DNA binding and modulation of gene expression by the latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus.
Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. The latency-associated nuclear antigen (LANA) is highly expressed in these malignancies and has been shown to play an important role in episomal maintenance, presumably by binding to a putative oriP. In addition, LANA modulates cellular and viral gene expression and interacts with the cellular tumor suppressors p53 and retinoblastoma suppressor protein. Many of these features are reminiscent of Epstein-Barr virus nuclear antigens (EBNAs), a family of six proteins expressed during latency. EBNA-1 is required for episome maintenance, binds to oriP, and strongly activates transcription from two promoters, including its own. We have previously shown that LANA can transactivate its own promoter and therefore asked whether LANA, like EBNA-1, activates transcription by direct binding to DNA. By using recombinant LANA expressed from vaccinia virus vectors for electrophoretic mobility shift assays, we found that LANA does not bind to its own promoter. In contrast, LANA binds specifically to sequences containing an imperfect 20-bp palindrome in the terminal repeat (TR) of KSHV. We further show that the C-terminal domain of LANA is sufficient for site-specific DNA binding. Unlike EBNA-1, which activates transcription through binding of oriP, we found that LANA inhibits transcription from a single TR binding site. A multimerized TR as found in the viral genome results in strong transcriptional suppression when linked to a heterologous promoter. These data suggest that LANA, although fulfilling functions similar to those of EBNA-1, does so by very different mechanisms.
Garber
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AC
Division of Hematology/Oncology and Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Shu
M A
MA
Hu
J
J
Renne
R
R
D000818:Animals; D000956:Antigens, Viral; D001665:Binding Sites; D002460:Cell Line; D004279:DNA, Viral / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D019288:Herpesvirus 8, Human / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology; D020079:Terminal Repeat Sequences / Q000235:genetics / Q000502:physiology; D014158:Transcription, Genetic; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins
D000956:Antigens, Viral; D004279:DNA, Viral; D009687:Nuclear Proteins; D014764:Viral Proteins; C109540:latency-associated nuclear antigen; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases