Judith Laffin

OKADAIC ACID INDUCES APPEARANCE OF THE MITOTIC EPITOPE MPM-2 IN SV40-INFECTED CV-1 CELLS WITH A >G2-PHASE DNA CONTENT

Simian virus 40 (SV40) infection of quiescent monkey kidney cells stimulates two successive rounds of cellular DNA synthesis without an intervening mitosis. This uncoupling of S phase and mitosis indicates that SV40 modulates pathways regulating the G2-to-M phase transition. To examine the integrity of mitotic initiation pathways in infected cells that have bypassed mitosis, SV40-infected CV-1 cells were treated with okadaic acid (OA), a known inducer of premature mitosis in other cell types. OA treatment triggered the appearance of the mitotic marker MPM-2 in SV40-infected CV-1 cells progressing through either the first (diploid) or second (tetraploid) S phases. These results demonstrate that a subset of mitotic pathways are intact but inactive in SV40-infected cells that have bypassed mitosis and initiated tetraploid S phase.

A subset of cells expressing SV40 large T antigen contain elevated p53 levels and have an altered cell cycle phenotype

Cells transformed by the simian virus 40 (SV40) large T antigen (Tag) contain elevated levels of cellular p53 protein. To quantify this relationship, levels of p53 were measured in NIH 3T3 cells that expressed different concentrations of Tag. Using immunoblotting, average p53 levels were shown to increase linearly with Tag concentrations in these cell lines. Single‐cell measurements were also performed using flow cytometry to measure p53 immunofluorescence. Surprisingly, the flow cytometry experiments showed that two distinct cell populations, based on p53 content, were present in cells expressing high levels of Tag. One cell population contained elevated p53 levels. A second population did not contain elevated p53, even though high concentrations of Tag were present in the cells. This latter cell population did not appear to arise because of mutations in either Tag or p53. The two cell populations also had phenotypic differences. In exponentially growing cells, Tag alters the cell cycle distribution (decreases the percentage of G1 phase cells and increases the percentages of S and G2 + M phase cells). This phenotype was maximum in the cell population containing elevated p53. A lesser phenotype was found in the cell population that did not contain elevated p53. These data show, firstly, that cells can express significant levels of Tag and not contain elevated levels of p53 and, secondly, that elevated p53 correlates with the altered cell cycle distribution produced by Tag in growing cells.

Isolation of poliovirus variants resistant to and dependent on arildone

Abstract Arildone has previously been shown to inhibit poliovirus replication by blocking uncoating of the virus in infected cells. The drug interacts directly with the virus particle so as to stabilize the capsid in vitro against the effects of heat or alkaline pH. We have isolated variants of poliovirus which are resistant to arildone at concentrations which inhibit native virus by greater than 99% and variants which require the presence of the drug for growth. Arildone interacts with both the drug-resistant and drug-dependent variants so as to prevent the inactivation of infectivity by heat. This suggests that the interaction of the drug with the virus particle per se is not sufficient to prevent uncoating in vivo . The drug resistance or drug dependence of the virus is not associated with gross changes in VP1, VP2, or VP3.

INCREASE IN TOTAL PROTEIN FOLLOWING INFECTION OF CV-1 CELLS WITH SV40 VIRUS AS ASSAYED BY FLOW CYTOMETRY

SummaryThe changes in cell size and total protein were determined for G1-arrested, contact-inhibited CV-1 cells infected with Simian virus 40 (SV40). The assays used were the Biorad total protein assays (Bradford and DC protein assays) on a standard number of cells, total protein as assayed by fluorescein isothiocyanate (FITC) and SR101 by flow cytometry, orthoganol (90°) light scatter by flow cytometry, and direct microscopic measurement with an ocular micrometer. Uninfected CV-1 cells and two cell lines with variations in DNA content (diploid vs. tetraploid) were used as controls for the studies presented. The results demonstrated a 40–60% increase in total protein at 32 to 42 h postinfection. These increases were similar to values obtained as control cells progress through the cell cycle. At later times postinfection (>42 h), total protein decreased due to cellular changes resulting from viral replication and cell death.

Chapter 26 Detection of Intracellular Virus and Viral Products

Publisher Summary Flow cytometric (FCM) analysis of viral infection can detect both viral and cellular events qualitatively and quantitatively. FCM technology has been used to gain an understanding of the events that are necessary in permissive and nonpermissive infections with simian virus 40 (SV40). The large tumor (T) antigen has received considerable attention, because this protein is known to initiate viral DNA replication in permissive cells and may have multiple functions in the transformation of nonpermissive cells. The development of monoclonal antibodies (mAb) to different epitopes of T antigen has permitted the FCM analysis of the appearance of this viral protein. Simultaneous measurements with other discriminating factors are made and this has allowed quantity, distribution, and population dynamics of T antigen to be correlated to the appearance of these macromolecules. Simian virus 40 is capable of a lytic infection (permissive) in monkey kidney cells and a transforming infection (nonpermissive) in numerous other cells. FCM studies have provided information in defining the relationship of the initiation of host cell DNA synthesis with the appearance and quantity of T antigen during the lytic cycle. The appearance, quantity, and correlation of T antigen have been reported for permissive cells and nonpermissive human cells.

Induction of tetraploid DNA content by mutant simian virus 40 T antigen that has reduced complex formation with p53.

The 402 mutants (DE, DH, DN) of simian virus (SV) 40 form reduced levels of p53-T antigen complexes or no complexes in lytically infected cells (CV-1 cells) relative to wild-type (wt) virus when assayed by immunoprecipitation. When CV-1 cells were infected with the 402 mutants, the cells were induced into multiple rounds of DNA synthesis without mitosis, resulting in a large population of cells with > G2 (tetraploid) DNA content similar to wt virus. The levels of T antigen and p53 per cell that were determined by flow cytometry were similar to wt lytically infected cells, with the levels of T antigen increasing as the infection proceeded. The p53 increased as the levels of T antigen increased, similar to a wt infection. These studies demonstrate that, in lytically infected cells with reduced p53-T antigen complex formation, the cells are induced into multiple rounds of DNA synthesis.

Analysis of Viral Infection and Viral and Cellular DNA and Proteins by Flow Cytometry

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.