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Inhibitors of prostaglandin synthesis inhibit growth of human cytomegalovirus and reactivation of latent virus in a productively and latently infected human cell line
VIROLOGY
163,205-208(1988)
Inhibitors
of Prostaglandin Synthesis Inhibit Growth of Human Cytomegalovirus and Reactivation of Latent Virus in a Productively and Latently Infected Human Cell Line
JUNJI TANAKA\.,*,’ *Department
TSUTOMU
OGURA,*
HIDETADA
IIDA,? HIROSHI SATO,” AND MOTOICHI
of virology, Cancer Research Institute, Kanazawa University, Kanazawa and tFukui Prefectural Institute of Public Health, Fukui 9 10, Japan Received June 25, 1987; accepted November
HATANO*
920, Japan,
2, 1987
lndomethacin and tetracaine, inhibitors of prostaglandin synthesis, inhibited production of infectious human cytomegalovirus (HCM\I) in a human thyroid papillary carcinoma cell line (TPC-1) by 99.9% when added to cultures at the concentration of 2 X lo-“ M during the first 24 hr after infection. Although immediate early virus proteins were synthesized at similar molar ratios in mock- and compound-treated cultures, induction of HCMV-specific DNA polymerase (one of the early virus proteins) was inhibited by treatment with these compounds, suggesting that the early stages of the virus growth cycle are most likely to be under the control of indomethacin or tetracaine action. We have previously developed an in vitro HCMV latency model system in TPC-1 cultures. This system was used to study the effect of these compounds on reactivation of the latent virus. When TPC-1 cultures preheated for 48 hr at 40.5” were infected with HCMV and incubated at 40.5”, the cultures could be maintained for 30 days without detection of infectious virus. The latent HCMV was reactivated within 10 days by reducing the incubation temperature from 40.5 to 37”. However, when the latently infected cultures were treated with indomethacin or tetracaine immediately after o 1988Academic PWS. hc. being shifted to 37”. reactivation of the latent virus was not observed.
As a consequence of primary infection in vivo, human cytomegalovirus (HCMV) usually persists in man in a latent form for the rest of life (7). Many different stimuli which cause physiological changes in the host cell may reactivate infectious virus from the latent state, thereby producing a serious disease such as interstitial pneumonia (2, 3). The physical state of the HCMV genome during the latent period and the mechanisms of its main.tenance in the latent state and virus reactivation are still poorly understood. For study of the molecular mechanisms of HCMV latent infection in viva, we have established an in vitro HCMV latency model system using a human thyroid papillary carcinoma cell line (TPC-1) as the host cell (4). In this laten8zy model, infectious virus was undetectable when HCMV-infected TPC-1 cultures were incubated at 40.!5”, while HCMV could be reactivated from the latent state by reducing the incubation temperature from 40.5 to 37”. The data obtained suggest that a critical cellular factor(s) is involved in the mechanism of the maintenance of the HCMV genome in the latent state and the reactivation of the latent virus (4). Since cer?ain inhibitors of prostaglandin synthesis can inhibit replication of RNA (5, 6) and DNA viruses in cells (5, 7, 8), the possibility has been suggested that arachidonic acid metabolites may play a role as a regulatory agent(s) for productive infections with various viruses. In the present experiments, therefore, we
have investigated the possibility that the inhibitors of prostaglandin synthesis, i.e., indomethacin and tetraCaine, could inhibit HCMV replication in productively infected TPC-1 cultures and suppress reactivation of latent virus in our in vitro HCMV latency model system. The stocks of HCMV (the Towne strain) were prepared as described previously (4). The 196 strain of herpes simplex virus type 2 (HSV-2) was propagated in TPC-1 cells and infectious virus titer was plaque assayed on the same cell. TPC-1 cells (4) were cultured and maintained after virus infection with Dulbecco’s modified Eagle’s medium (DMEM) containing 10 and 2.5% fetal calf serum, respectively. Human embryonic lung cells, used for HCMV plaque assay, were cultured with DMEM supplemented with 10% calf serum. Indomethacin, an