iPOND detects viral and cellular proteins associated with replicated HSV-1 genomes.

<p>(A) ICP4 was detected by western blot of protein eluates from iPOND carried out on viral genomes grown in the presence of EdU at 4–6, 6–8, and 8–12 hpi. The control was iPOND carried out on virus grown in the absence of EdU (−) and harvested 8 hpi. Purified ICP4 is shown. (B) DNA eluted from streptavidin-coated beads during iPOND experiments in (A) is viral. The amount of viral DNA present in cell lysates (input) and eluted from beads (bound) during iPOND experiments was measured by qRT-PCR of the viral thymidine kinase (TK) gene. The ratio of viral DNA (vDNA) to total DNA was calculated and is presented as log %vDNA. (C) Pie charts summarize proteins that were identified by mass spectrometry of protein eluates from iPOND carried out 6, 8, and 12 hpi with UL2/UL50 mutant virus. Values indicate the number of proteins identified for each functional category. (D) Venn diagrams depict the overlap of proteins identified by iPOND at each time point.</p

Chromatin remodeling factors and histones identified by iPOND and aniPOND.

<p>Experiments, conditions, and complexes are indicated in bold. Columns 1, 2, and 3 include protein name, description, and accession number. Values indicate spectral counts determined by mass spectrometry.</p><p>Chromatin remodeling factors and histones identified by iPOND and aniPOND.</p

RNA processing factors identified by iPOND and aniPOND.

<p>Experiments, conditions, and complexes are indicated in bold. Columns 1, 2, and 3 include protein name, description, and accession number. Values indicate spectral counts determined by mass spectrometry.</p><p>RNA processing factors identified by iPOND and aniPOND.</p

HSV-1 proteins identified by iPOND and aniPOND.

<p>Experiments, conditions, and complexes are indicated in bold. Columns 1, 2, and 3 include protein name, description, and accession number. Values indicate spectral counts determined by mass spectrometry. Identified viral replication, transcription, and RNA processing factors are listed in Tables <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004939#ppat.1004939.t002" target="_blank">2</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004939#ppat.1004939.t003" target="_blank">3</a>, and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004939#ppat.1004939.t006" target="_blank">6</a>, respectively.</p><p>HSV-1 proteins identified by iPOND and aniPOND.</p

Cellular repair proteins identified by iPOND and aniPOND.

<p>Experiments, conditions, and complexes are indicated in bold. Columns 1, 2, and 3 include protein name, description, and accession number. Values indicate spectral counts determined by mass spectrometry.</p><p>Cellular repair proteins identified by iPOND and aniPOND.</p

Histones H1 and H3 do not colocalize with replicated HSV genomes.

<p>(A) Infected Vero cells were maintained in the presence of EdU at 4–8 hpi. Cellular DNA was visualized by Hoechst staining, viral replication compartments by click chemistry with EdU, and histone H1 by immunofluorescence. Merged panels show the lack of colocalization of viral DNA with histone H1. Uninfected cells are shown as a control for normal histone distribution in the nucleus. (B) Imaging was carried out as in (A) except that immunofluorescence was carried out with antibodies specific for histone H3.</p

Replication factors identified by iPOND and aniPOND.

<p>Experiments, conditions, and complexes are indicated in bold. Columns 1, 2, and 3 include protein name, description, and accession number. Values indicate spectral counts determined by mass spectrometry. Cellular replication fork iPOND-MS data was reported previously as follows: A [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004939#ppat.1004939.ref028" target="_blank">28</a>], B [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004939#ppat.1004939.ref026" target="_blank">26</a>], C [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004939#ppat.1004939.ref023" target="_blank">23</a>].</p><p>Replication factors identified by iPOND and aniPOND.</p

Proteins identified to interact with HSV-1 genomes by iPOND and aniPOND relocalize to viral replication compartments during lytic infection.

<p>Vero cells infected with wild type KOS were grown in the presence of 10 μM EdU at 4–8 hpi. Cellular DNA was visualized by Hoechst staining, viral replication compartments (vDNA) by click chemistry with EdU, and cellular proteins by immunofluorescence at 8 hpi (panels KOS, 8 hpi). Mock infected cells (panels Mock) display the normal distribution of cellular proteins in the nucleus in the absence of HSV infection. For KOS infected cells, the first merged panel displays colocalization of viral DNA with cellular proteins. The second merged panel shows the localization of viral DNA and cellular proteins with respect to cellular DNA.</p

Schematic representation of procedures used in this paper.

<p>(A) Resting MRC-5 cells in G₀ were infected with either prelabeled (left) or unlabeled (right) virus. To assay unreplicated viral DNA (left), prelabeled genomes were processed less than four hpi. To assay viral replication compartments (right), EdU (orange stars) was added to the growth medium during viral DNA replication (≥ 4 hpi) and genomes were assayed 2–4 hours after the addition of EdU. EdU labeled DNA is orange. (B) Viral and cellular DNA, as well as viral and cellular proteins were labeled and visualized as described in the experimental procedures. DNA imaging experiments were carried out in proliferating Vero cells. Viral DNA is green. (C) and (D) iPOND and aniPOND experiments were carried out as described. aniPOND (accelerated native iPOND) is a modified version of iPOND that does not involve crosslinking and therefore requires less stringent wash conditions during purification.</p

aniPOND detects viral and cellular proteins associated with replicated viral genomes.

<p>(A) ICP4 was detected in protein eluates from aniPOND experiments carried out on wild type KOS or UL2/UL50 mutant virus by western blotting. AniPOND was carried out on virus grown in the presence (+) or absence (-) of 2.5 μM EdU at 4–8 hpi and/or 33 μM acycloguanosine (ACG) throughout infection. (B) DNA eluted from streptavidin-coated beads during aniPOND experiments in (A) is viral. The amount of viral DNA present in cell lysates (input) and eluted from beads (bound) during aniPOND experiments was measured by qPCR of the viral TK gene. The ratio of viral DNA (vDNA) to total DNA was calculated and is presented in log %vDNA. Values for virus grown in the presence of ACG are too small to be displayed on this graph. These experiments were carried out with the UL2/UL50 mutant virus. (C) Pie charts summarize proteins that were identified by mass spectrometry of protein eluates from aniPOND carried out on the UL2/UL50 mutant grown in the presence of EdU at 4–8 hpi. Pie charts represent proteins that were identified with high confidence in independent duplicate experiments. Values indicate the number of proteins identified for each functional category. (D) Venn diagrams depict the overlap of proteins identified by iPOND and aniPOND carried out on the UL2/UL50 mutant 8 hpi.</p