Leporipoxvirus infection does not increase the levels of H3K9me3 and H4K20me3 formation.

<p>BSC-40 cells were grown on coverslips and subsequently infected with VACV WR, MYXV, or SFV. Eighteen hours post infection the cells were fixed and stained to detect VACV I3 and (<b>A</b>) H3K9me3 or (<b>C</b>) H4K20me3. DNA was counterstained with DAPI. The presence of viral factories (stained with DAPI) was used to confirm infection with MYXV and SFV, as the I3 antibody does not cross-react in Leporipoxviruses. Representative images are shown (scale bar = 25 μm). Nuclear (<b>B</b>) H3K9me3 and (<b>D</b>) H4K20me3 signal intensities were quantified using FIJI imaging analysis software and normalized to mock-infected cells. We show the SEM of three independent experiments. Statistically significant differences are noted (<i>*P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001; **** <i>P</i><0.0001).</p

Orthopoxviruses promote H3K9me3 and H4K20me3 formation.

<p>BSC-40 cells were grown on coverslips and subsequently infected with VACV WR, VACV Cop, and CPXV. The cells were fixed and stained to detect I3 and (<b>A</b>) H3K9me3 or (<b>C</b>) H4K20me3 9hr post-infection. DNA was counterstained with DAPI. Images were acquired at 60x magnification (scale bar = 25 μm). The nuclear (<b>B</b>) H3K9me3 and (<b>D</b>) H4K20me3 signal intensities were quantified using FIJI imaging analysis software and normalized to mock-infected cells. Data represent the SEM of three independent experiments and any statistically significant differences relative to mock-infected cells, are noted (<i>*P</i><0.05; **<i>P</i><0.01).</p

The vaccinia virus K7 protein promotes histone methylation associated with heterochromatin formation

<div><p>It has been well established that many vaccinia virus proteins suppress host antiviral pathways by targeting the transcription of antiviral proteins, thus evading the host innate immune system. However, whether viral proteins have an effect on the host’s overall cellular transcription is less understood. In this study we investigated the regulation of heterochromatin during vaccinia virus infection. Heterochromatin is a highly condensed form of chromatin that is less transcriptionally active and characterized by methylation of histone proteins. We examined the change in methylation of two histone proteins, H3 and H4, which are major markers of heterochromatin, during the course of viral infection. Using immunofluorescence microscopy and flow cytometry we were able to track the overall change in the methylated levels of H3K9 and H4K20. Our results suggest that there is significant increase in methylation of H3K9 and H4K20 during <i>Orthopoxviruses</i> infection compared to mock-infected cells. However, this effect was not seen when we infected cells with <i>Leporipoxviruses</i>. We further screened several vaccinia virus single and multi-gene deletion mutant and identified the vaccinia virus gene K7R as a contributor to the increase in cellular histone methylation during infection.</p></div

Exogenous expression of K7 and rescue of K7R in XY-dBID-VACV partially restores H3K9me formation during virus infection.

<p><b>(A)</b> BSC-40 cells were grown on glass coverslips and infected with VACV WR or XY-dBID-VACV for 2h prior to transfection with plasmids expressing either K7-myc or N2-myc-tagged proteins. The samples were fixed 18h post infection and stained with antibodies to myc and nuclear H3K9me3. Representative images are shown (scale bar = 25 μm). (<b>B</b>) The levels of nuclear H3K9me3 were imaged as shown in panel (A), quantified using FIJI, and normalized to levels seen in mock-infected cells. Data represent the SEM of three independent experiments and statistically significant differences are noted (**<i>P</i><0.01; **<i>P</i><0.01, ****<i>P</i><0.0001). <b>(C)</b> BSC-40 cells were infected with VACV WR, XY-dBID-VACV, XY-dBID-VACVΔJ2R, or XY-dBID-VACVK7Rmyc1 viruses, the last encoding K7Rmyc inserted into the J2R locus. The cells were fixed and stained with myc and H3K9me3 specific antibodies and counterstained with DAPI to visualize nuclei. Nuclear H3K9me3 levels were quantified using FIJI and normalized to that seen in mock-infected cells. The data show the SEM of three independent experiments and statistically significant differences are noted (<i>*P</i> <0.05; **<i>P</i> <0.01; ***<i>P</i><0.001; **** <i>P</i><0.0001).</p

VACV ΔK7R-mutants promote less H3K9me3 and H4K20me3 formation.