inhibitor of cyclooxygenase (9), and tetracaine, an inhibitor of phospholipase A2 (70), were obtained from Sigma Chemical Co. and the stock solutions were prepared in 50% dimethyl sulfoxide (DMSO). The basic experiments revealed that the compounds at the concentrations of 1Oe3 M or higher exhibit a cytocidal effect on TPC-1 cells. However, these two compounds at the concentrations below 2 x 1Om4M used in this study did not alter either the rate of cell growth or cell viability when judged at 10 days after the compound addition (data not shown). In the first experiment, we studied the effect of indomethacin and tetracaine on the replication of HCMV in TPC-1 cultures. For comparison, the production of HSV-2 in the compound-treated TPC-1 cultures was
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also examined, because the inhibitory effect of the compounds on HSV growth has already been well documented (7, 8). Subconfluent (90%) TPC-1 cultures were infected with HCMV or HSV-2. After 1 hr of adsorption, maintenance medium containing various concentrations of the compounds was added. Control cultures were treated with the medium containing DMSO in an amount equal to that used in the highest concentration of the compound-treated cultures. The total amount of infectious virus was determined as previously described (4). The results, shown in Table 1, indicate that both compounds reduced HCMV yields at 6 days postinfection (p.i.) by 99.9% when added to the cultures at a concentration of 2 X 1Oe4M. Little or no reduction in virus yield was observed when the infected cultures were treated at concentrations below 2 x 1Om5M. The inhibitory effect by these compounds is reversible, since within 5 days after the removal of these compounds virus yields were recovered to a level near that at 6 days p.i. in mock-treated cultures (Table 1). lmmunofluorescence staining using HCMV immune human serum showed that in the mock-treated cultures 12% of the cells contained nuclear inclusion bodies at 6 days p.i., whereas only 0.002% of the cells in both the compound-treated cultures exhibited late virus antigens, On the other hand, synthesis of HSV-2 in TPC-1 cultures at 48 hr p.i. was inhibited by 88 and 72% by treatment with 2 x 1Oe4 M indomethacin and tetracaine, respectively. Thus the replication of HCMV in TPC-1 cultures seems to be more dependent on a factor(s) that is compromised by treatment with indomethacin or tetracaine than is the replication of HSV-2.
To elucidate the stages in the virus growth cycle that are affected by the action of these compounds, TPC-1 cultures were treated with indomethacin or tetracaine before and after virus infection and virus yield at 5 days p.i. was compared to that in mock-treated controls. Pretreatment of TPC-1 cultures with indomethacin or tetracaine for 48 hr prior to virus infection reduced virus yield by 89 or 85%, respectively. In addition, synthesis of infectious virus in the cultures treated at 1 or 24 hr after infection was consistently reduced by more than 99.9%. However, when the cultures were treated at 48 or 72 hr p.i. approximately 99 or 90% reduction in yields was observed, respectively (data not shown). These results suggest that the early stages of the virus growth cycle are most likely to be under the control of the action of these compounds. Consequently, to determine the molecular mechanism by which indomethacln or tetracaine inhibits virus replication, expression of immediate early (IE) and early virus functions in the compound-treated cultures was investigated. For study of the effect of indomethacin or tetracaine on expression of IE virus functions, synthesis of IE virus proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As shown in Flg. 1, two IE virus proteins with apparent mol wt of 72,000 and 76,000 were detectable at similar molar ratios in HCMV-infected mock(lane A), indomethacin- (lane B), and tetracaine-treated cultures (lane C), but not in mock-infected cultures (lanes D-F). The effect of the compounds on expression of an early virus function was assessed by induction of virus-specific DNA polymerase, one of the early virus functions (12). DNA polymerase activity in-
TABLE 1 INHIBITORYEFFECTOFINDOMETHACINORTETRACAINEONREPLICATIONOFHCMVOR HSV-2 INTPC-1 CULTURES~ Virus yield (PFU/ml) HCMVb Expt.
