249
J. gen. Virol. (1976), 33, 249-257 Printed in Great Britain
Inhibitory Effect of Herpes Simplex Virus Type 1 on Type 2 Virus Replication By D A N A Z E L E N A , J. R O U B A L AND V. V O N K A
Department of Experimental Virology, Institute of Sera and Vaccines', Ioi o3 Prague, Czechoslovakia (Accepted 14 July I976) SUMMARY
Simultaneous infection with herpes simplex type I and type 2 viruses of chick embryo fibroblasts (CEF), which are only permissive for type 2 virus, or rabbit embryo fibroblasts (REF), which are permissive for both virus types, resulted in a marked reduction of type 2 virus production. This effect was dependent on the m.o.i, of type ~, being expressed at a high rather than a low m.o.i. The rate of interference decreased with the prolongation of the interval between infection with type 2 and type I viruses. No evidence suggestive of interferon involvement was obtained. Partial inactivation of type 2 virus by ultraviolet irradiation enhanced the inhibitory effect of type t virus. On the other hand, u.v. irradiation of type I virus resulted in a progressive loss of inhibitory activity. The results of the present experiments suggest that a type I genome function is responsible for the interfering effect, and that an early step in the growth of type 2 virus is sensitive to the particular type I virus product involved. INTRODUCTION
The two antigenic types of herpes simplex virus (HSV) differ in a number of biological characters, among others in the capability of growing in chick embryo fibroblasts (CEF; Figueroa & Rawls, I969). Although type I viruses capable of growth in CEF have been described (Waterson, I958; Taniguchi & Yoshino, I963), the great majority of type I viruses do not replicate in these cells. On the other hand, CEF do support the replication of type 2 strains (Lowry, Melnick & RaMs, I97I). In tissue cultures derived from other animal species such type-dependent differences have not been described thus far. In the present work we followed the formation of infectious virus progeny capable of replicating in CEF after co-infection of chick or rabbit cells with type I and type 2 viruses. The data presented indicate that in either cell system, type I infection resulted in a decreased yield of infectious type 2 virus. METHODS
Cells. Chick embryo fibroblasts (CEF) released by trypsinization of Io-day-old embryos were grown and maintained in Parker's I99 medium supplemented with IO ~ of calf serum (CS), 5 ~ of tryptose phosphate broth (TPB; Oxoid) and 0"045 ~ of NaHCOa. The first passages of CEF, i.e. secondary cultures, were used throughout the present experiments. I7-2
250
D. ZELENIi., J. ROUBAL AND V. VONKA
REF cells derived from rabbit embryo tissues and H E F cells derived from hamster embryo tissues have been described in previous papers from this laboratory (Roubal & Vonka, 2973; Kutinovfi, Vonka & Brou~ek, I973). Plaque technique. Infected REF cells were overlaid with Parker's medium enriched with growth promoting proteins of CS (Michl, 2961), I ~oo Difco Bacto agar, o'x5 ~ NaHCO3 and antibiotics. For the CEF cells, Parker's medium supplemented with IO ~o of CS, 5 of TPB, 1 ~ agar, o'15 ~ NaHCO3 and antibiotics was employed. Viruses. HSV type I, strain Kupka, isolated from a patient suffering from herpes labialis was kindly supplied by Dr R. Benda (Military Institute of Hygiene, Epidemiology and Microbiology, Praba). HSV type 2, strain 196, was obtained through the courtesy of Dr J. L. Melnick (Baylor College, Houston, Texas). Vesicular stomatitis virus (VSV), Indiana strain, was kindly provided by Dr L. HronovskS' (Military Institute of Hygiene, Epidemiology and Microbiology, Praha). Both HSV strains were propagated at a m.o.i, less than I p.f.u./cell. They were u.v. irradiated as described elsewhere (Roubal & Vonka, 1973). VSV stocks were prepared in REF cells by the same procedure as for HSV. Virus titration. Virus stocks were serially diluted in tenfold steps and o.I mI amounts of each virus dilution were inoculated in 3 or 4 parallel tube cultures. The infected cultures were incubated at 37 °C for 7 days. Titres were determined according to K/irber (193I). Titration by the plaque technique in MUller bottles (surface area 15 cm2) was performed as described elsewhere (Roubal & Vonka, 1973). Single-virus-growth experiments in CEF cells. Tube cultures of confluent CEF cells were infected with HSV-1 and HSV-2 at a m.o.i, of about 3"5 p.f.u./cell. Virus was allowed to adsorb for 1 h at 37 °C. The cultures were then washed twice with medium, and 2.0 ml of fresh medium were added to each tube. Immediately after this, five parallel cultures were placed at - 70 °C to determine the infectious virus content at time o. The remaining cultures were incubated at 37 °C. At intervals three or four parallel cultures were withdrawn and placed at - 7 o °C. Prior to virus titration the cultures underwent three cycles of freezing and thawing. Parallel cultures were pooled and the fluids were clarified by low speed centrifugation (2000 g for 20 min). Infectious virus content was determined by titration in tube cultures from REF cells. Co-infection experiments. Six parallel tube cultures of confluent CEF or R E F cells were each infected with o.I ml of type 1 and o.1 ml of type 2 virus suspension. The m.o.i, used in the separate experiments are indicated in the Results section. Control cultures were infected with one virus only and concomitantly o. 2 ml of medium was added. The cultures were treated as indicated in the preceding paragraph. The viruses were harvested after 24 h incubation. The amount of virus capable of growing in CEF was determined by plaque titration. Interferon assay. The samples examined were prepared from CEF and R E F cells I8 h after infection with HSV-1, strain Kupka, at a m.o.i, of 5 p.f.u./cell. After three cycles of freezing and thawing the suspensions were clarified by low-speed centrifugation and the supernatant fluids were treated by the procedure used by Cortada de de la Pefia & De Lustig (I974). In brief, the preparations were adjusted to pH 2.0 with I M-HC1 and left at 4 °C for 48 h. They were then neutralized with I N-NaOH, centrifuged for 2 h at 2ooooog and filtered through Millipore H A W P filters. All samples were tested in tube cultures of CEF, REF and H E F cells. One ml amounts of each sample, undiluted and diluted in medium in twofold steps, were added to the cultures. After an 18-h incubation in a roller at 37 °C, the materials were infected either
Interference between H S V - I and H S V - 2 I
!
I
I
I
251
I
e~o
>
"o---------..o
0
I
I
5
10
I
i
I
15 20 25 Time after infection (h)
I
30
Fig. I. Virus growth in CEF cells: 0 - - 0 , HSV-2; O - - O , HSV-I ; m.o.i, for HSV-I and HSV-2 were about 3"5 p.f.u./cell, as determined by titration in R E F cells.
with VSV or HSV type 2. In the first assay, I ml of medium containing ioo TCDs0 of VSV was added to each culture. Cultures infected with the same amount of VSV which had been untreated or pre-treated with materials prepared from uninfected cells served as controls. The cultures were placed at 37 °C and were checked for cytopathic changes after a 48-h incubation. Their full development was considered evidence of the absence of interferon. In the second assay, the treated cells were infected with HSV type 2 at a m.o.i, of I p.f.u./cell. After 2o h of cultivation at 37 °C the viruses were harvested and titrated in R E F tube cultures. The virus yields in the pre-treated and control cultures were compared. The absence of a difference in virus titres was considered to be evidence of the absence of interferon. CEF interferon kindly supplied by D r H. Libikovfi (Institute of Virology, Bratislava) and rabbit interferon provided by Dr I. T~iborsk~ (Faculty of Medicine, Brno), treated as mentioned above, were used as positive controls. Under the described experimental conditions, the first preparation prevented the development of c.p.e, induced by VSV and reduced markedly the HSV-2 production at the dilution I : 64 and I : 32, respectively. The rabbit preparation was effective against VSV and HSV-2 at the dilution I : 16. RESULTS
Growth of type I and type 2 viruses in CEF cells The results of growth experiments with HSV type I and type 2 viruses in CEF cells are shown in Fig. ~. It can be seen that HSV-2, strain 'I96', multiplied in CEF reaching the maximum titre at 18 to 22 h post infection (p.i.). On the other hand, CEF did not support the replication of HSV-I, strain Kupka. Although the type 2 virus replication in CEF was not too efficient, the differences between the maximum type 2 virus titres and corresponding
252
D. Z E L E N / ~ , J. R O U B A L
AND
V. V O N K A
Table I. Influence of H S V type I presence on H S V type 2 production in CEF and REF cells HSV-2 production (p.f.u. × lo-a/ml) t-
Cells CEF
HSV-2 irradiation* None
60 s
I2OS
REF
None
60 s
Relative HSV-2 production in presence of HSV-I~" ~
r
Expt. no.
HSV-I absent
HSV-I present
I II III IV
2-3 5"7 43'0 52"0
o'3 0'36 8"3 9"5
13'o] 6"31 I9"3 18"2!
~
I
I, 7
o.14
8.2]
II III IV
4'5 I o-o 2'5
0"07 0"97 o.16
1'5 t 9"7 6"4
I
0"48
0"02
4'2]
II IlI IV
2"0 2"6 2'I
0"07 0"O7 0"03
3"71 2'6 1.5
I II III
38"o I8O-O 16o'o
JIM
2IO'O
I II III
3"o 2t'o 27"0 36"0
7"9] II.6 t I6'9| I7"2"
6"5
o'2
3"t]
25"0 3t.o
2"5
IO.O~
2"0
6" 5 )
Mean ~:~ i4.2 (3_2.9)
6 5 ( + I'75) -
3'0 (+0"55) -
I3"4 (3_2'22)
6"5 (3_2"03)
* The pre-irradiation HSV-2 titre was I- 3 x in 6 p.f.u./ml. The survival o f the 60 s irradiated virus was 3"1 x IO 2 in R E F and 7'o× IO ~ in C E F cells. The survival of 12o s irradiated virus was 3.7 x Io -3 in R E F and 2.I × In -2 in C E F cells. The m.o.i, of HSV-2, based upon pre-irradiation virus titre, was about o'7 p.f.u./cell. The approximate m.o.i, o f HSV-I virus was 3"5 p.f.u./cell. t As determined by titration in C E F cells. :~ Standard mean errors are given in parentheses.
type I virus contents, which most probably represented the residual inoculum infectivity, were 3 loglo or more in repeated experiments. In agreement with this, type I did not induce plaque formation in CEF monolayers, while type 2 formed large clear plaques.
Co-infection experiments CEF cells were infected concomitantly with both type I and type 2 viruses. The m.o.i. used is indicated in the legends to the tables and figures. Cultures infected with HSV-2 only served as controls. The titres of virus progenies in CEF cells determined 24 h p.i. are shown in Table I. In all four experiments the production of virus growing in CEF (i.e type 2 virus) was reduced in the presence of HSV-I, on an average amounting to I4 ~ of the type 2 virus production observed in control cultures. The inhibitory effect was increased when using type 2 virus partially inactivated by u.v., more so when the virus was irradiated for I2o s than when it was inactivated for 6o s. Type I also interfered with type 2 replication in R E F cells, which are permissive for both virus types. The results are shown in the lower part of Table I. It can be seen that the presence of type I virus decreased the production of type 2 virus at the same rate in both R E F and CEF cells. Also, the inhibitory effect was similarly enhanced in both cell types when the type 2 virus had been partially inactivated by u.v. light.
Interference between HSV-I and HSV-2 100:
I
I
I
I
l
I
I I
I
I
I
I
253 I
I
I
I J--
90 80 70 e-,
.o o
60 5O
'7> 40 30
20 10 0
I
0.l
I
I
~
1 i
Ill
1.0 m.o.i. (p.f,u./'cell)
I
[
]
t
~
t
10.0
Fig. 2. Relationship between HSV-z production in CEF cells and m.o.i, of HSV-I. M.o.i. for HSV-2 was o.6 p.f.u./cell as determined by titration in REF cells. We also followed the dependence between the degree of inhibition and the m.o.i, of both viruses. In the first experiment, infection of CEF cells with type 2 virus was constant and the m.o.i, of type I varied. Conversely, in the second experiment the type I m.o.i, remained constant but the type 2 m.o.i, varied. The results of the first test are shown in Fig. 2. Maximum inhibition occurred at the highest m.o.i, tested (6.6 p.f.u.[cell) of type I. However, at a m.o.i, of only o.I I p.f.u./cell the amount of virus growing in CEF was reduced by 6o in comparison with virus production in control cultures. On the other hand, the varying of type 2 m.o.i, from 4 × IO-4 to 4"o p.f.u.]cell, while type I m.o.i, was constant, had a negligible effect on the rate of interference (results not shown). In the subsequent experiments we tried to determine the relationship between the interference effect and the time of addition of type I virus after infection with type z virus. CEF cells were infected with type 2 virus and then superinfected with type I virus at different intervals. Twenty-four h after infection with type 2 virus the contents of viruses capable of growing in C E F cells were determined (Fig. 3). It can be seen that the maximum inhibition of type 2 virus production (i.e. minimum production of virus growing in CEF) was observed when cells were infected simultaneously with both viruses. With prolongation of the interval between infection and superinfection the inhibitory effect of type 1 virus gradually decreased. The effect of HSV-I became negligible when the interval reached 6 h. It is possible to conclude from these data that type I virus was only capable of interfering with type 2 production early after infection with type 2. We also investigated whether an interferon-like substance could be involved in the interference described. Extracts from HSV type I-infected CEF and R E F cells were tested for the presence of interferon, as described in Methods. None of the extracts prevented the development of c.p.e, induced by VSV in CEF, R E F and H E F cells, or reduced the pro-
254
D. Z E L E N , ~ , J. R O U B A L 100
I
'
A N D V. V O N K A
'
I
I
I
i
i
5
6
O
10
I
1
i
I
i
2 3 4 Time of HSVq addition (h)
-1-
I
Fig. 3. Relationship between the time of type ~ virus addition and type 2 virus production in CEF cells. M.o.i. for HSV-~ was 3'3, for HSV-2, 0"7 p.f.u./cell.
duction of HSV type 2 virus in these cells. Thus no evidence on the presence of interferon in the system was obtained. To check whether an expression of type I virus genetic information was needed for the interference effect, CEF cells were co-infected with untreated type 2 virus and type I virus which had been irradiated with different u.v. doses. It is shown in Fig. 4 that the maximum interference effect was achieved when using untreated type I virus (6 ~ of control virus production) and that the inhibitory effect of type I virus decreased when the u.v. dose was increased. When using 3oo s irradiated type I v i r u s , the production of virus capable of multiplying in CEF was reduced by only 5o ~ when compared with control cultures infected with type 2 virus only. DISCUSSION
In agreement with earlier observations (Figueroa & RaMs, I969; Lowry et al. I97I), the type I Kupka virus did not replicate in CEF cells, while type 2 ' I96' virus grew well in this system. This difference was utilized for monitoring the effects of simultaneous infection with these two viruses on the production of type 2 virus. In both CEF and REF cells, type I infection resulted in a marked decrease in the production of virus capable of growing in CEF cells. This virus is referred to as type 2 virus, although we realize that a small amount of recombinant viruses might have been present in the virus populations (Timbury & Subak-Sharpe, I973).
Interference between HSV-t and HSV-2 100 ~
I
I
i
I
E 2
255
i
$
lO
0
I
I
60
120
I
I
180 240 U.V. dose (s)
I
300
Fig. 4. Effect of u.v.-irradiation of HSV-I on production of type 2 virus in CEF cells. HSV-I and HSV-2 were used at rn.o.i. 3"3 and 0"7 p.f.u./cell, respectively. The pre-irradiation titre of HSV-~ in REF cells was 2x io 7 p.f.u./ml. Survivals after 6o, I8O and 30o s of irradiation were 2.4 x Io-z, 1 . 2 × IO-a and i x io -~, respectively. Several experiments were performed to obtain more information on this interference. The findings can be summarized as follows. First, the rate of interference was dependent on the m.o.i., more so on the m.o.i, of the interfering type ~ virus than of the interfered with type 2 virus under the present experimental conditions. Interestingly enough, the interference was expressed even when the m.o.i, of type ~ virus was as low as o.I p.f.u./cell. This might either be due to the capability of the non-infectious particles to induce the phenomenon or to the involvement of a diffusible factor produced in the type I infected cells and influencing non-infected ones. Second, the inhibition effect of type I virus decreased with prolonging the interval between type 2 infection and type I superinfection. This indicates that an early step in the type z virus growth cycle was sensitive to the interfering action of type I virus. Third, the interfering capacity of type I was markedly decreased by u.v. irradiation, suggesting that a type I genome function was responsible for the phenomenon. It has been shown that the irradiation of HSV-I results in a delay in the synthesis of virus-specific products (Ross, Wildy & Cameron, I97I). Thus, the delayed synthesis of the particular type I virus product involved could diminish the expression of the interference effect, imitating the effect of late type I virus addition. Fourth, partial inactivation of type 2 virus resulted in an enhancement of the interference. This observation might again be explained by the delay in the replication of HSV caused by u.v. irradiation (Ross, Cameron & Wildy, I972; Roubal & Vonka, t973). It is reasonable to presume that this
256
D. ZELENA, J. ROUBAL AND V. VONKA
