Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus Up-Regulates Transcription of Human Telomerase Reverse Transcriptase Promoter through Interaction with Transcription Factor Sp1

Abstract

Telomerase is required for the maintenance of telomere length and is an important determinant for cell immortalization. In human cells, telomerase activity is due to the expression of its enzymatic subunit, human telomerase reverse transcriptase (hTERT). The expression of hTERT is not typically detectable in healthy somatic human cells but is present in cancerous tissues and immortalized cells. We have previously shown that hTERT promoter activity is up-regulated by the Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA). LANA is expressed in all forms of human malignancies associated with KSHV. The hTERT promoter sequence located at positions ؊130 to ؉5 contains several Sp1 binding motifs and was shown be important for up-regulation by LANA. In this report, we demonstrate that hTERT promoter activity is due to the direct interaction of LANA with Sp1. The interaction of LANA with Sp1 was demonstrated through in vitro binding experiments and coimmunoprecipitation and is supported by the colocalization of these two molecules in the nuclei of KSHV-infected cells. Moreover, LANA modulates Sp1-mediated transcription in transient GAL4 fusion reporter assays. Mapping of the regions involved in binding and transcriptional activation showed that the amino terminus of LANA is the major site for interaction and up-regulation but that it can cooperate with the carboxy terminus to enhance these functions. An analysis of Sp1 binding to its cognate sequence corroborated the binding data. Together, our results suggest that the interaction of LANA with Sp1 up-regulates the telomerase promoter, potentially contributing to the immortalization of KSHV-infected cells.

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

“…Surprisingly, LANA containing both the amino and carboxyl termini fused in frame (LANA ⌬IR) was unable to repress K1p transcriptional activity in our reporter assays even though this contained the DNA binding domain. This may be due the fact that LANA lacking glutamine and glutamic acid residues has been demonstrated to have higher transcriptional activity than full-length LANA (54,59). Thus, the repressive effect due to the binding of LANA-C may be overridden by increased transcription regulation by LANA ⌬IR (Fig.…”

“…LANA mutants LANA N (amino acids 1 to 340), LANA C (amino acids 762 to 1162), and LANA ⌬IR (amino acids 327 to 929 deleted) used in reporter assay were described previously (54).…”

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).…”

Tumor Cell Development: A Role for Viruses and Telomerase Activity?

“…LANA is one of the major latent proteins detected in all forms of Kaposi's sarcoma-associated malignancies, including bodycavity-based lymphomas and multicentric Castleman's disease, an aggressive lymphoproliferative disorder. 46 Importantly, in contrast to up-regulating TERT expression, LANA was shown to interact with telomere-related proteins and ablate their binding to telomeric repeats. Moreover, LANA expression resulted in telomere shortening 47 in telomerase-immortalized endothelial cells, but not in U2OS cells, which uses the so-called alternative lengthening of telomeres (ALT) mechanism (discussed in the next section).…”

“…44,45 While HPV E6 activates TERT transcription through a Myc-dependent pathway, latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) activates TERT promoter through Sp1. 46 LANA serves as an origin binding protein to recruit the cellular replication machinery to the KSHV latency origin of replication. LANA is one of the major latent proteins detected in all forms of Kaposi's sarcoma-associated malignancies, including bodycavity-based lymphomas and multicentric Castleman's disease, an aggressive lymphoproliferative disorder.…”