Compound
1
lndomethacin
2
Tetracaine
Concentration W/I) 0 2 x 1om6 2 x 1om5 2 x 1om4 0 2 x 1o-6 2 x 1om5 2 x 1o-4
2 days 8.7 5.2 8.5 8.2 2.2 2.5 3.0 2.7
X X X X x X x X
10' 10' 10’ 10’ 10' 10' 10’ 10’
6 days 1.0 1.0 7.7 1.1 2.9 2.7 1.9 2.5
x x x x x X x X
lo4 lo4 lo3 10' lo4 lo4 lo4 10’
HSV-2= 11 da@
2 hr
48 hr
3.8 X lo5 ND" ND 8.5 X lo3 6.1 X lo5 ND ND 1.8 X lo4
2.7 x 10’ ND 3.1 x 10' 2.7 x 10’ 6.0 x 10' ND 4.5 x 10’ 4.9 x 10’
3.0 x lo8 ND 1.0 x lo* 3.6 x 10’ 4.2 X 10’ ND 1.6 x 10’ 1.2 x lo*
a TPC-1 cultures were infected with HCMV or HSV-2 at a multiplicity of 5 or 0.01 plaque-formrng units (PFU)/cell, respectively. After 1 hr of adsorption maintenance medium containing indicated concentration of indomethacin or tetracaine was added. b Total amount of infectious HCMV was determined at 2, 6, and 1 1 days after infection. c Total amount of infectious HSV-2 was determined at 2 and 48 hr after infection. d At 6 days p.~.,medium was replaced with maintenance medium lacking the compound and Incubated for an additronal 5 days. e ND, not determined.
SHORT COMMUNICATIONS
A
B
C
D
E
F
Mr 76000. 72000(
FIG. 1. Effect of indomethacin and tetracaine on synthesis of IE virus proteins in TPC-1 cultures. TPC-1 cultures were infected with HCMV at a multiplicity of 10 PFWcell (A-C), or mock-infected (D-F). The cultures Iwere treated with cycloheximide (50 pg/ml) 1 hr before infection, durlllg the 1-hr viral adsorption period, and from 1 to 6 hr after infectiorl The cultures were then labeled for 2 hr with [%]methionine (30 pCi/ml) in methionine-free maintenance medium containing actinomycin D (10 ,rg/ml). The IE virus proteins were extracted, Immunoprecipitated, and separated by electrophoresis as described previously (4, 11). (A,D) Mock-treated cultures. (B,E) Cultures treated ,continuously ounng transcription and translation with indomethaclr (2 X 1Om4M) or (C,F) with tetracaine (2 X 1Om4n/l).
creased with time after infection in mock-treated cultures (Fig. 2). However, appreciable amounts of enzyme actiblty were not induced in both indomethacinand tetracaine-treated cultures during the 72-hr period of this experiment. In previous experiments, we have been able to develop an ;,,I vitro HCMV latency model system using TPC-1 cultures as the host cell (4). In the latently infected cultures, the blockage of HCMV replication occurs at the early stages of the virus growth cycle, because t’le IE virus proteins and antigens were detectable, vvhereas HCMV-specific DNA polymerase was undeiectable in the cells during the latent period (4). It is, therefore, of interest to examine whether indomethac I or tetracaine could inhibit HCMV reactivation in this latency model system. To develop HCMV latently infected cultures, TPC-1 cells were preheated at 40.5” for 48 hr prior to virus infection and incubated at 40.5” after infection with medium changes at 5-day intervals. Under these experimental conditions, HCMV-infected TPC-1 cultures could be maintained for 30 days without detection of infectious virus (Fig. 3). When the cultures were shifted from 40.5 to 37” at 30 days p.i., infectious, virus became detectable within 10 days, indicating the reactivation of latent HCMV. However, in the cultures treated with indomethacin immediately after being shifted to 37”, infectious virus
207
remained undetectable during the remaining 40-day incubation period. Infectious HCMV could be reactivated within 10 days when indomethacin was removed from the cultures at 15 days after the compound addition (45 days p.i.), indicating that the inhibitory effect by indomethacin on reactivation of latent HCMV is reversible. A similar inhibitory effect on the reactivation of latent HCMV was also observed in the latently infected cultures treated with tetracaine. When HCMV-infected cultures maintained at 40.5” were shifted to 37” at 30 days p.i., infectious virus could be reactivated in all five cultures. The amount of infectious virus was found to range from 1 .O X 10’ to 9.7 x 10’ PFU/ml at 15 days after the temperature shift. However, in the cultures treated with tetracaine (2 X 10e4 n/l) when the incubation temperature was reduced to 37”, infectious virus was not reactivated within 15 days in all five cultures tested (data not shown). The results presented in this paper indicate that the inhibitors of prostaglandin synthesis, indomethacin and tetracaine, inhibit HCMV replication in the productively infected TPC-1 cultures and suppress reactivation of infectious HCMV in the latently infected TPC-1 cultures. These results are consistent with the reports by Newton (7), Harbour et al. (8), and Kurane et al. (73) who showed that the replication of HSV in conventional tissue culture was inhibited and the reactivation of latent HSV in the explanted murine trigeminal gan-
UY 0
0Time
0
24
after
virus
48 infection
72 (hr)
DNA polymerase in indomethFIG. 2. Induction of HCMV-specific acin- or tetracaine-treated TPC-1 cultures. TPC-1 cultures were infected with HCMV at a multiplicity of 5 PFU/cell. After 1 hr of adsorption, maintenance medium containing DMSO (O), indomethacin (0), or tetracaine (B) was added. At 24.hr intervals after infection, the total cell extracts were prepared and assayed for virus-specified DNA polymerase activity in the presence of 100 mM ammonium sulfate as previously described (7 1).
