Inhibition of Epstein-Barr virus OriP function by tankyrase, a telomere-associated poly-ADP ribose polymerase that binds and modifies EBNA1.
Tankyrase (TNKS) is a telomere-associated poly-ADP ribose polymerase (PARP) that has been implicated along with several telomere repeat binding factors in the regulation of Epstein-Barr virus origin of plasmid replication (OriP). We now show that TNKS1 can bind to the family of repeats (FR) and dyad symmetry regions of OriP by using a chromatin immunoprecipitation assay and DNA affinity purification. TNKS1 and TNKS2 bound to EBNA1 in coimmunoprecipitation experiments with transfected cell lysates and with purified recombinant proteins in vitro. Two RXXPDG-like TNKS-interacting motifs in the EBNA1 amino-terminal domain mediated binding with the ankyrin repeat domain of TNKS. Mutations of both motifs at EBNA1 G81 and G425 abrogated TNKS binding and enhanced EBNA1-dependent replication of OriP. Small hairpin RNA targeted knock-down of TNKS1 enhanced OriP-dependent DNA replication. Overexpression of TNKS1 or TNKS2 inhibited OriP-dependent DNA replication, while a PARP-inactive form of TNKS2 (M1045V) was compromised for this inhibition. We show that EBNA1 is subject to PAR modification in vivo and to TNKS1-mediated PAR modification in vitro. These results indicate that TNKS proteins can interact directly with the EBNA1 protein, associate with the FR region of OriP in vivo, and inhibit OriP replication in a PARP-dependent manner.
Deng
Zhong
Z
The Wistar Institute, Philadelphia, PA 19104, USA.
Atanasiu
Constandache
C
Zhao
Kehao
K
Marmorstein
Ronen
R
Sbodio
Juan I
JI
Chi
Nai-Wen
NW
Lieberman
Paul M
PM
D000595:Amino Acid Sequence; D002460:Cell Line; D016384:Consensus Sequence; D019309:Epstein-Barr Virus Nuclear Antigens / Q000378:metabolism*; D006367:HeLa Cells; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D010957:Plasmids / Q000378:metabolism; D018741:Replication Origin / Q000235:genetics; D038501:Tankyrases / Q000378:metabolism*; D014162:Transfection; D014779:Virus Replication
D019309:Epstein-Barr Virus Nuclear Antigens; D038501:Tankyrases; C437334:TNKS2 protein, human; C418109:TNKS protein, human; C417029:EBV-encoded nuclear antigen 1