 37 o C for 30 min. The DNase I-treated cell lysate was layered over 8 ml of the mixture consisting of 54% Percoll (Pharmacia Fine Chemicals AB), 1.1% calf serum and 0.25 M sucrose in 5 mM N-2-hydroxyethylpiperazine-N’-Zethanesulfonic acid (HEPES), pH 7.4, and centrifuged at 20 000 rpm for 90 min in an SS34-rotor (Sorvall). Both enveloped and naked nucleocapsids were banded at the same position in a Percoll gradient because of the steepness of the gradient formed under the above conditions. This procedure allows a rapid determination of the amount of all nucleocapsids synthesized in the infected cells. To determine the amount of the naked nucleocapsids separately from the enveloped ones, the DNase I-treated lysate was mixed with Percoll to make up a solution containing 55% Percoll, 0.25 M sucrose in 11.25 mM HEPES, pH 7.4, and centrifuged as described above. Radioactivity in fractions was measured after acid-precipitation with trichloroacetic acid. The kinetics of viral DNA synthesis is shown in Fig. 1. The incorporation of [ 3H]thymidine into HSV-1 DNA began at 3 h postinfection (p.i.) and reached nearly maximum level at 6 h p.i. These kinetics are similar to those reported previously (Roizman and Furlong, 1974). In contrast to the kinetics of viral DNA synthesis, nucleocapsid formation showed quite different kinetics under the same experimental conditions (Fig. 1). The amount of nucleocapsids began to increase at about 5 h p.i., indicating that there is a time lag between the synthesis of viral DNA and the formation of nucleocapsids. Since the formation of nucleocapsids was immediately followed by envelopment (as shown below), the encapsidation of viral DNA is a rate-limiting step in the morphogenesis of HSV-1 in the infected cells. Comparison of the amounts of [3H]thymidine incorporated into the viral DNA and that incorporated into the viral nucleocapsids showed that, at the end of nucleocapsid
283
Time (h) Tim-e (h) Fig. 1. Synthesis of viral DNA and formation of nucleocapsids in the HSV-l-infected cells. Vero cells in 35-mm dishes were allowed to adsorb HSV-1 at an MO1 of 12 for 30 min at room temperature. After removal of the inocula, the cells were incubated at 37 o C in MEM containing [ 3H]thymidine. At intervals the cells along with the culture fluid were harvested. The amount of viral DNA (0) was measured by centrifugation in a ccsium chloride minor modifications. The amount
density gradient as described by Ben-Porat and Kaplan (1963) with of all nucleocapsids synthesized in the cells (A) was assayed as described in the text.
Fig. 2. Formation of enveloped particles and infectious progeny virus in HSV-l-infected cells. Vero cells in 35-mm dishes were allowed to adsorb HSV-1 at an MO1 of 19 for 30 min at room temperature. After removal of the inocula the cells were washed with phosphate-buffered saline. Infection was initiated by incubating the cells at 37 o C in MEM containing [3H]thymidine. At intervals the cells along with the culture fluid were harvested and subjected to three cycles of freezing and thawing. The infectivities in the lysates (0) were assayed. The amounts of enveloped particles (0) as well as of naked nucleocapsids (A) were measured
as described
in the text.
