Download Inhibition of Replication of Herpes Simplex Virus

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J. gen. Virol. (1985), 66, 2231-2236. Printed in Great Britain
Key words: HS V-1/interJeron/mouse macrophages/HSV DNA polymerase
Inhibition of Replication of Herpes Simplex Virus in Mouse Macrophages
by Iuterferons
By I. D O M K E , * P. S T R A U B , H. J A C O B S E N ,
A. P A N E T 1
H. K I R C H N E R
AND
Institute o f Virus Research, German Cancer Research Center, Heidelberg, F.R.G. and
i Department o f Virology, The Hebrew University, Hadassah Medical School, Jerusalem, Israel
(Accepted 15 July 1985)
SUMMARY
The replication of herpes simplex virus (HSV) type 1 in macrophages grown from
spleen cells of mouse strains susceptible to HSV infection in vivo was very sensitive to
interferon (IFN). Different types of mouse IFN (c~, fl, ~,) exhibited similar antiviral
activities. However, treatment of cells with IFN-~ in combination with IEN-~ or IFN/3 resulted in a synergistic inhibition of virus growth. As shown by assaying HSV D N A
polymerase, IFN inhibited expression of the/3-genes. Inhibition of enzyme induction
correlated well with the reduction of viral yield. Induction of HSV DNA polymerase
was delayed by IFN in a dose-dependent manner. These results show that IFN inhibits
HSV replication at an early step prior to or during the synthesis of/3-proteins.
Mouse macrophages play an important role in non-specific host defence against herpes
simplex virus (HSV) (Hirsch et al., 1970; Mogensen, 1979; Morahan et al., 1980) and have been
identified as interferon (IFN)-producing cells (Kirchner et al., 1983). Pure cultures of peritoneal
or splenic macrophages have been shown to produce IFN after challenge with HSV (Briicher et
al., 1984). IFN is also induced in cultures of bone marrow macrophages by viruses and various
chemical compounds (Storch & Kirchner, 1982). It has been shown that precultured
macrophages of mouse strains susceptible to HSV in vivo produce only low IFN titres after virus
infection in vitro and are permissive for HSV replication (Br/icher et al., 1984).
Several studies have indicated that the replication of HSV is inhibited by IFN. However, high
IFN concentrations were required compared to other virus groups (Lerner & Bailey, 1976; Panet
& Falk, 1983). This makes it difficult to analyse the mechanism of IFN action. However,
treatment of precultured mouse macrophages with low doses of IFN results in a very effective
inhibition of HSV replication, and the effect of IFN on some parameters of replication of HSV
in macrophages is described in the present paper.
Macrophages were grown from spleens of 8- to 12-week-old male DBA/2 mice (Zentralinstitut
fiir Versuchstierkunde, Hannover, F.R.G.). After aseptic removal of the spleen, cells were suspended in culture media as described by Klimetzek & Remold (1980) with some modifications.
The medium consisted of RPMI 1640 (Gibco, code 041-1875) supplemented with 4% horse
serum, 12% foetal bovine serum (FBS), glutamine, gentamicin, and 16% L-cell supernatant
(from L-cells grown in DMEM 10% FBS after 7 days of culture). All other reagents were
obtained from Seromed (Mtinchen, F.R.G.). Cells were seeded in Falcon 35 mm Petri dishes
(code 3001 F). On day 5, cells were washed to remove non-adherent cells. Subsequently, media
were replaced daily. After 6 days of growth, cultures consisted of 100% macrophages as shown
by morphology, by phagocytosis of carbon particles, and by esterase staining (Koski et al., 1976).
A pool of HSV type 1 strain WAL was prepared as previously described (Zawatzky et al.,
1982) and will subsequently be referred to as HSV. Virus titrations were performed by a plaque
assay using RITA cells. Titres in macrophages ~vere determined after freezing and thawing.
Mouse IFN-~ (2.7 × l06 IU/mg) and mouse IFN-fl (4.7 × 107 IU/mg) were purchased from
Stratech Scientific Ltd. (London, U.K.). Mouse IFN-c~/fl (7 × 107 IU/mg) from the late K.