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208
104-
.a
/m-PI’
,p
:
lo=
F
{I
/
102-
i
:
: IO’ -
:
0
i i
: :
loo tect-
i
l
-
-
10
-
= I
20 Time
c--4--~--~=-~--~--~--o--~ I I I I
30 after
40
virus
I
50
infection
I
I
I
60
I
70
(day)
FIG. 3. Effect of indomethacin on reactivation of latent virus In an in vitro HCMV latency system. TPC-1 cultures preheated at 40.5” for 48 hr were infected with HCMV at a multiplicity of 5 PFU/cell and incubated at 40.5” with medium changes at 5-day intervals (O - 0). At 30 days p.i., the cultures were shifted to 37” and incubated in the presence (0 0) or absence (0 - - - 0) of indomethacin (2 X 1O-“ M). One group of indomethacin-treated cultures received the medium lacking the compound at 15 days after indomethacin addition $ -. - n), At the indicated time after infection, the total amount of infectious virus was determined (4).
glia was suppressed by treatment with these compounds. However, whether these phenomena have similar molecular mechanisms is presently not known. In the present experiment, we have found that indomethacin and tetracaine are most effective if added to cells during the first 24 hr after infection. In addition, gel electrophoresis analysis and DNA polymerase assay revealed that in the compound-treated cultures synthesis of the IE virus proteins was not affected, whereas induction of virus-specific DNA polymerase was inhibited. These observations suggest that the block of HCMV replication in the compound-treated cultures presumably occurs at the level of early transcription or translation of the viral genome. Because pretreatment of TPC-1 cultures with these compounds, which are then removed before infection, significantly reduced virus yield when compared to mock-treated control, it is most plausible that these compounds may act through some effect on a cellular metabolic system that in turn affects expression of the early virus gene. However, further studies are necessary to determine whether arachidonic acid metabolites including prostaglandins are involved directly in the replication of HCMV and the reactivation of the latent HCMV or whether the inhibitory effects by these compounds are due to some of their other functions. These studies will provide further insight into the role of arachidonic acid metabolites as a regulatory cellular
factor(s) for productive cultures with HCMV.
and latent infections
of TPC-1
ACKNOWLEDGMENT This work was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture of Japan.
REFERENCES 7. RAPP, F., and GEDER, L., ln “Persistent Viruses, ICN-UCLA Symposia on Molecular and Cellular Biology” (.I. G. Stevens, G. J. Todaro, and C. F. Fox, Eds.), pp. 767-785. Academic Press, New York, 1978. 2. PAGANO,J. S.,/. Infect. Dis. 132, 114-120(1975). 3. RUBIN, R. H., RUSSELL,P. S., LEVIN, M., and COHEN, C., /. Infecr. Dis. 139, 728-734 (1979). 4. TANAKA, J., OGURA, T., SATO, H., and HATANO. M., Virology 161, 62-72 (1987). 5. INGLOT, A. D., 1. Gen. Vkol. 4, 203-214 (1969). 6. MUKHERJEE,R. K., and SIMPSON, R. W., virology 135, 345-355 (1984). 7. NEWTON, A. A., Adv. Ophthalmol. 38, 58-63 (1979). 8. HARBOUR, D. R., BLYTH, W. A., and HILL, T. J., /. Gen. Viral. 41, 87-95 (1978). 9. FLOWER, R., and VANE, 1. R., Biochem. Pharmacol. 23, 1439-1450 (1974). 10. RUBIN, R. P., and LAYCHOCK,S. G., In “Calcium in Drug Action” (G. B. Weiss, Ed.), pp. 135-l 88. Plenum, New York, 1978. 11. TANAKA, J., KAMIYA, S., OGURA, T., SATO, H., OGURA. H., and HATANO, M., virology146, 165-176 (1985). 12. HUANG, E.-S.,/. Viral. 16, 298-310 (1975). 73. KURANE, I., TSUCHIYA, Y., SEKIZAWA,T., and KUMAGAI, K., /. Gen. l’kol. 65, 1665-l 674 (1984).
