Cytomegalovirus. for cell propagation in open containers in a 5% CO2 atmosphere, and 2% FCS and 0.15% sodium

Transcription

1 JOURNAL OF VIROLOGY, Feb. 1974, p Copyright American Society for Microbiology Vol. 13, No. 2 Printed in U.S.A. Stimulation of Cellular DNA Synthesis by Human Cytomegalovirus STEPHEN C. ST. JEOR, THOMAS B. ALBRECHT, FRED D. FUNK, AND FRED RAPP Department of Microbiology, College of Medicine, The Milton S. Hershey Medical Center of The Pennsylvania State University, Hershey, Pennsylvania Received for publication 11 September 1973 Human cytomegalovirus (CMV) is able to induce cellular DNA synthesis in both permissive (human embryonic lung) and nonpermissive (Vero) cells. The induction of cell DNA synthesis was assayed by the incorporation of [methyl- 3H Ithymidine into macromolecules having the buoyant density characteristics of cell DNA. The DNA synthesis induced by CMV infection appears to represent normal semiconservative replication as opposed to repair synthesis. Both strains of CMV tested were capable of inducing cell DNA synthesis. Virus exposed to heat or UV light prior to infection lost the ability to induce DNA synthesis, indicating that a virus-coded function expressed after infection is responsible for stimulation of cell DNA synthesis. Recent evidence indicates that certain members of the herpesvirus group might be potential oncogenic agents (1, 3, 5, 6, 10, 11, 25). Although human cytomegalovirus (CMV) has been associated with various human neoplastic diseases (8, 9), its role is felt to be that of an opportunist, secondarily infecting a debilitated host rather than contributing to the etiology of the neoplasia. Recently, human CMV has been shown to be capable of malignant transformation of hamster embryo fibroblasts (Albrecht and Rapp, in press). The latent characteristics of CMV infections (4, 15), particularly evident in patients with neoplastic disease (8, 9), and the oncogenic potential of human CMV in hamsters interested us in determining whether a characteristic common to oncogenic DNA viruses is associated with CMV. The ability to stimulate host cell DNA synthesis has been found with the majority of oncogenic DNA viruses (7, 13, 14, 21, 22). This report indicates that CMV does stimulate host cell DNA synthesis. Our data give added support to previous studies concerning the oncogenic potential of human CMV and strengthens the correlation between transformation by DNA viruses and stimulation of cell DNA synthesis. 353 MATERIALS AND METHODS Cell cultures and media. Eagle growth medium was used exclusively in these investigations. It was supplemented with 10% fetal calf serum (FCS) and 0.075% sodium bicarbonate for cell propagation in closed containers, 10% FCS and 0.225% sodium bicarbonate for cell propagation in open containers in a 5% CO2 atmosphere, and 2% FCS and 0.15% sodium bicarbonate for maintenance medium. The following antibiotics were added at the designated concentrations to all media: 100 U of penicillin per ml, 100 ug of streptomycin per ml, and 10,g of kanamycin per ml. All cell culture and virus studies were carried out at 37 C. Human embryonic lung (HEL) cells were obtained as primary cells from HEM Research Inc. (Rockville, Md.) and were used in experimental studies between passages 5 and 15. Vero cells, a stable line of African green monkey cells, are maintained in our laboratory. Cells were serially propagated in 16-oz (approximately 440-ml) glass prescription bottles and transferred to the appropriate containers for the experimental investigations. Virus strains, propagation, and assay. The strains of CMV used in these studies were Ad-169, obtained from Paul M. Feorino, Communicable Disease Center, Atlanta, Ga., and C-87, obtained from Benyesh-Melnick, Baylor College of Medicine, Houston, Tex. All virus stocks were prepared in HEL cells. Confluent monolayers of HEL cells were infected with CMV at a virus to cell ratio of as measured by plaque assay (see below) and incubated for 1 h at 37 C to allow virus adsorption. Maintenance medium was then added and replaced at 5-day intervals. Virus was harvested from infected cultures at 7 to 14 days postinfection (when 90 to 100% of the cells exhibited cytopathology) by freezing and thawing the cultures, sonicating for 2 min in a Branson sonifier (Cole-Palmer Instrument and Equipment Co.), and then clarifying the suspension by centrifugation at 16,000 x g for 10 min. The titer of virus stocks prepared in this manner varied over a range of 8 x 105 to 3 x 106 PFU per ml. Virus

