MecA dampens transitions to spore, biofilm exopolysaccharide and competence expression by two different mechanisms.

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 downregulates 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.

Convergence of Kaposi's sarcoma-associated herpesvirus reactivation with Epstein-Barr virus latency and cellular growth mediated by the notch signaling pathway in coinfected cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of primary effusion lymphoma (PEL). All PEL cell lines are infected with KSHV, and 70% are coinfected with Epstein-Barr virus (EBV). KSHV reactivation from latency requires promoter-specific transactivation by the KSHV Rta protein through interactions with RBP-Jk (CSL), the cellular DNA-binding component of the Notch signal transduction pathway. EBV transformation of primary B cells requires EBV nuclear antigen 2 (EBNA-2) to interact with RBP-Jk to direct the latent viral and cellular gene expression program. Although KSHV Rta and EBV EBNA-2 both require RBP-Jk for transactivation, previous studies have suggested that RBP-Jk-dependent transactivators do not function identically. We have found that the EBV latent protein LMP-1 is expressed in less than 5% of KSHV(+)/EBV(+) PEL cells but is induced in an Rta-dependent fashion when KSHV reactivates. KSHV Rta transactivates the EBV latency promoters in an RBP-Jk-dependent fashion and forms a ternary complex with RBP-Jk on the promoters. In B cells that are conditionally transformed by EBV alone, we show that KSHV Rta complements a short-term EBNA-2 growth deficiency in an autocrine/paracrine manner. Complementation of EBNA-2 deficiency by Rta depends on RBP-Jk and LMP-1, and Rta transactivation is required for optimal growth of KSHV(+)/EBV(+) PEL lines. Our data suggest that Rta can contribute to EBV-driven cellular growth by transactivating RBP-Jk-dependent EBV latency genes. However, our data also suggest that EBNA-2 and Rta induce distinct alterations in the cellular proteomes that contribute to the growth of infected cells.

Promoter- and cell-specific transcriptional transactivation by the Kaposi's sarcoma-associated herpesvirus ORF57/Mta protein.

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 Mta-mediated transactivation is a direct effect on transcription by direct and indirect promoter association.

Direct interactions of Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 ORF50/Rta protein with the cellular protein octamer-1 and DNA are critical for specifying transactivation of a delayed-early promoter and stimulating viral reactivation.

The Kaposi's sarcoma-associated herpesvirus (KSHV) delayed-early K-bZIP promoter contains an ORF50/Rta binding site whose sequence is conserved with the ORF57 promoter. Mutation of the site in the full-length K-bZIP promoter reduced Rta-mediated transactivation by greater than 80%. The K-bZIP element contains an octamer (Oct) binding site that overlaps the Rta site and is well conserved with Oct elements found in the immediate-early promoters of herpes simplex virus type 1(HSV-1). The cellular protein Oct-1, but not Oct-2, binds to the K-bZIP element in a sequence-specific fashion in vitro and in vivo and stimulates Rta binding to the promoter DNA. The coexpression of Oct-1 enhances Rta-mediated transactivation of the wild type but not the mutant K-bZIP promoter, and Oct-1 and Rta proteins bind to each other directly in vitro. Mutations of Rta within an amino acid sequence conserved with HSV-1 virion protein 16 eliminate Rta's interactions with Oct-1 and K-bZIP promoter DNA but not RBP-Jk. The binding of Rta to both Oct-1 and DNA contributes to the transactivation of the K-bZIP promoter and viral reactivation, and Rta mutants deficient for both interactions are completely debilitated. Our data suggest that the Rta/Oct-1 interaction is essential for optimal KSHV reactivation. Transfections of mouse embryo fibroblasts and an endothelial cell line suggest cell-specific differences in the requirement for Oct-1 or RBP-Jk in Rta-mediated transactivation of the K-bZIP promoter. We propose a model in which Rta transactivation of the promoter is specified by the combination of DNA binding and interactions with several cellular DNA binding proteins including Oct-1.

Kaposi's Sarcoma-associated herpesvirus lytic switch protein stimulates DNA binding of RBP-Jk/CSL to activate the Notch pathway.

Kaposi's sarcoma-associated herpesvirus (KSHV) lytic switch protein, Rta, is a ligand-independent inducer of the Notch signal transduction pathway, and KSHV cannot reactivate from latency in cells null for the Notch target protein RBP-Jk. Here we show that Rta promotes DNA binding of RBP-Jk, a mechanism that is fundamentally different from that established for the RBP-Jk-activating proteins, Notch intracellular domain (NICD) and Epstein-Barr virus EBNA2. Although constitutively active RBP-Jk and NICD do not transactivate KSHV promoters independently, cotransfection of an Rta mutant lacking its transactivation domain robustly restores transcriptional activation. Cooperation requires intact DNA binding sites for Rta and RBP-Jk and trimeric complex formation between the three molecules in vitro. In infected cells, RBP-Jk is virtually undetectable on a series of viral and cellular promoters during KSHV latency but is significantly enriched following Rta expression during viral reactivation. Accordingly, Rta, but not EBNA2 and NICD, reactivates the complete viral lytic cycle.