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Polyamine depletion of cells reduces the infectivity of herpes simplex virus but not the infectivity of sindbis virus
Pergamon Press
Life Sciences, Vol. 42, pp. 2011-2018 Printed in the U.S.A.
POLYAMINE DEPLETION OF CELLS REDUCES THE INFECTIVITY OF HERPES SIMPLEX VIRUS BUT NOT THE INFECTIVITYOF SINDBIS VIRUS Pirkko Pohjanpelto (l), Akseli Sekki (11, Veijo Hukkanen (3) and Carl-Henrik von Bonsdorff (1) (1) Department of Virology, University of Helsinki, Helsinki, Finland (3) Department of Virology, University of Turku, Turku, Finland (Received in final form March 17, 1988)
The effect of
on the viral growth examined using be effectively depleted of In order to the polyamines present in we used a polyamine auxotrophic Chinese P22 growing in and Vero cells in low serum medium. final yield of an enveloped RNA virus, in P22 cells was not decreased by of cellular polyamines although onset of viral replication was delayed. In the final yield of an enveloped DNA virus, was considerably reduced in Vero cells, depleted of polyamines by an inhibitor of the number of particles detected by was not decreased. Southern blot analysis of the polyamine depleted and the control cells in the relative abundance of the DNA fragments in DNA synthesis may have caused decreased infectivity of HSV. Polyamines putrescine, and spermine, are organic in all cells. Their is required for the optimal growth of and they may indispensable for the growth of is not are hampered by fact in most tissue of polyamines to interfere the experiments. In this paper we have explored the impact of of cells on growth of to multiply in or medium containing low concentration of The growth of containing enveloped Sindbis was studied in of Chinese hamster ovary has active ornithine decarboxylase in the synthesis of The P22 cells are especially suited for the experiments because they in serum-free medium and can be of polyamines by omitting polyamines the medium. Because Herpes an enveloped DNA virus, not in P22 cells it .OO Copyright (c)
plc
2012
Polyamine Depletion Reduces Infectivity
Vol. 42, No. 20, 1988
was cultivated in Vero cells which were depleted of polyamines by adifluoromethylornithine (DFMO), an irreversible inhibitor of ODC (4). The results show that polyamine depleted cells produced normal amounts of infectious Sindbis virus but the infectivity of the HSV-2 particles formed in polyamine depleted cells was considerably reduced as compared to the control. We give also evidence suggesting that the decrease in the infectivity of HSV-2 is due to disturbances in the synthesis of viral DNA. Materials and Methods Cells and viruses, Polyamine auxotrophic mutant cell strain P22, was derived from CHO cells and has been described earlier (3). The P22 cells were cultured on collagen coated petri dishes in a 1:l mixture of minimal essential medium (MEM) and nutrient mixture F12 supplemented with 0.1% bovine serum albumin (BSA) and putrescine lo5 M. Vero cells, originating from monkey kidney, and BHK cells, originating from hamster kidney, were routinely cultured in a 1:l mixture of MEM and nutrient mixture F12 supplemented with 5% foetal calf serum. The viruses used in the experiments were Herpes simplex type 2, strain Curtis and Sindbis virus, strain HR. Denletion of nolvamines of the cell% The P22 cells were cultured as described above except that no putrescine was added to the medium and nutrient mixture F12 without putrescine was used. The BSA was dialyzed, first against 1M NaCl plus 0.2 M MgC12 and finally against 0.9% NaCl (3). The Vero cells were cultured like the P22 cells except that medium had no BSA, but contained 0.5mM DFMO and 0.2% human serum, dialyzed like BSA. mation of viruses_ After adsorption of the virus for 1 hr at +37’ C the cells were washed twice with MEM. The Sindbis virus was titrated in BHK cells on petri dishes, diameter 5 cm, using 0.5% carboxymethyl cellulose in the medium. HSV was titrated in Vero cells in multiwell plates, each well 1 cm of diameter, in medium containing 1% human gammaglobulin (5). The cells were stained after 3-4 days with 0.25% crystal violet and the plaques were counted. Southern hvbridization The following plasmids containing HSV-2 sequences were used in the experiments: 1) pHSV218 containing 280 bp insert from Hind III F fragment, mapping between HSV-2 cordinates 0.39 - 0.41. 2) pHSV224 containing 0.3 and 2.5 kb fragments from the Hind III L and K fragments, and reacting with Eco RI fragments N, 0 and a part of the fragment M, mapping between coordinates 0.894 and 0.972. 3) pHSV246 containing 5 kb and 1.5 kb fragments from Hind III fragment H between map coordinates 0.29 and 0.329. The HSV-2 DNA was cloned as Hind III and Sal I fragments. HSV-2 DNA, prepared from cytoplasmic extracts was digested with restriction of polyamine depleted and control cells, endonucleases Taq I, Barn HI and Eco RI (5U/pg DNA) for 2 hr at 37OC (65OC for Taq I) and electrophoresed in 0.6% neutral agarose gels. After electrophoresis the DNA was transferred to a nylon membrane (GeneScreen Plus, NEN,USA) in 0.4 M NaOH, 0.6 M NaCl for 18 hr. Hybridization with the the plasmids containing HSV-DNA segments was carried out as recommended by the manufacturer of the membrane. 35S-methionine labeling of nroteins, Cells were incubated with MEM without methionine in the presence or absence of DFMO for 30 min, 35S-methionine lOl,Ci/ml was added and incubation was continued for another 30 min. After washing the cells twice with MEM the cells were scraped off the plates and
vol. 42, No. 20, 1988
Polyamine Depletion Reduces Infectivity
homogenized by in 50 Tris-HCl buffer, 7.4, NaCl, mM EDTA, Nonidet P-40 2 mM at 1OOOOOxg 30 min, supernatants SDS-polyacrylamide electrophoresis following standard gel autoradiographed.