2 354 ST. JEOR ET AL. J. VIROL. stocks were quick-frozen and stored at -65 C. Plaque assays to determine infectious virus titers were carried out by a method similar to the one described by Wentworth and French (24). HEL cells grown in plastic tissue culture dishes (60 by 15 mm; Falcon Plastic Co.) were infected and incubated for 2 weeks to allow for plaque development. The overlay medium used was Eagle medium supplemented with 5% FCS, % sodium bicarbonate, the normal antibiotics, and 0.25% agarose. Overlays of 5 ml were added immediately after virus adsorption and again at 1 week after infection. One week after the second overlay was added, the cells were fixed and stained and the plaques were counted. DNA analysis. The method used for DNA analysis was that described by Crouch and Rapp (2). Cells used for DNA studies were grown in 1-oz glass prescription bottles and were infected in the same manner used to prepare virus stocks. After virus adsorption, the cultures were overlaid with 3 ml of maintenance medium. Sham-infected control cultures were exposed to an equal volume of medium containing the same serum concentration as was present in the virus stocks. Infected and control cultures were incubated for sequential 24-h periods in the presence of 10 gci of [methyl-3h]thymidine ([3H ]TdR) per ml with a specific activity of 17 Ci/ mmole (Schwarz-Mann). A small volume of concentrated isotopic solution of [3H ]TdR was added directly to the medium. This avoided nonspecific stimulation of DNA synthesis due to addition of fresh serum (23). A sample of the medium was removed at the end of each 24-h labeling period and tested to insure that excess [3H JTdR was present throughout the labeling period. DNA was extracted for analysis by using Sarkosyl and Pronase as described (2). The DNA was then centrifuged to equilibrium in either neutral (density g/cm3) or alkaline (density g/cm3, ph 12.5) CsCl gradients. The samples were centrifuged in a Beckman preparatory ultracentrifuge by using either a 40.3 or an SW50.1 Beckman rotor. The velocities at which the gradients were centrifuged varied and are, therefore, indicated with each particular experiment. The gradients were collected (by bottom puncture) onto 2.4-cm Whatman no. 3 mm paper disks. Radioactivity incorporated into acid-insoluble material was determined as previously described (2). Samples of the gradient were removed at regular intervals, and the refractive index was measured with a refractometer for density determinations. Mammalian cell marker DNA was prepared by using HEL cells grown in the presence of [methyl- "4C ]thymidine (Schwarz-Mann, specific activity 36.6 mci/mmol). Virus marker DNA was prepared by continuously labeling HEL cells infected with CMV with [3H ]TdR from 2 to 5 days postinfection. The virus was harvested in the same manner as described for stock virus preparation. It was then pelleted at 30,000 rpm for 1 h in the Beckman 60 Ti rotor. The virus pellet was resuspended in isotonic Tris buffer in 1/20 of the original volume and exposed to DNase II (10 ug/ml in 10-3 M MgCl2) for 1 h at 37 C. The virus was then sedimented through a 15% sucrose solution and collected on a 60% sucrose cushion (Beckman Spinco SW27 rotor, 25,000 rpm, 2 h, 4 C). Purified virions were deproteinized with Pronase and Sarkosyl as described (2). Both virus and cell DNA markers were examined by isopycnic centrifugation as previously described and found to produce a homogeneous single DNA peak of the appropriate density reported for cell (density g/cm3) (2) and CMV DNA (density g/cm3) (19). The method used for determination of DNA synthesis by using 32P varied from the technique of DNA analysis previously described only in the method used for DNA extraction. Cultures were labeled with 32P (100 tci per ml) for 24 to 48 h post-inoculation. Cells were lysed by adding 0.3 ml of 10% Sarcosyl and 0.2 ml of 0.3 M EDTA per ml of culture fluid. The nucleic acid was extracted x5 by using hot (50 C) phenol. Residual phenol was removed by using anhydrous ether. The cultures were then exposed to pancreatic RNase (20 jag per ml, 37 C, 30 min). Virus and cell DNA were separated by isopycnic centrifugation in alkaline CsCl as described. The total counts of 32P incorporated into cell DNA were then determined by summing the counts in the cell DNA fraction. Incorporation of IUdR into cellular DNA. The method used for pretreatment of cells with 5-iodo-2'- deoxyuridine (IUdR) is that described previously by St. Jeor and Rapp (20). Replicating cells were exposed to 100 gg of IUdR per ml (Calbiochem) for 72 to 96 h. The unincorporated IUdR was then removed by washing the cells two times with isotonic Tris buffer (ph 7.4). RESULTS Effect of CMV infection on cellular DNA