formation (12 h p.i.), about 48 x lo3 cpm of [ 3H]thymidine molecules were incorporated into the nucleocapsids while about 227 X lo3 cpm of [3H]thymidine were incorporated into the viral DNA. These results indicate that approximately one fifth of the viral DNA synthesized was encapsidated to form the nucleocapsids. This value agrees with that for pseudorabies virus (Ben-Porat and Kaplan, 1963). Previously, Kaplan (1964) suggested that pseudorabies virus DNA was immediately encapsidated following the synthesis of viral DNA by showing the rapid conversion of newly synthesized DNA into DNase-resistant form. The discrepancy between our results and his can be attributed to the difference in cell culture conditions. He treated the cells with 5-fluorouracil for 18 h prior to the infection. This drug-treatment apparently affected the intracellular reactions toward .the nucleocapsid formation, since, for example, the kinetics of viral DNA synthesis in his report differ from that in this and other reports (Roizman and Furlong, 1974). As described below, the kinetics of nucleocapsid formation shown in Fig. 1 is in agreement with several morphological observations. The envelopment of nucleocapsids occurred simultaneously with the formation of nucleocapsids. As shown in Fig. 2, the enveloped particles appeared, increased, and reached a plateau with similar kinetics to the formation of naked nucleocapsids. There was no apparent time lapse between the formation of nucleocapsids and that of enveloped particles, indicating that the formation of nucleocapsids is immediately followed by envelopment. Comparison of radioactivity incorporated into the enveloped particle fractions with that incorporated into the naked nucleocapsid
284 fractions revealed that approximately 60% of the nucleocapsids were enveloped at 8 h p.i. and that the radioactivity ratio of enveloped to naked remained unchanged even at 25 h p.i. In addition, the infectious progeny virus appeared with kinetics quite similar to those of the formation of nucleocapsids and enveloped particles. As shown in Fig. 2, the infectious progeny virus appeared at 6 h p.i. and increased with time. The similarity between the kinetics of the formation of enveloped particles and that of infectious progeny virus indicates that the enveloped particles thus formed are infectious without any further processing of viral constituents. Although it is difficult by morphological methods to determine precisely the time of formation of capsids or of enveloped virus, the above conclusions are supported by previous studies with the electron microscope. On examination of HSV-l-infected cells at sequential intervals, Nii et al. (1968) found that capsids with dense cores were not seen until 6 h p.i. and appeared approximately at the same time as enveloped virus. Watson et al. (1964) also showed that naked capsids with DNA cores appeared in infected cells at the time of formation of infectious progeny virus. The molecular mechanism of virogenesis in HSV-l-infected cells is not well understood at the present time. During the middle stages of infection, expression of late genes begins immediately after the onset of viral DNA synthesis. Capsid proteins are synthesized in the cytoplasm and thereafter migrate to nuclei where they are assembled into capsids. These empty capsids bind tightly to the nuclear matrix wherein the encapsidation of viral DNA probably takes place. In agreement with our finding that the formation of nucleocapsids is immediately followed by envelopment, the nucleocapsids thus formed leave the nuclear matrix immediately, since only the empty capsids, not capsids with a dense core, are seen to be associated with the nuclear matrix (Bibor-Hardy et al., 1982). Envelopment is considered to occur by budding of the nucleocapsids from the modified region of the inner nuclear membrane into the perinuclear space (Roizman and Furlong, 1974). The present study shows that the encapsidation of viral DNA is a rate-limiting step in the multiplication of HSV-1 in Vero cells and that, once nucleocapsids are formed, they are converted quickly into infectious progeny virus. Since the lumen of the endoplasmic reticulum and the perinuclear space are continuous structures, egress of the progeny virus is likely to occur essentially in the same way as that of an excretion of secretory proteins via transport vesicles (Spear, 1985).
Acknowledgements We thank Fumi Tashiro for excellent technical assistance. This work was supported in part by a grant from the Ministry of Education, Science and Welfare of Japan.
References Ben-Porat, T. and Kaplan, AS. (1963) The synthesis and fate of pseudorabies mammalian cells in the stationary phase of growth. Virology 20, 310-317.
DNA
in infected
