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. 2014 Feb;88(4):2279-90.
doi: 10.1128/JVI.03423-13. Epub 2013 Dec 11.

A Novel DDB2-ATM Feedback Loop Regulates Human Cytomegalovirus Replication

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Free PMC article

A Novel DDB2-ATM Feedback Loop Regulates Human Cytomegalovirus Replication

Xiaofei E et al. J Virol. .
Free PMC article

Abstract

Human cytomegalovirus (HCMV) genome replication requires host DNA damage responses (DDRs) and raises the possibility that DNA repair pathways may influence viral replication. We report here that a nucleotide excision repair (NER)-associated-factor is required for efficient HCMV DNA replication. Mutations in genes encoding NER factors are associated with xeroderma pigmentosum (XP). One of the XP complementation groups, XPE, involves mutation in ddb2, which encodes DNA damage binding protein 2 (DDB2). Infectious progeny virus production was reduced by >2 logs in XPE fibroblasts compared to levels in normal fibroblasts. The levels of immediate early (IE) (IE2), early (E) (pp65), and early/late (E/L) (gB55) proteins were decreased in XPE cells. These replication defects were rescued by infection with a retrovirus expressing DDB2 cDNA. Similar patterns of reduced viral gene expression and progeny virus production were also observed in normal fibroblasts that were depleted for DDB2 by RNA interference (RNAi). Mature replication compartments (RCs) were nearly absent in XPE cells, and there were 1.5- to 2.0-log reductions in viral DNA loads in infected XPE cells relative to those in normal fibroblasts. The expression of viral genes (UL122, UL44, UL54, UL55, and UL84) affected by DDB2 status was also sensitive to a viral DNA replication inhibitor, phosphonoacetic acid (PAA), suggesting that DDB2 affects gene expression upstream of or events associated with the initiation of DNA replication. Finally, a novel, infection-associated feedback loop between DDB2 and ataxia telangiectasia mutated (ATM) was observed in infected cells. Together, these results demonstrate that DDB2 and a DDB2-ATM feedback loop influence HCMV replication.

