Analysis of simian virus 40 infection of CV‐1 cells by quantitative two‐color fluorescence with flow cytometry

Abstract

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...

Negative Regulation of Mitotic Promoting Factor by the Checkpoint Kinase Chk1 in Simian Virus 40 Lytic Infection

“…All cultures were maintained at 37°C in a humidified 5% CO 2 incubator. SV40 wild-type (wt) strain RH911 and small t mutant strain dl888 were grown and plaque assayed on CV-1 cells as previously described (35,70). The SV40 small t mutant dl888 was provided by Kathleen Rundell.…”

“…The infected cells progress through G 1 , S, and G 2 phases but do not enter mitosis (17,35,58). Rather, the DNA content of the infected cells increases beyond 4C (defined as ϾG 2 phase) because of replication of both viral and cellular DNA.…”

Induction and Utilization of an ATM Signaling Pathway by Polyomavirus

“…In SV40-infected cells, mitomycin C treatment leads to an increase in a 5S form of DNA ligase thought to be involved in DNA repair (36). Lytic infection by SV40 results in G 2 arrest and a negative regulation of CDC2 by CHK1, a checkpoint kinase downstream of ATR (ATM-Rad3-related kinase) (30,38). The SV40 T antigen disrupts nuclear DNA repair foci containing MRE11 and interacts with NBS1, a target of ATM (ataxia telangiectasia mutated) and an essential factor in the S-phase checkpoint that senses DNA damage (14,51).…”

Distinct patterns of MCM protein binding in nuclei of S phase and rereplicating SV40‐infected monkey kidney cells

“…S phase in lytically infected cells is not followed by G2 phase and mitosis, but rather by continued DNA synthesis and an increase in overall cellular DNA content (3). Several experimental approaches demonstrated that the DNA synthesized in cells with DNA content >4C is a combination of viral and host cell DNA.…”