DNA Binding and Modulation of Gene Expression by the Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus

Abstract

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.

Kaposi's Sarcoma-Associated Herpesvirus-Encoded Latency-Associated Nuclear Antigen Modulates K1 Expression through Its cis -Acting Elements within the Terminal Repeats

“…Since LANA is expressed in almost all KSHV-positive cells and important for tethering of the viral genome by binding to the LANA binding sequence (2,10,19,20,26), we hypothesized that LANA may have a role in regulation of K1 expression. We digested cosmid Z6 of KSHV with BamHI and NotI and cloned K1p as described in the schematic (Fig.…”

Regulation and Autoregulation of the Promoter for the Latency-associated Nuclear Antigen of Kaposi's Sarcoma-associated Herpesvirus

“…Plasmids expressing LANA mutants, LANA1 ⌬C50 (pDD770), LANA1 NLS ⌬DED (pDD771), LANA1 NLS LZ-C (pDD772), and LANA1 ⌬1002-1062 (pDD774) (courtesy of Joonho Choe, Korea Advanced Institute of Science and Technology, Korea), were described previously (70,83). Plasmids with larger deletions of LANA, LANA-A (pDD720), LANA-AB (pDD721), LANA-BC (pDD722), and LANA-C (pDD7230) were as described by Garber et al (29).…”

“…The LANA binding motif in the TR is a 20-bp GC-rich imperfect palindrome (5Ј-ccccatgcccgggcgggagg) (29,35), which also fits a minimal GC box/Sp1 consensus sequence 5Ј-GG(C/ A)G. Hence, we tested whether a highly purified LANA Cterminal fragment that was known to bind to the TR (28,29) was able to bind the GC box/Sp1 element in LANAp. Shown in Fig.…”

“…Autoregulation of the LANAp by LANA Protein-We previously reported that LANA transactivates its own promoter (47, 53) but had been unable to demonstrate full-length LANA binding to the LANAp ϩ271 to Ϫ279 fragment directly (29). This may have been due to technical difficulties of LANA-based electrophoretic mobility shift assay, but it left open the alternative possibility that LANA, like adenovirus E1A or EBV EBNA-2, regulated transcription via proteinprotein interactions and did not contact DNA directly (54,55).…”

The Kaposi Sarcoma Herpesvirus Latency-associated Nuclear Antigen DNA Binding Domain Dorsal Positive Electrostatic Patch Facilitates DNA Replication and Episome Persistence

“…LANA is a multifunctional protein; it regulates a variety of cellular activities including gene transcription, DNA replication, and signaling pathways involved in cell proliferation (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27).…”