delay resulted in the prolonging of the period during which type 2 virus growth was sensitive to the effect of the particular type ~ virus product involved in the interference. It seems unlikely that the observed interference was mediated by interferon. Herpesviruses are generally considered to be poor inducers of interferon and not very sensitive to its action (Lockart, ~973), although induction of interferon by HSV and its action on the virus have been reported (Waddell, Sigel & Wryk, ~963; Fruitstone, Waddell & Sigel, 1964; Libikov~i, I973; Hooks, x975; Rasmussen & Farley, I975). Our results failed to demonstrate the presence of detectable amounts of interferon-like substances in either C E F or R E F cultures infected with type ~ virus. Interference with HSV not mediated by interferon was described by Roizman 0965): the co-infection of dog kidney cells with HSV M P d k - incapable of growing in these cells, and the MPdk÷sp mutant, for which the dog kidney cells were permissive, resulted in a decrease of MPdk+sp virus production. It was assumed that this effect was due to the formation of non-functional heteropolymers between the polypeptides specified by the dk + virus and the non-functional products of the d k - virus. It does not seem probable that a similar type of interference was operative in the present system, because the same effects as in CEF cells were also observed in R E F cells, permissive for both type I and type 2 viruses. Another type of apparently interferon-independent interference has been described recently by Bronson et al. 0973) and by Frenkel et al. 0975), who reported that the defective HSV particles produced by undiluted passages blocked the replication of infectious HSV. It is not likely that this would be the present case, since both virus stocks were prepared by passing the virus at a low m.o.i. The mechanism causing the relative resistance of HSV-transformed cells to HSV replication as reported by Doller, Duff & Rapp (I973), Kutinov~t et al. (I973) and by Kimura et al. 0975) may, but need not, be related to those involved in the phenomenon presently reported. The nature of the interference described in this report is not understood. One can only speculate, at this stage, about the mode of action of the responsible type ~ virus product in co-infected cells. It has been demonstrated that the regulation of HSV-I polypeptide production is mediated by turning off the synthesis of the earlier products by the later groups of polypeptides (Honess & Roizman, ~974). Since differences between type I and type 2 viruses have been reported for both the transcriptional programmes (Roizman & Frenkel, r973) and the synthesis of virus polypeptides (Powell & Courtney, I975), it seems possible that some type ~ virus product(s) appearing in a sequence, or at a time, which is incorrect for the type 2 virus cycle, might prevent the expression of some function(s) essential for the growth of this virus. REFERENCES BRONSON,D. L., DREESMAN,~. R., BmWAL,N. & BENYESH-MELNICK,M. T. (I973). Defective virions of herpes simplex viruses. Intervirology x, I4I-I53. CORTADADEDELAPE~A,N. & DELUSTIG,E. S. 0974)- Interferon production by leukocytes cultured from neoplastic patients. European Journal of Cancer xo, I89-I92. DOLLER,E., DUFF,R. & RAPP,F. 0973). Resistance of hamster cells transformed by herpes simplex virus type 2 to superinfection by herpes simplex viruses. Intervirology I, I54-i67. FI~UEROA,M. E. & RAWLS,W. r. (I969). Biological markers for differentiation of herpes virus strains of oral and genital origin. Journal of General Virology 4, 259-267. FRENKEL, N., JACOB, R. J., HONESS, R. W., HAYWARD, G. S., LOCKER, H. & ROIZMAN, B. 0975). Anatomy of herpes simplex virus DNA. III. Characterization of defective DNA molecules and biological properties of virus populations containing them. Journal of Virology x6, I53-I67. FRUITSTONE,M. J., WADDELL,O. n. & SIGEL,M. M. 0964). An interferon produced in response to infection by herpes simplex virus. Proceedings of the Society for Experimental Biology and Medicine xx7, 8o4-8o7.
Interference between HSV-I and HSV-2
257
HONESS, R. W. & ROIZMAN,n. (1974). Regulation of herpesvirus macromolecular synthesis. I. Cascade regulation of the synthesis of three groups of viral proteins. Journal of Virology 14, 8 - I 9 . HOOKS, J. J. 0975). Mechanisms by which cellular immunity halts herpes simplex virus spread. In Abstracts of the 3rd International Congressfor Virology, p. I67. Edited by H. S. Bedson, R. N~jera, L. Valenciano & P. Wildy. Madrid. K~RBER, G. (I931). Beitrag zur Kollektiven Behandlung pharmakologischer Reihenversuche. Archives fiir experimentale Pathologic und Pharmakologie x6z, 48o-483. KIMLIRA, S., FLANNERY, V. L., LEVY, B. & SCHAFFER, P. A. (1975). Oncogenic transformation of primary hamster cells by herpes simplex virus type 2 (HSV-2) and an HSV-2 temperature-sensitive mutant. International Journal of Cancer 15, 786-798. KtJrINOV~., L., VONKA,V. & BROU6EK,J. (I973). Increased oncogenicity and synthesis of herpesvirus antigen in hamster cells exposed to herpes simplex type 2 virus. Journal of the National Cancer Institute 50, 759-766. LmiKOV~, H. (I973). Interferon in young and aged chick embryo cell cultures infected with herpes simplex and pseudorabies viruses. Acta Virologica x7, 464-47 I. LOCI~ART, R.Z. SUN. 0973). Interference and interferon with respect to herpesviruses. In The Herpesviruses, pp. 261-27o. Edited by A. S. Kaplan, New York & London: Academic Press. LOWRY, S. P., MELNICK,J. L. & RAWLS, W. E, 0971)- Investigation of plaque formation in chick embryo cells as a biological marker for distinguishing herpes virus type 2 from type I. Journal of General Virology IO, 1-9. MICIaL, J. (I96I). Metabolism of cells in tissue culture in vitro. 1. The influence of serum protein fractions on the growth of normal and neoplastic cells. Experimental Cell Research 23, 324-33o. POWELL, K. L. & COURTNEY, a. J. (1975). Polypeptides synthesized in herpes simplex virus type 2 infected HEp-2 cells. Virology 66, 2 1 7 - 2 2 8 . RASMUSSEN,L. & FARLEY, L. 13. 0975). Inhibition of herpesvirus hominis replication by human interferon. Infection and Immunity r2, IO4-io8. ROIZMAN, 13. (I965). Abortive infection of canine cells by herpes simplex virus. III. The interference of conditional lethal virus with an extended host range mutant. Virology 27, I I3-117. ROIZMAN, B. & I~RENKEL,N. (I973). The transcription and state of herpes simplex virus DNA in productive infection and in human cervical cancer tissue. Cancer Research 33, 14o2-1416. ROSS, g. J. N., CAMEROr~,K. a. & WILDY, e. (I972). Ultraviolet irradiation of herpes simplex virus: reactivation processes and delay in virus multiplication. Journal of General Virology 16, 299-311. ROSS, L. J. N., VV'ILDY, P. & CAMERON, K. R. 097I). Formation of small plaques by herpes viruses irradiated with ultraviolet light. Virology 45, 8o8-812. ROU13AL,J. & VONKA,V. 0973). Multiplicity reactivation in u.v.-irradiated herpes simplex type I virus. Intervirology x, 73-79. TANIGUCI-II, S. & YOSmNO, K. (1963). An analysis of the plaque assay of herpes simplex virus in chick embryo monolayers. Archiv fiir die gesamte Virusforschung 14, 537-552. TIMBURY, M. C. & SU13AK-SHARPE,J. H. (I973). Genetic interaction between temperature sensitive mutants of type I and type 2 herpes simplex viruses. Journal of General Virology 18, 347-357. WADDELL, G. H., SIGEL, M. M. & VqRYK,M. (I963). Factors associated with the response of the ceil to herpes simplex virus. Bacteriological Proceedings 88, 15o. WATERSON, A. P. (1958). Plaque formation by herpes simplex virus in chick embryo tissue. Archly fiir die gesamte Virusforschung 8, 592-599.
