Key terms: DNA content changes, permissive infection, viral antigens, T-antigen, V-antigen Considerable interest has been generated in the analysis of gene products obtained from both viral and cellular oncogenes, since these genes may be responsible for such activities as cell proliferation, differentiation, and the acquisition of the neoplastic phenotype (1). Simian Virus 40 (SV40) has been an important model system for studies of viral replication, viral transformation, and gene regulation (14,20,22). The early viral gene products large T and small t antigen are thought to be responsible for events in viral replication in permissive cells and also for the initiation and establishment of the neoplastic phenotype in susceptible nonpermissive cells (4). The SV40 T antigen is known to bind viral DNA at the origin of replication and initiate viral DNA synthesis in the permissive cell (19,26). Further, the T antigen activates late viral transcription and inhibits early viral transcription (2). The part that the T antigen plays in transformation may be at a number of levels which include gene regulation and the interactions with the plasma membrane and cellular proteins such as p53 (15,25,27). The molecular approach to the study of viral expression has provided much information concerning the role of these proteins in permissive and nonpermissive cells.One further method of investigation would utilize flow cytometry to quantitatively assay the appearance of nity to assay various parameters of a population of cells, such as DNA and specific viral and cellular proteins, and to correlate these parameters on a single-cell basis (3,5,17,29). The instrumentation also allows quantitation of each of these parameters singly and in concert. In previous studies, a technique was developed that allowed simultaneous staining of cellular DNA content and the viral T antigen using a specific monoclonal antibody (13). This technique has been applied to study SV40 infection of permissive CV-1 cells. Changes in host cell cycle distribution and the synthesis of both T antigen and the late viral proteins that encapsulate the viral DNA were followed in time. MATERIALS AND METHODSCells and Virus The cells utilized in this study were the CV-1 line of African green monkey kidney, passages 26 through 38, CV-1 cells were grown in modified Eagle's medium (MEM, GIBCO) with 2 x amino acids and vitamins. MEM with 10% fetal bovine serum (FBS) was used for growth and 5% FBS for maintenance. The CHE lines were grown in MEM with 5% FBS. Cultures were kept at 37°C in humidified 5% C 0 2 incubator (17).The SV40 stock virus used was RH911, grown and plaque-assayed for titer on CV-1 cells. All cells and virus stock tested negative for mycoplasma contamination (17).Infection was performed on 100-mm plastic petri dishes containing CV-1 cells that were maintained for 1-2 days after reaching confluence. At this time, media was removed and 1.0 ml of virus, diluted in MEM, was added to the cultures. The cells were infected with varying multiplicities of infecti...

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Human diploid fibroblasts (HDF) have a finite life span in cell culture which can be extended when transformed with simian virus 40 (SV40). Flow cytometric analysis of SV40-HDF transformation allowed DNA content changes to be correlated with the appearance, quantity, and distribution of T antigen, p53, and V antigen, three proteins associated with this process. These studies demonstrated a shift in the DNA content to tetraploidy, which was correlated with the age of the SV4O-HDF but not the time of infection. A significant increase of the epitope recognized by PAbl22 to host p53 and the epitope PAblOl to SV40 T antigen occurred at the same time the tetraploid population appeared. However, an antigen reactive with SV40 V antibody was present at high levels in most of the population early after infection, but the levels declined with time. The percentage of PAblOl-T antigen-positive cells increased more rapidly in cells infected at a late passage, and this was concomitant with the shift in DNA content to tetraploid. Analysis of the mean fluorescence of total, gated populations (GI, G2, and > Gt) demonstrated that a threshold level of p53 and T antigen was reached in each compartment of the cell cycle. As the transformed phenotype appeared, a population of cells was continually released into the supernatant, and although these cells had a DNA pattern similar to the monolayer cells, the T antigen and p53 levels were 3-5 times higher in the tetraploid 6 2 cells.These studies correlated the expression of proteins associated with viral transformation in HDF which vary with time and shift in DNA content.

These studies examined cell cycle progression and quantitative changes in T-antigen following infection by SV40. Single cells were assayed by multiparameter flow cytometric analysis (FCM) for DNA content and T-antigen expression. Conditions were used which permitted permissive, semi-permissive, and non-permissive cells to be monitored through two rounds of DNA synthesis induced by SV40. The permissive cells included the monkey kidney cell lines; CV-1, Vero and BSC-1 and the COS-1 and COS-7 which are CV-1 cells transformed with an origin defective SV40. The non-permissive cell strains included mouse embryo fibroblasts, Chinese hamster fibroblasts, and IMR-90, a human diploid fibroblast. Cell types differed in the maximal amount of T-antigen expressed per cell. Additionally, all cell types expressed a limited quantity of T-antigen for each cell cycle phase and the quantity increased in each successive phase. The level in each phase was increased only two-fold when 100 times more virus was used. Thus, for an infected population the quantity of T-antigen was dependent on cell cycle distribution. High levels of T-antigen were not required for permissive infection; however, permissive cells were distinguished from non-permissive cells by the G2 levels. Permissive G2 cells had more than double the T-antigen content expressed in G1, while nonpermissive G2 cells had less than a two-fold increase over G1 levels. The appearance of cells with tetraploid DNA content and the failure to undergo mitosis correlated to the higher Tantigen levels in the G2 of the permissive cells. Two other strains of SV40,776, and VA45 exhibit similar values for T-antigen expression and movement into tetraploid DNA content. This study establishes the levels of T-antigen correlated to the cell cycle and cell type. o 1993 Wiley-Liss, Inc.