The Latency-associated Nuclear Antigen of Kaposi's Sarcoma-associated Herpesvirus Transactivates the Telomerase Reverse Transcriptase Promoter

Jason S Knight,Murray A Cotter,Erle S Robertson

doi:10.1074/jbc.m101890200

Jason S Knight, Murray A Cotter + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m101890200

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Abstract

Telomerase is a multi-subunit ribonucleoprotein holoenzyme that stabilizes telomere length through the addition of new repeat sequence to the ends of chromosomes. Telomerase reverse transcriptase is the subunit of this complex responsible for the enzymatic activity of telomerase. Expression of the reverse transcriptase is regulated at the level of transcription through the action of transcription factors that target its promoter. Most Kaposi's sarcoma tumor cells are latently infected with the Kaposi's sarcoma-associated herpesvirus, and the constitutive expression of a viral-encoded latency-associated nuclear antigen has been shown to be important for the maintenance of the viral episome. The proliferative nature of Kaposi's sarcoma suggests that this antigen may also play a critical role in viral-mediated oncogenesis. In this study telomerase reverse transcriptase promoter elements cloned into a luciferase reporter plasmid were analyzed to determine the ability of the latency-associated nuclear antigen to regulate transcription. The latency-associated nuclear antigen transactivated the full-length promoter in 293T, 293, and BJAB cell lines. Furthermore, truncation promoter studies implicated sequence from -130 to +5 in viral-mediated activation. This region contains five Sp1 transcription factor-binding sites. Electrophoretic mobility shift assays indicated that the latency-associated nuclear antigen targets and affects the Sp1-DNA complex in the context of BJAB nuclear extracts.

Highlights

Telomeric DNA, found at the distal ends of chromosomes, consists of a 6-base pair repeat of the sequence TTAGGG [1]
Because expression of hTERT is primarily regulated at the transcriptional level, the effect of LANA on hTERT expression was investigated by testing the ability of LANA to regulate expression from luciferase reporter constructs containing the hTERT promoter in HEK 293 cells
Increasing the concentration of the LANA expression construct consistently augmented the activation to 7-fold. These data indicate that the transactivation of the hTERT promoter in the HEK 293 cell line is directly proportional to the quantity of LANA expressed as demonstrated by the observed dose-response relationship (Fig. 2)

Summary

Introduction

Telomeric DNA, found at the distal ends of chromosomes, consists of a 6-base pair repeat of the sequence TTAGGG [1]. LANA Activates Transcription of the Full-length hTERT Promoter in HEK 293 Cells—The proliferative nature of cells latently infected with KSHV as well as structural domains of LANA having the potential to mediate protein-protein and protein-DNA interactions suggest a role for LANA in regulating cellular gene expression (Fig. 1).

Results

Conclusion