Identification of Direct Transcriptional Targets of the Kaposi's Sarcoma-Associated Herpesvirus Rta Lytic Switch Protein by Conditional Nuclear Localization

Abstract

Lytic reactivation from latency is critical for the pathogenesis of Kaposi's sarcoma-associated herpesvirus (KSHV). We previously demonstrated that the 691-amino-acid (aa) KSHV Rta transcriptional transactivator is necessary and sufficient to reactivate the virus from latency. Viral lytic cycle genes, including those expressing additional transactivators and putative oncogenes, are induced in a cascade fashion following Rta expression. In this study, we sought to define Rta's direct targets during reactivation by generating a conditionally nuclear variant of Rta. Wild-type Rta protein is constitutively localized to cell nuclei and contains two putative nuclear localization signals (NLSs). Only one NLS (NLS2; aa 516 to 530) was required for the nuclear localization of Rta, and it relocalized enhanced green fluorescent protein exclusively to cell nuclei. The results of analyses of Rta NLS mutants demonstrated that proper nuclear localization of Rta was required for transactivation and the stimulation of viral reactivation. RTA with NLS1 and NLS2 deleted was fused to the hormone-binding domain of the murine estrogen receptor to generate an Rta variant whose nuclear localization and ability to transactivate and induce reactivation were tightly controlled posttranslationally by the synthetic hormone tamoxifen. We used this strategy in KSHV-infected cells treated with protein synthesis inhibitors to identify direct transcriptional targets of Rta. Rta activated only eight KSHV genes in the absence of de novo protein synthesis. These direct transcriptional targets of Rta were transactivated to different levels and included the genes nut-1/PAN, ORF57/Mta, ORF56/Primase, K2/viral interleukin-6 (vIL-6), ORF37/SOX, K14/vOX, K9/vIRF1, and ORF52. Our data suggest that the induction of most of the KSHV lytic cycle genes requires additional protein expression after the expression of Rta.Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus-8) is the etiologic agent of the human cancers primary effusion lymphoma (PEL) and Kaposi's sarcoma (KS) (8,22). Since B-cell infection predicts future KS development and latent KSHV infection is established before KS onset, the reactivation of productive (lytic) KSHV infection from the latently infected B-cell reservoir is a necessary step in KS development (2,51,54,78). Deciphering the mechanisms that function at the molecular level to control KSHV reactivation is therefore essential for understanding the pathogenesis of the virus.During KSHV reactivation in PEL tissue culture models, de novo expression of viral genes unfolds sequentially in a cascade fashion (31,44). A fraction of the cells in which the virus initiates reactivation support completion of the gene expression program, resulting in the production of progeny virus and lysis of the host cells (46,47,60). The reactivation program has thus been termed the KSHV lytic cycle. Similarly to other DNA viruses, the KSHV lytic cycle genes have been classified as immediate early (IE), delayed early (DE), or late (L...

Cooperation between Viral Interferon Regulatory Factor 4 and RTA To Activate a Subset of Kaposi's Sarcoma-Associated Herpesvirus Lytic Promoters

“…When protein synthesis is blocked with hygromycin, only 10% of lytic genes are activated in the presence of preexisting RTA, suggesting that in fact the majority of lytic genes rely on de novo protein synthesis for efficient transcription (4). The hygromycin experiment does not distinguish between viral and cellular proteins, but the findings presented in the current study argue that the absence of vIRF4 should account for some or many of the hygromycin-sensitive lytic genes.…”

The Single RBP-Jκ Site within the LANA Promoter Is Crucial for Establishing Kaposi's Sarcoma-Associated Herpesvirus Latency during Primary Infection

“…Interestingly, the KSHV genome contains 177 RBP-J sites, and RTA is capable of transactivating only eight KSHV genes in infected cells (6). Importantly, RBP-J is required for productive KSHV reactivation as well as for latent infection and transformation of primary B cells by the other human gammaherpesvirus, EBV (41 (25).…”

Kaposi's Sarcoma-Associated Herpesvirus Rta Tetramers Make High-Affinity Interactions with Repetitive DNA Elements in the Mta Promoter To Stimulate DNA Binding of RBP-Jk/CSL

“…Indeed, some reports propose that Rta binds only indirectly to the Mta promoter (and others), by piggybacking onto DNA-bound RBP-Jk (10, 21, 35, 42-44, 48, 51). Furthermore, the KSHV genome contains at least 260 predicted RBP-Jk sites (32; O. Gonzalez-Lopez and D. M. Lukac, unpublished observations), yet in the absence of de novo protein expression, Rta transactivates only 8 KSHV genes in infected cells (4).…”