The association of PML and DNA damage response foci with HSV-1 genomes.

<p>The uninfected human diploid fibroblast nucleus on the left shows a typical distribution of PML NBs (PML, red) with only faint staining of the DNA damage response protein γH2AX (blue), with the separated channels shown in grayscale below. The infected cell nucleus on the right is of a cell at the edge of a developing ICP0-null mutant HSV-1 plaque, which has been infected with a very high number of virus particles by spread from neighboring heavily infected cells. The viral genomes can be detected by the presence of the viral transcriptional activator ICP4 (green), which binds efficiently to viral DNA. PML (red) has been redistributed from its normal locations to sites that closely associated with the viral genomes. This association is stabilized by the absence of ICP0, which efficiently inhibits the formation of these foci in a normal wild type HSV-1 infection. A DNA damage response has been initiated to produce a marked increase in γH2AX (blue) in regions close to the viral genomes.</p

List of putative SUMO2 substrate proteins that are reduced in abundance in the purified infected compared to uninfected samples.

<p>The entries are shaded by degree of change, and all gave H/L ratios of 2 or greater with SigB values <0.1. The darkest shading indicates greater than 10-fold decrease, then in order of shading 7–10 fold, 5–7 fold, 3–5 fold and 2–3 fold.</p

Influence of a SUMO interaction motif within ICP0 on the stability of selected proteins during HSV-1 infection.

<p>A. HepaRG cells were infected with wt or mSLS4 mutant HSV-1 and samples taken at 3, 6 and 10 h after infection were analyzed for the abundance of NACC1, MORC3 and ZBTB38 as indicated. The MOI were adjusted to ensure similar levels of viral gene expression (10 for wt, 20 for mSLS4), as shown in the ICP0, ICP4 and UL42 panels. B and C. HepaRG cells transduced to express ZBTB20, MBD1 (B) and ZBTB10 and BEND3 (C) in an inducible manner were left uninduced or treated with doxycycline for the indicated times to induce expression of the myc tagged proteins. These cells were then infected with wt or mSLS4 mutant HSV-1 for 8 h (MOI 5 and 20 respectively), to ensure similar levels of viral protein expression (as indicated by the panels on the right) then analyzed for the relevant proteins by detection of the myc tag.</p

Analysis of potential sumoylation of HSV-1 proteins.

<p>(A) Tabulation of the nine HSV-1 proteins (excluding glycoproteins) whose apparent molecular weights in the purified fraction exceed those in the crude. App and Pred MW indicate apparent (based on gel slice data) and predicted (based on primary sequence) molecular weights, while reported gel MW indicates the established apparent MW based on gel mobility as extensively reported in the literature. (B) Western blot analysis of UL42 in crude and purified HA-HisSUMO2 fractions of cells infected with wt HSV-1 (MOI 10 at 12 h after infection; m indicates a purified mock infected sample prepared in parallel), showing probable sumoylated species in the purified infected sample (indicated by dashes on right). (C) Sumoylated forms of UL42 are below the level of detection in total protein extracts of wt HSV-1 infected HepaRG cells. The asterisk on the left indicates a non-specific background band. (D) The samples used in B were analyzed for UL6 and ICP0. Trace amounts of potential sumoylated forms of UL6 and ICP0 are indicted by dashes on the right. (E). The same samples were analyzed for ICP4 and UL12.</p

Degradation of selected cellular proteins by ICP0 in the absence of infection.

<p>A. HA-TetR and HA-cICP0 cells were treated with doxycycline (100 ng/ml) for 24 h (+) or left untreated (-), then whole cell extracts were prepared and analyzed by western blotting for the indicated proteins using antibodies directed against the endogenous proteins. The dashes on the right indicate the major presumed specific bands (determined on the basis of analyses in previous figures) in each case. Except in the cases of CITED2 and ZBTB4, these major bands decrease in the presence of ICP0. The upper dash in the ZBTB4 panel indicates a potential sumoylated form that is sensitive to ICP0. (B). Analysis of HA-cICP0 cells (left, lanes 1 and 2) and derivatives of HA-cICP0 cells that had been transduced with lentivirus vectors expressing myc-tagged BEND3 (left, lanes 3 and 4) and MBD1 (right), before and after induction of ICP0 expression, illustrating that ICP0 is sufficient to degrade BEND3 and MBD1.</p

List of putative SUMO2 substrate proteins that are reduced in abundance in the purified infected compared to uninfected samples.

<p>The entries are shaded by degree of change, and all gave H/L ratios of 2 or greater with SigB values <0.1. The darkest shading indicates greater than 10-fold decrease, then in order of shading 7–10 fold, 5–7 fold, 3–5 fold and 2–3 fold.</p

Brief information on sumoylated proteins validated in this study.

<p>Brief information on sumoylated proteins validated in this study.</p

A listing of the proteins showing greater than 3-fold increases in H/L ratios.

<p>The proteins are taken from entries in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005059#ppat.1005059.g003" target="_blank">Fig 3</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005059#ppat.1005059.s008" target="_blank">S4 Table</a>, grouped according to general function or characteristics and shaded according to degree of ratio change (as used in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005059#ppat.1005059.g003" target="_blank">Fig 3</a>). Asterisks indicate proteins from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005059#ppat.1005059.s008" target="_blank">S4 Table</a>. There is some overlap between some of the categories, for example many BTB proteins also have a zinc finger, while many zinc finger proteins could equally be described as transcription factors or chromatin related.</p

Time course of degradation of selected proteins of interest during wt HSV-1 infection.

<p>HFs were infected with wt HSV-1 (MOI 10) then samples harvested at 3, 6 and 10 h after infection were analyzed by western blot for the indicated proteins. For ZBTB4, the asterisk marks a potentially sumoylated species that decreases in abundance during infection. The panels on the lower right of the figure show the analysis of selected viral proteins in these samples.</p

Characterization of HA-HisSUMO2 cells.

<p>(A) Cells expressing His-tagged SUMO2 were infected with wt HSV-1 at MOI 10 and samples were harvested at 3, 6, and 10 h p.i. Proteins were analyzed by western blotting using 6xHis, PML 5E10, ICP0 11060, and tubulin antibodies. Mock infected HA-HisSUMO2 cells were included as a control. (B) Kinetics of HSV-1 protein expression are equivalent in HA-HisSUMO2, parental HepaRG and HA-His only cell lines. Cell extracts were analyzed by western blotting for viral proteins ICP4, ICP0 and UL42, following wt HSV-1 infection for 3, 6, and 10 h (MOI 10). Mock infected controls (m) were included for each cell line and blots probed for actin as a loading control. (C) Plaque forming efficiency of wt HSV-1 in HA-HisSUMO2 cells relative to the parental HepaRG cell line. Data from replicate experiments was averaged, normalized to the HepaRG cell data and represented as mean +/- standard deviation. (D) His affinity purification of His-SUMO2 conjugated proteins. Samples collected throughout the affinity purification were analyzed by western blot for purification and elution of His-SUMO2 proteins. I–initial extract; U–unbound fraction; E1 and E2 –sequential elution samples; B–a sample of the remaining beads after elution. 1% of the initial lysate, 16% of total elution fractions and 4% of the bead fraction were loaded. (E) The same samples were analyzed for the presence of sumoylated PML.</p