Stimulation of homology-directed repair at I-SceI-induced DNA breaks during the permissive life cycle of human cytomegalovirus

Abstract

Human cytomegalovirus (HCMV) selectively relocalizes many DNA repair proteins, thereby avoiding a potentially detrimental damage response. In the present study, we evaluated interactions between HCMV and the homology-directed repair (HDR) pathway. In permissive human foreskin fibroblasts, a fluorescence-based double-stranded break repair assay was used to determine that HCMV stimulated HDR. Repair of both stably integrated and extrachromosomal reporter substrates was observed to increase. HDR was also stimulated through individual expression of the viral immediate-early protein IE1-72, mimicking full virus infection. These experiments further demonstrate HCMV's role in modulating critical cellular processes during a permissive infection.

Fig. 1.

Efficient expression of I-SceI from an Ad-based system in HFFs with an integrated pDRGFP substrate. (A) Schematic diagram of reporter plasmid pDRGFP, depicting I-SceI-induced DSB-initiated HDR to produce functional GFP. (B) Representative IF staining of mock- or NGUS24i-infected clone 4. Cells were seeded onto glass coverslips and mock or Ad infected. Coverslips were harvested and processed at 72 hpi as described previously (27). GFP⁺ cells were scored as HDR competent. Cells were also stained with HA-specific Ab (mouse monoclonal antibody [MAb; IgG_2b] 12CA5; Abcam) to detect HA-I-SceI. In all IF experiments, isotype-specific secondary Abs conjugated with either tetramethyl rhodamine isothiocyanate (TRITC; Jackson ImmunoResearch Laboratories) or Alexa Fluor 488 (Molecular Probes) were used to detect mouse primary Abs. Nuclei were counterstained in all experiments with Hoechst dye. Scale bar, 5 μm for all panels in all figures.

Fig. 2.

HCMV infection enhanced HDR in HFFs in the presence of both integrated and transiently expressed pDRGFP. (A and B) Clone 4 was seeded onto glass coverslips and mock infected, infected with HCMV or Ad alone, or dually infected with HCMV and Ad in succession. Coverslips were harvested 96 h after Ad superinfection. GFP⁺ cells were scored as HDR competent. Clone 4 was also stained by IF for the presence of IE1-72 to monitor HCMV infection, using a mouse MAb (IgG_2a; a gift from Bill Britt, University of Alabama, Birmingham). (A) Representative IF staining for GFP⁺ and IE1-72⁺ cells. (B) Experimental results represent the averages of three experiments. In each experiment, 200 to 500 cells per coverslip were counted and scored. Values at the tops of the bars indicate fold increases in percentages of GFP⁺ cells in all figures. Error bars represent ± one standard deviation in all figures. The asterisks (***) indicate statistical significance at the level of P < 0.05, as determined by the unpaired Student t test. (C) Representative protein profile for steady-state levels of HA-tagged I-SceI in clone 4 infected with NGUS24i alone or dually infected with HCMV and NGUS24i. Cells were synchronized in G₀ prior to seeding and infection. Total cell lysates derived from equivalent numbers of cells were used for each lane (7, 28). Actin was used as a loading control and was detected using an anti-pan actin mouse MAb (IgG1; NeoMarkers). Secondary for both Abs was horseradish peroxidase (HRP)-linked sheep anti-mouse Ab (GE Healthcare, United Kingdom). (D) Percentages of GFP⁺ cells in HFFs transiently expressing pDRGFP, as described in the text and above. Data represent the averages of two experiments.

Fig. 3.

Following HCMV infection, steady-state levels of HDR proteins were increased in HFFs and were available for cellular DNA repair. (A) G₀-synchronized HFFs were mock or HCMV infected at an MOI of 5. Cells were harvested at the indicated times p.i. Total cellular lysates derived from equivalent numbers of mock-infected and virus-infected cells were separated by SDS-PAGE, transferred to Protran, and probed with anti-Rad51C MAb (IgG1) or anti-FANCG MAb (IgG_2a) (Abnova) followed by HRP-linked sheep anti-mouse secondary Ab. Representative protein profiles are shown. Actin was used as a loading control. Quantitation of this immunoblot was performed using Bio-Rad Quantity One software, and numbers underneath each lane represent the fold change relative to the value for the normalized mock-infected cells at a given time point. All lanes were normalized to their actin control. (B) Rad51 (mouse MAb 14B4; Abcam Ab213), Rad51C, and FANCG were localized during HCMV infection of HFFs at 48 hpi using either standard formaldehyde fixation (27) or extraction-first (28) conditions to reveal more tightly associated protein localization. Viral replication centers were localized using mouse MAbs to either UL44 (IgG1; Rumbaugh-Goodwin Institute for Cancer Research, Inc.) or UL112 (a gift from Misaki Shirakata, Japan).

Fig. 4.

Transient expression of IE1-72 alone was enough to enhance HDR in HFFs. Cells were nucleofected with either pSG5 or pSG5IE1-72 and seeded onto glass coverslips. After 24 h, cells were mock or Ad infected. Coverslips were harvested 72 h later. Cells were stained by IF for the presence of IE1-72 using an anti-IE1 and anti-IE2 MAb Ch16.0 (IgG1; Rumbaugh-Goodwin Institute for Cancer Research, Inc.). (A) Representative IF staining for IE1-72⁺ and GFP⁺ cells. (B) Percentages of GFP⁺ and double-positive (GFP⁺ plus IE1-72⁺) cells. Bars represent the averages of four experiments. The differences in the percentages of GFP⁺ cells were considered statistically significant (*** = P < 0.05).