0000-6593 © 1985 SGM
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Table 1. Effect of combinations o f dif/erent IFNs on the replication o f H S V after infection at an
m.o.i, o f 2
IFN type
/3
y
Mq~
c~ + tq
ct + y
fl + 7
Mq~ + y
+ fl + y
Hu-cL,
IFN dose (IU/rnl)
200
200
200
2OO
100 + 100
100 + 100
100 + 100
150 + 50
60 + 60 + 60
200
logi0 Reduction of
virus titre*
2.9
2.4
2-6
2.6
2-6
3.6
3.3
3.3
3.4
0' 1
* Virus yield in the absence of IFN treatment was 8 × 100 p.f.u./ml; cells were frozen 24 h after infection.
Paucker's laboratory was a gift of Dr R. M. Friedman to Dr H. Jacobsen. Mouse IFN-y
(3.6 x 106 IU/mg) purified according to Gribaudo et al. (1984) was kindly donated by Dr S.
Landolfo (Institute of Microbiology, University of Turin, Italy). Macrophage I F N (IFN-Mq~;
1 x 10s IU/mg) was induced in bone marrow macrophages by 10-carboxymethyl-9-acridanone
as described by Storch & Kirchner (1982). It was partially purified using the procedure of G.
Brehm et al. (unpublished). Recombinant human IFN-c~2 (1 x 107 IU/mg) was given by Dr S.
Hiemstra (Essex Pharma, Mtinchen, F.R.G.). Antiserum against mouse IFN-~/fl (5 x 104
IU/ml) was purchased from Stratech Scientific Ltd. Interferon titrations were performed as
described by Zawatzky et al. (1982) using L-cells and vesicular stomatitis virus (VSV).
Cells were used for experiments at about 90~o confluency. At this time, cell number per dish
was about 1 x 106. Cells were washed and incubated with I F N for 18 h. Prior to infection
macrophages were washed three times to remove I F N and infected with 0.2 ml virus at a
multiplicity of infection (m.o.i.) as indicated in each experiment. After virus adsorption for t h,
cells were washed again, supplemented with 2.0 ml of fresh medium and incubated for various
times. Infected cells were stored at - 7 0 ° C until assay of virus yield. All experiments were
carried out with duplicate cell cultures.
For assaying D N A polymerase activity, cell monolayers were washed twice with cold
phosphate-buffered saline and once with cold extraction buffer (10 mM-Tris HCI pH 8.0, 2 mM2-mercaptoethanol, 20~o glycerol). Cells were incubated for 5 min at 4 °C in 0.4 ml extraction
buffer, scraped off with a rubber policeman and stored at - 7 0 °C. After thawing, postmitochondrial supernatants were prepared by centrifugation at 12 000 g for 15 min at 4 °C. The
supernatants were kept at - 7 0 °C. Protein concentrations were determined according to the
Bio-Rad protein assay (Bradford, 1976).
Enzyme activities were determined as described by Panet & Falk (1983) and Purifoy &
Benyesh-Melnick (1975). The incorporation of [3H]TTP (sp. act. 50 Ci/mmol) into T C A insoluble material was measured with activated D N A as template primer. The assay mixture
contained KC1 (0-2 M) to suppress cellular D N A polymerases. Residual cellular enzyme
activities are included in the figures. All enzyme assays were performed in triplicate.
Macrophages grown from spleens of DBA/2 mice were permissive for HSV and HSV
replication exhibited a high sensitivity to IFN. Different types of mouse I F N (~, fl, 7) reduced
virus titres by more than 2 log10, whereas HSV replication was not affected by Hu-IFN-~2
(Table 1). After stimulation with HSV, macrophage cultures produced IFN-~/fl (Kirchner et al.,
1983). Compared to other IFNs, endogenous macrophage I F N (IFN-M4~) induced by 10carboxymethyl-9-acridanone in bone marrow-derived macrophage cultures and partially
purified (G. Brehm et al., unpublished results) did not differ in its activity against HSV.
