Human Cytomegalovirus Disrupts both Ataxia Telangiectasia Mutated Protein (ATM)- and ATM-Rad3-Related Kinase-Mediated DNA Damage Responses during Lytic Infection

Abstract

Many viruses (herpes simplex virus type 1, polyomavirus, and human immunodeficiency virus type 1) require the activation of ataxia telangiectasia mutated protein (ATM) and/or Mre11 for a fully permissive infection. However, the longer life cycle of human cytomegalovirus (HCMV) may require more specific interactions with the DNA repair machinery to maximize viral replication. A prototypical damage response to the double-stranded ends of the incoming linear viral DNA was not observed in fibroblasts at early times postinfection (p.i.). Apparently, a constant low level of phosphorylated ATM was enough to phosphorylate its downstream targets, p53 and Nbs1. p53 was the only cellular protein observed to relocate at early times, forming foci in infected cell nuclei between 3.5 and 5.5 h p.i. Approximately half of these foci localized with input viral DNA, and all localized with viral UL112/113 prereplication site foci. No other DNA repair proteins localized with the virus or prereplication foci in the first 24 h p.i. When viral replication began in earnest, between 24 and 48 h p.i., there were large increases in steady-state levels and phosphorylation of many proteins involved in the damage response, presumably triggered by ATM-Rad3-related kinase activation. However, a sieving process occurred in which only certain proteins were specifically sequestered into viral replication centers and others were particularly excluded. In contrast to other viruses, activation of a damage response is neither necessary nor detrimental to infection, as neither ATM nor Mre11 was required for full virus replication and production. Thus, by preventing simultaneous relocalization of all the necessary repair components to the replication centers, HCMV subverts full activation and completion of both double-stranded break and S-phase checkpoints that should arrest all replication within the cell and likely lead to apoptosis.Human cytomegalovirus (HCMV) is the major viral cause of birth defects. Each year approximately 1 to 2% of all newborns are congenitally infected, and of these infants, 5 to 10% manifest signs of serious neurological defects (6,8,16,31). HCMV infection is also a major medical problem in immunocompromised individuals (8).Recent literature points to HCMV as a potential causative agent for the development of certain types of cancers including malignant gliomas, prostate carcinomas, and colorectal cancers (17,35,74). Studies of HCMV infection in nonpermissive rodent cells indicate that the virus can act as a mutagen (1,5,78). In fact, in permissive human cells expression of the immediate-early (IE) gene products by themselves can drive cells into S phase (57). This has led to the suggestion that the IE proteins of HCMV may cause "hit-and-run" mutagenesis. In addition, our earlier findings showed that 15% of fibroblasts infected in S phase were specifically broken in the long arm of chromosome 1 when analyzed during the subsequent mitosis (28). Due to the clinical ramifications of HCMV infection, the study of its interactions w...

Kaposi's Sarcoma-Associated Herpesvirus Induces the ATM and H2AX DNA Damage Response Early during De Novo Infection of Primary Endothelial Cells, Which Play Roles in Latency Establishment

“…Furthermore, upon entry of the KSHV genome into the nucleus by 30 min (0.5 h) p.i., the formation and colocalization of ␥H2AX foci with the viral genome were also observed. Similar immunofluorescent colocalization studies have shown that ␥H2AX localized in foci juxtaposed to parental HCMV DNA (Towne strain) at early times of infection in human fibroblasts (20). Moreover, ␥H2AX accumulates at the intranuclear sites of HCMV replication during the late stages of infection (20), forming distinct foci that appear to surround the viral inclusions.…”

“…Similar immunofluorescent colocalization studies have shown that ␥H2AX localized in foci juxtaposed to parental HCMV DNA (Towne strain) at early times of infection in human fibroblasts (20). Moreover, ␥H2AX accumulates at the intranuclear sites of HCMV replication during the late stages of infection (20), forming distinct foci that appear to surround the viral inclusions. In contrast, ␥H2AX did not colocalize with late HSV-1 replication compartments (18), although it accumulated at sites associated with the incoming HSV-1 genome (49).…”

“…In contrast, although human cytomegalovirus (HCMV; strain AD169) infection of fibroblast cells induced H2AX phosphorylation, the resulting DDR was inhibited by mislocalization of checkpoint proteins (19) and ␥H2AX colocalized with HCMV replication compartments. Activation of ␥H2AX was dependent on viral replication, as it did not occur in p53 mutant cells, in which HCMV replication was abrogated (19,20).…”

Infection of a Single Cell Line with Distinct Strains of Human Cytomegalovirus Can Result in Large Variations in Virion Production and Facilitate Efficient Screening of Virus Protein Function

“…Numerous ATM-deficient (ATM Ϫ ) cell lines have been derived from ataxia telangiectasia (A-T) patients, and most harbor unique mutations (23,24). HCMV infection induces ATM to phosphorylate Nbs1 and p53 (4,5,7,8,25); however, the damage-signaling cascade is defective, Conflicting results regarding ATM's role in HCMV virion production have been reported. Studies from our lab performed in Towne-infected normal human foreskin fibroblasts (HFFs), an ATM Ϫ cell line (GM02530) and Mre11 Ϫ cells found that disruption of the DSB DDR did not diminish functional virion production at either a high or a low multiplicity of infection (MOI) (5).…”

“…In fibroblasts, large bipolar viral replication centers (RCs) are formed within 48 h postinfection (hpi) and certain host cellular proteins become strongly associated with these RCs (1; reviewed in reference 2). These proteins include the regulatory protein p53 (3), as well as numerous components of the host cellular DNA damage response (DDR) and repair pathways (4)(5)(6)(7)(8).…”