The histograms of the onset time show a much sharper curve (Figure ?(Figure4A)4A) compared to that of previous experiments, indicating simultaneous gene expression. fixed, labeled with propidium iodide and FACS analyzed for fluorescence levels in three replicative experiments (one panel for each experiment). In each histogram, the number of cells was plotted as the function of the fluorescence from the propidium iodide in arbitrary models (AU). Image2.TIFF (291K) GUID:?6DED4E57-B3A7-4820-BB80-53585F04BB37 Supplementary Figure 3: Correlation between fluorescence onset time and fluorescence levels. Cells were infected at MOI20 with OK11 recombinant computer virus and monitored for fluorescence levels every 10 min. The cells were divided into 10 groups according to the onset time. For each group the average onset time and common fluorescence level at 6 hpi were calculated and plotted. A trend line, calculated using the ordinary least squares (OLS) method and the measured Pearson correlation, is usually presented in the graph. Error bars represent the standard error of the mean for each group. Image3.TIFF (71K) GUID:?99BB998F-7AB4-4A35-8A45-0F22BD19610B Supplementary Movie 1: Cells infected with OK11 at MOI Pyrithioxin 5 were monitored for 12 h at 37C. Scale bar 20 M. Images were taken every 10 min and played at a rate of three frames per second. Video1.MP4 (2.9M) GUID:?A2787DE4-A23E-4F24-ADAB-2D965F5CBA9E Supplementary Movies 2 and 3: Cells infected with OK11 at MOI 20 were monitored for 12 h at 34C (2) or 39C (3). Scale bar 20 M. Images were taken every 10 min and played at a rate of three frames per second. Video2.MP4 (3.6M) GUID:?F965BDBC-81B1-420F-97A4-CAE6147055A6 Video3.MP4 (3.6M) GUID:?D4A766F6-324B-4175-AC9E-864DC7BF4F05 Abstract Synchronous viral infection facilitates the study of Pyrithioxin viral gene expression, viral host interactions, and viral replication processes. However, the protocols for achieving synchronous infections were hardly ever tested in proper temporal resolution at the single-cell level. We set up a fluorescence-based, time lapse microscopy assay to study sources of variability in the timing of gene expression during herpes simplex computer virus-1 (HSV-1) contamination. We found that with the common protocol, the onset of gene expression within different cells can vary by more than 3 h. We showed that simultaneous viral genome entry to the nucleus can be achieved with a Pyrithioxin derivative of the previously characterized heat sensitive mutant tsB7, however, this did not improve gene expression synchrony. We found that elevating the heat in which the contamination is done and increasing the multiplicity of contamination (MOI) significantly promoted simultaneous onset of viral gene expression among infected cells. Further, elevated heat Pyrithioxin result in a decrease in the coefficient of variation (a standardized measure of dispersion) of viral replication compartments (RCs) sizes among cells as well as a slight increment of viral late gene expression synchrony. We conclude that simultaneous viral gene expression can be improved by simple modifications to the contamination process and may reduce the effect of single-cell variability on population-based assays. < 0.05 by student = 0.026 Determine ?Physique2F).2F). We further compared the time for the interquartile range and found only a small difference between the MOIs (49.6 compared to 55.5 min, Determine ?Physique2G).2G). These results indicate that a higher MOI can improve the simultaneous onset of viral gene expression, however, even at MOI 20, the variability in onset time is substantial. Synchronized nuclear entry does not increase simultaneous gene expression For herpesviruses, both transcription and replication occur in the nucleus of the infected cell. We therefore hypothesize that movement toward the nucleus and nuclear entry might be an important source for variability in the onset of viral gene expression. Pyrithioxin To Rabbit polyclonal to HMGCL test this hypothesis, we decided to monitor the expression during contamination with a heat sensitive (ts) mutant computer virus that is unable to enter the nucleus at the NPT. By homologous recombination we obtained an HSV-1 strain carrying the single amino acid change, Y1453H, in the VP1/2 protein (that results in the ts phenomenon) and encoding the mCherry fluorescent protein (HSV-1 OK24). Next, we wanted to demonstrate that this heat shift can lead to a rapid entry into the nucleus. Cells were infected with either OK11 or OK24 and the viral DNA.