<p>BSC-40 cells were grown on glass coverslips and infected (or mock infected) at a MOI of 5.0 with VACV, XY-dBID-VACV, or VACV bearing deletions in the indicated <i>Hin</i>dIII K-fragment genes. The cells were fixed and processed at 9 hr post-infection as described above. Representative microscopy images are shown (<i>scale</i> bar = 25 μm). The intensities of the nuclear H3K9me3 were quantified using FIJI and normalized to mock-infected cells. Data represent the SEM of three independent experiments. Any statistically significant differences, relative to mock-infected cells, are noted (**<i>P</i><0.01; ***<i>P</i><0.001).</p

Reduced H3K9me3 and H4K20me3 formation is seen in cells infected with VACV strain XY-dBID.

<p>The strain designated as XY-dBID-VACV encodes a deletion from N1L to F4L (inclusive). BSC-40 cells were grown on coverslips and infected with VACV or XY-dBID-VACV for 9h or 18h. The cells were fixed and stained with antibodies specific to I3, H3K9me3, or H4K20me3 and for DNA (with DAPI). (<b>A</b>) Representative images showing the staining of nuclear histone markers (scale bar = 25 μm). The amounts of nuclear (<b>B</b>) H3K9me3 and (<b>C</b>) H4K20me3 were measured using FIJI and normalized relative to mock-infected cells. The data represent the SEM of three independent experiments and any statistically significant differences are noted (<i>*P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001; **** <i>P</i><0.0001).</p

H3K9me3 and H4K20me3 formation requires VACV early gene expression.

<p>BSC-40 cells were grown on coverslips and infected for 9 hr with VACV (<b>A</b>) with or without UV inactivation, (<b>B</b>) with or without cycloheximide, or (<b>C, D</b>) with or without of AraC. The cells were fixed and stained for I3 and for H3K9me3 <b>(A-C)</b> or H4K20me3 <b>(D)</b> using specific antibodies. After imaging, the levels of nuclear H3K9me3 and H4K20me3 were quantified using FIJI and normalized relative to amounts measured in mock-infected cells. The data show the SEM of three independent experiments.</p

VACV infection increases the levels of H3K9me3.

<p>(<b>A)</b> BSC-40 cells were grown on coverslips and infected at a MOI 5.0 with VACV strain WR. At various time points post infection, the coverslips were fixed and stained to detect the VACV I3 protein and H3K9me3. DNA was counterstained with DAPI. Images were acquired using an Olympus IX-71 inverted microscope at 60x magnification and deconvolved using Softworx software (GE Healthcare) (scale bar = 25 μm). The nuclear H3K9me3 signal intensities from (<b>B</b>) microscopy images and (<b>C</b>) flow cytometry were quantified using FIJI imaging analysis software and normalized to mock-infected cells. At least five images were analyzed per samples within an independent experiment. Data represent the standard error of the mean (SEM) of three independent experiments. GraphPad was used to determine significant differences in H3K9me3 levels following VACV infection. Statistically significant differences are noted, relative to time zero (<i>*P</i><0.05; **<i>P</i><0.01).</p

VACV infection increases the levels of H4K20me3.

<p>(<b>A)</b> BSC-40 cells were grown on coverslips and infected at a MOI 5.0 with VACV. The cells were fixed and stained to detect VACV I3 protein, H4K20me3, and DNA 9 hr post-infection. Images were acquired at 60x magnification (scale bar = 25 μm). The nuclear H3K9me3 signal intensities from (<b>B</b>) microscopy images and (<b>C</b>) flow cytometry were quantified using FIJI imaging analysis software and normalized to mock-infected cells. At least five images were analyzed per samples within an independent experiment. The experiment was performed three independent times and the SEM was then calculated relative to the mock infection <b>(B)</b> or time zero <b>(C)</b>. Statistically significant differences are noted (<i>*P</i><0.05; ***<i>P</i><0.001).</p