163,205-208(1988)
Inhibitors
of Prostaglandin Synthesis Inhibit Growth of Human Cytomegalovirus and Reactivation of Latent Virus in a Productively and Latently Infected Human Cell Line
JUNJI TANAKA\.,*,’ *Department
TSUTOMU
OGURA,*
HIDETADA
IIDA,? HIROSHI SATO,” AND MOTOICHI
of virology, Cancer Research Institute, Kanazawa University, Kanazawa and tFukui Prefectural Institute of Public Health, Fukui 9 10, Japan Received June 25, 1987; accepted November
HATANO*
920, Japan,
2, 1987
lndomethacin and tetracaine, inhibitors of prostaglandin synthesis, inhibited production of infectious human cytomegalovirus (HCM\I) in a human thyroid papillary carcinoma cell line (TPC-1) by 99.9% when added to cultures at the concentration of 2 X lo-“ M during the first 24 hr after infection. Although immediate early virus proteins were synthesized at similar molar ratios in mock- and compound-treated cultures, induction of HCMV-specific DNA polymerase (one of the early virus proteins) was inhibited by treatment with these compounds, suggesting that the early stages of the virus growth cycle are most likely to be under the control of indomethacin or tetracaine action. We have previously developed an in vitro HCMV latency model system in TPC-1 cultures. This system was used to study the effect of these compounds on reactivation of the latent virus. When TPC-1 cultures preheated for 48 hr at 40.5” were infected with HCMV and incubated at 40.5”, the cultures could be maintained for 30 days without detection of infectious virus. The latent HCMV was reactivated within 10 days by reducing the incubation temperature from 40.5 to 37”. However, when the latently infected cultures were treated with indomethacin or tetracaine immediately after o 1988Academic PWS. hc. being shifted to 37”. reactivation of the latent virus was not observed.
As a consequence of primary infection in vivo, human cytomegalovirus (HCMV) usually persists in man in a latent form for the rest of life (7). Many different stimuli which cause physiological changes in the host cell may reactivate infectious virus from the latent state, thereby producing a serious disease such as interstitial pneumonia (2, 3). The physical state of the HCMV genome during the latent period and the mechanisms of its main.tenance in the latent state and virus reactivation are still poorly understood. For study of the molecular mechanisms of HCMV latent infection in viva, we have established an in vitro HCMV latency model system using a human thyroid papillary carcinoma cell line (TPC-1) as the host cell (4). In this laten8zy model, infectious virus was undetectable when HCMV-infected TPC-1 cultures were incubated at 40.!5”, while HCMV could be reactivated from the latent state by reducing the incubation temperature from 40.5 to 37”. The data obtained suggest that a critical cellular factor(s) is involved in the mechanism of the maintenance of the HCMV genome in the latent state and the reactivation of the latent virus (4). Since cer?ain inhibitors of prostaglandin synthesis can inhibit replication of RNA (5, 6) and DNA viruses in cells (5, 7, 8), the possibility has been suggested that arachidonic acid metabolites may play a role as a regulatory agent(s) for productive infections with various viruses. In the present experiments, therefore, we
have investigated the possibility that the inhibitors of prostaglandin synthesis, i.e., indomethacin and tetraCaine, could inhibit HCMV replication in productively infected TPC-1 cultures and suppress reactivation of latent virus in our in vitro HCMV latency model system. The stocks of HCMV (the Towne strain) were prepared as described previously (4). The 196 strain of herpes simplex virus type 2 (HSV-2) was propagated in TPC-1 cells and infectious virus titer was plaque assayed on the same cell. TPC-1 cells (4) were cultured and maintained after virus infection with Dulbecco’s modified Eagle’s medium (DMEM) containing 10 and 2.5% fetal calf serum, respectively. Human embryonic lung cells, used for HCMV plaque assay, were cultured with DMEM supplemented with 10% calf serum. Indomethacin, an inhibitor of cyclooxygenase (9), and tetracaine, an inhibitor of phospholipase A2 (70), were obtained from Sigma Chemical Co. and the stock solutions were prepared in 50% dimethyl sulfoxide (DMSO). The basic experiments revealed that the compounds at the concentrations of 1Oe3 M or higher exhibit a cytocidal effect on TPC-1 cells. However, these two compounds at the concentrations below 2 x 1Om4M used in this study did not alter either the rate of cell growth or cell viability when judged at 10 days after the compound addition (data not shown). In the first experiment, we studied the effect of indomethacin and tetracaine on the replication of HCMV in TPC-1 cultures. For comparison, the production of HSV-2 in the compound-treated TPC-1 cultures was