containing Homogenates were procedures,
2013
mM were to the
Electron Polyamine depleted control Vero were infected HSV-2 and 8,15, and hrs post with 2.5% and processed thin sectioning described earlier For morphometric micrographs were by random at a of 3600 and paper at a magnification of x were for the The nuclear well as surface area determined using square lattice described by and by viral capsid in the areas. Chemicals, Center, Cincinnati, The restriction Boehringer Mannheim, L- 35S-methionine was from International, U.K.
was a
from Merrell were purchased activity 1120
polyamine auxotropbic cells can effectively depleted polyamines by polyamines from medium. After days of starvation putrescine spermidine were in these and spermine was reduced 80 % compared to control cells Prolonged starvation not further the spermine of the most likely the cells nearly ceased replicate, preventing dilution of The P22 were starved polyamines for days while control cells putrescine 10s5M. is notable after 6 of polyamine the cells viable as by trypan exclusion test by time cinematography. The depleted and control cells infected with virus and growth curves both kinds cells in presence or of putrescine were determined. results show the final of infectious virus was high in polyamine depleted in the control cells 1A). However, onset of replication of virus was by 2-3 in the depleted as to the cells. Vero can be of polyamines culturing in medium containing dialyzed serum 0.5 mM The depletion not, however, good as the P22 In 6 the content putrescine and in the treated cells about 100 as compared the control. spermine showed about 30 decrease (8). Vero cells the P22 were viable 6 days polyamine depletion. growth curve HSV-2 was in Vero incubated with without DFMO 6 days. 1B shows the control without DFMO about 100 more infectious than the treated with In the cells the of growth the virus also retarded. order to that DFMO not have side effects general inhibition virus multiplication growth of virus was in Vero in the and absence DFMO. The conditions were same as with HSV-2. hr post the DFMO cultures contained x lo* and the control cultures x108 pfu/ml that DFMO of the did not the yield infectious Sindbis
vol. 42, No. 20, 1988
Polyamine Depletion Reduces Infectivity
2014
the analysis the viral the cytoplasmic was isolated polyamine depleted control Vero 20 hr infection with About 30% cytoplasmic DNA obtained from control cultures than from polyamine depleted (56pg/107cells). The samples from polyamine depleted from the cells were with restriction Taq I, HI and RI and to three plasmids, all different regions the HSV The results presented in Fig.2. In experiments the from the depleted cells to be than those the control this in of the that equal of DNA submitted to electrophoresis in cases. Hybridization different plasmids the following in the from the depleted as to the cells. Hybridization pHSV224 indicated certain sequences the S-segment
6..
B
0
*
lP"
-P" DFMO
L
I
6
12
16
20
hr
I 5
10
15
20
is
A
30 hr
FIG. 1 Effect of polyamine depletion of cells on growth of Sindbis virus (A) and the Herpes simplex virus (B). The P22 cells were cultured for 6 days with putrescine (4.9x104cells/cm2) and without putrescine (4.6~10~ cells/cm2) and infected with 5 pfu/cell). Vero cells were cultured for 6 days with DFMO Sindbis virus and infected with HSV-2 (3.6x104cells/cm a ) and without DFMO (2.9x104cells/cm2) (7 pfu/cell). The titers are given per 10 6 cells. Each value is a mean of two (B) 0 no additions, (A) 0 putrescine 10s5M, 0 no additions. determinations. 0 DFMO 0.5 n-M.