Inhibition was shown to be synergistic with combinations of IFN-y and IFN-~, IFN-7 and I F N fl, or IFN-~, and IFN-M4~. For example, log10 reduction of virus titre was 3.3 for a mixture of 100
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I
I
I
0
E
0
'd
<
o
"d
2 o
3
0
I
I
I
1
2
3
3 _~
IFN (logto IU/ml)
Fig. 1. Dose-response of IFN for inhibition of HSV replication in macrophage cultures. Cells were
treated with IFN-~/fl for 18 h and infected at an m.o.i, of 4. Virus titres (O) were assayed 24 h after
infection. HSV yield in the absence of IFN treatment was 5 × 106 p.f.u./ml. For assaying HSV DNA
polymerase activity (O) cells were harvested 6 h after infection. Viral enzyme activity in macrophages
not treated with I FN (0~ inhibition) was 34190 c.p.m./~g protein. Residual cellular enzyme activity in
uninfected cultures was 1210 c.p.m./~tg protein.
I U / m l IFN-fl and 100 I U / m l IFN-y, As calculated from dose-response experiments an
inhibition of 3.3 loglo would have been expected with 2000 IU IFN-fl per ml (data not shown).
Thus, potentiation of antiviral activity in the mixtures of I F N - y and I F N - f l was 10-fold.
The effect of different doses of I F N on HSV replication in mouse macrophages is shown in
Fig. 1. Even after infection at an m.o.i, of 4 a 1.5 log10 reduction in virus titre was observed with
I F N doses as low as 10 IU/ml. The degree of protection was strictly dose-dependent. To study
the effect of ] F N on early parameters during the viral replication cycle we have analysed the
induction of HSV D N A polymerase. Production of this fl-protein precedes the synthesis of viral
D N A . A n inhibition of viral enzyme activity was observed even with low I F N doses. After
treatment of macrophages with high I F N concentrations HSV D N A polymerase activity
measured 6 h after infection was reduced to cellular background levels. The reduction of D N A
polymerase correlates well with the reduction of yield of infectious virions.
T h e kinetics of viral replication was analysed in I F N - t r e a t e d macrophages. As expected,
infection at a multiplicity of 4 resulted in high virus titres and cell death after the first round of
replication (Fig. 2a). C o m p a r e d to control cells only few infectious virions were produced in
I F N - t r e a t e d cells. Inhibition was similar with 20 and 500 I U I F N per ml 9 h after infection.
However, some hours later reduction of virus yield was more pronounced with higher I F N
doses. There was a marked effect of I F N on the induction of HSV fl-protein synthesis (Fig. 2b).
In untreated macrophages the m a x i m u m of viral D N A polymerase activity was reached 7 to 9 h
after infection and an increase of enzyme activity was first observed 3 h after infection, I F N
decreased the m a x i m u m of enzyme activity and delayed enzyme induction in a dose-dependent
manner. With 20 IU IFN-ct/fl per ml, the first increase of D N A polymerase was measured 5 h
after infection. After pretreatment of cells with 500 IU IFN-u/fl per ml, viral enzyme was first
detectable 7 h after infection.
So far, little is known about the molecular events leading to the inhibition of herpesviruses in
I F N - t r e a t e d cells. We have studied I F N - m e d i a t e d inhibition of HSV replication in splenic
mouse macrophages. One reason for selecting these cells was the sensitivity of the m a c r o p h a g e HSV system to I F N . Compared to other systems (Panet & Falk, 1983; Rasmussen et al., 1984)
inhibition of HSV in mouse macrophages by I F N was much more effective. Previously, most
experiments concerning I F N - m e d i a t e d inhibition of HSV replication were done in permanent
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107
I
I
I
I
I
I
I
I
I
I
(b)
5O
10 6
40 ~ x
30 >,~-
~ 105
20 Z ~
> 104
>~
r~
10 m
103
I
I
I
3
6
9
I
I
2
4
6
8 10
12
15
Time after infection (h)
Fig. 2. Kinetics of HSV replication in IFN-treated mouse macrophages. Macrophages treated with 500
IU IFN-~/fl per ml (O), 20 IU IFN-a/fl per ml (F]) and control cells ( 0 ) were infected with HSV at
an m.o.i, of 4. (a) For assaying virus titres cells were stored at -70°C at the indicated time points. (b)
For assaying HSV DNA polymerase activity cells were harvested at the indicated time points. Residual
cellular enzyme activity in uninfected cultures was 980 c.p.m./~tg protein.