synthesis in permissive cells. HEL cells were used as the permissive cell system in these investigations. In an earlier report, we demonstrated that pretreatment with IUdR converted human embryonic kidney (HEK) cells (which are normally nonpermissive for CMV replication) to a permissive state. In this same study, we reported that CMV appeared to stimulate cell DNA synthesis in HEK cells which had been pretreated with IUdR (St. Jeor and Rapp, in press). Consequently, cells both untreated and pretreated with IUdR were used in these investigations. Untreated HEL cells and cells pretreated with IUdR for 96 h were either infected with CMV (Ad-169) or sham-infected as described. The cultures were then labeled for consecutive 24-h periods with [3H ]TdR. The DNA from these cells was extracted, centrifuged to equilibrium in CsCl, and examined for incorporation of [3H ]TdR. Figure 1 represents the radioactivity in the CsCl gradients from the samples labeled for 24 to 48 h postinfection. Panel A represents the IUdR-treated cells and panel B represents the untreated cells. In the untreated cells only

3 VOL. 13, 1974 STIMULATION OF CELLULAR DNA SYNTHESIS BY CMV INFECTED NF EC T E D FRACTION NUMBER FIG. 1. Neutral isopycnic centrifugation of DNA extracted from CMV-infected and uninfected HEL cells. A, Cultures pretreated with IUdR; B, untreated cultures. Replicating cell cultures were exposed to either normal Eagle growth medium or medium containing 100 jig of IUdR per ml for 96 h prior to infection. Cultures were infected as described in the text and then labeled with [3H]TdR for 24 to 48 h after infection. DNA was extracted and centrifuged in CsCl gradients (initial density g/cm3) in a Beckman 40.3 rotor at 30,000 rpm for 60 h at 20 C. Symbols: 0, counts per minute from infected cultures; 0, counts per minute from uninfected cultures; A, density determined from refractive indices. cellular DNA was detectable during the 24- to 48-h labeling period. CMV DNA was first observed in the untreated cells beginning with the 48- to 72-h labeling period. There was little difference between infected and uninfected cells in the amount of [3H ]TdR incorporated into cell DNA. In infected cells pretreated with IUdR (panel A), three peaks were resolved. A dense peak (density g/cm3) near the bottom of the tube (fraction 9) is shown later to be cellular DNA substituted with IUdR. The peak at fraction 28 has a density similar to CMV DNA, and the peak at fraction 41 has a density similar to cell DNA. The densities of the three peaks (within experimental error) is equivalent to those reported by other investigators for cell (2), virus (19), and IUdR-substituted cell DNA (16, 20). The observation that the density of the CMV DNA in panel A is the same as that reported would indicate that the procedure used for eliminating unincorporated IUdR prior to labeling with [3H]TdR was effective. If excess IUdR had been present, it would have been incorporated into virus DNA, resulting in a density shift (16, 17). The sham-infected, IUdRpretreated cells incsporated [3H ]TdR primarily into macromolecules with a density of unsubstituted cell DNA. However, the amount of cell DNA synthesis was greatly reduced in the sham-infected cells as compared to the CMVinfected cells. To examine the kinetics of incorporation of [3H ]TdR into cellular DNA, we determined the total amount of [3H]TdR incorporated into cell DNA in both IUdR-treated and untreated cells which were infected with CMV or sham infected. The results with IUdR-pretreated cells are illustrated in Fig. 2, and data with the untreated cells are shown in Fig. 3. These results support the conclusion made from the data presented in Fig. 1; cells arrested with IUdR and infected with CMV have increased incorporation of [3H]TdR into cell DNA when compared to sham-infected cultures. However, in nonarrested cells there is no significant difference between sham- and virus-infected cultures. Effect of CMV on uptake of [3H ]TdR in cells arrested with low serum concentrations. From the previous experiment (Fig. 3), it was apparent that the large amount of cel- x 10 INFECTED a. (2 4 3 _ 2 UNFECTED HOURS POST-INOCULATION FIG. 2. Kinetics of cell DNA synthesis in HEL cells pretreated with IUdR and infected with CMV (Ad-169) or sham infected. The cultures were labeled for sequential 24-h periods with [3H]TdR, and the DNA was analyzed (see legend, Fig. 1). Data points are plotted at times corresponding to the end of that particular labeling period and represent the total counts of [3H]TdR incorporated into DNA with a density of cell DNA. Symbols: A, CMV-infected cultures; *, uninfected cultures.