Polyamine Depletion Reduces Infectivity
Vol. 42, No. 20, 1988
2015
HSV-DNA were significantly decreased (densitometrically 5-fold difference) (Fig.2A). Hybridization with pHSV246 showed decrease in the DNA fragments in the L-region of HSV-DNA (densitometrically a-fold difference) (Fig. 2B). The use of pHSV218 revealed also clear decreases in high molecular weight DNA fragments in the L-segment (Fig. 2C). The results suggest that polyamine depletion of the cells caused changes in both L and S segments of the HSV genome. The effect of polyamines on the synthesis of viral proteins was studied by giving a 35S-methionine pulse at different time intervals to the polyamine depleted and control Vero cells, infected with HSV-2. The labeled cells were collected 4,7 and 14 hr post infection, the proteins were separated in SDS-polyacrylamide gel electrophoresis and the gel was submitted to autoradiography. Since the synthesis of host proteins is shut off soon after HSV infection most of the newly synthesized, 35S-labeled proteins should be virus specific. No marked difference was found in the pattern of protein bands in the DFMO treated and the control cells ( Fig. 3).
DFMO
TAQ + -
Kb
A
BAM + -
EC0 + -
TAO + -
BAM + -
EC0 + -
2;.‘5 z
FIG. 2 2.0-m
TAQ + -
DFMO Kb 2379567L3-
c
BAM +-
EC0 +
TAQ +-+-+-
BAM
EC0
Southern blot analysis of HSV-2 DNA from polyamine depleted and control cells. Vero cells were cultured with and without DFMO (0.5 n&I) for 6 days in medium containing 0.2% dialyzed human serum and infected with HSV-2 (5 pfu/cell). The cytoplasmic DNA was isolated 22 hr post infection, and Southern blot analysis carried out using was radioactive probes pHSV224 (A), pHSV246 (B), pHSV218 (C) and total HSV genome (D). The quantity of DNA in each lane was 4ug. The dots indicate the restriction fragments decreased in the DFMO treated cells.
2016
Polyamine Depletion Reduces Infectivity
CELLS DFMO
m
96-
68--
46-
+
Vol. 42, No. 20, 1988
HSV-2 4 hr -+-+-+
7hr
qpmamQR~-
14 hr FIG. 3 Protein synthesis in polyamine depleted and control cells infected with Herpes simplex virus. Vero cells were cultured with and without DFMO (0.5mM) for 6 days and infected with HSV-2 (5 pfu/cell). After 4,7 and 14 hr the cells were labeled with 35S-methionine UOuCi/ml) for 0.5 hr and the labeled proteins were analyzed by SDS-gel electrophoresis using 8% polyacrylamide.
kd
For calculation of the number of virus particles the polyamine depleted and control Vero cells infected with HSV-2 were prepared for electron microscopy 8, 15 and 24 hr post infection. At 8 hr the nuclei were already found to contain assembled capsids, at 24 hr the cells showed marked degenerative changes with abundant nuclear and cytoplasmic virus particles. No obvious differences in the amount or in the appearance of intranuclear or cytoplasmic virus particles was found between the polyamine depleted and the control cells. The samples at 15 hr were selected for closer scrutiny. In the morphometric analysis of the infected cells the number of virus particles found in the nucleus of the control cells was 20/100l.~m~ and in the polyamine depleted cells 22/100pm2. The amount of viral particles in the cytoplasm of both kinds of cells was the same, 5/100um2.
