Model of DNA repair proteins at sites of DNA damage in the presence of the 4-protein complex.

<p>In the presence of ICP8 and helicase/primase, RPA can still coat ssDNA at sites of damage and recruit ATR/ATRIP. However, these four-proteins bind to the ss/dsDNA junction that would normally serve as the loading platform for the 9-1-1 complex and exclude it from binding the DNA. This serves to prevent all of the downstream proteins from being recruited to sites of DNA damage and effectively inhibits ATR signaling.</p

HSV-1 inhibition of ATR signaling requires ICP8 and UL8.

<p>(A) Vero cells were either mock-infected or infected with HSV-1 at an MOI of 10 PFU/cell. At 5 hours post infection cells were treated with HU for 1 hour or UV and allowed to recover for 1 hour. (B) Vero cells were either mock-infected or infected with HSV-1 at an MOI of 10 PFU/cell. Cells were treated with UV at the indicated time post infection and allowed to recover for 1 hour. (C) Vero cells were either mock-infected or infected with the indicated HSV-1 mutant viruses at an MOI of 10 PFU/cell. At 5 hours post infection cells were treated with UV and allowed to recover for one hour. All cell lysates were analyzed by Western blot with the indicated antibodies. The band marked with an asterisk (*) in the P-RPA-S33 blot corresponds to a non-specific band that does not cross react with antibodies to endogenous RPA and likely represents cross-reactivity with a viral protein.</p

ICP8 and UL8 are sufficient to inhibit ATR signaling.

<p>(A) U2OS or (B) Vero cells were transfected with ICP8, UL8, UL5, and UL52 (ICP8+H/P) or ICP8 and UL8 alone and then damaged with UV. Cells were fixed at 1 hour post damage and prepared for immunofluorescence as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003652#s4" target="_blank">materials and methods</a>. (C) Cells were treated as in A and stained for either P-RPA-S33 or P-RPA-S4/S8. At least 100 cells were counted between two independent experiments.</p

Essential ATR pathway proteins are excluded from the four-protein complex.

<p>Vero cells were transfected with ICP8, UL8, UL5, and UL52 and damaged with UV and allowed to recover for 1-RPA70 or Myc-TopBP1 were cotransfected with the viral proteins. To follow HA-Rad9 Vero cells stably expressing HA-Rad9 were transfected with the viral proteins prior to UV irradiation.</p

Summary of HSV-1 mutants ability to disable ATR signaling.

<p>HeLa cells were infected with the indicated HSV-1 mutants at an MOI of 10 PFU/cell. At 5 hours post infection cells were treated with HU for 2 hours. All cell lysates were analyzed by Western blot for P-Chk1 S345, total Chk1, and ICP4/ICP8 as described in the legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003652#ppat-1003652-g002" target="_blank">Figure 2</a>. The ΔUL30 mutant was used at an MOI of 2 PFU/cell. The helicase/primase inhibitor, BAY 57-1293 (BAY), and the polymerase inhibitor, phosphonoacetic acid (PAA), were used as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003652#s4" target="_blank">Materials and Methods</a>.</p

ATR can be activated in cells expressing the four-protein complex and the ATR Activation Domain of TopBP1.

<p>Vero cells were transfected with GFP-TopBP1-AAD alone or in combination with ICP8, UL8, UL5, and UL52 (ICP8+H/P). Cells were fixed at 18 hours post transfection and prepared for immunofluorescence as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003652#s4" target="_blank">materials and methods</a>.</p

UL8 mutants that do not support DNA replication still inhibit ATR signaling.

<p>(A) Schematic of UL8 mutants used in this study. (B) Vero cells were transfected with ICP8, UL5, UL5, and the indicated UL8 mutants and then damaged with UV. Cells were fixed at 1 hour post damage and prepared for immunofluorescence as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003652#s4" target="_blank">materials and methods</a>.</p

The four-protein complex localizes to sites of DNA damage.

<p>Vero cells were transfected with ICP8, UL8, UL5, and UL52 and BrdU was added at the time of transfection. Cells were treated with UV or HU at 24</p

HSV infection increases SSA and inhibits HR, A-NHEJ and NHEJ.

<p>HR, SSA, A-NHEJ and NHEJ reporter cell lines were transfected with an empty vector or I-SceI expression vector and infected with HSV or mock infected. The average frequency of repair from at least four independent experiments, each performed with three independent samples were normalized to the I-SceI- transfected and mock infected samples. The HR and SSA experiments include the data from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002862#ppat-1002862-g003" target="_blank">figures 3A</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002862#ppat-1002862-g004" target="_blank">4A and 4B</a> as well as two additional experiments. Error bars represent the standard error of the mean. The asterisk indicates statistically significant differences from samples transfected with I-SceI and mock infected (P≤0.02). The insert shows a representative western blot analysis of cell lysates from mock- or HSV-infected cells. Infected cell lysates expressed the viral ICP4 protein and actin served as a loading control.</p

Schematic of the DSBR reporter assays.

<p>(A) DR-GFP reporter used to monitor HR <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002862#ppat.1002862-Pierce1" target="_blank">[47]</a>. SceGFP is a modified GFP gene, which contains an I-SceI site and in-frame termination codons. An 812-bp internal GFP fragment (iGFP) can repair the DSB by HR (gene conversion) and results in a functional GFP gene. (B) SA-GFP reporter used to monitor SSA <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002862#ppat.1002862-Stark1" target="_blank">[26]</a>. 5′ GFP and SceGFP3 are GFP gene fragments which have 266 bp of homology (light gray). Repair of the DSB in SceGFP3 by SSA results in a functional GFP gene and a 2.7-kb deletion in the chromosome. Although an identical repair product could arise in this assay by HR with crossing over, this type of repair is estimated to be at least 30-fold less frequent <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002862#ppat.1002862-Stark1" target="_blank">[26]</a>. (C) EJ2 reporter used to monitor A-NHEJ <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002862#ppat.1002862-Bennardo1" target="_blank">[48]</a>. GFP is separated from an N-terminal tag (NLS/Zinc-finger) by an I-SceI site and stop codons in all three reading frames, which are flanked by 8 nts of microhomology. Repair of the DSB by A-NHEJ results in a functional GFP gene by restoring the coding frame between the tag and GFP as well as causing a 35 nt deletion. (D) EJ5 reporter used to monitor total NHEJ <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002862#ppat.1002862-Bennardo1" target="_blank">[48]</a>. GFP is separated from a promoter by a puromycin gene flanked by two I-SceI sites. Excision of the puromycin gene and repair of the DSB by NHEJ joins the promoter with GFP thus creating a functional GFP gene. Two repair products can be formed, one that restores the I-SceI site (Ku-dependent C-NHEJ) and one that is I-SceI resistant (Ku-independent A-NHEJ).</p