4 356 ST. JEOR ET AL. J. VIROL. 1* X the infected cultures: a peak in fraction 37 with a density corresponding to cell DNA, and a peak in fraction 20 with a density corresponding to CMV DNA. In the gradient from the uninfected cultures, the only peak detected (fraction 37) had the density of cell DNA. The data presented indicate that there was more than a fivefold increase in the amount of [3H ]TdR incorporated into cell DNA in infected cells as compared to sham-infected controls. Ability of inactivated CMV to induce host cell DNA synthesis. The next question examined was whether a functional virus genome is needed for the stimulation of [3H ]TdR incorporation into cellular DNA. For example, can CMV which has been inactivated by certain physical procedures, such as UV light (8,000 ergs per s per cm2 for 30 min) or heat (56 C for 30 min), stimulate cell DNA synthesis? We previously demonstrated that cells must be arrested in some manner to detect virus stimulation of cell DNA synthesis. We found IUdR pretreatment of cells to be the most reliable method for arresting DNA synthesis. Consequently, this method was used for all further HOURS POST-INOCULATION FIG. 3. Kinetics of cell DNA synthesis in nonarrested HEL cells infected with CMV (Ad-169) or sham infected. Confluent HEL cells were infected with CMV or sham infected and labeled for sequential 24-h periods with [3H]TdR, and cell DNA was analyzed (see legend, Fig. 2). Symbols: A, infected cultures; *, uninfected cultures. lular DNA synthesis in nonarrested but confluent HEL cells masked the effect of CMV on cell DNA synthesis. Previous investigators, in examining the effect of oncogenic DNA viruses on synthesis of cellular DNA, resolved this problem by arresting DNA synthesis using low concentrations of serum (12). We applied this technique to our system in the following manner. Confluent monolayers of HEL cells were incubated for 48 h in the presence of growth medium supplemented with 0.2% FCS. The cells were then either infected with CMV or sham infected with maintenance medium. One hour was allowed for virus adsorption, after which medium containing 0.2% FCS was added to the cultures. They were then labeled for 24-h periods with [3H]TdR, and the DNA was analyzed. A profile of radioactivity from a gradient of the 24- to 48-h labeling period is shown in Fig. 4. Two peaks were resolved in the gradient from C4 10 IL CMV-INFECTED o o UNINFECTED FRACTION NUMBER FIG. 4. CMV-induced stimulation of cellular DNA synthesis in HEL cells arrested by low serum concentrations. Confluent HEL cells were exposed to 0.2%/ FCS for 48 h. The cells were then infected with CMV (Ad-169) or sham infected with maintenance medium and labeled for sequential 24-h periods with [3H]TdR. The DNA was extracted and analyzed as described in the legend for Fig. 1. This figure represents the 24- to 48-h labeling period. Symbols: 0, counts per minute for the CMV-infected cultures; 0, counts per minute for the sham-infected cultures; A, density.

5 VOL. 13, 1974 STIMULATION OF CELLULAR DNA SYNTHESIS BY CMV 357 experiments concerning uptake of ['H ]TdR into cell DNA in CMV-infected cells. HEL cells pretreated with IUdR were inoculated with the following: infectious CMV (Ad-169), UV-inactivated CMV, heat-inactivated CMV, or maintenance medium. The titer of the virus stocks after inactivation was < 1 PFU per ml. Previous studies had indicated that simulation of ['H]TdR incorporation into cell DNA in CMV-infected cells occurs at 24 to 72 h postinfection. Hence, 24- to 48-h labeling periods were used to determine virus effects on cell DNA synthesis. The data from this study are presented in Table 1. The UV- and heatinactivated viruses were prepared from the same stock virus as the infectious virus and, consequently, the number of particles was the same. The data indicate that both UV light and heat inactivation of CMV reduced the ['H ]TdR uptake into cell DNA to approximately the same level as the sham-infected cultures. These results suggest that infectious virus is necessary for virus stimulation of cell DNA synthesis. Induction of host DNA synthesis by the C-87 strain of CMV. In all studies reported to this point, the Ad-169 strain of CMV was used. To determine if the ability to induce increased uptake of [3H ]TdR into cellular DNA is a characteristic of more than one strain of CMV, the C-87 strain of CMV was used to infect HEL cells arrested with IUdR. The experimental technique was the same as those previously described in this report. The results of this study are presented in Table 2. DNA induction in infected cells was first detectable during the 24- to 48-h labeling period. The data obtained from this experiment indicate that the C-87 strain of CMV also stimulates host DNA. Induction of cell DNA synthesis in an abortive infection. A virus is classified as a human cytomegalovirus based on several characteristics, one of which is that replication is limited to human fibroblasts. We were interested in determining whether human CMV could stimulate host DNA synthesis in an abortive system where no virus DNA or infectious virus is produced. We had previously observed that replication of CMV (Ad-169) DNA was not detectable in Vero cells (St. Jeor and Rapp, unpublished data). Confluent monolayers of Vero cells, in which normal cell DNA synthesis had been arrested