Inhibition of polyamine synthesis causes retardation of cellular proliferation in many different kinds of tissue cultures (2,9,11). It also suppresses the growth of cytomegalovirus which causes an increase in the concentration of polyamines, spermidine and spermine, in the infected cells. If polyamine synthesis is inhibited by methyl glyoxal(bis)guanyl hydrazone (12) or by DFMO (12,13) the amount of infectious cytomegalo virus produced by the cells is greatly reduced. On the other hand Tuomi et al. found that although the replication of Semliki forest virus, a close relative of Sindbis virus, was retarded by 2 hr in polyamine depleted, DFMO treated BHK cells as compared to the controls the final yield of the virus in both kinds of cells was the same (14). There is evidence suggesting that polyamine depletion inhibits replication of HSV. Treatment of BHK cells with DFMO caused 60% inhibition in the infectious titer of the virus 20 hr post infection (141, and DFMO treatment of human fibroblasts reduced the infectious titer of HSV by about 98% when measured 7 days post infection (13). In both cases the virus titers were
Vol. 42, No. 20, 1988
Polyamine Depletion Reduces Infectivity
2017
determined only at one time point, and the maximal virus titers are therefore not known with certainty. Moreover, in the latter work the number of cells in the DFMO treated and nontreated cells were not counted. This is relevant considering the considerable retardation of cellular growth usually found in the presence of DFMO. In this paper we have re-examined the effect of polyamine depletion on growth of an enveloped RNA virus, Sindbis, and of an enveloped DNA virus, HSV-2, using cell strains that could be effectively depleted of polyamines. We have also made growth curves of the viruses thus distinguishing the effect on the rate of growth and on the final yield of the virus. We found that the polyamine auxotrophic P22 cells, starved of polyamines, were able to produce the same amount of infectious Sindbis virus, and at the same rate, as the control cells although there was a delay of about 2 hr in the onset of the viral replication. The results show that the viral RNA and protein synthesis could proceed successfully in cells efficiently depleted of polyamines. On the other hand the final yield of the infectious HSV-2 was considerably reduced in DFMO treated Vero cells in spite of the fact that polyamine depletion was not as good as in P22 cells. However, the number of HSV-2 particles, composed mainly of proteins, was not decreased. Moreover, analysis of the 35S-labeled proteins from the two kinds of HSV-2 infected cells did not reveal any clear differences. These results suggest that failure of protein synthesis was not responsible for the reduction of infectious titer of HSV-2 in polyamine depleted cells. The fact that the DFMO treated Vero cells were able to produce normal quantities of infectious Sindbis virus indicated that this drug did not have toxic side effects causing general inhibition of viral growth. The next question we asked was whether polyamine depletion might affect the synthesis of HSV-DNA and thus cause the reduction in the infectivity of the virus particles. Southern blotting revealed overall decrease in the intensity of the bands of the HSV-DNA from the polyamine depleted cells as compared to the control. It is conceivable that DNA synthesis is more error prone in the polyamine depleted than in the control cells and in the former case loss of the sites for the restriction enzymes may have prevented a certain fraction of DNA molecules from migrating in the gel. The errors made in individual cells would probably not be identical, and therefore mostly changes in the relative abundance rather than total loss of the DNA fragments would be expected. The results obtained with HSV are compatible with the idea that the inhibition of growth of polyamine depleted cells is mainly due to impairment of DNA synthesis. This is supported by the excellent sterospecific fit between spermine and the DNA double helix (15), giving rise to speculations that polyamines might facilitate DNA synthesis by neutralizing the excessive negative charges in the DNA. In line with this assumption are the results indicating that a shortage of polyamines causes retardation of DNA synthesis in different kinds of cells (11,16,17). Furthermore it is notable that polyamine depletion induces chromosomal damage in CHO cells (18). The results presented in this paper suggest that disturbances in the DNA synthesis are responsible for the reduction of the infectious titer of HSV-2 in polyamine depleted cells. More generally this supports the idea that polyamines are important for unerroneous synthesis of DNA. However, it cannot be excluded that a complex virus like HSV requires cellular functions, other than DNA synthesis, which are dependent on polyamines, and which are not important for the structurally more simple Sindbis virus.
2018
Polyamine Depletion Reduces Infectivity
We thank
Ms. Irma Lantinen
for competent
Vol. 42, No. 20, 1988
technical
assistance
Merrell Research Center for the gift of a- difluoromethylornithine. was supported by a grant from the Sigrid Juselius foundation.
