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Octamer-binding sequence is a key element for the autoregulation of Kaposi's sarcoma-associated herpesvirus ORF50/Lyta gene expression.

ABSTRACT: The expression of the Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 50 (ORF50) protein, Lyta (lytic transactivator), marks the switch from latent KSHV infection to the lytic phase. ORF50/Lyta upregulates several target KSHV genes, such as K8 (K-bZip), K9 (vIRF1), and ORF57, finally leading to the production of mature viruses. The auto-upregulation of ORF50/Lyta is thought to be an important mechanism for efficient lytic viral replication. In this study, we focused on this autoregulation and identified the promoter element required for it. An electrophoretic mobility shift assay indicated that the octamer-binding protein 1 (Oct-1) bound to this element. Mutations in the octamer-binding motif resulted in refractoriness of the ORF50/Lyta promoter to transactivation by ORF50/Lyta, and Oct-1 expression enhanced this transactivation. These results suggest that the autoregulation of ORF50/Lyta is mediated by Oct-1.

SUBMITTER: Sakakibara S

PROVIDER: S-EPMC114417 | biostudies-literature | 2001 Aug

REPOSITORIES: biostudies-literature

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Octamer-binding sequence is a key element for the autoregulation of Kaposi's sarcoma-associated herpesvirus ORF50/Lyta gene expression.

Sakakibara S S Ueda K K Chen J J Okuno T T Yamanishi K K

Journal of virology 20010801 15

The expression of the Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 50 (ORF50) protein, Lyta (lytic transactivator), marks the switch from latent KSHV infection to the lytic phase. ORF50/Lyta upregulates several target KSHV genes, such as K8 (K-bZip), K9 (vIRF1), and ORF57, finally leading to the production of mature viruses. The auto-upregulation of ORF50/Lyta is thought to be an important mechanism for efficient lytic viral replication. In this study, we focused on this aut ...[more]

PMID: 11435569

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Project description:The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded open reading frame 50 (ORF50) protein is the key transactivator responsible for the latent-to-lytic switch. Here, we investigated the transcriptional activation of the ORF56 gene (encoding a primase protein) by ORF50 and successfully identified an ORF50-responsive element located in the promoter region between positions -97 and -44 (designated 56p-RE). This 56p-RE element contains a noncanonical RBP-Jκ-binding sequence and a nonconsensus Sp1/Sp3-binding sequence. Electrophoretic mobility shift assays revealed that RBP-Jκ, Sp3, and ORF50 could form stable complexes on the 56p-RE element. Importantly, transient-reporter analysis showed that Sp3, but not RBP-Jκ or Sp1, acts in synergy with ORF50 to activate the 56p-RE-containing reporter construct, and the synergy mainly depends on the Sp1/Sp3-binding region of the 56p-RE element. Sequence similarity searches revealed that the promoters for ORF21 (thymidine kinase), ORF60 (ribonucleotide reductase, small subunit), and cellular interleukin-10 (IL-10) contain a sequence motif similar to the Sp1/Sp3-binding region of the 56p-RE element, and we found that these promoters could also be synergistically activated by ORF50 and Sp3 via the conserved motifs. Noteworthily, the conversion of the Sp1/Sp3-binding sequence of the 56p-RE element into a consensus high-affinity Sp-binding sequence completely lost the synergistic response to ORF50 and Sp3. Moreover, transcriptional synergy could not be detected through other ORF50-responsive elements from the viral PAN, K12, ORF57, and K6 promoters. Collectively, the results of our study demonstrate that ORF50 and Sp3 can act in synergy on the transcription of specific gene promoters, and we find a novel conserved cis-acting motif in these promoters essential for transcriptional synergy.IMPORTANCE Despite the critical role of ORF50 in the KSHV latent-to-lytic switch, the molecular mechanism by which ORF50 activates its downstream target genes, especially those that encode the viral DNA replication enzymes, is not yet fully understood. Here, we find that ORF50 can cooperate with Sp3 to synergistically activate promoters of the viral ORF56 (primase), ORF21 (thymidine kinase), and ORF60 (ribonucleotide reductase) genes via similar Sp1/Sp3-binding motifs. Additionally, the same synergistic effect can be seen on the promoter of the cellular IL-10 gene. Overall, our data reveal an important role for Sp3 in ORF50-mediated transactivation, and we propose a new subclass of ORF50-responsive elements in viral and cellular promoters.

Dataset Information

Octamer-binding sequence is a key element for the autoregulation of Kaposi's sarcoma-associated herpesvirus ORF50/Lyta gene expression.

Publications

Octamer-binding sequence is a key element for the autoregulation of Kaposi's sarcoma-associated herpesvirus ORF50/Lyta gene expression.

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