Global Changes in Kaposi's Sarcoma-Associated Virus Gene Expression Patterns following Expression of a Tetracycline-Inducible Rta Transactivator

Abstract

An important step in the herpesvirus life cycle is the switch from latency to lytic reactivation. In order to study the life cycle of Kaposi's sarcoma-associated herpesvirus (KSHV), we developed a gene expression system in KSHV-infected primary effusion lymphoma cells. This system uses Flp-mediated efficient recombination and tetracycline-inducible expression. The Rta transcriptional activator, which acts as a molecular switch for lytic reactivation of KSHV, was efficiently integrated downstream of the Flp recombination target site, and its expression was tightly controlled by tetracycline. Like stimulation with tetradecanoyl phorbol acetate (TPA), the ectopic expression of Rta efficiently induced a complete cycle of viral replication, including a well-ordered program of KSHV gene expression and production of infectious viral progeny. A striking feature of Rtamediated lytic gene expression was that Rta induced KSHV gene expression in a more powerful and efficient manner than TPA stimulation, indicating that Rta plays a central, leading role in KSHV lytic gene expression. Thus, our streamlined gene expression system provides a novel means not only to study the effects of viral gene products on overall KSHV gene expression and replication, but also to understand the natural viral reactivation process.

Kaposi's Sarcoma-Associated Herpesvirus-Encoded Protein Kinase and Its Interaction with K-bZIP

“…Because the mode of action of the inducers (e.g., phorbol ester or butyrate) is complex and the fraction of cells affected is relatively small, we chose to measure the expression kinetics of vPK by K-Rta-mediated reactivation. In the TREx-K-Rta BCBL-1 cell line (36), a derivative of the BCBL cell line, the expression of K-Rta is induced by DOX, resulting in a well-ordered KSHV gene expression program and a relatively synchronized cycle of viral replication in nearly all cells (19,36). RNA from induced cells was analyzed by Northern blot analysis, which showed that vPK is encoded in two transcripts (4.2 and 3.6 kb), together with ORF37 and ORF38.…”

“…Human embryonic kidney epithelial 293 cells and 293T cells were grown in monolayer culture in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (FBS) in the presence of 5% CO 2 . The TREx-KRta BCBL-1 cell line, generated by Nakamura et al (36), was cultured in RPMI 1640 medium supplemented with 15% FBS, 100 g/ml of blasticidin (Invitrogen), and 100 g/ml of hygromycin (Invitrogen).…”

Caspase-7 Cleavage of Kaposi Sarcoma-associated Herpesvirus ORF57 Confers a Cellular Function against Viral Lytic Gene Expression

“…To induce ORF57 expression and virus lytic replication, KSHV-infected cells were treated with 0.6 mM valproate (VA), 0.3 mM butyrate, or 20 ng/ml of 12-O-tetradecanoylphorbol-13-acetate. Doxycycline (dox)-inducible TREx BCBL-1 RTA and vector control cell lines were cultured as described (14).…”

“…1B). To exclude the possible nonspecific effects of chemical inducers, this observation was further verified in TREx BCBL-1 RTA cells (14), which inducibly express RTA (ORF50), a potent transactivator for KSHV lytic induction (24). RTA induction by dox in these cells led to the production of both full-length and cleaved ORF57, along with the production of cleaved PARP (Fig.…”

The pleiotropic effects of Kaposi's sarcoma herpesvirus

“…Also expressed during latency are transcripts derived from the K12 gene of KSHV [103], although expression of this gene is markedly up-regulated during the early stages of the lytic cycle, leading to its classification as a 'latent-lytic' [104] or even 'late' [105,106] gene. One of these transcripts encodes a protein, 'kaposin A', which has been reported to have transforming activity in rodent fibroblasts [107] and induces lymphocyte aggregation and adhesion possibly mediated through its direct interaction with cytohesin-1, a guanine nucleotide exchange factor for ARF GTPases and regulator of integrin-mediated cell adhesion [108,109].…”