and the
This research
References 1. 2. 3. 4. :: 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
E.W. HAFNER, C.W. TABOR and H. TABOR, J. Biol. Chem. &% 12419-12426 (1979). C.W. TABOR, H. TABOR, A.K. TYAGI and M.S. COHN, Fed. Proc. 41 3084-3088 (1982). P. POHJANPELTO, E. HCLTTA and O.A. JANNE, Mol. Cell. Biol. 5 1385-1390 0985). B.W. METCALF, P. BEY, C. DANZIN, M.J. JUNG, P. CASARA and J.P. VERVERTS, J. Am. Chem. Sot. Isn 2551-2553 (1978). B.ROIZMAN and P.R. ROANE, Jr. Virology u 75-79 (1961). C-H. v.BONSDORFF, S.D. FULLER and K. SIMONS, EMBO J. 4 2781-2792 (1985). E.R. WEIBEL, Stereological methods 1. Practical methods for biological morphometry. Acad.Press London (1985). P. POHJANPELTO, E. HOLTTA, A. MAICHE and S. KNUUTILA, Hereditas (in press). P.S. MAMONT, M-C. DUCHESNE, J. GROVE and P. BEY, Biochem. Biophys. Res. Commun. & 58-66 (1978). A. PEGG, Biochem. J. 224 29-38 (1984). C. SEYFRID and D. MORRIS Cancer Res. Xj 4861-4867 (1979). A.S. TYMS and J.D. WILLIAMSON Nature m 690-691 (1982). W. GIBSON, R. van BREEMEN, A. FIELDS, R. LaFEMINA and A. IRMIERE, J. Virol. a 145-154 (1984). K. TUOMI, A. RAINA and R. MANTYJARVI, Biochem. J. a 113-119 (1982). B.G. FEUERSTEIN, N. PATTABIRAMAN and L.J. MARTON, Proc. Natl. Acad. Sci. USA & 5948-5952 (1986). S. ANEHUS, P. POHJANPELTO, P. BALDETORP, E. LANGSTROM and 0. HEBY, Mol. Cell. Biol. 4 915-922 (1984). P. POHJANPELTO, Biomedicine (Paris) 2 350-352 (1975). P. POHJANPELTO and S. KNWTILA, Cancer Res. 44 4535-4539 (1984).
Life Sciences, Vol. 42, pp. 2011-2018 Printed in the U.S.A.
POLYAMINE DEPLETION OF CELLS REDUCES THE INFECTIVITY OF HERPES SIMPLEX VIRUS BUT NOT THE INFECTIVITYOF SINDBIS VIRUS Pirkko Pohjanpelto (l), Akseli Sekki (11, Veijo Hukkanen (3) and Carl-Henrik von Bonsdorff (1) (1) Department of Virology, University of Helsinki, Helsinki, Finland (3) Department of Virology, University of Turku, Turku, Finland (Received in final form March 17, 1988)
The effect of
on the viral growth examined using be effectively depleted of In order to the polyamines present in we used a polyamine auxotrophic Chinese P22 growing in and Vero cells in low serum medium. final yield of an enveloped RNA virus, in P22 cells was not decreased by of cellular polyamines although onset of viral replication was delayed. In the final yield of an enveloped DNA virus, was considerably reduced in Vero cells, depleted of polyamines by an inhibitor of the number of particles detected by was not decreased. Southern blot analysis of the polyamine depleted and the control cells in the relative abundance of the DNA fragments in DNA synthesis may have caused decreased infectivity of HSV. Polyamines putrescine, and spermine, are organic in all cells. Their is required for the optimal growth of and they may indispensable for the growth of is not are hampered by fact in most tissue of polyamines to interfere the experiments. In this paper we have explored the impact of of cells on growth of to multiply in or medium containing low concentration of The growth of containing enveloped Sindbis was studied in of Chinese hamster ovary has active ornithine decarboxylase in the synthesis of The P22 cells are especially suited for the experiments because they in serum-free medium and can be of polyamines by omitting polyamines the medium. Because Herpes an enveloped DNA virus, not in P22 cells it .OO Copyright (c)
plc
2012
Polyamine Depletion Reduces Infectivity
Vol. 42, No. 20, 1988
was cultivated in Vero cells which were depleted of polyamines by adifluoromethylornithine (DFMO), an irreversible inhibitor of ODC (4). The results show that polyamine depleted cells produced normal amounts of infectious Sindbis virus but the infectivity of the HSV-2 particles formed in polyamine depleted cells was considerably reduced as compared to the control. We give also evidence suggesting that the decrease in the infectivity of HSV-2 is due to disturbances in the synthesis of viral DNA. Materials and Methods Cells and viruses, Polyamine auxotrophic mutant cell strain P22, was derived from CHO cells and has been described earlier (3). The P22 cells were cultured on collagen coated petri dishes in a 1:l mixture of minimal essential medium (MEM) and nutrient mixture F12 supplemented with 0.1% bovine serum albumin (BSA) and putrescine lo5 M. Vero cells, originating from monkey kidney, and BHK cells, originating from hamster kidney, were routinely cultured in a 1:l mixture of MEM and nutrient mixture F12 supplemented with 5% foetal calf serum. The viruses used in the experiments were Herpes simplex type 2, strain Curtis and Sindbis virus, strain HR. Denletion of nolvamines of the cell% The P22 cells were cultured as described above except that no putrescine was added to the medium and nutrient mixture F12 without putrescine was used. The BSA was dialyzed, first against 1M NaCl plus 0.2 M MgC12 and finally against 0.9% NaCl (3). The Vero cells were cultured like the P22 cells except that medium had no BSA, but contained 0.5mM DFMO and 0.2% human serum, dialyzed like BSA. mation of viruses_ After adsorption of the virus for 1 hr at +37’ C the cells were washed twice with MEM. The Sindbis virus was titrated in BHK cells on petri dishes, diameter 5 cm, using 0.5% carboxymethyl cellulose in the medium. HSV was titrated in Vero cells in multiwell plates, each well 1 cm of diameter, in medium containing 1% human gammaglobulin (5). The cells were stained after 3-4 days with 0.25% crystal violet and the plaques were counted. Southern hvbridization The following plasmids containing HSV-2 sequences were used in the experiments: 1) pHSV218 containing 280 bp insert from Hind III F fragment, mapping between HSV-2 cordinates 0.39 - 0.41. 