ABSTRACT
The latency-associated nuclear antigen (LANA) encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) plays a major role in maintaining latency and is critical for the perpetual segregation of viral episomes to the progeny nuclei of newly divided cells. LANA binds to KSHV terminal repeat (TR) DNA and tethers the viral episomes to host chromosomes through the association of chromatin-bound cellular proteins. TR elements serve as potential origin sites of KSHV replication and have been shown to play important roles in latent DNA replication and transcription of adjacent genes. Affinity chromatography and proteomics analysis using KSHV TR DNA and the LANA binding site as the affinity column identified topoisomerase IIß (TopoIIß) as a LANA-interacting protein. Here, we show that TopoIIß forms complexes with LANA that colocalize as punctuate bodies in the nucleus of KSHV-infected cells. The specific TopoIIß binding region of LANA has been identified to its N terminus and the first 32 amino acid residues containing the nucleosome-binding region crucial for binding. Moreover, this region could also act as a dominant negative to disrupt association of TopoIIß with LANA. TopoIIß plays an important role in LANA-dependent latent DNA replication, as addition of ellipticine, a selective inhibitor of TopoII, negatively regulated replication mediated by the TR. DNA break labeling and chromatin immunoprecipitation assay using biotin-16-dUTP and terminal deoxynucleotide transferase showed that TopoIIß mediates a transient DNA break on viral DNA. These studies confirm that LANA recruits TopoIIß at the origins of latent replication to unwind the DNA for replication.
Subject(s)
Antigens, Viral/genetics , Antigens, Viral/metabolism , DNA Replication , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Herpesvirus 8, Human/genetics , Herpesvirus 8, Human/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Amino Acid Sequence , Animals , Antigens, Viral/chemistry , Base Sequence , Cell Line , Cell Nucleus/metabolism , Cell Nucleus/virology , DNA Replication/drug effects , DNA Topoisomerases, Type II/chemistry , DNA, Viral/biosynthesis , DNA, Viral/genetics , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/chemistry , Ellipticines/pharmacology , HEK293 Cells , Herpesvirus 8, Human/pathogenicity , Host-Pathogen Interactions , Humans , Mice , Models, Biological , Molecular Sequence Data , Nuclear Proteins/chemistry , Protein Interaction Domains and Motifs , Terminal Repeat Sequences , Topoisomerase II Inhibitors/pharmacology , Virus LatencyABSTRACT
Pathological as well as physiological angiogenesis is known to be regulated by such factors as nucleotides and Vascular Endothelial Growth Factor (VEGF). Activated P2Y nucleotide receptors have been observed to associate and transactivate VEGF Receptor 2 (VEGFR2), suggesting a cooperation between nucleotide and VEGF signaling in angiogenesis. P2YR mediated VEGFR2 signaling therefore may be important in describing the angiogenic signaling of nucleotides such as ATP. Here, we provide evidence that supports the notion of P2YR-VEGFR2 signaling. The significant angiogenic effect of P2Y1/2 receptor agonists (100 microM ATP and 10 microM 2MS-ATP) on endothelial cell tubulogenesis was suppressed back to near control levels upon addition of 1 microM SU1498 (specific VEGFR2 tyrosine kinase inhibitor). We believe that this P2YR-VEFGR2 signaling is an important component of pathological, as well as physiological angiogenesis.