by pretreatment with IUdR, were inoculated with either the Ad-169 strain of CMV or maintenance medium containing the same serum concentration as the virus inoculum. Replicate cultures were labeled for four consecutive 24-h periods beginning at 24 h post-inoculation. The DNA was then isolated and centrifuged in CsCl gradients, and the TABLE 2. Stimulation of HEL cellular DNA synthesis by the C-87 strain of CMVG Determination Time postinfection (h) Sham infected Counts/min of ['H JTdR incorporated into cell DNA CMV infected Counts/min of ['H]-TdR incorporated into cell DNA Fraction increase in incorporation of ['H JTdR into cell DNA of CMVinfected over shaminfected cultures ahel cells were arrested by pretreatment with IUdR. Cultures were infected with CMV or sham infected and labeled with ['HJTdR for sequential 24-h periods. The DNA was analyzed as described (Fig. 1), and total counts of ['H JTdR incorporated into cell DNA per 0.2 ml of cell extract were determined. TABLE 1. Effect of inactivation of CMV on its ability to stimulate host DNA synthesisa Sham cmv ~UV inacti- Heat inacti- Determination csham infecmv vated and vated and infected infected CMV infected CMV infected Counts/min of [H ]TdR incorporated into cell DNA... 7,878 31,452 6,548 3,666 Relative incorporation of ['H JTdR into cell DNA in CMV-infected vs. sham-infected cultures a HEL cells were arrested by pretreatment with IUdR for 96 h. The cells were then exposed to equal volumes of either maintenance medium, CMV (Ad-169), UV-inactivated (300 lethal hits per PFU) CMV, or heat-inactivated (56 C for 30 min) CMV. Twenty-four hours after infection, ['H JTdR was added to the culture media. At 48 h postinfection, the DNA was extracted and analyzed as described (legend, Fig. 1), and the total counts of ["H]TdR incorporated into cell DNA per 0.2 ml of cell digest were determined.

6 358 ST. JEOR ET AL. J. VIROL. gradients were analyzed for radioactivity. The results of this study are shown in Table 3 and Fig. 5. The results found in the CMV-Vero cell system are similar to those obtained in the permissive cell studies. There was increased incorporation of [3H ]TdR into cellular DNA with maximum stimulation of host cell DNA occurring 24 to 48 h post-inoculation (Table 3). No CMV DNA was detectable in the Vero cell extract (Fig. 5), and, as will be shown later, the peak in fraction 3 is apparently cell DNA substituted with IUdR, whereas the peak in fraction 38 is normal cell DNA. Apparent rate of DNA synthesis as measured by incorporation of [3H ]TdR versus 82P. The practice of estimating the rate of DNA synthesis based on the rate of incorporation of [3H]TdR from the media is commonly used. However, the method will give misleading results if the relative amount of exogenous thymidine versus endogenous thymidine incorporated into DNA varies between control and virusinfected cells. For example, the relative amount of exogenous thymidine incorporated into DNA might increase in the virus-infected cell due to the induction of higher activities of thymidine kinase. We have assumed that the incorporation of [3H]TdR into cell DNA accuratelv reflected the rate of DNA synthesis. To test this assumption, cell DNA synthesis was compared in control and CMV-infected HEL cells by labeling separate but identical cultures with either 32p or [3H ]TdR. Since 32P will be incorporated into both RNA and DNA, all nucleic acid preparations containing 32P had to be freed of contaminating RNA. However, all phosphorous incorporated into DNA must be derived from the medium (ex- Determination TABLE 3. cluding nucleic acid degradation products), and hence the use of "P represents an accurate if somewhat awkward means of measuring the rate of DNA synthesis. The experimental design was identical to that used when [3H ]TdR was the labeled precursor. At 24 h postinfection either 32P or [3H ]TdR were added to the cultures, and 48 h postinfection the DNA was analyzed, as described, for incorporation of Stimulation of Vero cell DNA synthesis by CMVa 'M 'C FRACTION NUMBER FIG. 5. CMV-induced stimulation of DNA synthesis in Vero cells. Vero cells were arrested with IUdR, infected with CMV (Ad-169), or sham infected with maintenance medium. The cultures were pulsed for sequential 24-h periods with [3H]TdR. The DNA was extracted and analyzed as described in the legend to Fig. 1. This figure represents the radioactivity from cultures labeled for 24 to 48 h. Symbols: 0, counts per minute from CMV-infected cells; 0, counts per minute from sham-infected cells; A, density. Time postinfection (h) Sham infected Counts/min of [3H ]TdR incorporated into cell DNA... 13,400 9,400 4,500 4,900 CMV infected Counts/min of [3H ]TdR incorporated into cell DNA ,600 27,400 23,600 10,100 Fraction increase in incorporation of [3H ]TdR into cell DNA of CMVinfected over sham-infected cultures a Vero cells were pretreated with 100 jg of IUdR per ml for 72 h as described. The cells were either sham infected or infected with the Ad-169 strain of CMV. Twenty-four hours after infection, cultures were labeled with [3H ]TdR for sequential 24-hour periods. The DNA was analyzed by isopycnic centrifugation as described (legend, Fig. 1), and the total counts of [3H]TdR incorporated into cell DNA per 0.1 ml of cell digest were determined. CMV DNA was not detectable in the Vero cell extracts.