cell lines (Lerner & Bailey, 1976; Fish et al., 1983). Treatment of macrophages with a mixture of
IFN-y and other I F N types resulted in a more than additive inhibition of virus growth. Similar
findings have been made for inhibition of Mengovirus in L-cells (Fleischmann et al., 1979) or
cell growth inhibition and inhibition of HSV in human melanoma cells (Czarniecki et al., t984).
The phenomenon of potentiation may indicate that IFN-y and IFN-a//3 activate cells by
different mechanisms. It has been shown that an additional set of polypeptides is induced by
treatment of cells with IFN-7 compared to IFN-c~ or IFN-/3 (Weil et al., 1983).
Analysis of cell supernatants after virus adsorption showed that more than 99 ~ of infectious
virus particles were adsorbed to the cell membrane in IFN-treated as well as in control cells (data
not shown). Thus, viral adsorption was unaffected by IFN. However, induction of the HSV/3protein D N A polymerase was inhibited by IFN. Additionally, there was a delay in expression of
/3-genes. This suggests that I F N might act on a-protein synthesis, because sufficient amounts of
HSV immediate early a-proteins are required to trigger the synthesis of/3-proteins (Honess &
Roizman, 1975).
Our results indicate that I F N inhibits HSV replication at an early step. Similar findings have
been made with mouse L-cells (Panet & Falk, 1983) and human HeLa cells (Gloger & Panet,
1984). Contrary to these findings Mufioz & Carrasco (1984) reported no inhibition of HSV
protein synthesis by I F N in HeLa cells which led them to suggest that I F N acts via the
production of defective virions during the first virus cycle. Because of its high sensitivity to I F N
the macrophage-HSV systemis useful to analyse further the step in viral replication cycle that is
blocked by IFN. This might be during penetration of HSV through the cell membrane as shown
for VSV (Whitaker-Dowling et al., 1983) or during the synthesis of a-proteins as indicated by
Gloger & Panet (1984).
Despite the fact that exogenous I F N was removed at the time of infection, there was no
increase of virus yield in IFN-treated macrophages up to 3 days after infection. This continued
restriction of viral growth is due to the production of endogenous IFN. Macrophages start
producing I F N 9 h after infection and I F N titres of 100 IU/ml are still measurable 72 h after
infection. After the addition of an antiserum against IFN-c~//3, virus yield increases 2 to 3 log10
(data not shown). Thus, the antiserum is active in vitro on splenic macrophages cultured for 1
week. In contrast, Belardelli et al. (1984) have shown with fresh peritoneal macrophages that
anti-IFN globulin acts only in viz~o.
Short communication
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I F N is k n o w n to a c t i v a t e m a c r o p h a g e f u n c t i o n s (Bielefeldt O h m a n n et al., 1984; Blasi et al.,
1984). T h e efficient a n t i v i r a l activity could be due to d i f f e r e n t m e c h a n i s m s s w i t c h e d o n by I F N .
