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

  • ️Sun Jan 01 2006

. 2006 Jul 1;119(Pt 13):2695-703.

doi: 10.1242/jcs.02981. Epub 2006 Jun 6.

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

Dianna E Wilkinson et al. J Cell Sci. 2006.

Abstract

Like other DNA viruses, herpes simplex virus type 1 (HSV-1) interacts with components of the cellular response to DNA damage. For example, HSV-1 sequesters endogenous, uninduced, hyperphosphorylated RPA (replication protein A) away from viral replication compartments. RPA is a ssDNA-binding protein that signals genotoxic stress through the ATR (ataxia telangiectasia-mutated and Rad3-related) pathway. The sequestration of endogenous hyperphosphorylated RPA away from replicating viral DNA suggests that HSV-1 prevents the normal ATR-signaling response. In this study we examine the spatial distribution of endogenous hyperphosphorylated RPA with respect to ATR, its recruitment factor, ATRIP, and the cellular dsDNA break marker, gammaH2AX, during HSV-1 infection. The accumulation of these repair factors at DNA lesions has previously been identified as an early event in signaling genotoxic stress. We show that HSV-1 infection disrupts the ATR pathway by a mechanism that prevents the recruitment of repair factors, spatially uncouples ATRIP from ATR and sequesters ATRIP and endogenous hyperphosphorylated RPA within virus-induced nuclear domains containing molecular chaperones and components of the ubiquitin proteasome. The HSV-1 immediate early protein ICP0 is sufficient to induce the redistribution of ATRIP. This is the first report that a virus can disrupt the usually tight colocalization of ATR and ATRIP.

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Figures

Fig. 1
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-γH2AX (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 γH2AX clearly surround replication compartments. Mock-infected cells (D–F) and HSV-1-infected cells (G–I) were double labeled using mouse anti-γH2AX (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 γH2AX staining. Images were obtained at 100× magnification with 2× zoom.

Fig. 2
Fig. 2

P-RPA and γH2AX 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 γH2AX (green) and P-RPA (red). The merged image shows a significant colocalization of the two signals (yellow). 100× magnification with 2× zoom.

Fig. 3
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. 100× magnification with 2× zoom.

Fig. 4
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-γH2AX (green) and rat anti-Hsc70 (blue) to detect the localization of replication compartments, cellular DSBs and VICE domains, respectively. Images were obtained at 100× magnification with 2× 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 γH2AX staining.

Fig. 5
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. 100× magnification with 2× zoom.

Fig. 6
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. 100× magnification with 2× zoom.

Fig. 7
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. 100× magnification with 2× zoom.

Fig. 8
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. 100× magnification with 2× zoom.

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