Noise in timing and precision of gene activities in a genetic cascade

Biological developmental pathways require proper timing of gene expression. We investigated timing variations of defined steps along the lytic cascade of bacteriophage λ. Gene expression was followed in individual lysogenic cells, after induction with a pulse of UV irradiation. At low UV doses, some cells undergo partial induction and eventually divide, whereas others follow the lytic pathway. The timing of events in cells committed to lysis is independent of the level of activation of the SOS response, suggesting that the lambda network proceeds autonomously after induction. An increased loss of temporal coherence of specific events from prophage induction to lysis is observed, even though the coefficient of variation of timing fluctuations decreases. The observed temporal variations are not due to cell factors uniformly dilating the timing of execution of the cascade. This behavior is reproduced by a simple model composed of independent stages, which for a given mean duration predicts higher temporal precision, when a cascade consists of a large number of steps. Evidence for the independence of regulatory modules in the network is presented

Phage Lambda CIII: A Protease Inhibitor Regulating the Lysis-Lysogeny Decision

The ATP-dependent protease FtsH (HflB) complexed with HflKC participates in post-translational control of the lysis-lysogeny decision of bacteriophage lambda by rapid degradation of lambda CII. Both phage-encoded proteins, the CII transcription activator and the CIII polypeptide, are required for efficient lysogenic response. The conserved CIII is both an inhibitor and substrate of FtsH. Here we show that the protease inhibitor CIII is present as oligomeric amphipathic α helical structures and functions as a competitive inhibitor of FtsH by preventing binding of the CII substrate. We identified single alanine substitutions in CIII that abolish its activity. We characterize a dominant negative effect of a CIII mutant. Thus, we suggest that CIII oligomrization is required for its function. Real-time analysis of CII activity demonstrates that the effect of CIII is not seen in the absence of either FtsH or HflKC. When CIII is provided ectopically, CII activity increases linearly as a function of the multiplicity of infection, suggesting that CIII enhances CII stability and the lysogenic response. FtsH function is essential for cellular viability as it regulates the balance in the synthesis of phospholipids and lipopolysaccharides. Genetic experiments confirmed that the CIII bacteriostatic effects are due to inhibition of FtsH. Thus, the early presence of CIII following infection stimulates the lysogenic response, while its degradation at later times ensures the reactivation of FtsH allowing the growth of the established lysogenic cell

A Dual Infection Pseudorabies Virus Conditional Reporter Approach to Identify Projections to Collateralized Neurons in Complex Neural Circuits

Replication and transneuronal transport of pseudorabies virus (PRV) are widely used to define the organization of neural circuits in rodent brain. Here we report a dual infection approach that highlights connections to neurons that collateralize within complex networks. The method combines Cre recombinase (Cre) expression from a PRV recombinant (PRV-267) and Cre-dependent reporter gene expression from a second infecting strain of PRV (PRV-263). PRV-267 expresses both Cre and a monomeric red fluorescent protein (mRFP) fused to viral capsid protein VP26 (VP26-mRFP) that accumulates in infected cell nuclei. PRV-263 carries a Brainbow cassette and expresses a red (dTomato) reporter that fills the cytoplasm. However, in the presence of Cre, the dTomato gene is recombined from the cassette, eliminating expression of the red reporter and liberating expression of either yellow (EYFP) or cyan (mCerulean) cytoplasmic reporters. We conducted proof-of-principle experiments using a well-characterized model in which separate injection of recombinant viruses into the left and right kidneys produces infection of neurons in the renal preautonomic network. Neurons dedicated to one kidney expressed the unique reporters characteristic of PRV-263 (cytoplasmic dTomato) or PRV-267 (nuclear VP26-mRFP). Dual infected neurons expressed VP26-mRFP and the cyan or yellow cytoplasmic reporters activated by Cre-mediated recombination of the Brainbow cassette. Differential expression of cyan or yellow reporters in neurons lacking VP26-mRFP provided a unique marker of neurons synaptically connected to dual infected neurons, a synaptic relationship that cannot be distinguished using other dual infection tracing approaches. These data demonstrate Cre-enabled conditional reporter expression in polysynaptic circuits that permits the identification of collateralized neurons and their presynaptic partners

Gene Expression Correlates with the Number of Herpes Viral Genomes Initiating Infection in Single Cells

<div><p>Viral gene expression varies significantly among genetically identical cells. The sources of these variations are not well understood and have been suggested to involve both deterministic host differences and stochastic viral host interactions. For herpesviruses, only a limited number of incoming viral genomes initiate expression and replication in each infected cell. To elucidate the effect of this limited number of productively infecting genomes on viral gene expression in single cells, we constructed a set of fluorescence-expressing genetically tagged herpes recombinants. The number of different barcodes originating from a single cell is a good representative of the number of incoming viral genomes replicating (NOIVGR) in that cell. We identified a positive correlation between the NOIVGR and viral gene expression, as measured by the fluorescent protein expressed from the viral genome. This correlation was identified in three distinct cell-types, although the average NOIVGR per cell differed among these cell-types. Among clonal single cells, high housekeeping gene expression levels are not supportive of high viral gene expression, suggesting specific host determinants effecting viral infection. We developed a model to predict NOIVGR from cellular parameters, which supports the notion that viral gene expression is tightly linked to the NOIVGR in single-cells. Our results support the hypothesis that the stochastic nature of viral infection and host cell determinants contribute together to the variability observed among infected cells.</p></div

Cells with higher expression of a housekeeping gene have a decreased expression of viral genes.

