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.

Simian virus 40 induces multiple S phases with the majority of viral DNA replication in the G2 and second S phase in CV-1 cells.

The infection of permissive monkey kidney cells (CV-1) with simian virus 40 induces G1 growth-arrested cells into the cell cycle. After completion of the first S phase and movement into G2, mitosis was blocked and the cells entered another DNA synthesis cycle (second S phase). Growth-arrested CV-1 cells replicated significant amounts of viral DNA in the G2 phase with the majority of synthesis occurring during the second S phase. When mimosine-blocked (G1/S) infected cells were released into the cell cycle, a major portion of the viral DNA was detected in G2 with the largest accumulation in the second S phase. The total DNA produced per infected cell was 10-12C with approximately 0.5-2C of viral DNA replicated per cell. Therefore the majority of the DNA per cell was cellular, 4C from the first S phase and approximately 4-6C from the second cellular synthesis 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.

SV40 small T antigen enhances progression to >G2 during lytic infection.

The infection of monkey kidney (CV-1) cells with simian virus 40 (SV40) stimulates the cells into successive rounds of DNA synthesis without an intervening mitosis, leading to the acquisition of a >G2 DNA content. To elucidate the role of small t antigen in cell cycle progression and in viral replication during infection, studies were performed using an SV40 mutant (dl888) that lacks the ability to produce small t. Initially dl888-infected cells move through the first S phase at roughly the same rate as wild-type infected cells. Upon reaching G2, however, the dl888-infected cells progressed to >G2 at a reduced rate relative to wild-type. The slower rate of entry into >G2 of dl888-infected cells is associated with a decrease in total pRb and an increase in the ratio of hypophosphorylated to hyperphosphorylated pRb. The expression of cyclin D1 and p27(kip1) were elevated in dl888-infected cells compared to wild-type-infected CV-1 cells. Taken together, these results indicate that small t antigen plays a role in stimulating entry into >G2 in SV40-infected CV-1 cells, possibly by affecting the regulation of key cell cycle proteins.

Microdeletions at chromosome bands 1q32-q41 as a cause of Van der Woude syndrome.

Van der Woude syndrome (VWS) is an autosomal dominant disorder comprising cleft lip and/or cleft palate and lip pits. We reported previously a family whose underlying mutation is a 500-800 kb deletion localized to chromosome bands 1q32-q41 [Sander et al., 1994: Hum Mol Genet 3:576-578]. Along with cleft lip/palate and lip pits, affected relatives exhibit developmental delays, suggesting that the function of a gene nearby may also be disrupted. To further localize the VWS gene we searched for other deletions that cause VWS. An allele loss assay was performed using a novel highly polymorphic marker, D1S3753. From a panel of 37 unrelated individuals, we detected an allele loss in one family, indicating the presence of a deletion. In this family, the phenotype in three generations of affected individuals was confined to the cardinal signs of VWS. Surprisingly, mapping of the new deletion showed that it extended 0.2-1 Mb beyond the proximal breakpoint for the deletion described previously. No deletions were detected in seven cases of popliteal pterygia syndrome, 76 cases of mixed syndromic forms of cleft lip and palate, and 178 cases of nonsyndromic cleft lip and palate. These observations suggest that genetic searches for microdeletions should be routine in screening patients for causes of VWS and may facilitate the positional cloning efforts of the VWS gene and of a nearby gene or genes that may be involved in brain development.

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.

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.

Increase in total protein following infection of CV-1 cells with SV40 virus as assayed by flow cytometry.

The 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 degrees) 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 due to cellular changes resulting from viral replication and cell death.

DNA content distribution of mouse cells following infection with polyoma virus.

Infection of primary to tertiary mouse embryo fibroblasts or mouse kidney cells with polyoma virus leads to stimulation of cellular DNA synthesis. When either confluent or growing mouse cells were infected, the monolayer cells were found to accumulate cells with a DNA content of S and G2/M phases of the cell cycle as assayed by flow cytometry. A similar pattern of DNA content was also observed in cells in the supernatant, which are probably cells replicating virus and dying. When compared with control cells, the infected monolayer and supernatant cells exhibited a population (5-27%) with a > G2 DNA content. The increase in DNA content of these > G2 cells was calculated to be an average of 26.7%, which is probably due to viral DNA. Polyoma contrasts with another papovavirus, SV40, which stimulates cells into DNA synthesis, with the majority of cells attaining a > G2/tetraploid DNA content, suggesting that there are differences in polyploidization between these two viruses.

Induction of tetraploid DNA content by simian virus 40 is dependent on T-antigen function in the G2 phase of the cell cycle.

Previous experiments with the simian virus 40 mutant tsA357R-K (tsA30) demonstrated a T-antigen function that is required for production of cells with a greater-than-G2-phase DNA content. In this study, temperature shift experiments indicated that the temperature-sensitive function of tsA357R-K, which is necessary for entry into the greater-than-G2 phase, is not required in G1 or S but must be supplied in the G2 phase.

Simian virus 40 prevents activation of M-phase-promoting factor during lytic infection.

