A Novel DDB2-ATM Feedback Loop Regulates Human Cytomegalovirus Replication

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)-associatedfactor 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.

Identification of Host Factors Involved in Human Cytomegalovirus Replication, Assembly, and Egress Using a Two-Step Small Interfering RNA Screen

“…This in turn results in activation of IκB kinase (IKK), degradation of IKBA, and activation of NF-kB. Previous studies have shown that ATM signaling is required for efficient HCMV replication, and knockdown of ATM resulted in a similar phenotype as knockdown of ERC1 in our screen, supporting a role for ERC1 in proviral DNA damage signaling during HCMV infection ( 31 ). Studies are ongoing to define whether either or both ERC1 pathways are involved in HCMV replication.…”

PUL21a-Cyclin A2 Interaction is Required to Protect Human Cytomegalovirus-Infected Cells from the Deleterious Consequences of Mitotic Entry

“…5B ) in UL21a-RXL-mutant-infected cells indicate that HCMV overrides these checkpoints, most likely by abrogating p53 and Chk2-dependent signaling [85] , [86] . This and the partitioning of DNA repair enzymes to viral replication compartments predisposes the host cells to the accumulation of DNA damage [49] , [87] , [88] . It is yet unclear to what extent the intrinsic genotoxicity of HCMV contributes to the severe chromosomal instability seen after mitotic entry of infected cells.…”

OR14I1 is a receptor for the human cytomegalovirus pentameric complex and defines viral epithelial cell tropism

“…Cellular DNA and cell-associated viral DNA were isolated using a DNeasy Blood & Tissue Kit (Qiagen). Viral DNA ( UL83 ) and host DNA (β -ACTIN ) were quantified by real-time quantitative PCR as described (72).…”