HSV-1 ICP0 activation of Chk2 kinase. (A) 293T cells were infected at an MOI of 2 with TOZ22R or QOZ22R and harvested at 0–24 hours post-infection (hpi). Cell lysates were electrophoresed and examined by immunoblotting with the indicated general or phospho-specific antibodies. The lanes are labeled by time point (hpi). (B) 293T cells were transfected with plasmid E110 expressing ICP0 or with empty plasmid. Cells were harvested 24 h later and processed for immunoblotting with the indicated antibodies.

Chk2 kinase requirement for ICP0-induced G2/M arrest. (A) HCT116 and HCT116(Chk2^−/−) cells were mock infected or infected with TOZ22R or QOZ22R at an MOI of 2. At 24 hpi, the cells were fixed, stained with propidium iodide and subjected to flow cytometric analysis. The numbers inside the panels show the percentage of cells in G1, S, and G2/M phase. Since the total number of viable cells differs between the panels, absolute peak surfaces can not be compared between the panels. (B) Chk2-deficient HCT15 cells and HCT15 cells complemented with either wild-type Chk2 [HCT15(Chk2⁺)] or kinase-deficient Chk2 [HCT15(Chk2-T68A)] were mock infected or infected with TOZ22R and cell cycle progression analyzed at 24 hpi by flow cytometry. The percentage of cells in each phase is indicated inside the panels. (C) HCT116 and HCT116(Chk2^−/−) cells were mock infected or infected with TOZ22R and harvested at 24 hpi for immunoblotting with the indicated antibodies.

ATM requirement for ICP0-induced Chk2 activation. (A, C) HCT116 cells and A-T fibroblast lines GM05849, GM02052 and GM09607 were mock-infected or infected with TOZ22R at an MOI of 2. At 24 hpi, cells were collected and lysates analyzed by immunoblotting with general and phospho-specific antibodies, as indicated. (B) 293T cells were transfected with ICP0 expression plasmid E110 or eGFP-expressing control plasmid pEGFP-C1. As additional controls, cells were untreated (Control) or treated with ionizing radiation (IR; 5 Gy, ¹³⁷Cs source). Lysates were prepared 24 h later and analyzed by immunoblotting. (D) 293T cells were incubated for 1 h with or without 10 μM KU-55933 and transfected with plasmid E110 in the presence or absence of the drug. The cells were washed and replenished with fresh medium with or without the drug. Lysates were prepared 24 h later and analyzed by immunoblotting with the indicated antibodies.

Chk2 requirement for efficient virus growth. (A, B) HCT116 and HCT116(Chk2^−/−) cells were infected at an MOI of 0.2 with either wild-type HSV-1 KOS (A) or ICP0-deficient mutant virus ICP0:lacZ (B). Cells plus media were harvested at various times post-infection and viral yields assessed by plaque assays on Vero cells (A) or 7B0-104 cells (B). All infections were carried out at least in duplicate. Error bars in B and for certain data points in A are smaller than the data-point symbols and thus are not visible here. (C) AD-293 cells were incubated for 1 h with 0–30 μM KU-55933 and infected in duplicate with HSV-1 KOS (MOI=0.2) for 4 h in the continued presence or absence of the drug. Cells were washed and fed with fresh medium with or without the drug. Cells and media were harvested at 18 hpi for determination of virus yields by plaque assay on Vero cells. (D) Mock-infected AD-293 cells were incubated with KU-55933 as in (C) and cell viability measured by MTS assay at 18 hpi for 10 wells per drug concentration. (E) 293T cells were infected with either HSV-1 KOS (MOI=0.5) or ICP0:lacZ (MOI=5) and harvested at the times indicated in hpi above the lanes. Immunoblotting was performed with the antibodies shown. (D) 293T cells were mock infected or infected with ICP0:lacZ at an MOI of 2 and processed 24 h later for flow cytometry.

Virus constructs. (A) Structure of the ICP0⁺ (TOZ22R) and ICP0⁻ (QOZ22R) HSV-1 strain KOS mutant viruses compared to wild type HSV-1 KOS and parental mutant virus TOZ.1. The HSV-1 genome is a linear, double-stranded DNA of ~152 kb characterized by unique long (U_L) and unique short (U_S) segments, each flanked by inverted repeats (IR_L, internal repeat of the long segment; IR_S, internal repeat of the short segment; TR_L, terminal repeat of the long segment; TR_S, terminal repeat of the short segment). TOZ.1 contains a lacZ expression construct in the U_L41 gene, has complete deletions of the essential IE genes ICP4 and ICP27, and has an internal portion of the ICP22 gene replaced with the HCMV IE promoter fused to the bovine growth hormone polyadenylation region (Krisky et al., 1998). TOZ22R was derived from TOZ.1 by substitution of the interrupted ICP22 coding sequence with the DsRed2 gene. QOZ22R was derived from TOZ22R by replacement of both copies of the ICP0 coding region with an eGFP expression construct. Black bars, gene deletions; colored bars, gene substitutions. Blue, lacZ; red, DsRed2; green, eGFP. IE genes are identified by their numbers. (B) Southern blot demonstrating deletion of the ICP0 gene in QOZ22R. Viral DNAs were digested with DraI and BamHI and the blot reacted with a probe for the ICP0 gene (top). Fragment sizes (in kbp) are indicated at the left of the autoradiograph. The target locus is illustrated underneath with the location of the probe and relevant restriction sites indicated. Plain numbers refer to HSV-1 strain 17 map positions (GenBank accession no. X14112) while italicized numbers preceded by a + sign indicate the distance in bp to the left boundary (SspI site) of the eGFP insertion in QOZ22R. D, DraI; B, BamHI; H, HpaI; St, StuI; S, SspI; A, AseI. Restriction sites that were lost in constructing the eGFP substitution of ICP0 in QOZ22R are shown between parentheses. Restriction sites derived from the eGFP donor plasmid are shown in italics. The two copies of the ICP0 locus are identical in the region represented here.