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First published online 6 June 2006
doi: 10.1242/jcs.02981

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Herpes simplex virus type I disrupts the ATR-dependent DNA-damage response during lytic infection

Department of Molecular, Microbial and Structural Biology MC3205, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA

View larger version (115K): [in a new window]   Fig. 1. Endogenous hyperphosphorylated RPA32 (P-RPA) does not mark sites of DSBs in HSV-1-infected cells. Vero cells were infected with HSV-1 (A-C,G-I) or mock-infected (D-F) and fixed with 4% PFA as described in the Materials and Methods. (A-C) HSV-1 infected cells were double labeled with mouse anti-H2AX (green) and rabbit anti-UL29 (red) to detect the nuclear localization of DSBs with respect to HSV-1 replication compartments, respectively. The merged image shown in C indicates that H2AX clearly surround replication compartments. Mock-infected cells (D-F) and HSV-1-infected cells (G-I) were double labeled using mouse anti-H2AX (green) and rabbit phosphospecific anti-P-RPA (red) to determine the nuclear localization of cellular DSBs and endogenous P-RPA, respectively. (J-L) Digital enlargements of an area of the infected cell nucleus show in G-I. Arrows indicate typical P-RPA foci that are exclusive of H2AX staining. Images were obtained at 100x magnification with 2x zoom.

View larger version (40K): [in a new window]   Fig. 2. P-RPA and H2AX accumulate at camptothecin-induced DNA repair foci in Vero cells. Vero cells were treated with 1 µM camptothecin (CPT) for 5 hours and prepared for IF analysis as described in the legend to Fig. 1. Cells were double labeled to detect the localization of H2AX (green) and P-RPA (red). The merged image shows a significant colocalization of the two signals (yellow). 100x magnification with 2x zoom.

View larger version (50K): [in a new window]   Fig. 3. Endogenous hyperphosphorylated RPA32 (P-RPA) is found within VICE domains during HSV-1 infection. Vero cells were mock-infected (A-D), or infected with HSV-1 (E-I). Pre-extracted cells were triple labeled with mouse anti-UL29 (green), rabbit anti-P-RPA (red) and rat anti-Hsc70 (blue) to detect the nuclear localization of replication compartments, endogenous P-RPA and VICE domains, respectively. (D,H) Merged images for P-RPA and Hsc70 with purple indicating the colocalization of the two proteins. (I) Merged image of UL29, P-RPA and Hsc70 for the HSV-1-infected cell. 100x magnification with 2x zoom.

View larger version (66K): [in a new window]   Fig. 4. VICE domains are excluded from cellular DSBs during HSV-1 infection. Vero cells were mock infected (A-D), or infected with HSV-1, strain KOS (E-I). Pre-extracted cells were triple labeled with mouse anti-UL29 (red), rabbit anti-H2AX (green) and rat anti-Hsc70 (blue) to detect the localization of replication compartments, cellular DSBs and VICE domains, respectively. Images were obtained at 100x magnification with 2x zoom. (J-L) Digital enlargements of an area of the cell shown on the left in panels F-H. Arrows indicate typical VICE domains that are exclusive of H2AX staining.

View larger version (98K): [in a new window]   Fig. 5. The staining pattern observed for ATR/ATRIP is dependent on the method used for immunolabeling. Mock-infected (A-F) or HSV-1-infected (G-L) cells were either fixed in 4% PFA to visualize total cellular proteins (Fixed, A-C,G-I) or extracted with 0.5% triton X-100 to visualize chromatin-bound and/or matrix-associated proteins (Pre-extracted, D-F,J-L). Cells were double stained using rabbit anti-ATR (red) and mouse anti-ATRIP (green) antibodies. Yellow color indicates a colocalization of the two proteins in the merged images. 100x magnification with 2x zoom.

View larger version (38K): [in a new window]   Fig. 6. ATR is retained in the nucleolus whereas ATRIP is redistributed to VICE domains during HSV-1 infection. Vero cells were infected with HSV-1 (A-D,F-I) or mock infected (E,J). Pre-extracted cells were triple labeled with mouse (A,E) or rabbit (F,J) anti-UL29 (green), rabbit anti-ATR (B,E; red) or mouse anti-ATRIP (G,J; red) and rat anti-Hsc70 (C,E,H,J; blue) to detect the localization of replication compartments, cellular DSBs and VICE domains, respectively. Lavender/pink color indicates the colocalization of ATR or ATRIP with Hsc70 in the merged images. 100x magnification with 2x zoom.

View larger version (30K): [in a new window]   Fig. 7. ATRIP and P-RPA colocalize in VICE domains during HSV-1 infection. HSV-1-infected Vero cells were pre-extracted with 0.5% Triton X-100 as described in the Materials and Methods. Cells were triple labeled with mouse anti-ATRIP (green), rabbit anti-P-RPA (red) and rat anti-Hsc70 (blue). White foci shown in the merged image indicate the colocalization of the three proteins within VICE domains. 100x magnification with 2x zoom.

View larger version (43K): [in a new window]   Fig. 8. ICP0 is sufficient to redistribute ATRIP in transfected cells. Vero cells were transfected with an expression plasmid encoding the ICP0 gene (pICP0) and fixed in 4% PFA as described in the Materials and Methods. (A) Merged image of a transfected cell double labeled with mouse anti-ICP0 (green) and rabbit anti-P-RPA (red). No colocalization of the two proteins was observed. (B) Merged image of a transfected cell double labeled with rat anti-Hsc70, a cellular marker that stains the interior of ICP0 inclusions (green), and mouse anti-ATRIP (red). Note the ATRIP-stained ICP0-like nuclear inclusions surrounding Hsc70. (C) Merged image of a transfected cell double labeled with mouse anti-ICP0 (green) and rabbit anti-ATR (red). No colocalization of the two proteins was observed. 100x magnification with 2x zoom.

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