285 Bibor-Hardy, V., Pouchelet, M., St-Pierre, E., Herzberg, M. and Simard, R. (1982) The nuclear matrix is involved in herpes simplex virogenesis. Virology 121, 296-306. Kaplan, AS. (1964) Studies on the replicating pool of viral DNA in cells infected with pseudorabies virus. Virology 24, 19-25. Koyama, A.H. and U&da, T. (1984) Inhibition of multiplication of herpes simplex virus type 1 by ammonium chloride and chloroquine. Virology 138, 332-335. Koyama, A.H. and Uchida, T. (1987) The mode of entry of herpes simplex virus type 1 into Vero cells. Microbial. Immunol. 31, 123-130. Nii, S., Morgan, C. and Rose, H.M. (1968) Electron microscopy of herpes simplex virus. II. Sequence of development. J. Virol. 2, 517-536. Roizman, B. (1980) Herpes simplex viruses. In: DNA Tumor Viruses, 2nd ed. (Part 2) (Tooze, J., ed.), pp. 615-745. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Roizman, B. and Furlong, D. (1974) The replication of herpesviruses. In: Comprehensive Virology, Vol. 3 (Fraenkel-Conrat, H. and Wagner, R.R., eds.), pp. 229-403. Plenum Press, New York. Spear, P.G. (1985) Glycoproteins specified by herpes simplex viruses. In: Herpesviruses, Vol. 3 (Roizman, B., ed.), pp. 315-356. Plenum Press, New York. Watson, D.H. (1973) Replication of viruses-morphological aspects. In: The Herpesviruses (Kaplan, A., ed.), pp. 133-161, Academic Press, New York. Watson, D.H., Wildy, P. and Russell, W.C. (1964) Quantitative electron microscope studies on the growth of herpes virus using the techniques of negative staining and ultramicrotomy. Virology 24, 523-538. (Received
21 October
1987; revision
received
5 January
1988)
281
10 (1988) 281-286
VRR 00408
Short
Communication
Quantitative studies on the maturation process of herpes simplex virus type 1 in Vero cells A. Hajime Department
Koyama
of Vwology, School of Medicine, (Accepted
and Takahiro
Uchida
The Vnioersity of Tokushima,
for publication
12 January
Tokushima,
770 Japan
1988)
Summary We examined the time courses of viral DNA synthesis, the formation and envelopment of viral nucleocapsids, and the formation of infectious progeny virus in Vero cells infected with herpes simplex virus type 1 (HSV-1). The results showed that the formation of nucleocapsids coincided with the appearance of enveloped particles as well as of infectious progeny virus, although the synthesis of viral DNA took place approximately 2 h prior to the beginning of nucleocapsid formation. These results indicate that the rate-limiting step in the virogenesis of HSV-l-infected cells is the encapsidation of viral DNA and that the enveloped virus is formed immediately after the formation of nucleocapsids and is infectious without any further processing of virion constituents. Herpes simplex virus; Viral DNA of viral DNA; Envelopment
replication;
Maturation
of virus;
Encapsidation
The morphogenesis of herpes virus has been studied extensively (Roizman and Furlong, 1974; Watson, 1973). However, the chronological relationships between DNA synthesis, formation and envelopment of nucleocapsids, and the appearance of infectious progeny virus have not been conclusively determined. All we know is the sequence of the reactions, i.e., viral DNA is synthesized and encapsidated to form nucleocapsids in the nuclei of infected cells. These nucleocapsids are then enveloped by budding through the inner nuclear membrane into the perinuclear space to form an enveloped virus (Roizman and Furlong, 1974; Roizman, 1980).
Correspondence to: A.H. Koyama, Department of Virology, Tokushima Kuramotocho-3, Tokushima, 770 Japan.
016%1702/88/$03.50
0 1988 Elsevier Science Publishers
School
of Medicine.
B.V. (Biomedical
Division)
The
University
of
282 To understand the manner of the sequential events in the middle stage of HSV-1 multiplication, we examined the intracellular events in virus-infected cells chronologically and quantitatively. Vero cells grown in Eagle’s minimum essential medium (MEM) containing 10% calf serum were used. Herpes simplex virus type 1 (HSV-l), strain HF, was propagated in Vero cells and infectivity was measured by a plaque assay as described in previous papers (Koyama and Uchida, 1984, 1987). Infection was carried out by the following procedures. Monolayer cells in 35-mm dishes were washed with phosphate-buffered saline (PBS), and received 0.3 ml of the virus preparations in PBS containing 1% calf serum. The cells were incubated with the virus for 30 min at room temperature with continuous rocking. After removal of the inocula, the infection was started by incubating the infected monolayers at 37°C in an appropriate medium. We repeated the experiments several times with different multiplicities of infection (MOI). The difference in the MO1 between 10 and 20 PFU/cell did not affect the kinetics shown below. Determination of the amount of all nucleocapsids in the virus-infected cells was carried out as follows: virus-infected monolayer cells in 35-mm dishes were incubated at 37” C in 1 ml of MEM containing 2.5 pCi of [ 3H]thymidine (2 mCi/mmol). At intervals, the