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Successful viral replication is dependent on a conducive cellular environment; thus, viruses must be sensitive to the state of their host cells. We examined the idea that an interplay between viral and cellular regulatory factors determines the switch from Kaposi's sarcoma-associated herpesvirus (KSHV) latency to lytic replication. The immediate-early gene product K-Rta is the first viral protein expressed and an essential factor in reactivation; accordingly, this viral protein is in a key position to serve as a viral sensor of cellular physiology. Our approach aimed to define a host transcription factor, i.e., host sensor, which modulates K-Rta activity on viral promoters. To this end, we developed a panel of reporter plasmids containing all 83 putative viral promoters for a comprehensive survey of the response to both K-Rta and cellular transcription factors. Interestingly, members of the NF-kappaB family were shown to be strong negative regulators of K-Rta transactivation for all but two viral promoters (Ori-RNA and K12). Recruitment of K-Rta to the ORF57 and K-bZIP promoters, but not the K12 promoter, was significantly impaired when NF-kappaB expression was induced. Many K-Rta-responsive promoters modulated by NF-kappaB contain the sequence of the RBP-Jkappa binding site, a major coactivator which anchors K-Rta to target promoters via consensus motifs which overlap with that of NF-kappaB. Gel shift assays demonstrated that NF-kappaB inhibits the binding of RBP-Jkappa and forms a complex with RBP-Jkappa. Our results support a model in which a balance between K-Rta/RBP-Jkappa and NF-kappaB activities determines KSHV reactivation. An important feature of this model is that the interplay between RBP-Jkappa and NF-kappaB on viral promoters controls viral gene expression mediated by K-Rta.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma. K-Rta and K-bZIP are two major viral transcription factors that control reactivation of this virus. Here we report a genome-wide analysis of transcriptional capacity by evaluation of a comprehensive library of 83 putative KSHV promoters. In reporter assays, 34 viral promoters were activated by K-Rta, whereas K-bZIP activated 21 promoters. When K-Rta and K-bZIP were combined, 3 K-Rta responsive promoters were repressed by K-bZIP. The occupancy of K-Rta and K-bZIP across KSHV promoters was analyzed by chromatin immunoprecipitation with a viral promoter-chip in BCBL-1 cells. In addition, acetylation of local histones was examined to determine accessibility of promoters during latency and reactivation. Finally, 10 promoters were selected to study the dynamics of transcription factor recruitment. This study provides a comprehensive overview of the responsiveness of KSHV promoters to K-Rta and K-bZIP, and describes key chromatin changes during viral reactivation.

Kaposi sarcoma-associated herpesvirus (KSHV; also known as HHV8) is the causative agent of two B cell tumors, multicentric Castleman disease (MCD) and primary effusion lymphoma (PEL). However, little is known about the nature of the specific B cell subtype(s) most susceptible to infection. Identifying these cells would provide direct insight into KSHV transmission and virus-induced transformation. To identify this subset and to determine whether infection alters its cellular phenotype, we exposed human tonsillar cells toKSHV and characterized infected cells using high-throughput multispectral imaging flow cytometry (MIFC). Stable expression of the virally encoded latency-associated nuclear antigen (LANA), a marker of latent KSHV infection, was observed predominantly in cells expressing the λ light chain of the B cell receptor. These [LANA.sup.+] B cells proliferated and exhibited similarities to the cells characteristic of MCD (IgMλ-expressing plasmablasts), including blasting morphology with elevated expression of Ki67, variable expression of CD27, and high levels of IgM and IL-6 receptor. Furthermore, the proportion of infected cells showing a blasting phenotype increased upon addition of exogenous IL-6. Our data lead us to propose that oral transmission of KSHV involves the latent infection of a subset of tonsillar IgMλ-expressing B cells, which then proliferate as they acquire the plasmablast phenotype characteristic of MCD., Introduction Kaposi sarcoma-associated herpesvirus (KSHV/HHV8) is the causative agent of two B cell tumors, multicentric Castleman disease (MCD) and primary effusion lymphoma (PEL), as well as its namesake, Kaposi sarcoma. [...]

Valyi Institute for Plastic Forming University of Massachusetts Lowell Lowell, Massachusetts Arthur Delusky ValTek LLC Sterling Heights, Michigan THE PROCESS The Valyi SFC molding process for surface finishing/compression (SFC) molding [...]

