Synchronized murine 3T3 cells have been used to investigate the possible dependency of murine cytomegalovirus replication upon the cell cycle. The normal latent period of 12 h characteristic of asynchronous 3T3 cells was protracted to more than 24 h after an early G1 infection in synchronous cells. In this case viral progeny were not detected until after the initiation of the host Sphase. Cells maintained in the G1 phase did not replicate virus. This failure could not be explained by a decrease

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... in virus penetration but was apparently due to a requirement for an event associated with the host S-phase. Thymidineinduced inhibition of cell cycle traverse also blocked virus replication. Viral DNA synthesis did not initiate until after the initiation of host DNA. In contrast, herpes simplex virus type 1 replicated in 3T3 cells independently of the cell cycle. Studies in this laboratory, as well as earlier reports, indicate certain unique features of the murine cytomegalovirus (MCV). These include centrifugal enhancement of infectivity (10, 16), multicapsid morphology (9), and genome size (14). In addition, we have observed that permissive viral replication is contingent upon exponential host cell growth. The dependence of viral growth on the physiological status of the cell suggests that MCV has a predilection for cells in some phase of the cell cycle other than G1. Although the majority of studies on herpesviruses have involved asynchronously growing cells, a few reports have documented interactions between virus and synchronized cells (4, 5, 12). For example, herpes simplex virus type 1 (HSV-1) was shown to replicate independently of the cell cycle using double-thymidine(TdR)induced synchrony in human KB cells (4). In another report, equine abortion virus was shown to be dependent upon the S-phase in vitro (12). Thus, the ability of a herpesvirus to replicate independently ofthe cell cycle appears to be unrelated to genome size. Our analysis with MCV indicates that, like equine abortion virus in vitro, MCV requires events associated with the host S-phase for initiation of viral DNA synthesis. MATERIALS AND METHODS Cells. 3T3 cells (Flow Laboratories), HEp-2 cells (Microbiological Associates), and cells obtained from freshly explanted mouse embryos (ME) were 267 grown in Dulbecco modified minimal essential medium supplemented with 10% fetal bovine serum plus 20 ,ug of gentamicin per ml. Cells were incubated in a humidified atmosphere containing 5% CO2-95% air at 370C or in roller bottles at 370C (Bellco). 3T3 cells were passed a maximum of 20 times, after which fresh cells were obtained from frozen stocks. Viruses. The Smith strain of MCV was used. Virus stocks were prepared by low-multiplicity passage (<1 PFU/cell) in ME cultures. The P strain of HSV-1 was used after plaque purification on HEp-2 cells. MCV and HSV-1 were assayed by plaque formation on either freshly plated ME or 3T3 cells and HEp-2 cells, respectively, as described before (14) . The multiplicity of infection in these experiments was 20 to 30 PFU/cell. Virus titers are expressed as PFU per milliliter of cells plus supernatant fluid. Intracellular virus was released by freeze-thawing the cells three times. Synchronization of 3T3 cells. 3T3 cells were synchronized by the "serum-split" method, which entailed serum activation of quiescent G1-arrested cells. 3T3 cells in 90-mm Falcon plates were allowed to reach confluence and left without medium change for a total of 4 to 10 days. Stimulation of quiescent cells was achieved by rinsing the cell sheet with prewarmed medium, trypsinizing for 1 to 2 min, and replating in a fiveto eightfold larger volume of fresh minimal essential medium plus 10% fetal bovine serum. The degree of synchrony achieved was determined by 30-min pulses with 1 ,uCi of [methyl-3HI-TdR (New England Nuclear Corp., 20 Ci/mmol) per ml. The pulse was terminated by the addition of chilled (4°C) TNE (0.01 M Tris-hydrochloride, pH 7.5, 0.1 M NaCl, 0.001 M EDTA). To the resuspended on May 9, 2020 by guest