The SUMO Ligase Protein Inhibitor of Activated STAT 1 (PIAS1) is a constituent PML-NB protein that contributes to the intrinsic antiviral immune response to herpes simplex virus 1 (HSV-1)

Aspects of intrinsic antiviral immunity are mediated by promyelocytic leukaemia (PML)-nuclear body (PML-NB) constituent proteins. During herpesvirus infection, these antiviral proteins are independently recruited to nuclear domains that contain infecting viral genomes to cooperatively promote viral genome silencing. Central to the execution of this particular antiviral response is the small ubiquitin-like modifier (SUMO) signalling pathway. However, the participating SUMOylation enzymes are not fully characterized. We identify the SUMO ligase Protein Inhibitor of Activated STAT1 (PIAS1) as a constituent PML-NB protein. We show that PIAS1 localizes at PML-NBs in a SUMO interaction motif (SIM)-dependent manner that requires SUMOylated or SUMOylation competent PML. Following infection with herpes simplex virus 1 (HSV-1), PIAS1 is recruited to nuclear sites associated with viral genome entry in a SIM-dependent manner, consistent with the SIM-dependent recruitment mechanisms of other well characterized PML-NB proteins. In contrast to Daxx and Sp100, however, the recruitment of PIAS1 is enhanced by PML. PIAS1 promotes the stable accumulation of SUMO1 at nuclear sites associated with HSV-1 genome entry, whereas the accumulation of other evaluated PML-NB proteins occurs independently of PIAS1. We show that PIAS1 cooperatively contributes to HSV-1 restriction through mechanisms that are additive to those of PML and cooperative with those of PIAS4. The antiviral mechanisms of PIAS1 are counteracted by ICP0, the HSV-1 SUMO-targeted ubiquitin ligase, which disrupts the recruitment of PIAS1 to nuclear domains that contain infecting HSV-1 genomes through mechanisms that do not directly result in PIAS1 degradation

Chromatin Dynamics during Lytic Infection with Herpes Simplex Virus 1

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The differential mobilization of histones H3.1 and H3.3 by herpes simplex virus 1 relates histone dynamics to the assembly of viral chromatin.

During lytic infections, HSV-1 genomes are assembled into unstable nucleosomes. The histones required for HSV-1 chromatin assembly, however, are in the cellular chromatin. We have shown that linker (H1) and core (H2B and H4) histones are mobilized during HSV-1 infection, and proposed that the mobilized histones are available for assembly into viral chromatin. However, the actual relevance of histone mobilization remained unknown. We now show that canonical H3.1 and variant H3.3 are also mobilized during HSV-1 infection. Mobilization required no HSV-1 protein expression, although immediate early or early proteins enhanced it. We used the previously known differential association of H3.3 and H3.1 with HSV-1 DNA to test the relevance of histone mobilization. H3.3 binds to HSV-1 genomes first, whereas H3.1 only binds after HSV-1 DNA replication initiates. Consistently, H3.3 and H3.1 were differentially mobilized. H3.1 mobilization decreased with HSV-1 DNA replication, whereas H3.3 mobilization was largely unaffected by it. These results support a model in which previously mobilized H3.1 is immobilized by assembly into viral chromatin during HSV-1 DNA replication, whereas H3.3 is mobilized and assembled into HSV-1 chromatin throughout infection. The differential mobilizations of H3.3 and H3.1 are consistent with their differential assembly into viral chromatin. These data therefore relate nuclear histone dynamics to the composition of viral chromatin and provide the first evidence that histone mobilization relates to viral chromatin assembly

Correction:The Differential Mobilization of Histones H3.1 and H3.3 by Herpes Simplex Virus 1 Relates Histone Dynamics to the Assembly of Viral Chromatin

<p>Correction:The Differential Mobilization of Histones H3.1 and H3.3 by Herpes Simplex Virus 1 Relates Histone Dynamics to the Assembly of Viral Chromatin</p

KM110 infection only marginally mobilizes H3.1.

<p>(<b>A</b>) Average normalized levels of free GFP-H3.1 relative to mock-infected cells at 4 or 7 hpi, respectively. Vero cells were transfected with plasmids expressing GFP-H3.1. Transfected cells were mock-infected or infected with 30 PFU/cell of strain KM110. Mobilization of GFP-H3.1 was examined from 4 to 5 (<b>4</b>) or 7 to 8 (<b>7</b>) hpi by FRAP; error bars, SEM; dashed line, average normalized levels of free H3.1 in mock-infected cells. (<b>B</b>) Frequency distribution plots of the percentage of free GFP-H3.1 per individual cell at 4 or 7 hpi; dotted line, one SD above the average level of free GFP-H3.1 in mock-infected cells. (<b>C</b>) Average initial rates of normalized fluorescence recovery relative to mock-infected cells at 4 hpi; error bars, SEM. (<b>D</b>) Frequency distribution plots of the initial rate of normalized fluorescence recovery of GFP-H3.1 per individual cell; dotted line, one SD above the average initial rate of normalized fluorescence recovery in mock-infected cells. *, P<0.05; <i>n.s.</i>, not significant.</p

HSV-1 DNA replication decreases mobilization of H3.1 but not of H3.3.

