IL-10 promoter transactivation by the viral K-RTA protein involves the host-cell transcription factors, specificity proteins 1 and 3

Masanori Miyazawa,Kohji Noguchi,Mana Kujirai,Kazuhiro Katayama,Satoshi Yamagoe,Yoshikazu Sugimoto

doi:10.1074/jbc.m117.802900

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 (HHV-8) causes a persistent infection, presenting latent and lytic replication phases during its life cycle. KSHV-related diseases are associated with deregulated expression of inflammatory cytokines, including IL-6 and IL-10, but the mechanisms underlying this dysregulation are unclear. Herein, we report a molecular mechanism for KSHV-induced IL-10 gene expression. KSHV replication and transcription activator (K-RTA) is a molecular switch for the initiation of expression of viral lytic genes, and we describe, for the first time, that K-RTA significantly activates the promoter of the human IL-10 gene. Of note, mutations involving a basic region of K-RTA reduced the association of K-RTA with the IL-10 promoter. Moreover, the host-cell transcription factors, specificity proteins (SP) 1 and 3, play a pivotal cooperative role in K-RTA-mediated transactivation of the IL-10 promoter. K-RTA can interact with SP1 and SP3 directly in vitro, and electrophoresis mobility shift assays (EMSAs) revealed co-operative interaction involving K-RTA, SP1, and SP3 in binding to the IL-10 promoter. As DNase I footprinting assays indicated that K-RTA did not affect SP3 binding to the IL-10 promoter, SP3 can function to recruit K-RTA to the IL-10 promoter. These findings indicate that K-RTA can directly contribute to IL-10 up-regulation via a functional interplay with the cellular transcription factors SP1 and SP3.

Highlights

Kaposi’s sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 (HHV-8) causes a persistent infection, presenting latent and lytic replication phases during its life cycle
The KSHV-infected primary effusion lymphoma (PEL) cell line JSC-1 is reported to show higher basal and induced expression of lytic phase genes and to produce higher titers of infectious viruses than KSHV-infected BC3 cells [40]. In line with those results, luciferase reporter assays confirmed that promoter activities for the lytic phase initiator ORF50/KSHV replication and transcription activator (K-RTA) and human IL-10 genes were higher in the KSHV-infected PEL cell line JSC-1 than in BC3 cells (Fig. 1A)
These results suggest that lytic replication and IL-10 mRNA expression are correlated

Summary

To whom correspondence should be addressed

K-RTA has been shown to regulate a number of cellular genes [15], and an indirect mechanism is involved in K-RTA–mediated gene expression [16] Various cellular proteins, such as RBP-J␬ [17, 18], CCATT/ enhancer-binding protein C/EBP␣ [19], OCT-1 [20], STAT3 [21], K-RBP [22], and CBP [23] are demonstrated to interact with K-RTA at the sites of various DNA elements. We show that specific residues such as Lys-152, Lys-154, and His-145 on K-RTA play a role in K-RTA–mediated IL-10 promoter activation These data indicate that the functional activity of K-RTA can contribute to IL-10 induction in KSHV-associated diseases

Results

Discussion

Experimental procedures