monolayer cells along with the culture fluid were subjected to two or three cycles of freezing and thawing, followed by an incubation with 40 pg of DNase I (Sigma) at 37 o C for 30 min. The DNase I-treated cell lysate was layered over 8 ml of the mixture consisting of 54% Percoll (Pharmacia Fine Chemicals AB), 1.1% calf serum and 0.25 M sucrose in 5 mM N-2-hydroxyethylpiperazine-N’-Zethanesulfonic acid (HEPES), pH 7.4, and centrifuged at 20 000 rpm for 90 min in an SS34-rotor (Sorvall). Both enveloped and naked nucleocapsids were banded at the same position in a Percoll gradient because of the steepness of the gradient formed under the above conditions. This procedure allows a rapid determination of the amount of all nucleocapsids synthesized in the infected cells. To determine the amount of the naked nucleocapsids separately from the enveloped ones, the DNase I-treated lysate was mixed with Percoll to make up a solution containing 55% Percoll, 0.25 M sucrose in 11.25 mM HEPES, pH 7.4, and centrifuged as described above. Radioactivity in fractions was measured after acid-precipitation with trichloroacetic acid. The kinetics of viral DNA synthesis is shown in Fig. 1. The incorporation of [ 3H]thymidine into HSV-1 DNA began at 3 h postinfection (p.i.) and reached nearly maximum level at 6 h p.i. These kinetics are similar to those reported previously (Roizman and Furlong, 1974). In contrast to the kinetics of viral DNA synthesis, nucleocapsid formation showed quite different kinetics under the same experimental conditions (Fig. 1). The amount of nucleocapsids began to increase at about 5 h p.i., indicating that there is a time lag between the synthesis of viral DNA and the formation of nucleocapsids. Since the formation of nucleocapsids was immediately followed by envelopment (as shown below), the encapsidation of viral DNA is a rate-limiting step in the morphogenesis of HSV-1 in the infected cells. Comparison of the amounts of [3H]thymidine incorporated into the viral DNA and that incorporated into the viral nucleocapsids showed that, at the end of nucleocapsid
283
Time (h) Tim-e (h) Fig. 1. Synthesis of viral DNA and formation of nucleocapsids in the HSV-l-infected cells. Vero cells in 35-mm dishes were allowed to adsorb HSV-1 at an MO1 of 12 for 30 min at room temperature. After removal of the inocula, the cells were incubated at 37 o C in MEM containing [ 3H]thymidine. At intervals the cells along with the culture fluid were harvested. The amount of viral DNA (0) was measured by centrifugation in a ccsium chloride minor modifications. The amount
density gradient as described by Ben-Porat and Kaplan (1963) with of all nucleocapsids synthesized in the cells (A) was assayed as described in the text.
Fig. 2. Formation of enveloped particles and infectious progeny virus in HSV-l-infected cells. Vero cells in 35-mm dishes were allowed to adsorb HSV-1 at an MO1 of 19 for 30 min at room temperature. After removal of the inocula the cells were washed with phosphate-buffered saline. Infection was initiated by incubating the cells at 37 o C in MEM containing [3H]thymidine. At intervals the cells along with the culture fluid were harvested and subjected to three cycles of freezing and thawing. The infectivities in the lysates (0) were assayed. The amounts of enveloped particles (0) as well as of naked nucleocapsids (A) were measured
as described
in the text.
formation (12 h p.i.), about 48 x lo3 cpm of [ 3H]thymidine molecules were incorporated into the nucleocapsids while about 227 X lo3 cpm of [3H]thymidine were incorporated into the viral DNA. These results indicate that approximately one fifth of the viral DNA synthesized was encapsidated to form the nucleocapsids. This value agrees with that for pseudorabies virus (Ben-Porat and Kaplan, 1963). Previously, Kaplan (1964) suggested that pseudorabies virus DNA was immediately encapsidated following the synthesis of viral DNA by showing the rapid conversion of newly synthesized DNA into DNase-resistant form. The discrepancy between our results and his can be attributed to the difference in cell culture conditions. He treated the cells with 5-fluorouracil for 18 h prior to the infection. This drug-treatment apparently affected the intracellular reactions toward .the nucleocapsid formation, since, for example, the kinetics of viral DNA synthesis in his report differ from that in this and other reports (Roizman and Furlong, 1974). As described below, the kinetics of nucleocapsid formation shown in Fig. 1 is in agreement with several morphological observations. The envelopment of nucleocapsids occurred simultaneously with the formation of nucleocapsids. As shown in Fig. 2, the enveloped particles appeared, increased, and reached a plateau with similar kinetics to the formation of naked nucleocapsids. There was no apparent time lapse between the formation of nucleocapsids and that of enveloped particles, indicating that the formation of nucleocapsids is immediately followed by envelopment. Comparison of radioactivity incorporated into the enveloped particle fractions with that incorporated into the naked nucleocapsid
