Stimulation of Homology-Directed Repair at I-SceI-Induced DNA Breaks during the Permissive Life Cycle of Human Cytomegalovirus

Kulkarni, Amit; Fortunato, Elizabeth A.

doi:10.1128/jvi.02514-10

Cited by 18 publications

(37 citation statements)

References 52 publications

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“…These papers have examined the capacity of the cell's homologous recombination, base excision, nucleotide excision and non-homologous endjoining repair machinery to function, with the very large majority of the investigations finding decreased capacity of the cell to repair damage after viral protein expression (or full infection) commenced. Only four of these studies have reported evidence of an increase in repair capacity of the cell after infection or viral protein overexpression [21], [23], [36], [40]. Our results extend this analysis and separate the two genomes within an infected cell.…”

Section: Discussionsupporting

confidence: 80%

HCMV-Infected Cells Maintain Efficient Nucleotide Excision Repair of the Viral Genome while Abrogating Repair of the Host Genome

O’Dowd¹,

Zavala²,

Brown³

et al. 2012

PLoS Pathog

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Many viruses subvert the host cell's ability to mount and complete various DNA damage responses (DDRs) after infection. HCMV infection of permissive fibroblasts activates host DDRs at the time of viral deposition and during replication, but the DDRs remain uncompleted without arrest or apoptosis. We believe this was in part due to partitioning of the damage response and double strand break repair components. After extraction of soluble proteins, the localization of these components fell into three groups: specifically associated with the viral replication centers (RCs), diffused throughout the nucleoplasm and excluded from the RCs. Others have shown that cells are incapable of processing exogenously introduced damage after infection. We hypothesized that the inability of the cells to process damage might be due to the differential association of repair components within the RCs and, in turn, potentially preferential repair of the viral genome and compromised repair of the host genome. To test this hypothesis we used multiple strategies to examine repair of UV-induced DNA damage in mock and virus-infected fibroblasts. Comet assays indicated that repair was initiated, but was not completed in infected cells. Quantitative analysis of immunofluorescent localization of cyclobutane pyrimidine dimers (CPDs) revealed that after 24 h of repair, CPDs were significantly reduced in viral DNA, but not significantly changed in the infected host DNA. To further quantitate CPD repair, we developed a novel dual-color Southern protocol allowing visualization of host and viral DNA simultaneously. Combining this Southern methodology with a CPD-specific T4 endonuclease V alkaline agarose assay to quantitate repair of adducts, we found efficient repair of CPDs from the viral DNA but not host cellular DNA. Our data confirm that NER functions in HCMV-infected cells and almost exclusively repairs the viral genome to the detriment of the host's genome.

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Section: Discussionsupporting

confidence: 80%

HCMV-Infected Cells Maintain Efficient Nucleotide Excision Repair of the Viral Genome while Abrogating Repair of the Host Genome

O’Dowd¹,

Zavala²,

Brown³

et al. 2012

PLoS Pathog

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“…Cells were trypsinized, washed, counted, and prepared as described previously (Kulkarni and Fortunato, 2011; Luo et al, 2007). Equivalent amounts of cell lysates (10 5 cell equivalents unless otherwise noted) were run on SDS-polyacrylamide gels, and then transferred to a Protran membrane (GE Healthcare Life Sciences).…”

Section: Methodsmentioning

confidence: 99%

The absence of p53 during Human Cytomegalovirus infection leads to decreased UL53 expression, disrupting UL50 localization to the inner nuclear membrane, and thereby inhibiting capsid nuclear egress

2016

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Our electron microscopy study found HCMV nuclear capsid egress was significantly reduced in p53 knockout cells (p53KOs), correlating with inhibited formation of infoldings of the inner nuclear membrane (IINMs). Molecular examination of these phenomena has found p53KOs expressed UL97 and phosphorylated lamins, however the lamina failed to remodel. The nuclear egress complex (NEC) protein UL50 was expressed in almost all cells. UL50 re-localized to the inner nuclear membrane (INM) in ~90% of wt cells, but only ~35% of p53KOs. UL53 expression was significantly reduced in p53KOs, and cells lacking UL50 nuclear staining, expressed no UL53. Re-introduction of p53 into p53KOs largely recovered UL53 positivity and UL50 nuclear re-localization. Nuclear rim located UL50/53 puncta, which co-localized with the major capsid protein, were largely absent in p53KOs. We believe these puncta were IINMs. In the absence of p53, UL53 expression was inhibited, disrupting formation of the NEC/IINMs, and reducing functional virion secretion.

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“…The proposed model offers a mechanistic explanation for our previously reported results in wt HFFs [12]. The focus of the earlier HFF study was the recognition of viral manipulation of a cellular repair mechanism to its own ends in the context of a fully permissive infection.…”

Section: Resultsmentioning

confidence: 69%

“…( A ) Schematic diagram of reporter plasmid pDRGFP as described in [12]. ( B ) Representative IF staining of Ad-infected Clone 10.…”

Section: Resultsmentioning

confidence: 99%

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Modulation of Homology-Directed Repair in T98G Glioblastoma Cells Due to Interactions between Wildtype p53, Rad51 and HCMV IE1-72

Kulkarni

Fortunato

2014

Viruses

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Human cytomegalovirus (HCMV) is a ubiquitous pathogen capable of causing life threatening consequences in neonates and immune-compromised individuals. HCMV inflicts site-specific double strand breaks (DSBs) in the cellular genome. DNA damage infliction raises the corollary question of virus modulation of DNA repair. We recently reported HDR was stimulated in wt human foreskin fibroblasts (HFFs) during fully permissive infection or expression of the HCMV protein IE1-72 (IE72). These studies have been extended into semi-permissive T98G glioblastoma cells. T98Gs encode a mutant p53, which may contribute to their high baseline rate of HDR. We fully expected HCMV infection to increase HDR in T98Gs, similar to its effects in HFFs. Surprisingly in T98Gs HCMV infection, or sole expression of IE72, decreased HDR by two-fold. Transient expression of wt p53 in T98Gs also reduced HDR by two-fold. Dual transient expression of wt p53 and IE72 restored high baseline HDR levels. GST pulldown experiments revealed that both IE72 and wt p53 bound the important HDR protein, Rad51. We conclude that the expression of certain HCMV proteins can modulate HDR in an infected cell, dependent upon p53 status. We propose a model of the protein interactions explaining this behavior.

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Stimulation of Homology-Directed Repair at I-SceI-Induced DNA Breaks during the Permissive Life Cycle of Human Cytomegalovirus

Cited by 18 publications

References 52 publications

HCMV-Infected Cells Maintain Efficient Nucleotide Excision Repair of the Viral Genome while Abrogating Repair of the Host Genome

HCMV-Infected Cells Maintain Efficient Nucleotide Excision Repair of the Viral Genome while Abrogating Repair of the Host Genome

The absence of p53 during Human Cytomegalovirus infection leads to decreased UL53 expression, disrupting UL50 localization to the inner nuclear membrane, and thereby inhibiting capsid nuclear egress

Modulation of Homology-Directed Repair in T98G Glioblastoma Cells Due to Interactions between Wildtype p53, Rad51 and HCMV IE1-72

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