(Received 22 March I976)
J. gen. Virol. (1976), 33, 249-257 Printed in Great Britain
Inhibitory Effect of Herpes Simplex Virus Type 1 on Type 2 Virus Replication By D A N A Z E L E N A , J. R O U B A L AND V. V O N K A
Department of Experimental Virology, Institute of Sera and Vaccines', Ioi o3 Prague, Czechoslovakia (Accepted 14 July I976) SUMMARY
Simultaneous infection with herpes simplex type I and type 2 viruses of chick embryo fibroblasts (CEF), which are only permissive for type 2 virus, or rabbit embryo fibroblasts (REF), which are permissive for both virus types, resulted in a marked reduction of type 2 virus production. This effect was dependent on the m.o.i, of type ~, being expressed at a high rather than a low m.o.i. The rate of interference decreased with the prolongation of the interval between infection with type 2 and type I viruses. No evidence suggestive of interferon involvement was obtained. Partial inactivation of type 2 virus by ultraviolet irradiation enhanced the inhibitory effect of type t virus. On the other hand, u.v. irradiation of type I virus resulted in a progressive loss of inhibitory activity. The results of the present experiments suggest that a type I genome function is responsible for the interfering effect, and that an early step in the growth of type 2 virus is sensitive to the particular type I virus product involved. INTRODUCTION
The two antigenic types of herpes simplex virus (HSV) differ in a number of biological characters, among others in the capability of growing in chick embryo fibroblasts (CEF; Figueroa & Rawls, I969). Although type I viruses capable of growth in CEF have been described (Waterson, I958; Taniguchi & Yoshino, I963), the great majority of type I viruses do not replicate in these cells. On the other hand, CEF do support the replication of type 2 strains (Lowry, Melnick & RaMs, I97I). In tissue cultures derived from other animal species such type-dependent differences have not been described thus far. In the present work we followed the formation of infectious virus progeny capable of replicating in CEF after co-infection of chick or rabbit cells with type I and type 2 viruses. The data presented indicate that in either cell system, type I infection resulted in a decreased yield of infectious type 2 virus. METHODS
Cells. Chick embryo fibroblasts (CEF) released by trypsinization of Io-day-old embryos were grown and maintained in Parker's I99 medium supplemented with IO ~ of calf serum (CS), 5 ~ of tryptose phosphate broth (TPB; Oxoid) and 0"045 ~ of NaHCOa. The first passages of CEF, i.e. secondary cultures, were used throughout the present experiments. I7-2
250
D. ZELENIi., J. ROUBAL AND V. VONKA
REF cells derived from rabbit embryo tissues and H E F cells derived from hamster embryo tissues have been described in previous papers from this laboratory (Roubal & Vonka, 2973; Kutinovfi, Vonka & Brou~ek, I973). Plaque technique. Infected REF cells were overlaid with Parker's medium enriched with growth promoting proteins of CS (Michl, 2961), I ~oo Difco Bacto agar, o'x5 ~ NaHCO3 and antibiotics. For the CEF cells, Parker's medium supplemented with IO ~o of CS, 5 of TPB, 1 ~ agar, o'15 ~ NaHCO3 and antibiotics was employed. Viruses. HSV type I, strain Kupka, isolated from a patient suffering from herpes labialis was kindly supplied by Dr R. Benda (Military Institute of Hygiene, Epidemiology and Microbiology, Praba). HSV type 2, strain 196, was obtained through the courtesy of Dr J. L. Melnick (Baylor College, Houston, Texas). Vesicular stomatitis virus (VSV), Indiana strain, was kindly provided by Dr L. HronovskS' (Military Institute of Hygiene, Epidemiology and Microbiology, Praha). Both HSV strains were propagated at a m.o.i, less than I p.f.u./cell. They were u.v. irradiated as described elsewhere (Roubal & Vonka, 1973). VSV stocks were prepared in REF cells by the same procedure as for HSV. Virus titration. Virus stocks were serially diluted in tenfold steps and o.I mI amounts of each virus dilution were inoculated in 3 or 4 parallel tube cultures. The infected cultures were incubated at 37 °C for 7 days. Titres were determined according to K/irber (193I). Titration by the plaque technique in MUller bottles (surface area 15 cm2) was performed as described elsewhere (Roubal & Vonka, 1973). Single-virus-growth experiments in CEF cells. Tube cultures of confluent CEF cells were infected with HSV-1 and HSV-2 at a m.o.i, of about 3"5 p.f.u./cell. Virus was allowed to adsorb for 1 h at 37 °C. The cultures were then washed twice with medium, and 2.0 ml of fresh medium were added to each tube. Immediately after this, five parallel cultures were placed at - 70 °C to determine the infectious virus content at time o. The remaining cultures were incubated at 37 °C. At intervals three or four parallel cultures were withdrawn and placed at - 7 o °C. Prior to virus titration the cultures underwent three cycles of freezing and thawing. Parallel cultures were pooled and the fluids were clarified by low speed centrifugation (2000 g for 20 min). Infectious virus content was determined by titration in tube cultures from REF cells. Co-infection experiments. Six parallel tube cultures of confluent CEF or R E F cells were each infected with o.I ml of type 1 and o.1 ml of type 2 virus suspension. The m.o.i, used in the separate experiments are indicated in the Results section. Control cultures were infected with one virus only and concomitantly o. 2 ml of medium was added. The cultures were treated as indicated in the preceding paragraph. The viruses were harvested after 24 h incubation. The amount of virus capable of growing in CEF was determined by plaque titration. Interferon assay. The samples examined were prepared from CEF and R E F cells I8 h after infection with HSV-1, strain Kupka, at a m.o.i, of 5 p.f.u./cell. After three cycles of freezing and thawing the suspensions were clarified by low-speed centrifugation and the supernatant fluids were treated by the procedure used by Cortada de de la Pefia & De Lustig (I974). In brief, the preparations were adjusted to pH 2.0 with I M-HC1 and left at 4 °C for 48 h. They were then neutralized with I N-NaOH, centrifuged for 2 h at 2ooooog and filtered through Millipore H A W P filters. All samples were tested in tube cultures of CEF, REF and H E F cells. One ml amounts of each sample, undiluted and diluted in medium in twofold steps, were added to the cultures. After an 18-h incubation in a roller at 37 °C, the materials were infected either
Interference between H S V - I and H S V - 2 I
!