284 fractions revealed that approximately 60% of the nucleocapsids were enveloped at 8 h p.i. and that the radioactivity ratio of enveloped to naked remained unchanged even at 25 h p.i. In addition, the infectious progeny virus appeared with kinetics quite similar to those of the formation of nucleocapsids and enveloped particles. As shown in Fig. 2, the infectious progeny virus appeared at 6 h p.i. and increased with time. The similarity between the kinetics of the formation of enveloped particles and that of infectious progeny virus indicates that the enveloped particles thus formed are infectious without any further processing of viral constituents. Although it is difficult by morphological methods to determine precisely the time of formation of capsids or of enveloped virus, the above conclusions are supported by previous studies with the electron microscope. On examination of HSV-l-infected cells at sequential intervals, Nii et al. (1968) found that capsids with dense cores were not seen until 6 h p.i. and appeared approximately at the same time as enveloped virus. Watson et al. (1964) also showed that naked capsids with DNA cores appeared in infected cells at the time of formation of infectious progeny virus. The molecular mechanism of virogenesis in HSV-l-infected cells is not well understood at the present time. During the middle stages of infection, expression of late genes begins immediately after the onset of viral DNA synthesis. Capsid proteins are synthesized in the cytoplasm and thereafter migrate to nuclei where they are assembled into capsids. These empty capsids bind tightly to the nuclear matrix wherein the encapsidation of viral DNA probably takes place. In agreement with our finding that the formation of nucleocapsids is immediately followed by envelopment, the nucleocapsids thus formed leave the nuclear matrix immediately, since only the empty capsids, not capsids with a dense core, are seen to be associated with the nuclear matrix (Bibor-Hardy et al., 1982). Envelopment is considered to occur by budding of the nucleocapsids from the modified region of the inner nuclear membrane into the perinuclear space (Roizman and Furlong, 1974). The present study shows that the encapsidation of viral DNA is a rate-limiting step in the multiplication of HSV-1 in Vero cells and that, once nucleocapsids are formed, they are converted quickly into infectious progeny virus. Since the lumen of the endoplasmic reticulum and the perinuclear space are continuous structures, egress of the progeny virus is likely to occur essentially in the same way as that of an excretion of secretory proteins via transport vesicles (Spear, 1985).
Acknowledgements We thank Fumi Tashiro for excellent technical assistance. This work was supported in part by a grant from the Ministry of Education, Science and Welfare of Japan.
References Ben-Porat, T. and Kaplan, AS. (1963) The synthesis and fate of pseudorabies mammalian cells in the stationary phase of growth. Virology 20, 310-317.
DNA
in infected
285 Bibor-Hardy, V., Pouchelet, M., St-Pierre, E., Herzberg, M. and Simard, R. (1982) The nuclear matrix is involved in herpes simplex virogenesis. Virology 121, 296-306. Kaplan, AS. (1964) Studies on the replicating pool of viral DNA in cells infected with pseudorabies virus. Virology 24, 19-25. Koyama, A.H. and U&da, T. (1984) Inhibition of multiplication of herpes simplex virus type 1 by ammonium chloride and chloroquine. Virology 138, 332-335. Koyama, A.H. and Uchida, T. (1987) The mode of entry of herpes simplex virus type 1 into Vero cells. Microbial. Immunol. 31, 123-130. Nii, S., Morgan, C. and Rose, H.M. (1968) Electron microscopy of herpes simplex virus. II. Sequence of development. J. Virol. 2, 517-536. Roizman, B. (1980) Herpes simplex viruses. In: DNA Tumor Viruses, 2nd ed. (Part 2) (Tooze, J., ed.), pp. 615-745. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Roizman, B. and Furlong, D. (1974) The replication of herpesviruses. In: Comprehensive Virology, Vol. 3 (Fraenkel-Conrat, H. and Wagner, R.R., eds.), pp. 229-403. Plenum Press, New York. Spear, P.G. (1985) Glycoproteins specified by herpes simplex viruses. In: Herpesviruses, Vol. 3 (Roizman, B., ed.), pp. 315-356. Plenum Press, New York. Watson, D.H. (1973) Replication of viruses-morphological aspects. In: The Herpesviruses (Kaplan, A., ed.), pp. 133-161, Academic Press, New York. Watson, D.H., Wildy, P. and Russell, W.C. (1964) Quantitative electron microscope studies on the growth of herpes virus using the techniques of negative staining and ultramicrotomy. Virology 24, 523-538. (Received
21 October
1987; revision
received
5 January
1988)