Inflammatory monocytes require type I interferon receptor signaling to activate NK cells via IL-18 during a mucosal viral infection

The requirement of type I interferon (IFN) for natural killer (NK) cell activation in response to viral infection is known, but the underlying mechanism remains unclear. Here, we demonstrate that type I IFN signaling in inflammatory monocytes, but not in dendritic cells (DCs) or NK cells, is essential for NK cell function in response to a mucosal herpes simplex virus type 2 (HSV-2) infection. Mice deficient in type I IFN signaling, Ifnar(-/-) and Irf9(-/-) mice, had significantly lower levels of inflammatory monocytes, were deficient in IL-18 production, and lacked NK cell-derived IFN-gamma. Depletion of inflammatory monocytes, but not DCs or other myeloid cells, resulted in lower levels of IL-18 and a complete abrogation of NK cell function in HSV-2 infection. Moreover, this resulted in higher susceptibility to HSV-2 infection. Although Il18(-/-) mice had normal levels of inflammatory monocytes, their NK cells were unresponsive to HSV-2 challenge. This study highlights the importance of type I IFN signaling in inflammatory monocytes and the induction of the early innate antiviral response

Cytoplasmic factories, virus assembly, and DNA replication kinetics collectively constrain the formation of poxvirus recombinants.

Poxviruses replicate in cytoplasmic structures called factories and each factory begins as a single infecting particle. Sixty-years ago Cairns predicted that this might have effects on vaccinia virus (VACV) recombination because the factories would have to collide and mix their contents to permit recombination. We've since shown that factories collide irregularly and that even then the viroplasm mixes poorly. We've also observed that while intragenic recombination occurs frequently early in infection, intergenic recombination is less efficient and happens late in infection. Something inhibits factory fusion and viroplasm mixing but what is unclear. To study this, we've used optical and electron microscopy to track factory movement in co-infected cells and correlate these observations with virus development and recombinant formation. While the technical complexity of the experiments limited the number of cells that are amenable to extensive statistical analysis, these studies do show that intergenic recombination coincides with virion assembly and when VACV replication has declined to ≤10% of earlier levels. Along the boundaries between colliding factories, one sees ER membrane remnants and other cell constituents like mitochondria. These collisions don't always cause factory fusion, but when factories do fuse, they still entrain cell constituents like mitochondria and ER-wrapped microtubules. However, these materials wouldn't seem to pose much of a further barrier to DNA mixing and so it's likely that the viroplasm also presents an omnipresent impediment to DNA mixing. Late packaging reactions might help to disrupt the viroplasm, but packaging would sequester the DNA just as the replication and recombination machinery goes into decline and further reduce recombinant yields. Many factors thus appear to conspire to limit recombination between co-infecting poxviruses

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

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

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

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 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