2) pHSV224 containing 0.3 and 2.5 kb fragments from the Hind III L and K fragments, and reacting with Eco RI fragments N, 0 and a part of the fragment M, mapping between coordinates 0.894 and 0.972. 3) pHSV246 containing 5 kb and 1.5 kb fragments from Hind III fragment H between map coordinates 0.29 and 0.329. The HSV-2 DNA was cloned as Hind III and Sal I fragments. HSV-2 DNA, prepared from cytoplasmic extracts was digested with restriction of polyamine depleted and control cells, endonucleases Taq I, Barn HI and Eco RI (5U/pg DNA) for 2 hr at 37OC (65OC for Taq I) and electrophoresed in 0.6% neutral agarose gels. After electrophoresis the DNA was transferred to a nylon membrane (GeneScreen Plus, NEN,USA) in 0.4 M NaOH, 0.6 M NaCl for 18 hr. Hybridization with the the plasmids containing HSV-DNA segments was carried out as recommended by the manufacturer of the membrane. 35S-methionine labeling of nroteins, Cells were incubated with MEM without methionine in the presence or absence of DFMO for 30 min, 35S-methionine lOl,Ci/ml was added and incubation was continued for another 30 min. After washing the cells twice with MEM the cells were scraped off the plates and
vol. 42, No. 20, 1988
Polyamine Depletion Reduces Infectivity
homogenized by in 50 Tris-HCl buffer, 7.4, NaCl, mM EDTA, Nonidet P-40 2 mM at 1OOOOOxg 30 min, supernatants SDS-polyacrylamide electrophoresis following standard gel autoradiographed.
containing Homogenates were procedures,
2013
mM were to the
Electron Polyamine depleted control Vero were infected HSV-2 and 8,15, and hrs post with 2.5% and processed thin sectioning described earlier For morphometric micrographs were by random at a of 3600 and paper at a magnification of x were for the The nuclear well as surface area determined using square lattice described by and by viral capsid in the areas. Chemicals, Center, Cincinnati, The restriction Boehringer Mannheim, L- 35S-methionine was from International, U.K.
was a
from Merrell were purchased activity 1120
polyamine auxotropbic cells can effectively depleted polyamines by polyamines from medium. After days of starvation putrescine spermidine were in these and spermine was reduced 80 % compared to control cells Prolonged starvation not further the spermine of the most likely the cells nearly ceased replicate, preventing dilution of The P22 were starved polyamines for days while control cells putrescine 10s5M. is notable after 6 of polyamine the cells viable as by trypan exclusion test by time cinematography. The depleted and control cells infected with virus and growth curves both kinds cells in presence or of putrescine were determined. results show the final of infectious virus was high in polyamine depleted in the control cells 1A). However, onset of replication of virus was by 2-3 in the depleted as to the cells. Vero can be of polyamines culturing in medium containing dialyzed serum 0.5 mM The depletion not, however, good as the P22 In 6 the content putrescine and in the treated cells about 100 as compared the control. spermine showed about 30 decrease (8). Vero cells the P22 were viable 6 days polyamine depletion. growth curve HSV-2 was in Vero incubated with without DFMO 6 days. 1B shows the control without DFMO about 100 more infectious than the treated with In the cells the of growth the virus also retarded. order to that DFMO not have side effects general inhibition virus multiplication growth of virus was in Vero in the and absence DFMO. The conditions were same as with HSV-2. hr post the DFMO cultures contained x lo* and the control cultures x108 pfu/ml that DFMO of the did not the yield infectious Sindbis
vol. 42, No. 20, 1988
Polyamine Depletion Reduces Infectivity
2014
the analysis the viral the cytoplasmic was isolated polyamine depleted control Vero 20 hr infection with About 30% cytoplasmic DNA obtained from control cultures than from polyamine depleted (56pg/107cells). The samples from polyamine depleted from the cells were with restriction Taq I, HI and RI and to three plasmids, all different regions the HSV The results presented in Fig.2. In experiments the from the depleted cells to be than those the control this in of the that equal of DNA submitted to electrophoresis in cases. Hybridization different plasmids the following in the from the depleted as to the cells. Hybridization pHSV224 indicated certain sequences the S-segment
6..