<p>GFP expressing HeLa cells were infected with one of the barcoded viruses. (A). A representative image at 8 HPI is shown. Scale bar 10μm. (B). Fluorescence levels from GFP (shades of green) and mCherry (shade of red) were monitored during HeLa cells infection every 30 minutes for 10 HPI. Infection at MOI 100 (darkest color) or MOI 10 were compared to uninfected cells (lightest color). An average fluorescence of nine frames from cells infected with one of three viral recombinants (three from each recombinant) is shown. Standard deviation for each time point is represented. (C, D) A combination of 780 cells infected with one of three viral recombinants, (two frames from each recombinant, 130 cells per frame) were analyzed from images taken at 10 HPI. Cells were grouped according to the relative fluorescence levels in both GFP and mCherry as indicated for MOI 10 (C) and MOI 100 (D). Each number in the Heatmap represent the proportions of cells out of the total cells analyzed. (E, F) A combination of 1200 cells from 12 different LARCs infected with one viral recombinant, (two frames from each LARC, 50 cells per frame) were analyzed from images taken at 8 HPI. Cells were grouped according to the relative fluorescence levels in both GFP and mCherry as indicated for MOI 10 (E) and MOI 100 (F). Each number in the Heatmap represents the proportions of cells out of the total cells analyzed. The color scheme reflects the relative proportions as indicated in the side bar (log 2 scale).</p

The number of barcodes replicating in individual Vero cells correlates with fluorescence.

<p>(A) The relative proportion of each barcode of the total number of barcodes detected from all infectious centers analyzed. The results are presented according to MOI, and each barcode is represented by a different color. (B-E) The output infectious centers, each originated from one individual infected cell, were lysed and analyzed with qPCR. A total of 137 cells from three experiments are presented. (B, C) The distribution of the number of barcodes per cell is depicted at MOI 10 (B, open bars) and MOI 100 (C, solid bars). Each MOI is categorized according to the fluorescence intensity of the cells, from low (blue), through intermediate (purple) and high (red). (D, E) The number of replicated barcode progeny from individual cells, infected at MOI 10 (D) and MOI 100 (E), was plotted against expressed fluorescence, as measured by the cell sorter. Each point is color coded as above. A trend line that was calculated using the ordinary least squares (OLS) method and the measured Pearson correlation, are presented in each graph.</p

Relative fluorescence is maintained throughout infection.

<p>(A-E) Cells infected with a mixture of the barcode-viruses at MOI 100 were monitored for 18 hours. (A) Snapshots of cells at different time points (as indicated) are presented. Scale bar 20μm. (B-E) The infected cells were divided according to the fluorescence intensity of the cells at 4 HPI, from lowest (blue 30% of total), through middle (purple 40%) and highest (red 30%). (B) The fluorescent profile of 97 individual cells from a representative single well (4 different frames) is presented as a function of time. (C) The mean of the cell fluorescent profiles, for each of the groups of fluorescence intensity, was calculated for each well. A mean of three wells (with the standard deviation between the wells-stripe lines) is presented. (D-E) At each time point indicated, cell profiles were sorted according to fluorescence levels. The 30% lowest population (D) and 30% highest population (E) were compared to the 4 HPI time point. Each bar represents the ratio of cells from the 4 HPI segregation (colored as above), as found at the indicated time point. An average of the ratios from three different wells is presented. (F-G) Cells were infected with three different fluorescence expressing viral recombinants (each expressing one of the following fluorescent genes: mCherry, EYFP or mTurq2) at MOI of 100. Ten representative cells were plotted according to their hues on triangular barymetric plot. Each vertex represents a different pure color (red, blue and green), edges represent a combination of two colors and the inside of the triangle represent mixture of three colors. (F) The ten cells (dots) are shown at 3 HPI as indicated. (G) A three dimensional plot of the changes in hue over time (z-axis) for the ten cells (lines) is presented. Each cell is plotted in a representative color.</p

The correlation between the number of barcodes replicating in cells and viral fluorescence is maintained in individual human fibroblasts.

<p>(A) The relative proportion of each barcode of the total number of barcodes detected from all infectious centers analyzed. The results are presented according to MOI, and each barcode is represented by a different color. (B-E) The output infectious centers, each originated from one individual infected cell, were lysed and analyzed with qPCR. A total of 135 cells from three experiments are presented. (B, C) A distribution of the number of barcodes per cell is depicted at MOI 10 (B, open bars) and MOI 100 (C, solid bars). Each MOI is categorized according to the fluorescence intensity of the cells, from low (pink), through intermediate (light red) and high (red). (D, E) The number of replicated barcode progeny from individual cells, infected at MOI 10 (D) and MOI 100 (E), was plotted against expressed fluorescence, as measured by the cell sorter. Each point is color coded as above. A trend line that was calculated using the ordinary least squares (OLS) method and the measured Pearson correlation, are presented in each graph.</p

Relative fluorescence intensity of infected cells correlates with viral gene expression.

<p>(A-B) Cells were infected with the barcode-viruses at MOI 10 (A) or 100 (B). At 3 HPI, the cells were sorted into three groups according to their fluorescence intensity from lowest (blue 30% of total), through middle (base line levels 40%) and highest (red 30%). Total RNA was collected from each group and RT-qPCR was performed for several viral genes as indicated. In each experiment, expression levels from highest and lowest groups were normalized to that in the middle group. Box plots of five biological repeats generated by SPSS are presented. Outliers (1.5</p