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0 1988 by Academic Press. Inc of reproductlan I” any form reserved
SHORT COMMUNICATIONS
206
also examined, because the inhibitory effect of the compounds on HSV growth has already been well documented (7, 8). Subconfluent (90%) TPC-1 cultures were infected with HCMV or HSV-2. After 1 hr of adsorption, maintenance medium containing various concentrations of the compounds was added. Control cultures were treated with the medium containing DMSO in an amount equal to that used in the highest concentration of the compound-treated cultures. The total amount of infectious virus was determined as previously described (4). The results, shown in Table 1, indicate that both compounds reduced HCMV yields at 6 days postinfection (p.i.) by 99.9% when added to the cultures at a concentration of 2 X 1Oe4M. Little or no reduction in virus yield was observed when the infected cultures were treated at concentrations below 2 x 1Om5M. The inhibitory effect by these compounds is reversible, since within 5 days after the removal of these compounds virus yields were recovered to a level near that at 6 days p.i. in mock-treated cultures (Table 1). lmmunofluorescence staining using HCMV immune human serum showed that in the mock-treated cultures 12% of the cells contained nuclear inclusion bodies at 6 days p.i., whereas only 0.002% of the cells in both the compound-treated cultures exhibited late virus antigens, On the other hand, synthesis of HSV-2 in TPC-1 cultures at 48 hr p.i. was inhibited by 88 and 72% by treatment with 2 x 1Oe4 M indomethacin and tetracaine, respectively. Thus the replication of HCMV in TPC-1 cultures seems to be more dependent on a factor(s) that is compromised by treatment with indomethacin or tetracaine than is the replication of HSV-2.
To elucidate the stages in the virus growth cycle that are affected by the action of these compounds, TPC-1 cultures were treated with indomethacin or tetracaine before and after virus infection and virus yield at 5 days p.i. was compared to that in mock-treated controls. Pretreatment of TPC-1 cultures with indomethacin or tetracaine for 48 hr prior to virus infection reduced virus yield by 89 or 85%, respectively. In addition, synthesis of infectious virus in the cultures treated at 1 or 24 hr after infection was consistently reduced by more than 99.9%. However, when the cultures were treated at 48 or 72 hr p.i. approximately 99 or 90% reduction in yields was observed, respectively (data not shown). These results suggest that the early stages of the virus growth cycle are most likely to be under the control of the action of these compounds. Consequently, to determine the molecular mechanism by which indomethacln or tetracaine inhibits virus replication, expression of immediate early (IE) and early virus functions in the compound-treated cultures was investigated. For study of the effect of indomethacin or tetracaine on expression of IE virus functions, synthesis of IE virus proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As shown in Flg. 1, two IE virus proteins with apparent mol wt of 72,000 and 76,000 were detectable at similar molar ratios in HCMV-infected mock(lane A), indomethacin- (lane B), and tetracaine-treated cultures (lane C), but not in mock-infected cultures (lanes D-F). The effect of the compounds on expression of an early virus function was assessed by induction of virus-specific DNA polymerase, one of the early virus functions (12). DNA polymerase activity in-
TABLE 1 INHIBITORYEFFECTOFINDOMETHACINORTETRACAINEONREPLICATIONOFHCMVOR HSV-2 INTPC-1 CULTURES~ Virus yield (PFU/ml) HCMVb Expt.