O n e m i g h t be u n s p e c i f i c p h a g o c y t o s i s ; a n o t h e r m e t h o d m i g h t be t h e (2' 5 ' ) o l i g o a d e n y l a t e
s y s t e m a n d t h e p r o t e i n kinase. T h e latter m e c h a n i s m s h a v e b e e n s h o w n to b e i n v o l v e d in I F N m e d i a t e d i n h i b i t i o n o f lytic R N A viruses (for r e v i e w , see Lengyel, 1982). R e c e n t s t u d i e s w i t h
h u m a n cells i n d i c a t e t h a t t h e ( 2 ' - 5 ' ) o l i g o a d e n y l a t e s y s t e m is n o t i n v o l v e d in I F N - m e d i a t e d
i n h i b i t i o n o f H S V (Cayley et al., 1984). W e h a v e i n i t i a t e d studies to test this s y s t e m in m o u s e
m a c r o p h a g e s . So far w e h a v e s h o w n a n I F N - d e p e n d e n t i n d u c t i o n o f ( 2 ' - 5 ' ) o l i g o a d e n y l a t e
s y n t h e t a s e a n d t h e p r o t e i n k i n a s e ( d a t a n o t s h o w n ) . F u r t h e r w o r k is n e e d e d to c h a r a c t e r i z e the
m e c h a n i s m a c t i v a t e d by I F N in m a c r o p h a g e s t h a t is r e s p o n s i b l e for t h e i n h i b i t i o n o f H S V .
The authors appreciate the outstanding editorial assistance of Mrs M. Kasamasch.
REFERENCES
BELARDELLI,F., VIGNAUX,F., PROIETTI,E. &GRESSER,I. (1984). Injection of mice with antibody to interferon renders
peritoneal macrophages permissive for vesicular stomatitis virus and encephalomyocarditis virus.
Proceedings oJ the National Academy of Sciences, U.S.A. 81, 602-606.
BIELEFELDTOHMANN,H., GILCHRIST, J. E. & BABIUK,L. A. (1984). Effect of recombinant DNA-produced bovine
interferon alpha (BolFN<q) on the interaction between bovine alveolar macrophages and bovine
herpesvirus type 1. Journal of General Virology 65, 1487-1495.
BLASI,E., HERBERMAN,R. B. & VARESIO,L. (1984). Requirement for protein synthesis for induction of macrophage
tumoricidal activity by IFN-ct and IFN-fl hut not by IFN-~,. Journal of Immunology 132, 3226 3228.
BRADFORD, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein
utilizing the principle of the protein-dye-binding. Analytical Biochemistry 72, 248-254.
BRUCHER,J., DOMKE,1., SCHRODER,C. H. & KIRCHNER,H. (1984). Experimental infection of inbred mice with HSV.
VI. Effect of IFN on in vitro virus replication in macrophages. Archives of Virology 82, 83-93.
CAYLEY, P. J., DAVIES,J. A., McCULLAGH,K. G. & KERR, I. M. (1984). Activation of the ppp(A2'p), A system in
interferon-treated, herpes simplex virus-infected cells and evidence for novel inhibitors of the ppp(A2'p), Adependent R Nase. European Journal of Biochemistry 143, 165-174.
CZARNIECKI,C. W., FENNIE,C. W., POWERS,D. B. & ESTELL,D. A. (1984). Synergistic antiviral and antiproliferative
activities of Escherichia coli-derived human alpha, beta, and gamma interferons. Journal of Virology 49, 490496.
FISH, E. N., BANERJEE,K. & STEBBING,N. (1983). Antiviral activities of cloned human leucocyte IFN's against HSV
type 2 infections of mice. Journal of General Virology 64, 2317-2321.
FLEISCHMANN,W. R., JR., GEORGIADES,J. A., OSBORNE,L. C. & JOHNSON, H. M. (1979). Potentiation of interferon
activity by mixed preparations of fibroblast and immune interferon. Injection and Immunity 26, 248-253.
GLOGER,I. & PANET,A, (1984). Synthesis of herpes simplex virus proteins in interferon-treated human ceils. Journal
of General Virology 65, 1107 1111.
GRIBAUDO, G., COFANO, F., NEGRO-PONZI,A. & LANDOLFO,S. (1984). Mouse y-IFN produced by a cloned T-cell
lymphoma. I. Purification and physicochemical characterization. Journal oJlnterJeron Research 1, 91-99.