7 VOL. 13, 1974 STIMULATION OF CELLULAR DNA SYNTHESIS BY CMV either 31P or [3H ]TdR into cell DNA. The results of this investigation are presented in Table 4. These data indicate that the apparent stimulation of DNA synthesis is approximately the same when either [3H]TdR or 32P was the labeled precursor. Alkaline gradients of IUdR-substituted cell DNA. We were still concerned with the following questions. Is the IUdR-substituted heavy DNA peak found at the bottom of the neutral CsCl gradients cell DNA? Is the increase in uptake of [3H ]TdR into cell DNA due to the synthesis of new DNA or repair of DNA damage caused by the infecting virus? To answer these questions, the following points were considered. Only DNA synthesized in the presence of IUdR would have IUdR substituted for thymidine. The parental cellular DNA present prior to the addition of IUdR would not have IUdR substituted for thymidine. Consequently, there could be three species of duplex DNA molecules present, those containing none, one, or two individual strands substituted with IUdR. Unincorporated IUdR was removed prior to infection, and hence all cell DNA synthesis occurring after infection would involve only [3H ]TdR and no IUdR nucleotides. If we assume that the heavy DNA found near the bottom of the neutral CsCl gradients represents a hybrid molecule arising from normal semiconservative synthesis, then it would contain IUdR substituted into one strand and [3H ]TdR in the other. If this DNA were to be isopycnically banded in alkaline CsCl, the IUdR-labeled and radioactive-labeled strands would separate. The strand containing the [3H ]TdR should co-band with light cell DNA marker at a normal density. Since the IUdR-substituted strand should contain no [3H ]TdR, radioactivity should be seen in only one homogeneous peak at the marker DNA position. However, in contrast, if the [3H ]TdR is incorporated by a repair process the [3H ]TdR would be incorporated in both light and heavy strands. Then, an alkaline isopycnic gradient would contain two bands of radioactivity at both light and heavy positions. To insure that our technique of CsCl isopycnic centrifugation was adequate to separate single-stranded cellular and CMV DNA, we mixed equal volumes of CMV marker DNA substituted with [3H ]TdR and cell DNA substituted with ["C ]TdR. This mixture was centrifuged in both neutral and alkaline (ph 12.5) CsCl gradients with initial densities of g/cm3 and g/cm3, respectively (Beckman 40.3 rotor, 33,000 rpm, 60 h, 20 C). The gradients were analyzed for radioactivity as described previously, and the results are graphically presented in Fig. 6. The data presented indicated that we were able to effectively separate both single- and double-stranded cell and CMV DNA. We then used this procedure to ascertain whether the CMV infection stimulated semiconservative DNA replication or only repair synthesis of cell DNA. Our expectation, from the rationale outlined above, was that semiconservative replication would give rise to one band in the alkaline gradient, whereas repair synthesis would give two bands. HEL cells which had been pretreated with IUdR were infected or sham infected and labeled for 12 to 36 h x 359 x9 TABLE 4. Cdmparison of cell DNA synthesis in infected and uninfected HEL cells as determined by the incorporation of either [9H]TdR or S2p into cell DNAa Counts/min Infected/ Infected Uninfected uninfected 32p 4, H 5, Determination C sinfected a HEL cells were pretreated with IUdR and either sham infected or infected with the Ad-169 strain of CMV. The cells were then labeled from 24 to 48 h postinfection with either 32P or [3H]TdR. DNA was extracted and analyzed as described FRACTON NUMBER FIG. 6. Separation of double- and single-stranded cell and virus DNA in alkaline and neutral isopycnic CsCI gradients. CMVand HEL cell marker DNA were prepared as described in Materials and Methods. Virus DNA labeled with [3H]TdR and HEL cell DNA labeled with [14C]TdR were mixed and placed in both neutral and alkaline CsCI. The samples were centrifuged at 33,000 rpm for 60 h in a Beckman 40.3 rotor at 20 C. Gradients were collected and radioactivity was determined as described. Symbols: *, CMV DNA; 0, cell DNA.

