John M. Lehman

Background Simian Virus 40 (SV40) infection of growth‐arrested monkey kidney cells stimulates S phase entry and the continued synthesis of both viral and cellular DNA. Infected cells can attain total DNA contents as high as DNA Index, DI = 5.0–6.0 (10–12C), with host cell DNA representing 70–80% of the total. In this study, SV40‐infected and uninfected control cells were compared to determine whether continued DNA replication beyond DI = 2.0 was associated with rebinding of the minichromosome maintenance (MCM) hexamer, the putative replicative helicase, to chromatin. Method Laser scanning cytometry was used to measure the total expression per cell and the chromatin/matrix‐association of two MCM subunits in relation to DNA content. Results MCM2 and MCM3 proteins that were associated with the chromatin/matrix fraction in G1 phase of both uninfected and SV40‐infected cells were gradually released during progression through S phase. However, in SV40‐infected cells that progressed beyond DI = 2.0, chromatin/matrix‐associated MCM2 and MCM3 remained at the low levels observed at the end of S phase. Rereplication was not preceded by an obvious rebinding of MCM proteins to chromatin, as was observed in G1 phase. Conclusions The rereplication of host cell DNA in the absence of the reassociation of MCM proteins with chromatin indicates that SV40 infection induces a novel mechanism of licensing cellular DNA replication. © 2005 International Society for Analytical Cytology

Lytic infection of African green monkey kidney (CV-1) cells by simian virus 40 (SV40) is characterized by stimulation of DNA synthesis leading to bypass of mitosis and replication of cellular and viral DNA beyond a 4C DNA content. To define mechanisms underlying the absence of mitosis, the expression levels of upstream regulatory molecules of mitosis-promoting factor (MPF) were compared in parallel synchronized cultures of SV40-infected and uninfected CV-1 cells. The DNA replication/damage checkpoint kinase Chk1 was phosphorylated in both uninfected and SV40-infected cultures arrested at G ₁ /S by mimosine, consistent with checkpoint activation. Following release of uninfected cultures from G ₁ /S, Chk1 phosphorylation was lost even though Chk1 protein levels were retained. In contrast, G ₁ /S-released SV40-infected cultures exhibited dephosphorylation of Chk1 in S phase, followed by an increase in Chk1 phosphorylation coinciding with entry of infected cells into >G ₂ . Inhibitors of Chk1, UCN-01 and caffeine, induced mitosis and abnormal nuclear condensation and increased the protein kinase activity of MPF in SV40-infected CV-1 cells. These results demonstrate that SV40 lytic infection triggers components of a DNA damage checkpoint pathway. In addition, chemical inhibition of Chk1 activity suggests that Chk1 contributes to the absence of mitosis during SV40 lytic infection.

Mortal human fibroblasts can be partially transformed by the bovine papillomavirus E5 oncoprotein through activation of the platelet-derived growth factor beta receptor. Here, we report that these cells undergo massive apoptosis 2 weeks after confluence. Although activation of caspase 3 was observed in the apoptotic cells, it was not required for apoptosis. The appearance of the mitochondrial proteins cytochrome c and apoptosis-inducing factor in cytosolic and nuclear compartments, respectively, provided a basis for mitochondrial dysfunction and a caspase-independent mechanism of apoptosis in these cells. Although an activating conformational change in Bax also was evident in the apoptotic cells, enforced overexpression of Bcl-2 was insufficient to prevent apoptosis. Finally, a small peptide present in the conditioned medium from dying transformed cells appeared responsible for inducing apoptosis through affecting a conformational change in Bax and eventual relocalization of apoptosis-inducing factor to the nucleus. Thus, an atypical apoptotic pathway is activated in mortal human fibroblasts in response to transformation induced by sustained receptor tyrosine kinase activation.

Viruses are obligate intracellular parasites that require the host cell replication, transcription, and translation machinery for reproduction. Each viral group provides a unique series of viral-cellular interactions. Studies have provided insight not only into viral replication and control of host functions, but also into cellular functions such as eukaryotic replication, transcription, and translation as well as the regulation of these events. This unit presents a protocol for flow cytometric monitoring of viral infection and quantitating viral-cellular events. The availability of monoclonal and/or polyclonal antibodies directed to both viral and cellular proteins offers the ability to assay a specific molecule in the intact fixed cell and the opportunity to correlate viral events with cellular processes such as progression through the cell cycle.