VIROLOGY

85,622~625

(1978,

Enhanced Poliovirus Replication in Cytomegalovirus-Infected Fi broblasts TORU FURUKAWA,’

JONG-HO

JEAN,

AND

STANLEY

The Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia,

Human

A. PLOTKIN Pennsylvania

19104

Accepted December 7,1977 Replication of poliovirus in human cytomegalovirus (CMV)-infected cells is enhanced 5to lo-fold over replication in uninfected cells. Enhanced poliovirus replication in dually infected cells was not due to a difference in adsorption on infected cells and was supported by evidence of increased synthesis of polio-specific RNA. A functional CMV genome appeared to be required for the enhancement of polio replication since enhanced replication was not seen in cells infected with uv-irradiated CMV or in cultures treated with the inhibitors of CMV replication. Enhanced polio replication in CMV-infected cells may be due to the enhanced cellular metabolism in these cells.

Poliovirus type 1 (CHAT strain) was carried in this laboratory. Stocks were prepared by inoculating virus at a multiplicity of infection (m.0.i.) of 0.1 into con&rent Vero cells and incubating cultures at 37” for 24 hr. Viral stock usually contained 7-9 x 10’ plaque forming units (PFU)/ml of poliovirus. Stocks of VSV, Indiana strain, were similarly prepared in MRC-5 cells. Infectivity of CMV was assayed on MRC-5 cells by the method described by Wentwort and French (8). Polio and VSV viruses were assayed on Vero cells, a green monkey kidney cell line, by the plaque method ( 9). To determine whether these viruses replicate in CMV-infected MRC-5 cells, monolayers of MRC-5 cells were infected with CMV at a m.o.i. of 10; then 1,24, and 48 hr later the cells were superinfected with poliovirus or VSV at m.o.i. of 1. At the same time noninfected MRC-5 cells were singly infected with those viruses as controls. Under the conditions used for CMV infection, infection of greater than 99% of the cells was ensured by the demonstration of nuclear inclusion bodies at the third day p.i. The infectivities obtained 24 hr after superinfection are shown in Table 1. Poliovirus replication was more than fivefold greater in dual-infected cells than in singly infected

For at least 96 hr postinfection (p.i.) with human cytomegalovirus (CMV), host cell metabolism, including synthesis of cellular DNA (1, 2) and RNA (3), is stimulated. Whether activated cellular metabolism is necessary for the replication of CMV is unknown. In contrast, infection with many other viruses, such as herpes simplex, vesicular stomatitis virus (VSV) and poliomyelitis, quickly leads to an inhibition of host cell metabolism (4-6). Since single infection with these viruses and single infection with CMV apparently have opposite effects on host cell macromolecular synthesis, it was of interest to investigate their interaction in dually infected cells. MRC-5 human lung fibroblasts were obtained from the Medical Research Council of the United Kingdom. The cells were grown at 37” as monolayer cultures in minimum essential medium (MEM) containing 10 and 2% fetal calf serum (FCS) for growth and maintenance, respectively. The CMV “Towne” strain used in this study was isolated in our laboratory from an infant with congenital infection and passaged on WI-38 and MRC-5 human fibroblasts. The growth characteristics of the strain have been described elsewhere ( 7). ’ To whom

requests

for reprints

should

be sent. 622

0042-6822/78/0852-0622$02.00/O Copyright All rights

0 1978 by Academic Press, Inc. of reproduction in any form reserved.

SHORT

control cultures. The enhancement of replication was most distinct when the second infection was performed 48 hr after infection with CMV. For this reason, in subsequent experiments using superinfected cells, the second infection was always performed 48 hr p.i. with CMV. In contrast with these findings, the replication of VSV was inhibited at all times tested. The greatest degree of inhibition was observed when super-infection with VSV was carried out at 48 hr p.i. with CMV. To see whether the difference between viral replication of CMV-infected cells and that of noninfected cells might reflect a difference in the ability to replicate polio, adsorption of polio to both cells was performed. Monolayers of both infected and noninfected cells adsorbed poliovirus from the supernatant medium at nearly identical rates. This result suggested that the enhanced replication of polio we observed was not due to a difference in the adsorption of the polio inoculum, but rather that it reflected alterations of some later process involved in replication. To observe polio RNA synthesis in dualinfected cells, the synthesis of actinomycin D-resistant RNA was measured. Infected cultures were labeled for 8 hr with [3H]uridine in the presence of actinomycin D (5 pg/ml) immediately after polio infection TABLE REPLICATION