8 360 ST. JEOR ET AL. J. VIROL. post-inoculation with [3H]TdR. The 12- to 36-h labeling period was used to insure maximum cell DNA stimulation with minimum CMV DNA synthesis. The DNA was extracted, mixed with 14C cell marker DNA, and centrifuged in neutral and alkaline CsCl (Beckman SW50.1 rotor, 29,000 rpm, 60 h, 20 C). The gradients were analyzed for radioactivity, and the results with infected cultures are presented in Fig. 7. The heavy and light cell DNA molecules were easily separable in the neutral gradient, and in the alkaline gradient a single peak containing [3H]TdR was found which co-banded with 14C cell marker DNA. There was no detectable CMV DNA because of the early [3H JTdR labeling period (12 to 36 h). The uninfected control cells did not incorporate significant amounts of [3H]TdR, indicating that they had been effectively arrested (unpublished data). The total number of counts recovered in the two peaks in the neutral gradient was equal to the total counts present in the single peak in the alkaline gradient. Thus, [3H ]TdR and IUdR were never incorporated into the same single strand of DNA, indicating that [3H JTdR had been incorporated into cell DNA by semiconservative replication and not by cell DNA repair. We were also interested in whether the stimulation of cell DNA synthesis in an abortive infection, where no detectable CMV DNA is synthesized, represents new DNA replication or cell DNA repair. Vero cells which had been arrested with IUdR were infected with the Ad-169 strain of CMV and labeled for 24 to 48 h postinfection with [3H ]TdR. The DNA was extracted and centrifuged in both alkaline and neutral gradients, and the gradients were analyzed for radioactivity. The results of this experiment are illustrated in Fig. 8. The results obtained were similar to those in the previous experiments. The neutral gradient contained two peaks, presumably a heavy IUdR-substituted peak and a lighter peak, the latter with a density of normal cellular DNA. The alkaline gradient contained a single peak of [3H]TdRlabeled DNA which co-banded with the [IC ]TdR cell DNA marker. As in the previous experiment, the total counts in the two peaks resolved in the neutral gradient were equal to the total counts present in the single peak resolved in the alkaline gradient. These data indicate that, in an abortive infection, CMV can stimulate semiconservative replication of cell DNA. DISCUSSION The data presented in these investigations indicate that infection with human cytomegalovirus stimulates the incorporation of [3H]TdR A 4NEUTRAL B ALKALINE A NEUTRAL B ALKALINE 4 3 x U FRACTION NUMBER FIG. 7. Analysis of DNA from CMV-infected HEL cells in neutral and alkaline isopycnic CsCl gradients. HEL cells, pretreated with IUdR, were infected with CMV (Ad -169). They were then labeled with [3H]TdR for 12 to 36 h postinfection. The DNA was extracted as described and mixed with ['4C]TdR-labeled HEL cell DNA marker. The mixture was added to neutral and alkaline CsCl solutions and centrifuged in a Beckman SW50.1 rotor at 29,000 rpm for 60 h at 20 C. The gradient was then collected and the fractions were analyzed for radioactivity. Symbols: 0, ['H]TdRlabeled CMV-infected HEL cells; 0, [14C]TdRlabeled cell DNA marker FRACTION NUMBER FIG. 8. Analysis of DNA from CMV-infected Vero cells in neutral and alkaline isopycnic CsCl gradients. Vero cells which had been pretreated with IUdR were infected with the Ad-169 strain of CMV. The cells were then labeled with [3H]TdR for 24 to 48 h postinfection. The DNA was extracted, mixed with a [14C TdR-labeled, HEL cell marker DNA, and isopycnically centrifuged in both alkaline and neutral CsCl gradients in a Beckman 40.3 rotor at 33,000 rpm for 60 h at 20 C. The gradients were then collected and analyzed for radioactivity. Symbols: 0, infected Vero cell DNA. 0, marker DNA.