B
0
*
lP"
-P" DFMO
L
I
6
12
16
20
hr
I 5
10
15
20
is
A
30 hr
FIG. 1 Effect of polyamine depletion of cells on growth of Sindbis virus (A) and the Herpes simplex virus (B). The P22 cells were cultured for 6 days with putrescine (4.9x104cells/cm2) and without putrescine (4.6~10~ cells/cm2) and infected with 5 pfu/cell). Vero cells were cultured for 6 days with DFMO Sindbis virus and infected with HSV-2 (3.6x104cells/cm a ) and without DFMO (2.9x104cells/cm2) (7 pfu/cell). The titers are given per 10 6 cells. Each value is a mean of two (B) 0 no additions, (A) 0 putrescine 10s5M, 0 no additions. determinations. 0 DFMO 0.5 n-M.
Polyamine Depletion Reduces Infectivity
Vol. 42, No. 20, 1988
2015
HSV-DNA were significantly decreased (densitometrically 5-fold difference) (Fig.2A). Hybridization with pHSV246 showed decrease in the DNA fragments in the L-region of HSV-DNA (densitometrically a-fold difference) (Fig. 2B). The use of pHSV218 revealed also clear decreases in high molecular weight DNA fragments in the L-segment (Fig. 2C). The results suggest that polyamine depletion of the cells caused changes in both L and S segments of the HSV genome. The effect of polyamines on the synthesis of viral proteins was studied by giving a 35S-methionine pulse at different time intervals to the polyamine depleted and control Vero cells, infected with HSV-2. The labeled cells were collected 4,7 and 14 hr post infection, the proteins were separated in SDS-polyacrylamide gel electrophoresis and the gel was submitted to autoradiography. Since the synthesis of host proteins is shut off soon after HSV infection most of the newly synthesized, 35S-labeled proteins should be virus specific. No marked difference was found in the pattern of protein bands in the DFMO treated and the control cells ( Fig. 3).
DFMO
TAQ + -
Kb
A
BAM + -
EC0 + -
TAO + -
BAM + -
EC0 + -
2;.‘5 z
FIG. 2 2.0-m
TAQ + -
DFMO Kb 2379567L3-
c
BAM +-
EC0 +
TAQ +-+-+-
BAM
EC0
Southern blot analysis of HSV-2 DNA from polyamine depleted and control cells. Vero cells were cultured with and without DFMO (0.5 n&I) for 6 days in medium containing 0.2% dialyzed human serum and infected with HSV-2 (5 pfu/cell). The cytoplasmic DNA was isolated 22 hr post infection, and Southern blot analysis carried out using was radioactive probes pHSV224 (A), pHSV246 (B), pHSV218 (C) and total HSV genome (D). The quantity of DNA in each lane was 4ug. The dots indicate the restriction fragments decreased in the DFMO treated cells.
2016
Polyamine Depletion Reduces Infectivity
CELLS DFMO
m
96-
68--
46-
+
Vol. 42, No. 20, 1988
HSV-2 4 hr -+-+-+
7hr
qpmamQR~-
14 hr FIG. 3 Protein synthesis in polyamine depleted and control cells infected with Herpes simplex virus. Vero cells were cultured with and without DFMO (0.5mM) for 6 days and infected with HSV-2 (5 pfu/cell). After 4,7 and 14 hr the cells were labeled with 35S-methionine UOuCi/ml) for 0.5 hr and the labeled proteins were analyzed by SDS-gel electrophoresis using 8% polyacrylamide.
kd
For calculation of the number of virus particles the polyamine depleted and control Vero cells infected with HSV-2 were prepared for electron microscopy 8, 15 and 24 hr post infection. At 8 hr the nuclei were already found to contain assembled capsids, at 24 hr the cells showed marked degenerative changes with abundant nuclear and cytoplasmic virus particles. No obvious differences in the amount or in the appearance of intranuclear or cytoplasmic virus particles was found between the polyamine depleted and the control cells. The samples at 15 hr were selected for closer scrutiny. In the morphometric analysis of the infected cells the number of virus particles found in the nucleus of the control cells was 20/100l.~m~ and in the polyamine depleted cells 22/100pm2. The amount of viral particles in the cytoplasm of both kinds of cells was the same, 5/100um2.