I
I
I
251
I
e~o
>
"o---------..o
0
I
I
5
10
I
i
I
15 20 25 Time after infection (h)
I
30
Fig. I. Virus growth in CEF cells: 0 - - 0 , HSV-2; O - - O , HSV-I ; m.o.i, for HSV-I and HSV-2 were about 3"5 p.f.u./cell, as determined by titration in R E F cells.
with VSV or HSV type 2. In the first assay, I ml of medium containing ioo TCDs0 of VSV was added to each culture. Cultures infected with the same amount of VSV which had been untreated or pre-treated with materials prepared from uninfected cells served as controls. The cultures were placed at 37 °C and were checked for cytopathic changes after a 48-h incubation. Their full development was considered evidence of the absence of interferon. In the second assay, the treated cells were infected with HSV type 2 at a m.o.i, of I p.f.u./cell. After 2o h of cultivation at 37 °C the viruses were harvested and titrated in R E F tube cultures. The virus yields in the pre-treated and control cultures were compared. The absence of a difference in virus titres was considered to be evidence of the absence of interferon. CEF interferon kindly supplied by D r H. Libikovfi (Institute of Virology, Bratislava) and rabbit interferon provided by Dr I. T~iborsk~ (Faculty of Medicine, Brno), treated as mentioned above, were used as positive controls. Under the described experimental conditions, the first preparation prevented the development of c.p.e, induced by VSV and reduced markedly the HSV-2 production at the dilution I : 64 and I : 32, respectively. The rabbit preparation was effective against VSV and HSV-2 at the dilution I : 16. RESULTS
Growth of type I and type 2 viruses in CEF cells The results of growth experiments with HSV type I and type 2 viruses in CEF cells are shown in Fig. ~. It can be seen that HSV-2, strain 'I96', multiplied in CEF reaching the maximum titre at 18 to 22 h post infection (p.i.). On the other hand, CEF did not support the replication of HSV-I, strain Kupka. Although the type 2 virus replication in CEF was not too efficient, the differences between the maximum type 2 virus titres and corresponding
252
D. Z E L E N / ~ , J. R O U B A L
AND
V. V O N K A
Table I. Influence of H S V type I presence on H S V type 2 production in CEF and REF cells HSV-2 production (p.f.u. × lo-a/ml) t-
Cells CEF
HSV-2 irradiation* None
60 s
I2OS
REF
None
60 s
Relative HSV-2 production in presence of HSV-I~" ~
r
Expt. no.
HSV-I absent
HSV-I present
I II III IV
2-3 5"7 43'0 52"0
o'3 0'36 8"3 9"5
13'o] 6"31 I9"3 18"2!
~
I
I, 7
o.14
8.2]
II III IV
4'5 I o-o 2'5
0"07 0"97 o.16
1'5 t 9"7 6"4
I
0"48
0"02
4'2]
II IlI IV
2"0 2"6 2'I
0"07 0"O7 0"03
3"71 2'6 1.5
I II III
38"o I8O-O 16o'o
JIM
2IO'O
I II III
3"o 2t'o 27"0 36"0
7"9] II.6 t I6'9| I7"2"
6"5
o'2
3"t]
25"0 3t.o
2"5
IO.O~
2"0
6" 5 )
Mean ~:~ i4.2 (3_2.9)
6 5 ( + I'75) -
3'0 (+0"55) -
I3"4 (3_2'22)
6"5 (3_2"03)
* The pre-irradiation HSV-2 titre was I- 3 x in 6 p.f.u./ml. The survival o f the 60 s irradiated virus was 3"1 x IO 2 in R E F and 7'o× IO ~ in C E F cells. The survival of 12o s irradiated virus was 3.7 x Io -3 in R E F and 2.I × In -2 in C E F cells. The m.o.i, of HSV-2, based upon pre-irradiation virus titre, was about o'7 p.f.u./cell. The approximate m.o.i, o f HSV-I virus was 3"5 p.f.u./cell. t As determined by titration in C E F cells. :~ Standard mean errors are given in parentheses.
type I virus contents, which most probably represented the residual inoculum infectivity, were 3 loglo or more in repeated experiments. In agreement with this, type I did not induce plaque formation in CEF monolayers, while type 2 formed large clear plaques.
Co-infection experiments CEF cells were infected concomitantly with both type I and type 2 viruses. The m.o.i. used is indicated in the legends to the tables and figures. Cultures infected with HSV-2 only served as controls. The titres of virus progenies in CEF cells determined 24 h p.i. are shown in Table I. In all four experiments the production of virus growing in CEF (i.e type 2 virus) was reduced in the presence of HSV-I, on an average amounting to I4 ~ of the type 2 virus production observed in control cultures. The inhibitory effect was increased when using type 2 virus partially inactivated by u.v., more so when the virus was irradiated for I2o s than when it was inactivated for 6o s. Type I also interfered with type 2 replication in R E F cells, which are permissive for both virus types. The results are shown in the lower part of Table I. It can be seen that the presence of type I virus decreased the production of type 2 virus at the same rate in both R E F and CEF cells. Also, the inhibitory effect was similarly enhanced in both cell types when the type 2 virus had been partially inactivated by u.v. light.
Interference between HSV-I and HSV-2 100:
I
I
I
I
l
I
I I
I
I
I
I
253 I
I
I
I J--
90 80 70 e-,
.o o
60 5O
'7> 40 30
20 10 0
I
0.l
I
I
~
1 i
Ill
1.0 m.o.i. (p.f,u./'cell)
I
[
]
t
~
t
10.0
Fig. 2. Relationship between HSV-z production in CEF cells and m.o.i, of HSV-I. M.o.i. for HSV-2 was o.6 p.f.u./cell as determined by titration in REF cells. We also followed the dependence between the degree of inhibition and the m.o.i, of both viruses. In the first experiment, infection of CEF cells with type 2 virus was constant and the m.o.i, of type I varied. Conversely, in the second experiment the type I m.o.i, remained constant but the type 2 m.o.i, varied. The results of the first test are shown in Fig. 2. Maximum inhibition occurred at the highest m.o.i, tested (6.6 p.f.u.[cell) of type I. However, at a m.o.i, of only o.I I p.f.u./cell the amount of virus growing in CEF was reduced by 6o in comparison with virus production in control cultures. On the other hand, the varying of type 2 m.o.i, from 4 × IO-4 to 4"o p.f.u.]cell, while type I m.o.i, was constant, had a negligible effect on the rate of interference (results not shown). In the subsequent experiments we tried to determine the relationship between the interference effect and the time of addition of type I virus after infection with type z virus. CEF cells were infected with type 2 virus and then superinfected with type I virus at different intervals. Twenty-four h after infection with type 2 virus the contents of viruses capable of growing in C E F cells were determined (Fig. 3). It can be seen that the maximum inhibition of type 2 virus production (i.e. minimum production of virus growing in CEF) was observed when cells were infected simultaneously with both viruses. With prolongation of the interval between infection and superinfection the inhibitory effect of type 1 virus gradually decreased. The effect of HSV-I became negligible when the interval reached 6 h. It is possible to conclude from these data that type I virus was only capable of interfering with type 2 production early after infection with type 2. We also investigated whether an interferon-like substance could be involved in the interference described. Extracts from HSV type I-infected CEF and R E F cells were tested for the presence of interferon, as described in Methods. None of the extracts prevented the development of c.p.e, induced by VSV in CEF, R E F and H E F cells, or reduced the pro-
254
D. Z E L E N , ~ , J. R O U B A L 100
I
'
A N D V. V O N K A
'
I
I
I
i
i
5
6
O
10
I
1
i
I
i
2 3 4 Time of HSVq addition (h)
-1-
I
Fig. 3. Relationship between the time of type ~ virus addition and type 2 virus production in CEF cells. M.o.i. for HSV-~ was 3'3, for HSV-2, 0"7 p.f.u./cell.
duction of HSV type 2 virus in these cells. Thus no evidence on the presence of interferon in the system was obtained. To check whether an expression of type I virus genetic information was needed for the interference effect, CEF cells were co-infected with untreated type 2 virus and type I virus which had been irradiated with different u.v. doses. It is shown in Fig. 4 that the maximum interference effect was achieved when using untreated type I virus (6 ~ of control virus production) and that the inhibitory effect of type I virus decreased when the u.v. dose was increased. When using 3oo s irradiated type I v i r u s , the production of virus capable of multiplying in CEF was reduced by only 5o ~ when compared with control cultures infected with type 2 virus only. DISCUSSION
In agreement with earlier observations (Figueroa & RaMs, I969; Lowry et al. I97I), the type I Kupka virus did not replicate in CEF cells, while type 2 ' I96' virus grew well in this system. This difference was utilized for monitoring the effects of simultaneous infection with these two viruses on the production of type 2 virus. In both CEF and REF cells, type I infection resulted in a marked decrease in the production of virus capable of growing in CEF cells. This virus is referred to as type 2 virus, although we realize that a small amount of recombinant viruses might have been present in the virus populations (Timbury & Subak-Sharpe, I973).
Interference between HSV-t and HSV-2 100 ~
I
I
i
I
E 2
255
i
$
lO
0
I
I
60
120
I
I
180 240 U.V. dose (s)
I
300
Fig. 4. Effect of u.v.-irradiation of HSV-I on production of type 2 virus in CEF cells. HSV-I and HSV-2 were used at rn.o.i. 3"3 and 0"7 p.f.u./cell, respectively. The pre-irradiation titre of HSV-~ in REF cells was 2x io 7 p.f.u./ml. Survivals after 6o, I8O and 30o s of irradiation were 2.4 x Io-z, 1 . 2 × IO-a and i x io -~, respectively. Several experiments were performed to obtain more information on this interference. The findings can be summarized as follows. First, the rate of interference was dependent on the m.o.i., more so on the m.o.i, of the interfering type ~ virus than of the interfered with type 2 virus under the present experimental conditions. Interestingly enough, the interference was expressed even when the m.o.i, of type ~ virus was as low as o.I p.f.u./cell. This might either be due to the capability of the non-infectious particles to induce the phenomenon or to the involvement of a diffusible factor produced in the type I infected cells and influencing non-infected ones. Second, the inhibition effect of type I virus decreased with prolonging the interval between type 2 infection and type I superinfection. This indicates that an early step in the type z virus growth cycle was sensitive to the interfering action of type I virus. Third, the interfering capacity of type I was markedly decreased by u.v. irradiation, suggesting that a type I genome function was responsible for the phenomenon. It has been shown that the irradiation of HSV-I results in a delay in the synthesis of virus-specific products (Ross, Wildy & Cameron, I97I). Thus, the delayed synthesis of the particular type I virus product involved could diminish the expression of the interference effect, imitating the effect of late type I virus addition. Fourth, partial inactivation of type 2 virus resulted in an enhancement of the interference. This observation might again be explained by the delay in the replication of HSV caused by u.v. irradiation (Ross, Cameron & Wildy, I972; Roubal & Vonka, t973). It is reasonable to presume that this
256
D. ZELENA, J. ROUBAL AND V. VONKA