9 VOL. 13, 1974 STIMULATION OF CELLULAR DNA SYNTHESIS BY CMV 361 into cell DNA. We interpret this observation to suggest that CMV can stimulate cell DNA synthesis. When cells which are confluent but not arrested are infected with CMV, little difference in the amount of [3H ]TdR incorporated into cell DNA is observed between control and infected cultures. However, CMV infection of cells arrested either by low serum concentrations or IUdR pretreatment results in up to a 10-fold increase in the amount of [3H ]TdR incorporated into cell DNA. The virus infection must either induce new semiconservative replication of cell DNA or damage the cell DNA resulting in stimulation due to repair mechanisms. Isopycnic centrifugation in neutral and alkaline CsCl of labeled cell DNA from IUdR-arrested, CMV-infected cells was used to distinguish between these possibilities. Cells permissive (HEL) and nonpermissive (Vero) for CMV replication were used in these investigations. In neutral gradients, two radioactive bands were observed. One band occurred at the density of cell DNA (1.692 g/cm3), and the other occurred at a greater density. If it is assumed, from the rationale detailed in the Results section, that both bands represent cell DNA, one can anticipate the results obtained by alkaline isopycnic centrifugation of this DNA. If the CMV infection stimulates semiconservative replication, only one band would occur in the alkaline gradient, whereas CMV stimulation of repair synthesis would result in two bands. The DNA from both permissive and abortive CMV infections gave a single band in the alkaline isopycnic gradients; hence, the CMV infection stimulates semiconservative replication of cell DNA. Furthermore, the sum of the radioactivity in both bands in the neutral gradient was recovered in the single peak in the alkaline gradient, which co-banded with a cell DNA marker. Hence, the previous assumption, that the denser band in the neutral gradients represented cell DNA, is substantiated. If the dense band had represented virus DNA, it would have been resolved from the cell DNA marker, as demonstrated in Fig. 6. This conclusion is consistent with the results of other investigators (16). The incorporation of 32p was also used to measure the extent of CMV-induced cell DNA replication. The data (Table 4) indicate approximately equivalent stimulation of cell DNA synthesis by using either radioactive precursor. Further experiments would be required to accurately determine if the two precursors give identical or slightly different results. The data in Table 4 do, however, strongly support the assumption that the rate of incorporation of [3H JTdR is a reasonably accurate reflection of the rate of DNA synthesis under the conditions used in this study. Other data support the observation that CMV infection stimulates host DNA replication. A recent report (St. Jeor and Rapp, in press) indicated that CMV infection of human embryonic kidney cells, pretreated with IUdR, stimulated cell DNA replication. Lang (personal communication) detected an increase in mitotic figures in cells abortively infected with CMV. The observation that both the C-87 and Ad-169 strains of CMV stimulate cell DNA replication suggests that this may be a characteristic common to many strains of CMV. The data suggesting that either heat or UV inactivation of CMV eliminates the potential to stimulate host DNA replication in HEL cells (Table 1) indicate that the presence of the virion alone is not sufficient for such stimulation. It would appear that one or more virus functions must be expressed for the stimulation of DNA synthesis to occur. A relatively large dose of UV radiation (1.44 x 107 ergs/cm2, 300 mean lethal hits per PFU) was used. This insured that nearly all virus functions, rather than just the ability to form a plaque, would be inactivated. The data indicating that CMV infection stimulates DNA replication in Vero cells are of particular interest. Vero cells are nonpermissive for CMV, and tests for CMV DNA replication were negative (Fig. 5). If some virus gene expression is required for stimulation of cell DNA synthesis in Vero cells, CMV must be capable of penetration and partial genetic expression in the nonpermissive Vero system. CMV is often isolated from patients with neoplastic disease (8, 9); although this association has not been considered an etiologic relationship. However, recent data indicate that CMV can malignantly transform hamster fibroblasts (Albrecht and Rapp, in press), and the ability to stimulate cell DNA replication is an important characteristic related to oncogenic DNA viruses (7, 13, 14, 21, 22). These observations thus suggest that a possible association between CMV and neoplastic disease should be carefully considered. ACKNOWLEDGMENTS We express our thanks to Joseph Mele for his valuable suggestions and to Linda Mele for her excellent technical assistance. This study was conducted under Public Health Service contract no within the Virus Cancer Program of the National Cancer Institute.

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