Inhibition of polyamine synthesis causes retardation of cellular proliferation in many different kinds of tissue cultures (2,9,11). It also suppresses the growth of cytomegalovirus which causes an increase in the concentration of polyamines, spermidine and spermine, in the infected cells. If polyamine synthesis is inhibited by methyl glyoxal(bis)guanyl hydrazone (12) or by DFMO (12,13) the amount of infectious cytomegalo virus produced by the cells is greatly reduced. On the other hand Tuomi et al. found that although the replication of Semliki forest virus, a close relative of Sindbis virus, was retarded by 2 hr in polyamine depleted, DFMO treated BHK cells as compared to the controls the final yield of the virus in both kinds of cells was the same (14). There is evidence suggesting that polyamine depletion inhibits replication of HSV. Treatment of BHK cells with DFMO caused 60% inhibition in the infectious titer of the virus 20 hr post infection (141, and DFMO treatment of human fibroblasts reduced the infectious titer of HSV by about 98% when measured 7 days post infection (13). In both cases the virus titers were
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Polyamine Depletion Reduces Infectivity
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determined only at one time point, and the maximal virus titers are therefore not known with certainty. Moreover, in the latter work the number of cells in the DFMO treated and nontreated cells were not counted. This is relevant considering the considerable retardation of cellular growth usually found in the presence of DFMO. In this paper we have re-examined the effect of polyamine depletion on growth of an enveloped RNA virus, Sindbis, and of an enveloped DNA virus, HSV-2, using cell strains that could be effectively depleted of polyamines. We have also made growth curves of the viruses thus distinguishing the effect on the rate of growth and on the final yield of the virus. We found that the polyamine auxotrophic P22 cells, starved of polyamines, were able to produce the same amount of infectious Sindbis virus, and at the same rate, as the control cells although there was a delay of about 2 hr in the onset of the viral replication. The results show that the viral RNA and protein synthesis could proceed successfully in cells efficiently depleted of polyamines. On the other hand the final yield of the infectious HSV-2 was considerably reduced in DFMO treated Vero cells in spite of the fact that polyamine depletion was not as good as in P22 cells. However, the number of HSV-2 particles, composed mainly of proteins, was not decreased. Moreover, analysis of the 35S-labeled proteins from the two kinds of HSV-2 infected cells did not reveal any clear differences. These results suggest that failure of protein synthesis was not responsible for the reduction of infectious titer of HSV-2 in polyamine depleted cells. The fact that the DFMO treated Vero cells were able to produce normal quantities of infectious Sindbis virus indicated that this drug did not have toxic side effects causing general inhibition of viral growth. The next question we asked was whether polyamine depletion might affect the synthesis of HSV-DNA and thus cause the reduction in the infectivity of the virus particles. Southern blotting revealed overall decrease in the intensity of the bands of the HSV-DNA from the polyamine depleted cells as compared to the control. It is conceivable that DNA synthesis is more error prone in the polyamine depleted than in the control cells and in the former case loss of the sites for the restriction enzymes may have prevented a certain fraction of DNA molecules from migrating in the gel. The errors made in individual cells would probably not be identical, and therefore mostly changes in the relative abundance rather than total loss of the DNA fragments would be expected. The results obtained with HSV are compatible with the idea that the inhibition of growth of polyamine depleted cells is mainly due to impairment of DNA synthesis. This is supported by the excellent sterospecific fit between spermine and the DNA double helix (15), giving rise to speculations that polyamines might facilitate DNA synthesis by neutralizing the excessive negative charges in the DNA. In line with this assumption are the results indicating that a shortage of polyamines causes retardation of DNA synthesis in different kinds of cells (11,16,17). Furthermore it is notable that polyamine depletion induces chromosomal damage in CHO cells (18). The results presented in this paper suggest that disturbances in the DNA synthesis are responsible for the reduction of the infectious titer of HSV-2 in polyamine depleted cells. More generally this supports the idea that polyamines are important for unerroneous synthesis of DNA. However, it cannot be excluded that a complex virus like HSV requires cellular functions, other than DNA synthesis, which are dependent on polyamines, and which are not important for the structurally more simple Sindbis virus.
2018
Polyamine Depletion Reduces Infectivity
We thank
Ms. Irma Lantinen
for competent
Vol. 42, No. 20, 1988
technical
assistance
Merrell Research Center for the gift of a- difluoromethylornithine. was supported by a grant from the Sigrid Juselius foundation.
and the
This research
References 1. 2. 3. 4. :: 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
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