Compound
1
lndomethacin
2
Tetracaine
Concentration W/I) 0 2 x 1om6 2 x 1om5 2 x 1om4 0 2 x 1o-6 2 x 1om5 2 x 1o-4
2 days 8.7 5.2 8.5 8.2 2.2 2.5 3.0 2.7
X X X X x X x X
10' 10' 10’ 10’ 10' 10' 10’ 10’
6 days 1.0 1.0 7.7 1.1 2.9 2.7 1.9 2.5
x x x x x X x X
lo4 lo4 lo3 10' lo4 lo4 lo4 10’
HSV-2= 11 da@
2 hr
48 hr
3.8 X lo5 ND" ND 8.5 X lo3 6.1 X lo5 ND ND 1.8 X lo4
2.7 x 10’ ND 3.1 x 10' 2.7 x 10’ 6.0 x 10' ND 4.5 x 10’ 4.9 x 10’
3.0 x lo8 ND 1.0 x lo* 3.6 x 10’ 4.2 X 10’ ND 1.6 x 10’ 1.2 x lo*
a TPC-1 cultures were infected with HCMV or HSV-2 at a multiplicity of 5 or 0.01 plaque-formrng units (PFU)/cell, respectively. After 1 hr of adsorption maintenance medium containing indicated concentration of indomethacin or tetracaine was added. b Total amount of infectious HCMV was determined at 2, 6, and 1 1 days after infection. c Total amount of infectious HSV-2 was determined at 2 and 48 hr after infection. d At 6 days p.~.,medium was replaced with maintenance medium lacking the compound and Incubated for an additronal 5 days. e ND, not determined.
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A
B
C
D
E
F
Mr 76000. 72000(
FIG. 1. Effect of indomethacin and tetracaine on synthesis of IE virus proteins in TPC-1 cultures. TPC-1 cultures were infected with HCMV at a multiplicity of 10 PFWcell (A-C), or mock-infected (D-F). The cultures Iwere treated with cycloheximide (50 pg/ml) 1 hr before infection, durlllg the 1-hr viral adsorption period, and from 1 to 6 hr after infectiorl The cultures were then labeled for 2 hr with [%]methionine (30 pCi/ml) in methionine-free maintenance medium containing actinomycin D (10 ,rg/ml). The IE virus proteins were extracted, Immunoprecipitated, and separated by electrophoresis as described previously (4, 11). (A,D) Mock-treated cultures. (B,E) Cultures treated ,continuously ounng transcription and translation with indomethaclr (2 X 1Om4M) or (C,F) with tetracaine (2 X 1Om4n/l).
creased with time after infection in mock-treated cultures (Fig. 2). However, appreciable amounts of enzyme actiblty were not induced in both indomethacinand tetracaine-treated cultures during the 72-hr period of this experiment. In previous experiments, we have been able to develop an ;,,I vitro HCMV latency model system using TPC-1 cultures as the host cell (4). In the latently infected cultures, the blockage of HCMV replication occurs at the early stages of the virus growth cycle, because t’le IE virus proteins and antigens were detectable, vvhereas HCMV-specific DNA polymerase was undeiectable in the cells during the latent period (4). It is, therefore, of interest to examine whether indomethac I or tetracaine could inhibit HCMV reactivation in this latency model system. To develop HCMV latently infected cultures, TPC-1 cells were preheated at 40.5” for 48 hr prior to virus infection and incubated at 40.5” after infection with medium changes at 5-day intervals. Under these experimental conditions, HCMV-infected TPC-1 cultures could be maintained for 30 days without detection of infectious virus (Fig. 3). When the cultures were shifted from 40.5 to 37” at 30 days p.i., infectious, virus became detectable within 10 days, indicating the reactivation of latent HCMV. However, in the cultures treated with indomethacin immediately after being shifted to 37”, infectious virus
207
remained undetectable during the remaining 40-day incubation period. Infectious HCMV could be reactivated within 10 days when indomethacin was removed from the cultures at 15 days after the compound addition (45 days p.i.), indicating that the inhibitory effect by indomethacin on reactivation of latent HCMV is reversible. A similar inhibitory effect on the reactivation of latent HCMV was also observed in the latently infected cultures treated with tetracaine. When HCMV-infected cultures maintained at 40.5” were shifted to 37” at 30 days p.i., infectious virus could be reactivated in all five cultures. The amount of infectious virus was found to range from 1 .O X 10’ to 9.7 x 10’ PFU/ml at 15 days after the temperature shift. However, in the cultures treated with tetracaine (2 X 10e4 n/l) when the incubation temperature was reduced to 37”, infectious virus was not reactivated within 15 days in all five cultures tested (data not shown). The results presented in this paper indicate that the inhibitors of prostaglandin synthesis, indomethacin and tetracaine, inhibit HCMV replication in the productively infected TPC-1 cultures and suppress reactivation of infectious HCMV in the latently infected TPC-1 cultures. These results are consistent with the reports by Newton (7), Harbour et al. (8), and Kurane et al. (73) who showed that the replication of HSV in conventional tissue culture was inhibited and the reactivation of latent HSV in the explanted murine trigeminal gan-
UY 0
0Time
0
24
after
virus
48 infection
72 (hr)
DNA polymerase in indomethFIG. 2. Induction of HCMV-specific acin- or tetracaine-treated TPC-1 cultures. TPC-1 cultures were infected with HCMV at a multiplicity of 5 PFU/cell. After 1 hr of adsorption, maintenance medium containing DMSO (O), indomethacin (0), or tetracaine (B) was added. At 24.hr intervals after infection, the total cell extracts were prepared and assayed for virus-specified DNA polymerase activity in the presence of 100 mM ammonium sulfate as previously described (7 1).