TABLE IN CMV-

Yield

@‘W/ml) 1 24 48

10SJ (los.‘)b w7 (10s.‘) log” (lO=y

of CMV-infected cells. Synthesis of actinomycin D-resistant RNA in dual-infected cells increased eightfold over that in cells singly infected with polio (Table 2). The rate of presumed polio RNA synthesis correlated well with the rate of increased infectivity in dually infected cells. In order to determine what forms of polio RNA were increased in dually infected cells, acrylamide gel electrophoresis was performed using the method described by Nobel and Lewintow (10). Superinfected cultures were labeled for 3 hr with 20 $!i/ ml of [3H]uridine in the presence of actinomycin D. Phenol-extracted RNA was analyzed by gel electrophoresis. Three peaks were resolved in the same positions as multistranded replicative intermediate, doublestranded replicative, and single-stranded RNA from cells singly infected with poliovirus. These results indicate that increased synthesis of actinomycin D-resistant RNA reflects an increase in three species of poliospecific RNA. To determine whether the replication of CMV is necessary for the enhancement of polio replication, cells were infected with CMV and with CMV inactivated by uv (10 min) and heat (56’, 30 min), then superinfected with polio. The dual-infected cultures with infectious CMV were treated with inhibitors of CMV replication such as actinomycin D (5 pg/ml) and cytosine ara-

1

OF POLIOVIRUS OR VSV INFECTED CELLS D

Polio

623

COMMUNICATIONS

2

RNA SNYTHESIS IN CMV-INFECTED FIBROBLASTS WITH OR WITHOUT SUPERINFECTION~

[3H]Uridine

Treatment VW

106.’ ( 108.3) 107.2 (lo-) 106.2 (log.‘)

a Monolayers of MRC-5 cells in petri dishes containing 8 x 105 cells/dish were infected with CMV at a m.o.i. of 10. At 1, 24, and 48 hr later cells were superinfected with polio or VSV at a m.o.i. of 1. At the same times, noninfected cells were singly infected with polio or VSV as controls. Twenty-four hours after superinfection, yields of polio or VSV were assayed on Vero cells by the plaque method ( 9). ’ Value within parentheses indicates yield from control.

CMV CMV + actinomycin D Actinomycin D CMV + polio Polio + actinomycin D CMV + polio + actinomycin

HUMAN POLIO

(cpm)

D

20,034 241 116 16,666 1,033 8,513

n After the labeling period, cultures were washed three times with cold PBS. Cells were solubilized with a 1% solution of sodium dodecyl sulfate. Then cold trichloroacetic acid (TCA) was added to a final concentration of 5%, and the mixture was kept at 4’. The precipitates were collected on Millipore filter disks and washed with 5% TCA. Radioactivity was assayed in a Beckman scintillator.

624

SHORT

COMMUNICATIONS

binoside (100 pg/ml). When these drugs were added to the medium, CMV replication was inhibited (3, 11,12). There was no enhancement of poliovirus replication in cells infected with uv-irradiated, heat-inactivated CMV but enhancement occurred in the cultures treated with actinomycin D and cytosine arabinoside. Therefore, it appeared that the replication of infectious CMV was not a prerequisite for enhanced poliovirus replication. To analyze the DNA synthesized in superinfected cells, CMV-infected cultures were labeled for 24 hr with [3H]thymidine after superinfection with polio. A comparison of the gradients containing DNA from CMV-infected and double-infected cultures indicated that the incorporation of [3H]thymidine into CMV DNA was almost the same in singly and dually infected cells (Fig. 1). Analysis of DNA synthesized from 0 to 12 hr and from 12 to 24 hr after superinfection (i.e., 48-60 and 60-72 hr p.i.) was done after isopycnic centrifugation. In superinfected cultures, most of the CMV DNA was synthesized during the first 12 hr and ccllular DNA increased as much as in singly infected cultures. Less CMV DNA was synthesized in the double-infected cultures during the second 12-hr period, and there was a concomitant

IO SOTTON

20

30

FRACTION

decrease in the synthesis of cellular DNA. In contrast, cultures singly infected with CMV showed an increasing rate of both cellular and CMV DNA synthesis at 24 hr postsuperinfection. The evidence presented here shows that poliovirus replication is 5- to lo-fold greater in CMV-infected human fibroblasts than in uninfected cells. Enhanced poliovirus replication in dually infected cells was not due to a difference in adsorption on infected cells and was supported by evidence of increased synthesis of polio-specific RNA. A functional CMV genome appeared to be required for the enhancement of polio replication since enhanced replication was not seen in cells infected with uv-irradiated CMV or in cultures treated with the inhibitors of CMV replication. Although the molecular basis of the mechanism for enhanced replication of poliovirus was not elucidated in these studies, it seems that replication of CMV genome was not necessary for enhancement since intensified polio replication in super-infected cells was observed before the onset of CMV DNA synthesis. In the host cell human CMV induces synthesis of such cellular macromolecules as DNA, RNA, thymidine kinase, and DNA polymerase before the onset of viral DNA synthesis. The role of increased cellular metabolism in relation to

40 NUMBER

50

60 TOP

FIG. 1. Cells infected with CMV only or cells dually infected with CMV and polio were incubated for 24 hr after superinfection with media containing [3H]thymidine (5 @i/ml). After a 24-hr incubation, infected cells were washed in buffer (0.01 M Tris, pH 7.4, 0.01 M KCl, and 0.0015 M MgC12) and then incubated in the same buffer plus 1% SarkosylS7 for 15 min at 60”. Pronase (final concentration, 5 mg/ml) was added to the lysed cell suspension which was then incubated for a further 2 hr at 37’. The treated cell lysates were mixed with cesium chloride (4.55 g/3.5-ml sample). Isopycnic centrifugation was performed in an SW 50.1 rotor at 35,000 rpm for 64 hr. The fractions were precipitated with 5% TCA, precipitates were filtered and counted. (0) CMV only; (0) CMV and polio.