delay resulted in the prolonging of the period during which type 2 virus growth was sensitive to the effect of the particular type ~ virus product involved in the interference. It seems unlikely that the observed interference was mediated by interferon. Herpesviruses are generally considered to be poor inducers of interferon and not very sensitive to its action (Lockart, ~973), although induction of interferon by HSV and its action on the virus have been reported (Waddell, Sigel & Wryk, ~963; Fruitstone, Waddell & Sigel, 1964; Libikov~i, I973; Hooks, x975; Rasmussen & Farley, I975). Our results failed to demonstrate the presence of detectable amounts of interferon-like substances in either C E F or R E F cultures infected with type ~ virus. Interference with HSV not mediated by interferon was described by Roizman 0965): the co-infection of dog kidney cells with HSV M P d k - incapable of growing in these cells, and the MPdk÷sp mutant, for which the dog kidney cells were permissive, resulted in a decrease of MPdk+sp virus production. It was assumed that this effect was due to the formation of non-functional heteropolymers between the polypeptides specified by the dk + virus and the non-functional products of the d k - virus. It does not seem probable that a similar type of interference was operative in the present system, because the same effects as in CEF cells were also observed in R E F cells, permissive for both type I and type 2 viruses. Another type of apparently interferon-independent interference has been described recently by Bronson et al. 0973) and by Frenkel et al. 0975), who reported that the defective HSV particles produced by undiluted passages blocked the replication of infectious HSV. It is not likely that this would be the present case, since both virus stocks were prepared by passing the virus at a low m.o.i. The mechanism causing the relative resistance of HSV-transformed cells to HSV replication as reported by Doller, Duff & Rapp (I973), Kutinov~t et al. (I973) and by Kimura et al. 0975) may, but need not, be related to those involved in the phenomenon presently reported. The nature of the interference described in this report is not understood. One can only speculate, at this stage, about the mode of action of the responsible type ~ virus product in co-infected cells. It has been demonstrated that the regulation of HSV-I polypeptide production is mediated by turning off the synthesis of the earlier products by the later groups of polypeptides (Honess & Roizman, ~974). Since differences between type I and type 2 viruses have been reported for both the transcriptional programmes (Roizman & Frenkel, r973) and the synthesis of virus polypeptides (Powell & Courtney, I975), it seems possible that some type ~ virus product(s) appearing in a sequence, or at a time, which is incorrect for the type 2 virus cycle, might prevent the expression of some function(s) essential for the growth of this virus. REFERENCES BRONSON,D. L., DREESMAN,~. R., BmWAL,N. & BENYESH-MELNICK,M. T. (I973). Defective virions of herpes simplex viruses. Intervirology x, I4I-I53. CORTADADEDELAPE~A,N. & DELUSTIG,E. S. 0974)- Interferon production by leukocytes cultured from neoplastic patients. European Journal of Cancer xo, I89-I92. DOLLER,E., DUFF,R. & RAPP,F. 0973). Resistance of hamster cells transformed by herpes simplex virus type 2 to superinfection by herpes simplex viruses. Intervirology I, I54-i67. FI~UEROA,M. E. & RAWLS,W. r. (I969). Biological markers for differentiation of herpes virus strains of oral and genital origin. Journal of General Virology 4, 259-267. FRENKEL, N., JACOB, R. J., HONESS, R. W., HAYWARD, G. S., LOCKER, H. & ROIZMAN, B. 0975). Anatomy of herpes simplex virus DNA. III. Characterization of defective DNA molecules and biological properties of virus populations containing them. Journal of Virology x6, I53-I67. FRUITSTONE,M. J., WADDELL,O. n. & SIGEL,M. M. 0964). An interferon produced in response to infection by herpes simplex virus. Proceedings of the Society for Experimental Biology and Medicine xx7, 8o4-8o7.
Interference between HSV-I and HSV-2
257
HONESS, R. W. & ROIZMAN,n. (1974). Regulation of herpesvirus macromolecular synthesis. I. Cascade regulation of the synthesis of three groups of viral proteins. Journal of Virology 14, 8 - I 9 . HOOKS, J. J. 0975). Mechanisms by which cellular immunity halts herpes simplex virus spread. In Abstracts of the 3rd International Congressfor Virology, p. I67. Edited by H. S. Bedson, R. N~jera, L. Valenciano & P. Wildy. Madrid. K~RBER, G. (I931). Beitrag zur Kollektiven Behandlung pharmakologischer Reihenversuche. Archives fiir experimentale Pathologic und Pharmakologie x6z, 48o-483. KIMLIRA, S., FLANNERY, V. L., LEVY, B. & SCHAFFER, P. A. (1975). Oncogenic transformation of primary hamster cells by herpes simplex virus type 2 (HSV-2) and an HSV-2 temperature-sensitive mutant. International Journal of Cancer 15, 786-798. KtJrINOV~., L., VONKA,V. & BROU6EK,J. (I973). Increased oncogenicity and synthesis of herpesvirus antigen in hamster cells exposed to herpes simplex type 2 virus. Journal of the National Cancer Institute 50, 759-766. LmiKOV~, H. (I973). Interferon in young and aged chick embryo cell cultures infected with herpes simplex and pseudorabies viruses. Acta Virologica x7, 464-47 I. LOCI~ART, R.Z. SUN. 0973). Interference and interferon with respect to herpesviruses. In The Herpesviruses, pp. 261-27o. Edited by A. S. Kaplan, New York & London: Academic Press. LOWRY, S. P., MELNICK,J. L. & RAWLS, W. E, 0971)- Investigation of plaque formation in chick embryo cells as a biological marker for distinguishing herpes virus type 2 from type I. Journal of General Virology IO, 1-9. MICIaL, J. (I96I). Metabolism of cells in tissue culture in vitro. 1. The influence of serum protein fractions on the growth of normal and neoplastic cells. Experimental Cell Research 23, 324-33o. POWELL, K. L. & COURTNEY, a. J. (1975). Polypeptides synthesized in herpes simplex virus type 2 infected HEp-2 cells. Virology 66, 2 1 7 - 2 2 8 . RASMUSSEN,L. & FARLEY, L. 13. 0975). Inhibition of herpesvirus hominis replication by human interferon. Infection and Immunity r2, IO4-io8. ROIZMAN, 13. (I965). Abortive infection of canine cells by herpes simplex virus. III. The interference of conditional lethal virus with an extended host range mutant. Virology 27, I I3-117. ROIZMAN, B. & I~RENKEL,N. (I973). The transcription and state of herpes simplex virus DNA in productive infection and in human cervical cancer tissue. Cancer Research 33, 14o2-1416. ROSS, g. J. N., CAMEROr~,K. a. & WILDY, e. (I972). Ultraviolet irradiation of herpes simplex virus: reactivation processes and delay in virus multiplication. Journal of General Virology 16, 299-311. ROSS, L. J. N., VV'ILDY, P. & CAMERON, K. R. 097I). Formation of small plaques by herpes viruses irradiated with ultraviolet light. Virology 45, 8o8-812. ROU13AL,J. & VONKA,V. 0973). Multiplicity reactivation in u.v.-irradiated herpes simplex type I virus. Intervirology x, 73-79. TANIGUCI-II, S. & YOSmNO, K. (1963). An analysis of the plaque assay of herpes simplex virus in chick embryo monolayers. Archiv fiir die gesamte Virusforschung 14, 537-552. TIMBURY, M. C. & SU13AK-SHARPE,J. H. (I973). Genetic interaction between temperature sensitive mutants of type I and type 2 herpes simplex viruses. Journal of General Virology 18, 347-357. WADDELL, G. H., SIGEL, M. M. & VqRYK,M. (I963). Factors associated with the response of the ceil to herpes simplex virus. Bacteriological Proceedings 88, 15o. WATERSON, A. P. (1958). Plaque formation by herpes simplex virus in chick embryo tissue. Archly fiir die gesamte Virusforschung 8, 592-599.
(Received 22 March I976)
