Differential requirements of the C terminus of Nbs1 in suppressing adenovirus DNA replication and promoting concatemer formation.
Adenoviruses (Ad) with the early region E4 deleted (E4-deleted virus) are defective for DNA replication and late protein synthesis. Infection with E4-deleted viruses results in activation of a DNA damage response, accumulation of cellular repair factors in foci at viral replication centers, and joining together of viral genomes into concatemers. The cellular DNA repair complex composed of Mre11, Rad50, and Nbs1 (MRN) is required for concatemer formation and full activation of damage signaling through the protein kinases Ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR). The E4orf3 and E4orf6 proteins expressed from the E4 region of Ad type 5 (Ad5) inactivate the MRN complex by degradation and mislocalization, and prevent the DNA damage response. Here we investigated individual contributions of the MRN complex, concatemer formation, and damage signaling to viral DNA replication during infection with E4-deleted virus. Using virus mutants, short hairpin RNA knockdown and hypomorphic cell lines, we show that inactivation of MRN results in increased viral replication. We demonstrate that defective replication in the absence of E4 is not due to concatemer formation or DNA damage signaling. The C terminus of Nbs1 is required for the inhibition of Ad DNA replication and recruitment of MRN to viral replication centers. We identified regions of Nbs1 that are differentially required for concatemer formation and inhibition of Ad DNA replication. These results demonstrate that targeting of the MRN complex explains the redundant functions of E4orf3 and E4orf6 in promoting Ad DNA replication. Understanding how MRN impacts the adenoviral life cycle will provide insights into the functions of this DNA damage sensor.
Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California 920371, USA.
Seema S
SS
Lakdawala
Rachel A
RA
Schwartz
Kevin
K
Ferenchak
Christian T
CT
Carson
Brian P
BP
McSharry
Gavin W
GW
Wilkinson
Matthew D
MD
Weitzman
D000260:Adenoviruses, Human / Q000145:classification* / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000166:cytology; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D014162:Transfection
D018797:Cell Cycle Proteins; D004279:DNA, Viral; C112156:NBN protein, human; D009687:Nuclear Proteins