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FIG. 3. Effect of indomethacin on reactivation of latent virus In an in vitro HCMV latency system. TPC-1 cultures preheated at 40.5” for 48 hr were infected with HCMV at a multiplicity of 5 PFU/cell and incubated at 40.5” with medium changes at 5-day intervals (O - 0). At 30 days p.i., the cultures were shifted to 37” and incubated in the presence (0 0) or absence (0 - - - 0) of indomethacin (2 X 1O-“ M). One group of indomethacin-treated cultures received the medium lacking the compound at 15 days after indomethacin addition $ -. - n), At the indicated time after infection, the total amount of infectious virus was determined (4).
glia was suppressed by treatment with these compounds. However, whether these phenomena have similar molecular mechanisms is presently not known. In the present experiment, we have found that indomethacin and tetracaine are most effective if added to cells during the first 24 hr after infection. In addition, gel electrophoresis analysis and DNA polymerase assay revealed that in the compound-treated cultures synthesis of the IE virus proteins was not affected, whereas induction of virus-specific DNA polymerase was inhibited. These observations suggest that the block of HCMV replication in the compound-treated cultures presumably occurs at the level of early transcription or translation of the viral genome. Because pretreatment of TPC-1 cultures with these compounds, which are then removed before infection, significantly reduced virus yield when compared to mock-treated control, it is most plausible that these compounds may act through some effect on a cellular metabolic system that in turn affects expression of the early virus gene. However, further studies are necessary to determine whether arachidonic acid metabolites including prostaglandins are involved directly in the replication of HCMV and the reactivation of the latent HCMV or whether the inhibitory effects by these compounds are due to some of their other functions. These studies will provide further insight into the role of arachidonic acid metabolites as a regulatory cellular
factor(s) for productive cultures with HCMV.
and latent infections
of TPC-1
ACKNOWLEDGMENT This work was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture of Japan.
REFERENCES 7. RAPP, F., and GEDER, L., ln “Persistent Viruses, ICN-UCLA Symposia on Molecular and Cellular Biology” (.I. G. Stevens, G. J. Todaro, and C. F. Fox, Eds.), pp. 767-785. Academic Press, New York, 1978. 2. PAGANO,J. S.,/. Infect. Dis. 132, 114-120(1975). 3. RUBIN, R. H., RUSSELL,P. S., LEVIN, M., and COHEN, C., /. Infecr. Dis. 139, 728-734 (1979). 4. TANAKA, J., OGURA, T., SATO, H., and HATANO. M., Virology 161, 62-72 (1987). 5. INGLOT, A. D., 1. Gen. Vkol. 4, 203-214 (1969). 6. MUKHERJEE,R. K., and SIMPSON, R. W., virology 135, 345-355 (1984). 7. NEWTON, A. A., Adv. Ophthalmol. 38, 58-63 (1979). 8. HARBOUR, D. R., BLYTH, W. A., and HILL, T. J., /. Gen. Viral. 41, 87-95 (1978). 9. FLOWER, R., and VANE, 1. R., Biochem. Pharmacol. 23, 1439-1450 (1974). 10. RUBIN, R. P., and LAYCHOCK,S. G., In “Calcium in Drug Action” (G. B. Weiss, Ed.), pp. 135-l 88. Plenum, New York, 1978. 11. TANAKA, J., KAMIYA, S., OGURA, T., SATO, H., OGURA. H., and HATANO, M., virology146, 165-176 (1985). 12. HUANG, E.-S.,/. Viral. 16, 298-310 (1975). 73. KURANE, I., TSUCHIYA, Y., SEKIZAWA,T., and KUMAGAI, K., /. Gen. l’kol. 65, 1665-l 674 (1984).