<p>(<b>A</b>) Relative normalized fluorescence intensity of the photobleached nuclear region plotted against time. Vero cells were transfected with plasmids expressing GFP-H3.3 (<b>H3.3</b>) or -H3.1 (<b>H3.1</b>). Transfected cells were mock-infected or infected with 30 PFU/cell of strain KOS in the presence of 400 µg/ml of PAA (<b>PAA+KOS</b>) or no drug (<b>KOS</b>). Mobilization of GFP-H3.3 or -H3.1 was examined from 7 to 8 hpi by FRAP; error bars, SEM. (<b>B</b>) Average normalized levels of free GFP-H3.3 or -H3.1 relative to untreated mock-infected cells; error bars, SEM; dashed line, normalized levels in untreated mock-infected cells. (<b>C</b>) Frequency distribution plots of the percentage of free GFP-H3.3 or -H3.1 in individual cells; dotted line, one SD above the average level of free GFP-H3.3 or -H3.1 in untreated KOS infected cells. (<b>D</b>) Average initial rates of normalized fluorescence recovery relative to untreated mock-infected cells; error bars, SEM; dashed line, average initial rates of normalized fluorescence recovery in untreated mock-infected cells. (<b>E</b>) Frequency distribution plots of the initial rate of normalized fluorescence recovery of GFP-H3.3 or -H3.1 in each cell; dotted line, one SD above the average initial rate of normalized fluorescence recovery of GFP-H3.3 or -H3.1 in untreated KOS infected cells. KOS data re-plotted from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003695#ppat-1003695-g002" target="_blank">Figure 2</a> for comparison. **, P<0.01; <i>n.s.</i>, not significant.</p

FRAP of GFP-H3 fusion proteins.

<p>(<b>A</b>) Digital fluorescent micrographs of the nucleus of a cell expressing GFP-H3.3 before and after photobleaching. Vero cells were transfected with plasmids expressing GFP-H3.3. A region passing across the long nuclear axis was photobleached, and the fluorescence recovery in the photobleached region was evaluated. The photobleached region was selected independently of the presence or absence of replication compartments, and thus includes nuclear domains containing cellular and viral DNA. Fluorescence in the photobleached region recovers as the photobleached GFP-histones within this region exchange with the non-photobleached GFP-histones from outside of this region. FRAP was evaluated for only 100 s; therefore, potential contributions from newly synthesized GFP-histones to fluorescence recovery are negligible. The enlargements in the lower panel highlight the photobleached region. (<b>B</b>) Line graph of a representative GFP-H3.3 FRAP. The fluorescence intensity of the photobleached region at a given time is normalized to the fluorescence of the entire nucleus at that same time, expressed as a ratio of the normalized fluorescence prior to photobleaching, and plotted against time. The fluorescence intensity is therefore independent of GFP-H3 expression levels. The first data point after photobleaching is a surrogate measure for the levels of free GFP-H3. The subsequent fluorescence recovery is biphasic. The initial faster phase represents those histones that are weakly bound in chromatin and undergoing faster chromatin exchange. As a surrogate measure for this population, we calculated the initial rate of normalized fluorescence recovery (the slope between the normalized fluorescence at the first and second data points after photobleaching; shown in the inset). The second slower phase of fluorescence recovery represents those histones that are more stably bound in chromatin and undergoing slower chromatin exchange.</p

VP16 or ICP0 modulate H3.3 mobilization.

<p>(<b>A</b>) Average normalized levels of free GFP-H3.3 relative to mock-infected cells at 4 or 7 hpi, respectively. U2OS cells were transfected with plasmids expressing GFP-H3.3. Transfected cells were mock-infected or infected with 30 PFU/cell of strain KM110 (<b>KM110</b>) or 6 PFU/cell of strain KOS (<b>KOS</b>). Mobilization of GFP-H3.3 was examined from 4 to 5 (<b>4</b>) or 7 to 8 (<b>7</b>) hpi by FRAP; error bars, SEM; dashed line, average normalized levels of free GFP-H3.3 in mock-infected cells. (<b>B</b>) Frequency distribution plots of the percentage of free GFP-H3.3 per individual cell at 4 or 7 hpi; dotted line, one SD above the average level of free GFP-H3.3 in mock-infected cells. (<b>C</b>) Average initial rates of normalized fluorescence recovery relative to mock-infected cells at 4 hpi; error bars, SEM. (<b>D</b>) Frequency distribution plots of the initial rate of normalized fluorescence recovery of GFP-H3.3 per individual cell; dotted line, one SD above the average initial rate of normalized fluorescence recovery in mock-infected cells. **, P<0.01; <i>n.s.</i>, not significant.</p