DNA Binding by Kaposi's Sarcoma-Associated Herpesvirus Lytic Switch Protein Is Necessary for Transcriptional Activation of Two Viral Delayed Early Promoters

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus-8) establishes latent and lytic infections in both lymphoid and endothelial cells and has been associated with diseases of both cell types. The KSHV open reading frame 50 (ORF50) protein is a transcriptional activator that plays a central role in the reactivation of lytic viral replication from latency. Here we identify and characterize a DNA binding site for the ORF50 protein that is shared by the promoters of two delayed early genes (ORF57 and K-bZIP). Transfer of this element to heterologous promoters confers on them high-level responsiveness to ORF50, indicating that the element is both necessary and sufficient for activation. The element consists of a conserved 12-bp palindromic sequence and less conserved sequences immediately 3 to it. Mutational analysis reveals that sequences within the palindrome are critical for binding and activation by ORF50, but the presence of a palindrome itself is not absolutely required. The 3 flanking sequences also play a critical role in DNA binding and transactivation. The strong concordance of DNA binding in vitro with transcriptional activation in vivo strongly implies that sequence-specific DNA binding is necessary for ORF50-mediated activation through this element. Expression of truncated versions of the ORF50 protein reveals that DNA binding is mediated by the amino-terminal 272 amino acids of the polypeptide.Infection by Kaposi's sarcoma-associated herpesvirus (KSHV) also known as human herpesvirus 8 (HHV-8) is associated with malignancies of both endothelial and lymphoid cells in humans. KSHV has been well established as the etiologic agent responsible for Kaposi's sarcoma (KS), an endothelial neoplasm frequent in homosexual men with AIDS and highly prevalent in sub-Saharan Africa (reviewed in reference 25). KSHV is also linked to two other AIDS-related malignancies, primary effusion lymphoma and multicentric Castleman's disease (5, 26). The presence of viral DNA in CD19 ϩ B cells and other mononuclear cells of the peripheral blood of KS/ AIDS patients (1, 2, 31), even prior to full-blown KS, suggests that infection of the lymphoid compartment is antecedent to the development of the endothelial disease.KSHV infection, similar to infection by other herpesviruses, displays two life cycle modes, latency and lytic replication. Latency is established by the virus both in endothelial and B cells and is detectable in such cell types both in vitro and in infected hosts (1,3,7,19,22,27,31). KSHV latency-associated genes are expressed in most spindle cells of KS tumors and are thought to contribute to their survival and proliferation (25). However, many viral genes (e.g., vGCR and vMIPs I and II) encoding homologs of cellular signaling proteins which have been suspected of roles in the histogenesis of KS are expressed strictly as lytic cycle products (13,20,23,24,28). This suggests that the KSHV lytic cycle may also contribute to KS lesion formation. Additional support for this notion comes from stud...

The role of Kaposi's sarcoma-associated herpesvirus/human herpesvirus-8 regulator of transcription activation (RTA) in control of gene expression

“…Removal of all four sequences had little effect on RTA-activated expression from the ORF57 promoter; however, a 40 bp region between nt 81904 and 81944 was found to be essential for RTA responsiveness. A significant 16 bp homology between the ORF57 region and the 5 0 sequences preceding the HHV-8 K8, basic leucine zipper (bZip) gene were reported (Wang et al, 2001a); moreover, a near-identical 25 bp sequence was subsequently described by Lukac et al (Lukac et al, 2001). Mutational analyses revealed that both the K8 and ORF57 sequences were responsive to RTA and conferred RTA responsiveness to heterologous promoters.…”

“…However, RTA does not recognize the same sequence element in all responsive promoters. This diversity of recognition suggests that on some promoters, other viral and host factors may direct or augment DNA binding and may influence the diversity of RTA transactivation (Seaman et al, 1999;Lukac et al, 2001;Sakakibara et al, 2001;Wang et al, 2001a;Chang et al, 2002;Deng et al, 2002b;Saveliev et al, 2002).…”

The lytic switch protein of KSHV activates gene expression via functional interaction with RBP-Jκ (CSL), the target of the Notch signaling pathway

“…Figure 3A schematically depicts the organization of the relevant portions of the MTA and SSB promoters, indicating the location of the predicted RBP-J recognition element within each. In the MTA promoter, our earlier work (Lukac et al 2001) established that deletion to −106 preserves RTA-inducibility, while deletion to −54 abolishes it, thereby defining a 52-bp sequence that confers RTA responsiveness. The RBP-J site falls within this region, in the antisense orientation relative to the MTA ORF.…”

“…Radioactive probes were diluted in water to a final concentration of 105 cpm/µL. DNA binding reactions were performed in a manner similar to that described (Lukac et al 2001). Briefly, proteins from RRL were mixed with 1 µg poly (dI-dC) (Sigma) in 1× DNA binding buffer for 30 min on ice.…”

“…Not surprisingly, KSHV RTA also acts to promote transcriptional activation of a variety of viral promoters, including those for delayed-early viral genes like TK, SSB, the posttranscriptional activator MTA, and the noncoding polyadenylated nuclear RNA, PAN (Lukac et al 1998). KSHV RTA contains an N-terminal DNA-binding domain and a C-terminal activation domain (Lukac et al 1998(Lukac et al , 2001Seaman et al 1999), and direct, high-affinity binding of purified, recombinant RTA to a site in the PAN promoter has been reported (Song et al 2001). However, other sites that are activated by RTA in vivo-for example, those in the MTA and RAP (K8, K-bZIP) promoters-display no homology to the PAN promoter site, raising the question of whether RTA might have other modes of DNA interaction, such as binding to cellular DNA binding factors.…”