SHORT

625

COMMUNICATIONS

viral replication has not been well understood, but it might be due to the activation of the cell genome by CMV infection. The kinetics of poliovirus RNA synthesis and virus production have been shown to be dependent upon the cell cycle phase of the host ( 13). Thus, the greater yield of virus might result from a greater availability of cellular synthetic processes. Considering these results together, it is conceivable that enhanced polio replication in CMV-infected cells could be due to the enhanced cellular metabolism in these cells. However, comparison of gradients containing DNA from CMV-infected and double-infected cultures (Fig. 1) introduced another interesting aspect. DeMarchi and Kaplan (14) reported that CMV DNA synthesis proceeded at the same rate in cells treated with 5-fluorouracil as in untreated cells and that replication of CMV DNA does not depend on cellular DNA synthesis. On the other hand, evidence of the dependence of CMV growth on cellular DNA synthesis was suggested by findings that CMV growth occurs concomitantly with the stimulation of cellular DNA, and CMV growth in growing cells is faster and greater than that in resting cells. The same amount of CMV DNA was synthesized in dually infected cells as in singly infected cells. It was noted that DNA corresponding to cellular DNA was lacking in the superinfected cultures. Since polio infection gradually leads to the shutoff of host DNA synthesis (4), it is likely that cellular DNA was not necessary for the synthesis of CMV DNA. Although we cannot exclude the possibility that, in cells superinfected with poliovirus, host DNA synthesis might occur at a level that allows CMV DNA synthesis, our data suggest that host and CMV DNA synthesis need not occur simultaneously. However, analysis of DNA synthesized from 0 to 12

hr and from 12 to 24 hr after superinfection revealed a sharp decrease in viral DNA synthesis in superinfected cells during the second period. Concomitantly, there was a parallel decrease in cellular DNA synthesis. Super-infection will provide a good tool to study further the role of cellular DNA in CMV replication. ACKNOWLEDGMENTS We acknowledge with thanks the excellent cal assistance of Ruth Herold. This work ported, in part, by USPHS Research Grants from the National Institute for Allergy and Diseases and RR-05540 from the Division of Resources.

techniwas supAI-12151 Infectious Research

REFERENCES

5. 6. 7. 8. 9.

IO.

11. 12. 13. 14.

ST. JEOR, S. C., ALBRECHT, T. B., FUNK, F. D., and RAPP, F., J. Viral. 13, 353-362 (1974). FURUKAWA, T., TANAKA, S., and PLOTKIN, S. A., Proc. Sot. Exp. Biol. Med. 148,211-214 (1975). TANAKA, S., FURUKAWA, T., and PLOTKIN, S. A., J. Virol. 15, 297-304 (1975). HOLLAND, J. J., and PETERSON, J. A., J. Mol. Biol. 8,556-573 (1964). SYDISKIS, R. J., and ROIZMAN, B., Virology 42, 1043-1053 (1967). HUANG, A. S., and WAGNER, R. R., Proc. Nat. Acad. Sci. USA 54,1579-15&t (1965). FURUKAWA, T., FIORETTI, A., and PLOTKIN, S. A., J. Viral. 11, 991-997 (1973). WENTWORT, B. B., and FRENCH, L., Proc. SOC. Exp. Biol. Med. 135,253-258 (1970). BISHOP, J. M., and KOCH, G., In “Fundamental Techniques in Virology” (K. Habel and N. P. Salzman, eds.). Academic Press, New York, 1969. NOBEL, J., and LEWINTOW, L., Virology 40, 634-642 (1970). THE, T. H., KLEIN, G., and LAGENHUYSEN, M. M. A. C., CZin. Exp. Immunol. 16, 1-12 (1974). VILAGINES, R. J., and MCAUSLAN, B. R., Virology 42, 1043-1053 (1970). EREIUENKO, T., BENEDETTO, A., and VOLPE, P., Nature New Biol. 237, 114-116 (1972). DEMARCHI, M. J., and KAPLAN, A. S., J. Viral. l&1063-1070 (1976).

Enhanced poliovirus replication in cytomegalovirus-infected human fibroblasts.

VIROLOGY 85,622~625 (1978, Enhanced Poliovirus Replication in Cytomegalovirus-Infected Fi broblasts TORU FURUKAWA,’ JONG-HO JEAN, AND STANLEY...
368KB Sizes 0 Downloads 0 Views