The relationship between perceived self-environment similarity and satisfaction with the environment was tested in two differing kinds of organizational structures. It was hypothesized that the perceived similarity-satisfaction relationship was mediated by the salience or importance of the particular reference group involved. Two kinds of public high schools-traditional and alternative-were selected as examples of organizations where the same reference groups (e.g., teachers and students) play differentially salient organizational roles. Using a semantic differential technique, 46 traditional school students and 40 alternative school students rated various reference groups and school satisfactions. Differing patterns of perceived similarity and satisfaction found in the two schools support the notion that organizational structure-as embodied in organizational roles-causes the perceived similarity-satisfaction relationship to be different in different settings. The relationship of the findings to prior research is discussed and the importance of evolving more sophisticated notions of person-environment relationships is stressed. [ABSTRACT FROM AUTHOR]

In order to cope with hostile host environments, many viruses have developed strategies to perturb the cellular machinery to suit their replication needs. Some herpesvirus genes protect cells from undergoing apoptosis to prolong the lives of infected cells, while others, such as Epstein-Barr virus Zta, slow down the G[sub 1]/S transition phase to allow ample opportunity for transcription and translation of viral genes before the onset of cellular genomic replication. In this study, we investigated whether Kaposi's sarcoma-associated herpesvirus (KSHV) K-bZIP, a homologue of the Epstein-Barr virus transcription factor BZLF1 (Zta), plays a role in cell cycle regulation. Here we show that K-bZIP physically associates with cyclin-CDK2 and downmodulates its kinase activity. The association can be detected in the natural environment of KSHV-infected cells without artificial overexpression of either component. With purified protein, it can be shown that the interaction between K-bZIP and cyclin-CDK2 is direct and that K-bZIP alone is sufficient to inhibit CDK2 activity. The interacting domain of K-bZIP has been mapped to the basic region. The result of these associations is a prolonged G[sub 1] phase, accompanied by the induction of p21 and p27 in a naturally infected B-cell line. Thus, in addition to the previously described transcription and genome replication functions, a new role of K-bZIP in KSHV replication is identified in this report. [ABSTRACT FROM AUTHOR]

Sumoylation has emerged as a major post-translational modification of cellular proteins, affecting a variety of cellular processes. Viruses have exploited the sumoylation pathway to advance their own replication by evolving several ways to perturb the host sumoylation apparatus. However, there has been no report of virally encoded enzymes directly involved in catalyzing the sumoylation reaction. Here, we report that the K-bZIP protein encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) is a SUMO E3 ligase with specificity toward SUMO2/3. K-bZIP is a nuclear factor that functions to modulate viral gene expression and to prolong the G1 phase, allowing viral transcription and translation to proceed at the early stage of infection. In addition to functioning as a transcriptional factor, we show that K-bZIP carries a SIM (SUMO-interacting motif), which specifically binds to SUMO-2/3 but not SUMO-1. K-bZIP catalyzes its own SUMO modification as well as that of its interacting partners such as the cellular tumor suppressor proteins p53 and Rb, both in vitro and in vivo. This reaction depends on an intact SIM. Sumoylation of p53 leads to its activation and K.-bZIP is recruited to several p53 target chromatin sites in a SIM-dependent manner. In addition to the identification of a viral SUMO-2/3 E3 ligase, our results provide additional insights into the mechanisms whereby K-bZIP induces cell cycle arrest. [ABSTRACT FROM AUTHOR]

Successful viral replication is dependent on a conducive cellular environment; thus, viruses must be sensitive to the state of their host cells. We examined the idea that an interplay between viral and cellular regulatory factors determines the switch from Kaposi's sarcoma-associated herpesvirus (KSHV) latency to lytic replication. The immediate-early gene product K-Rta is the first viral protein expressed and an essential factor in reactivation; accordingly, this viral protein is in a key position to serve as a viral sensor of cellular physiology. Our approach aimed to define a host transcription factor, i.e., host sensor, which modulates K-Rta activity on viral promoters. To this end, we developed a panel of reporter plasmids containing all 83 putative viral promoters for a comprehensive survey of the response to both K-Rta and cellular transcription factors. Interestingly, members of the NF-κB family were shown to be strong negative regulators of K-Rta transactivation for all but two viral promoters (Ori-RNA and K12). Recruitment of K-Rta to the ORF57 and K-bZIP promoters, but not the K12 promoter, was significantly impaired when NF-κB expression was induced. Many K-Rta-responsive promoters modulated by NF-κB contain the sequence of the RBP-Jκ binding site, a major coactivator which anchors K-Rta to target promoters via consensus motifs which overlap with that of NF-κB. Gel shift assays demonstrated that NF-κB inhibits the binding of RBP-Jκ and forms a complex with RBP-Jκ. Our results support a model in which a balance between K-Rta/RBP-Jκ and NF-κB activities determines KSHV reactivation. An important feature of this model is that the interplay between RBP-Jκ and NF-κB on viral promoters controls viral gene expression mediated by K-Rta. [ABSTRACT FROM AUTHOR]