Figures

FIG 1
FIG 1
DDB2 is required for efficient HCMV replication. (A to D) Replication of HCMV in normal and XPE (ddb2) cells. Normal (BJ) and XPE telomerase-life-extended dermal fibroblasts were infected at an MOI of 0.1. (A) Cell supernatants were assayed for infectious virus production by plaque assay. (B) Viral protein expression is altered in XPE cells. Immunoblot analyses for viral IE (IE1/IE2), E (pp65), and E/L (gB55) protein expression in BJ and XPE (lanes E) dermal fibroblasts are shown. (C and D) DDB2 depletion by siRNA compromises HCMV replication. HEL fibroblasts were transfected with siRNAs specific for DDB2 (siDDB2) or with a control siRNA (siCON) 24 h prior to infection with HCMV at an MOI of 0.1. (C) Cell supernatants were assayed for infectious virus production by plaque assay. (D) Transient depletion of DDB2 alters viral expression. The levels of DDB2 and viral IE (IE1/IE2), E (pp65), and E/L (gB55) protein expression were assessed by immunoblot analysis. (E and F) Effect of rescuing ddb2 on HCMV replication. XPE cells were infected with a retrovirus containing a DDB2 cDNA, and BJ and XPE cells were infected with a retrovirus containing an empty vector. Stably transduced cells were infected with HCMV at an MOI of 0.1. (E) Cell supernatants were assayed for infectious virus production by plaque assay. (F) Protein levels of DDB2 and actin were measured by immunoblot analysis. (A, C, and E) Mean values are shown, with bars denoting 1 standard deviation, for three independent experiments. For certain data points, error bars may be too tight to be visible.
FIG 2
FIG 2
Reduced formation of “mature” viral replication compartments (RCs) in XPE fibroblasts. Normal (BJ) and XPE dermal fibroblasts were infected at an MOI of 0.1. Cells were fixed each day p.i., and IE1 and IE2 proteins and pUL44 were detected by immunostaining. (A) Localization of IE proteins and pUL44. Immunofluorescent images of normal (BJ) and XPE dermal fibroblasts infected with HCMV. Cells with “immature” RCs were defined as those with multiple, small pUL44 compartments (yellow arrows), and cells with “mature” RCs were identified as those composed of single, larger pUL44 compartments (white arrows). 4′,6-Diamidino-2-phenylindole (DAPI) staining was used to define nuclei. (B) The percentage of fibroblasts with mature RCs was plotted relative to those lacking or having immature RCs. More than 200 cells were scored per sample. Mean values are shown, with bars denoting 1 standard deviation, for three independent experiments. For certain data points, error bars may be too tight to be visible.
FIG 3
FIG 3
Localization of DDB2 to RCs during infection. HEL fibroblasts were transiently transfected with a plasmid that expresses HA-DDB2. Immunofluorescence detection of pUL44 and HA-DDB2 in HCMV-infected (MOI = 1.0) and mock-infected HEL fibroblasts is shown. DAPI staining was used to define nuclei.
FIG 4
FIG 4
Viral DNA levels are reduced in XPE fibroblasts. (A) BJ and XPE fibroblasts were infected with HCMV at an MOI of 0.1. Viral DNA levels were determined using real-time quantitative PCR with samples collected at 2-day intervals p.i. (dpi, days p.i.). The amount of viral DNA assayed is represented as copies of the viral gene, UL83, per copy of the cellular gene, β-ACTIN. (B) Transient depletion of DDB2 alters DNA replication. HEL fibroblasts were transfected with the indicated siRNAs and infected with HCMV at an MOI of 0.1 24 h later. Viral DNA levels were determined using real-time quantitative PCR on samples collected each day p.i. for 5 days. Viral DNA levels were determined as described for panel A. In both panels, mean values are shown, with bars denoting 1 standard deviation, for three independent experiments. For certain data points, error bars may be too tight to be visible.
FIG 5
FIG 5
DDB2 influences the expression of early genes involved in viral DNA replication. (A and B) BJ and XPE fibroblasts were infected with HCMV at an MOI of 0.1. (A) Measurement of HCMV UL44, UL54, and UL84 transcript levels by qRT-PCR. BJ and XPE fibroblasts were infected with HCMV at an MOI of 0.1. Levels of HCMV gene transcript levels in XPE fibroblasts relative to those in BJ fibroblasts are shown. (B) Accumulation of early proteins associated with viral DNA replication is altered in XPE fibroblasts. Cells infected in panel A were assayed for pUL44 and pUL84 protein expression by immunoblot analysis. (C and D) Transient depletion of DDB2 affects UL44, UL54, and UL84 gene expression. HEL fibroblasts were transfected with the indicated siRNAs and infected with HCMV at an MOI of 0.1 24 h later. (C) Measurement of HCMV UL44, UL54, and UL84 transcript levels by qRT-PCR. HCMV gene transcript levels in siDDB2-treated fibroblasts relative to those in siCON-treated fibroblasts are shown. (D) Early protein expression associated with viral DNA replication is altered in siDDB2-treated cells. Cells infected in panel C were assayed for pUL44 and pUL84 protein expression by immunoblot analysis. (A and C) Mean values are shown, with bars denoting 1 standard deviation, for three independent experiments.
FIG 6
FIG 6
The effect of a viral DNA replication inhibitor on the expression of viral genes affected by DDB2 status. BJ and XPE fibroblasts were infected with HCMV at an MOI of 0.1 in the presence (+) or absence (−) of the viral DNA synthesis inhibitor phosphonoacetic acid (PAA) (100 μg/ml). Samples were harvested at the indicated times p.i. (A) Viral protein expression is altered in XPE and PAA-treated cells. The levels of IE1, IE2, pp65, pUL44, pUL84, and gB55 protein expression were assessed by immunoblot analysis. (B) Replication of HCMV in BJ and XPE fibroblasts with or without PAA. Cell supernatants were assayed for infectious virus production by plaque assay. (C to H) Measurement of UL123 (C), UL122 (D), UL44 (E), UL54 (F), UL55 (G), UL84 (H), and GAPDH transcript levels by qRT-PCR. Total RNA was isolated at 24 to 120 hpi, and the amounts of viral transcripts were measured by qRT-PCR and normalized to that of GAPDH. The fold change in transcript levels of each open reading frame (ORF) and for different cell types with or without PAA is plotted relative to the corresponding transcript levels measured in BJ fibroblasts. Mean values are shown, with bars denoting 1 standard deviation, for three independent experiments.
FIG 7
FIG 7
DDB2 and ATM function in the same genetic pathway to influence HCMV replication. (A) Viral replication when ATM is depleted in BJ and XPE fibroblasts. BJ and XPE fibroblasts were transfected with siRNAs specific for ATM (siATM) or with a control siRNA (siCON) 24 h prior to infection with HCMV at an MOI of 0.1. Infected cell supernatants were assayed for infectious virus production by plaque assay. The mean values are shown, with bars denoting 1 standard deviation, for three independent experiments. For certain data points, error bars may be too tight to be visible. (B) Transient depletion of ATM in BJ and XPE fibroblasts. BJ and XPE fibroblasts were transfected with siATM or siCON and infected with HCMV at an MOI of 0.1. The levels of ATM protein expression were assessed by immunoblot analysis.
FIG 8
FIG 8
An infection-associated DDB2-ATM negative feedback loop. (A and B) DDB2 contributes to the kinetics of ATM accumulation. HEL fibroblasts were transiently transfected with siCON or siDDB2 and subsequently infected with HCMV at an MOI of 0.1 24 h later. Samples were harvested at the indicated times p.i. (A) ATM and DDB2 levels were assessed by immunoblot analyses. (B) ATM transcript levels were measured by qRT-PCR. The fold change in ATM transcript levels in siDDB2-treated fibroblasts is shown relative to ATM transcript levels measured in siCON-treated fibroblasts. Mean values are shown, with bars denoting 1 standard deviation, for three independent experiments. (C and D) Influence of ATM on DDB2 transcript levels. HEL fibroblasts were treated with siCON or siATM and infected with HCMV at an MOI of 0.1 24 h later. Cells were harvested at the indicated times p.i. For panel C, DDB2 transcript levels were measured by qRT-PCR. The fold change in DDB2 transcript levels in siATM-treated fibroblasts is plotted relative to DDB2 transcript levels measured in siCON-treated fibroblasts. Mean values are shown, with bars denoting 1 standard deviation, for three independent experiments. For panel D, the levels of ATM protein expression in siCON- and siATM-treated fibroblasts were assessed by immunoblot analysis. (E) DDB2-dependent ATM accumulation in response to HCMV infection is independent of viral DNA replication. HEL fibroblasts were treated with siCON or siDDB2 and infected with HCMV at an MOI of 0.1 24 h later in the presence (+) or absence (−) of the viral DNA synthesis inhibitor phosphonoacetic acid (PAA) (100 μg/ml). The levels of ATM protein expression in siCON- and siDDB2-treated cells with or without PAA were assessed by immunoblot analyses.
FIG 9
FIG 9
Model depicting the relationship among HCMV infection, DDB2, ATM, and virus replication.

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