Simian virus 40 (SV40) infection stimulates confluent cultures of monkey kidney cells into successive rounds of cellular DNA synthesis without intervening mitosis. As an initial step in defining the mechanisms responsible for viral inhibition of mitosis, M-phase-promoting factor (MPF) was examined in SV40-infected CV-1 cells passing from G2 phase into a second S phase. MPF is a serine-threonine protein kinase that is essential for mitosis in eukaryotic cells. In SV40-infected cells exiting G2 phase, there was a reduced amount of MPF-associated H1 kinase activity relative to that of uninfected cells passing through mitosis. Both subunits of MPF, cyclin B and the p34cdc2 catalytic subunit, were present and in a complex in infected cells. In uninfected cultures, passage through mitosis was associated with the dephosphorylation of the p34cdc2 subunit, which is characteristic of MPF activation. In contrast, the p34cdc2 subunit remained in the tyrosine-phosphorylated, inactive form in SV40-infected cells passing from G2 phase into a second S phase. These results suggest that although the MPF complex is assembled and modified normally, SV40 interferes with pathways leading to MPF activation.

Hypophosphorylated retinoblastoma gene product accumulates in SV40-infected CV-1 cells acquiring a tetraploid DNA content.

Simian virus 40 (SV40) infection of monkey kidney cells induces successive rounds of cellular DNA synthesis without intervening mitosis. To gain an understanding of the mechanisms responsible for disruption of cell cycle control during lytic infection, pRB phosphorylation and cell cycle distribution were examined following SV40 infection of CV-1 cells. The hypophosphorylated pRB present in confluent CV-1 cells was phosphorylated within 14 h following SV40 infection. Phosphorylated pRB then remained the predominant form as cells progressed from late G1 through S phase. Hypophosphorylated pRB reappeared as cells moved through G2 and acquired a tetraploid (> G2) DNA content. The reappearance of hypophosphorylated pRB in a population with decreasing numbers of cells in G1 phase and increasing numbers of cells in > G2 suggests that accumulation of hypophosphorylated pRB may be involved in T antigen-induced tetraploidy.

Quantitation of simian virus 40 T-antigen correlated with the cell cycle of permissive and non-permissive cells.

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 T-antigen 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.

Simian virus 40 large T-antigen function is required for induction of tetraploid DNA content during lytic infection.

Infection of quiescent CV-1 cells with simian virus 40 mutant tsA30 at 37 degrees C resulted in the induction of two rounds of cellular DNA synthesis in T-antigen-positive cells, as previously described for wild-type simian virus 40. Following infection with tsA30 at 40.5 degrees C, T-antigen-positive cells were induced into S phase and reached a diploid G2 DNA content; however, a second S phase was not initiated. The failure of tsA30-infected CV-1 cells to enter tetraploid S phase at 40.5 degrees C identifies a T-antigen function, distinct from T-antigen functions responsible for stimulation of cell DNA synthesis, which is required for initiation of a second round of DNA synthesis without mitosis.

Correlation of DNA content, p53, T antigen, and V antigen in simian virus 40-infected human diploid cells.

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 SV40-HDF but not the time of infection. A significant increase of the epitope recognized by PAb122 to host p53 and the epitope PAb101 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 PAb101-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 (G1, G2, and greater than G2) 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 G2 cells. These studies correlated the expression of proteins associated with viral transformation in HDF which vary with time and shift in DNA content.

Detection of an early cytomegalovirus antigen with two-color quantitative flow cytometry.

An early cytomegalovirus (CMV) antigen was detected with a monoclonal antibody by two-color fluorescent flow cytometry. With the aid of a human diploid fibroblast cell strain, FLOW 2000, infected with the AD169 strain of CMV, the viral antigen and the DNA content of infected or uninfected cells were measured. There was no evidence of change in the cell-cycle distribution of the infected cells. The viral antigen was detected within 30 minutes following virus adsorption at 0.1 and 1.0 plaque-forming units/cells; and the percentage of positive cells increased with time and viral dosage. All stages of the cell cycle were susceptible to viral infection and the average fluorescence was greater than the background fluorescence. Flow cytometry detected the viral antigen earlier than conventional immunofluorescent microscopy and cell culture for CMV cytopathological effect (CPE). Ten bronchoalveolar lavages assayed by flow cytometry and conventional diagnostic procedures demonstrated that flow cytometry might be useful in early diagnosis for CMV infection.

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

Quantitative two-color fluorescent analysis of Simian virus (SV40) infection of permissive CV-1 cells was investigated. Analysis included by quantitation of cellular DNA, the early viral tumor (T) antigen with a monoclonal antibody, and late viral (V) antigens with a polyclonal antibody. T antigen was detected in all phases of the cell cycle at 6 and 12 h, after SV40 infection of growth arrested cells. At later time intervals, the percentage of T-antigen-positive cells increased with the induction of the cells into successive rounds of DNA synthesis and an increase in tetraploid-polyploid cells. The amount of T antigen per cell increased as the cells entered the successive stages of the cell cycle (G0/G1----G2 + M----tetraploid S and G2 + M). The V antigen from adsorbed virus was detected immediately after infection. Synthesis of V antigen began in late S and G2 + M phases of the cell cycle. This quantitative analysis allows a definitive determination of antigen per cell in a population correlated with the cell cycle and may be useful in correlating viral and cellular events with transformation.