The mechanisms by which mammalian cells recognize and epigenetically restrict viral DNA are not well defined. We used herpes simplex virus with bioorthogonally labeled genomes to detect host factors recruited to viral DNA shortly after its nuclear entry and found that the cellular IFI16, PML, and ATRX proteins colocalized with viral DNA by 15 min post infection. HSV-1 infection of ATRX-depleted fibroblasts resulted in elevated viral mRNA and accelerated viral DNA accumulation. Despite the early association of ATRX with vDNA, we found that initial viral heterochromatin formation is ATRX-independent. However, viral heterochromatin stability required ATRX from 4-8 h post infection. Inhibition of transcription blocked viral chromatin loss in ATRX-knockout cells; thus, ATRX is uniquely required for heterochromatin maintenance during chromatin stress. These results argue that the initial formation and the subsequent maintenance of viral heterochromatin are separable mechanisms, a concept that likely extrapolates to host cell chromatin and viral latency.
Colocalization Distribution Data Analysis Script
MATLAB script uses nuclear masks and foci center x,y coordinate data from each channel (results from Foci Detection and Colocalization software) to determine distances of nearest neighbor foci in a second channel from foci in the reference channel and generate distance frequency data. The script then generates the same number of random x,y points within the nuclear mask as were detected in the second channel. The distances from the centers of reference channel foci to nearest neighbor random points is then calculated and reported to within a user defined user defined radius of each reference focus. The resulting frequency distributions of reference-to-test channel distances and reference-to-random points are compared by the non-parametric Kolmogorov-Smirnov test. If the test rejects the null hypothesis at 5% significance, the test returns a value of 1, and 0 if the null hypothesis is not rejected. The test also returns an asymptotic p-value and a list of distances in pixel values which can then be plotted and further analyzed using GraphPad Prism (as it was here) or other data analysis packages.
ColocCode_V02.zip
Foci Detection and Colocalization 1052 Software.
Foci Detection and Colocalization Software. The Image and Data Analysis Core (IDAC) at Harvard Medical School developed a custom MATLAB based software for nuclear foci and colocalization detection in microscopy images based upon their previously described nuclear foci detection software. In brief, unaltered images are analyzed by the software to detect signal intensity over a set threshold to isolate foci from background. The software then uses DAPI staining to generate a mask that defines nuclear areas in the image. Only foci above the fluorescence threshold and within the nuclear masks are scored. The software determines colocalization based on the distance between the centers of nuclear foci in a reference channel to foci in other channels of the image.
ColocMatlabScripts.zip
