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The Kaposi's sarcoma-associated herpesvirus (KSHV) Mta protein, encoded by open reading frame 57, is a transactivator of gene expression that is essential for productive viral replication. Previous studies have suggested both transcriptional and posttranscriptional roles for Mta, but little is known regarding Mta's transcriptional function. In this study, we demonstrate that Mta cooperates with the KSHV lytic switch protein, Rta, to reactivate KSHV from latency, but Mta has little effect on reactivation when expressed alone. We demonstrate that the Mta and Rta proteins are expressed with similar but distinct kinetics during KSHV reactivation. In single-cell analyses, Mta expression coincides tightly with progression to full viral reactivation. We demonstrate with promoter reporter assays that while Rta activates transcription in all cell lines tested, Mta's ability to transactivate promoters, either alone or synergistically with Rta, is cell and promoter specific. In particular, Mta robustly transactivates the nut-1/PAN promoter independently of Rta in 293 and Akata-31 cells. Using nuclear run-on assays, we demonstrate that Mta stimulates transcriptional initiation in 293 cells. Rta and Mta physically interact in infected cell extracts, and this interaction requires the intact leucine repeat and central region of Rta in vitro. We demonstrate that Mta also binds to the nut-1/PAN promoter DNA in vitro and in infected cells. An Mta mutant with a lesion in a putative A/T hook domain is altered in DNA binding and debilitated in transactivation. We propose that one molecular mechanism of Mtamediated transactivation is a direct effect on transcription by direct and indirect promoter association.Reactivation of Kaposi's-sarcoma associated herpesvirus (KSHV) (also known as human herpesvirus 8) from latency is a crucial step in Kaposi's sarcoma development (7,11,27,43,65,76,81,87,104). It is likely that reactivation of KSHV contributes to Kaposi's sarcoma development by facilitating dissemination of the virus from its B-cell reservoir and permitting the expression of lytic cycle genes with direct roles in cancer progression. Most of the candidate pathogenic genes of KSHV (encoding proteins with growth-deregulatory and immunomodulatory functions) are expressed in the delayed early class of the lytic gene expression program (8,14,17,25,26,28,30,41,46,49,75,86,94). Therefore, a complete understanding of KSHV pathogenesis demands elucidation of the mechanisms that regulate viral reactivation and progression through the lytic cycle.The members of the Herpesviridae all encode multiple lytic cycle transactivators that cooperate to promote viral replication. For herpes simplex virus type 1 (HSV-1) infection, the two essential transactivating proteins are called infected cell protein 4 (ICP4) and ICP27. ICP4 is a transcriptional transactivator necessary for activation of early and late genes (80, 103). ICP27 is a posttranscriptional and transcriptional activator that stimulates the switch from early-to late-gene expression (66,67,...

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Summary The adapter protein MecA targets the transcription factor ComK for degradation by the ClpC/ClpP proteolytic complex, thereby negatively regulating competence in Bacillus subtilis. Here we show that MecA also decreases the frequency of transitions to the sporulation pathway as well as the expression of eps, which encodes synthesis of the biofilm matrix exopolysaccharide. We present genetic and biophysical evidence that MecA down-regulates eps expression and spore formation by directly interacting with Spo0A. MecA does not target Spo0A for degradation, and apparently does not prevent the phosphorylation of Spo0A. We propose that it inhibits the transcriptional activity of Spo0A~P by direct binding. Thus, in its interaction with Spo0A, MecA differs from its role in the regulation of competence where it targets ComK for degradation. MecA acts as a general buffering protein for development, acting by two distinct mechanisms to regulate inappropriate transitions to energy-intensive pathways.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of primary effusion lymphoma (PEL).Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). PEL is a body cavity based lymphoma that is rapidly fatal (15,24,37,56,63). Multiple, continuous PEL cell lines were established by culturing clinical samples from PEL patients (13,54,60). These cell lines were the first tissue culture models for KSHV infection (53,60). Approximately 70% of PEL cell lines are coinfected with KSHV and EpsteinBarr virus (EBV) (17).The KSHV lytic switch protein, replication and transcriptional activator (Rta), encoded by open reading frame 50 (ORF50), is both necessary and sufficient for viral reactivation in PEL cells (47,48,65,70). Lytic reactivation requires the formation of a ternary complex between Rta, delayed early promoter DNA, and the host cell's recombination signal binding protein (RBP)-Jk (also known as CSL-1 and CBF1) (11,44,45). RBP-Jk is a sequence-specific DNA-binding protein that is the nuclear effector of the canonical Notch signal transduction pathway (23).Whereas RBP-Jk is required for productive KSHV reactivation (45), it is also required for latent (nonproductive) transformation of primary B cells by EBV (39). In this program, termed latency III, EBV nuclear antigen 2 (EBNA-2) transactivates two EBV promoters by interacting with RBP-Jk. The two promoters express transcripts that encode seven proteins that promote viral persistence by stabilizing the EBV genome, stimulating B-cell growth and expansion, and blocking B-cell apoptosis (39). One of the transforming EBV latency proteins is latent membrane protein 1 (LMP-1). LMP-1 is a constitutively active ortholog of the cellular tumor necrosis factor (TNF) receptor CD40 (55, 68). LMP-1 induces cell proliferation and transformation by engaging multiple signaling pathways including NF-B, TNF receptor-associated factors (TRAFs) 1 to 3, Akt kinase, Jun kinase, c-Rel, and p38 (16, 18-20, 27, 31, 40, 43, 46, 51, 52, 55). EBV transformation also requires transactivation of cellular genes by EBNA-2 in an RBP-Jk-dependent fashion (22,34,38,62,64,71).RBP-Jk's principal role in KSHV and EBV infection is to specify transcriptional targets of Rta and EBNA-2. RBP-Jk also specifies transcriptional targets for the activated form of the cellular Notch receptor (Notch intracellular domain 1 [NICD-1]); despite the apparent mechanistic similarity of NICD-1 transactivation to that of Rta and EBNA-2, these proteins are not always phenotypically interchangeable. For example, NICD-1 and EBNA-2 do not productively reactivate KSHV from latency (11,14,45), and NICD-1 does not fully complement EBNA-2 deficiency in long-term outgrowth of lymphoblastoid cell lines (LCLs) (25,28). Furthermore, the KSHV genome contains 177 RBP-Jk sites and yet, in the absence of de novo protein expression, Rta only transactivates eight KSHV genes * Corresponding author. Mailing address: ICPH E350C, 225 Warren St.,