HIRSCH, M. S., ZISMAN,B. & ALLISON,A. C. (1970). Macrophages and age-dependent resistance to HSV in mice.
Journal of Immunology 104, 1160-I 165.
HONESS,R. W. & ROIZMAN,B. (1975). Regulation of herpesvirus macromolecular synthesis: sequential transition of
polypeptide synthesis required functional viral polypeptides. Proceedings of the National Academy of Sciences,
U.S.A. 72, 1276-1280.
KtRCHNER, H., ENGLER, H., SCHRODER,C. H., ZAWATZKY,R. & STORCH, E. (1983). Herpes simplex virus type 1induced interferon production and activation of natural killer cells in mice. Journal of General Virology 64,
437-441.
KLIMETZEK,V. & REMOLD,H. G. (1980). The murine bone marrow macrophage, a sensitive indicator cell for murine
migration inhibitory factor and a new method for their harvest. Cellular Immunology 53, 257 266.
KOSKI,I. R., POPLACK,D. G. & BLAESE,R. M. (1976). A nonspecific esterase stain for the identification of monocytes
and macrophages. In In Vitro Methods in Cell-mediated and Tumor Immunity, pp. 359 362. Edited by B. R.
Bloom. New York & London: Academic Press.
LENGYEL, P. (1982). Biochemistry of IFN's and their action. Annual Rev&w of Biochemistry 51, 251-282.
LERNER, M. A. & BAILEY,E. J. (1976). Differential sensitivity of HSV types 1 and 2 to HulFN. Antiviral effects of
IFN plus 9-fl-arabinofuranosyladenine. Journal of InJectious Diseases 134, 400-404.
MOGENSEN,S. C. (1979). Role of macrophages in natural resistance to virus infections. Microbiological Reviews 43,
1 26.
MORAHAN, P. S., MORSE, S. S. & McGEORGE, M. B. (1980). Macrophage extrinsic antiviral activity during HSV
infection. Journal ~?['General Virology 46, 291-300.
MUNOZ, A. & CARRASCO,L. (1984). Formation of non-infective herpesvirus particles in cultured cells treated with
human interferon. Journal of General Virology 65, 1069-I078.
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PANET, A. & FALK, H. (1983). Inhibition by interferon of herpes simplex virus thymidine kinase and D N A
polymerase in infected and biochemically transformed cells. Journal of General Virology 64, 1999-2006.
PURIFOY, D. J. M. & BENYESH-MELNICK, M. (1975). D N A polymerase induction by DNA-negative temperaturesensitive m u t a n t s of herpes simplex virus type 2. Virology 68, 374-386.
RASMUSSEN, L. E., CHEN, P. T. & MERIGAN, T. C. (1984). Comparison of antiviral activities of cloned and native
h u m a n interferons against herpes simplex virus types I and 2 and h u m a n cytomegalovirus. Antimicrobial
Agents and Chemotherapy 26, 599-600.
STORCH, E. & KIRCHNER, H. (1982). Induction of I F N in murine bone marrow-derived macrophage cultures by 10carboxymethyl-9-acridanone. European Journal oflmmunology 12, 793 796.
WEIL, J., EPSTEIN, C. J., EPSTEIN, L. B., SEDMAK, J. J., SABRAN, J. L. & GROSBERG, S. E. (1983). A unique set of
polypeptides is induced by ~ interferon in addition to those induced in c o m m o n with ~ and fl interferons.
Nature, London 301, 437 439.
WHITAKER-DOWLING,P. A., WILCOX, D. K., WIDNELL,C. C. & YOUNGNER, J. S. (1983). Interferon-mediated inhibition
of virus penetration. Proceedings of the National Academy of Sciences, U.S.A. 80, 1083-1086.
ZAWATZKY,R., GRESSER, I., DEMAEYER,E. & KIRCHNER,H. (1982). Role of I F N in the resistance of C57BL/6 mice to
different doses of HSV type I. Journal of InJectious Diseases 146, 405 410.
(Received 6 March 1985)