Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), or human herpesvirus 8, is a lymphotropic virus strongly linked to several AIDS-related neoplasms. The primary reservoir of infection consists of latently infected B lymphocytes and possibly other mononuclear cells. Viral reactivation from latency and spread from this lymphoid reservoir is presumably required for development of nonlymphoid tumors like KS. Here we show that deregulated expression of a single viral gene, ORF 50, which encodes a transactivator able to selectively upregulate delayed-early viral genes, suffices to disrupt latency and induce the lytic gene cascade in latently infected B cells. The identification of this gene opens the way to studies of the physiologic mechanisms controlling reactvation of KSHV from latency.

Kaposi's sarcoma had been recognized as unique human cancer for a century before it manifested as an AIDS-defining illness with a suspected infectious etiology. The discovery of Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, in 1994 by using representational difference analysis, a subtractive method previously employed for cloning differences in human genomic DNA, was a fitting harbinger for the powerful bioinformatic approaches since employed to understand its pathogenesis in KS. Indeed, the discovery of KSHV was rapidly followed by publication of its complete sequence, which revealed that the virus had coopted a wide armamentarium of human genes; in the short time since then, the functions of many of these viral gene variants in cell growth control, signaling apoptosis, angiogenesis, and immunomodulation have been characterized. This critical literature review explores the pathogenic potential of these genes within the framework of current knowledge of the basic herpesvirology of KSHV, including the relationships between viral genotypic variation and the four clinicoepidemiologic forms of Kaposi's sarcoma, current viral detection methods and their utility, primary infection by KSHV, tissue culture and animal models of latent- and lytic-cycle gene expression and pathogenesis, and viral reactivation from latency. Recent advances in models of de novo endothelial infection, microarray analyses of the host response to infection, receptor identification, and cloning of full-length, infectious KSHV genomic DNA promise to reveal key molecular mechanisms of the candidate pathogeneic genes when expressed in the context of viral infection

The RTA protein of the Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is responsible for the switch from latency to lytic replication, a reaction essential for viral spread and KS pathogenesis. RTA is a sequence-specific transcriptional activator, but the diversity of its target sites suggests it may act via interaction with host DNA-binding proteins as well. Here we show that KSHV RTA interacts with the RBP-J protein, the primary target of the Notch signaling pathway. This interaction targets RTA to RBP-J recognition sites on DNA and results in the replacement of RBP-J 's intrinsic repressive action with activation mediated by the C-terminal domain of RTA. Mutation of such sites in target promoters strongly impairs RTA responsiveness. Similarly, such target genes are induced poorly or not at all by RTA in fibroblasts derived from RBP-J −/− mice, a defect that can be reversed by expression of RBP-J . In vitro, RTA binds to two adjacent regions of RBP-J , one of which is identical to the central repression domain that binds the Notch effector fragment. These results indicate that KSHV has evolved a ligand-independent mechanism for constitutive activation of the Notch pathway as a part of its strategy for reactivation from latency.

Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus-8) establishes latent and lytic infections in both lymphoid and endothelial cells and has been associated with diseases of both cell types. The KSHV open reading frame 50 (ORF50) protein is a transcriptional activator that plays a central role in the reactivation of lytic viral replication from latency. Here we identify and characterize a DNA binding site for the ORF50 protein that is shared by the promoters of two delayed early genes (ORF57 and K-bZIP). Transfer of this element to heterologous promoters confers on them high-level responsiveness to ORF50, indicating that the element is both necessary and sufficient for activation. The element consists of a conserved 12-bp palindromic sequence and less conserved sequences immediately 3 to it. Mutational analysis reveals that sequences within the palindrome are critical for binding and activation by ORF50, but the presence of a palindrome itself is not absolutely required. The 3 flanking sequences also play a critical role in DNA binding and transactivation. The strong concordance of DNA binding in vitro with transcriptional activation in vivo strongly implies that sequence-specific DNA binding is necessary for ORF50-mediated activation through this element. Expression of truncated versions of the ORF50 protein reveals that DNA binding is mediated by the amino-terminal 272 amino acids of the polypeptide.Infection by Kaposi's sarcoma-associated herpesvirus (KSHV) also known as human herpesvirus 8 (HHV-8) is associated with malignancies of both endothelial and lymphoid cells in humans. KSHV has been well established as the etiologic agent responsible for Kaposi's sarcoma (KS), an endothelial neoplasm frequent in homosexual men with AIDS and highly prevalent in sub-Saharan Africa (reviewed in reference 25). KSHV is also linked to two other AIDS-related malignancies, primary effusion lymphoma and multicentric Castleman's disease (5, 26). The presence of viral DNA in CD19 ϩ B cells and other mononuclear cells of the peripheral blood of KS/ AIDS patients (1, 2, 31), even prior to full-blown KS, suggests that infection of the lymphoid compartment is antecedent to the development of the endothelial disease.KSHV infection, similar to infection by other herpesviruses, displays two life cycle modes, latency and lytic replication. Latency is established by the virus both in endothelial and B cells and is detectable in such cell types both in vitro and in infected hosts (1,3,7,19,22,27,31). KSHV latency-associated genes are expressed in most spindle cells of KS tumors and are thought to contribute to their survival and proliferation (25). However, many viral genes (e.g., vGCR and vMIPs I and II) encoding homologs of cellular signaling proteins which have been suspected of roles in the histogenesis of KS are expressed strictly as lytic cycle products (13,20,23,24,28). This suggests that the KSHV lytic cycle may also contribute to KS lesion formation. Additional support for this notion comes from stud...

Open reading frame (ORF) 57 of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a homolog of known posttranscriptional regulators that are essential for replication in other herpesviruses. Here, we examined the expression of this gene and the function(s) of its product. KSHV ORF 57 is expressed very early in infection from a 1.6-kb spliced RNA bearing several in-frame initiation codons. Its product is a nuclear protein that, in transient assays, has little effect on the expression of luciferase reporter genes driven by a variety of KSHV and heterologous promoters. However, ORF 57 protein enhances the accumulation of several viral transcripts, in a manner suggesting posttranscriptional regulation. These transcripts include not only known cytoplasmic mRNAs (e.g., ORF 59) but also a nuclear RNA (nut-1) that lacks coding potential. Finally, ORF 57 protein can also modulate the effects of the ORF 50 gene product, a classical transactivator known to be required for lytic induction. The expression from some (e.g., nut-1) but not all (e.g., tk) ORF 50-responsive promoters can be synergistically enhanced by coexpression of ORF 50 and ORF 57. This effect is not due to upregulation of ORF 50 expression but rather to a posttranslational enhancement of the transcriptional activity of ORF 50. These data indicate that ORF 57 is a powerful pleiotropic effector that can act on several posttranscriptional levels to modulate the expression of viral genes in infected cells.Kaposi's sarcoma (KS), an endothelial tumor with neoangiogenic and inflammatory components, is a common neoplasm of AIDS patients. Recent evidence strongly implicates a novel lymphotropic herpesvirus, KS-associated herpesvirus (KSHV; also called human herpesvirus 8), in the pathogenesis of KS (5, 12; for reviews, see references 14 and 41). KSHV DNA is found in virtually all of the spindle (endothelial) cells of clinically apparent KS lesions (8, 46), as well as in tumorinfiltrating monocytes (6) and circulating B cells (1,27,50). Consistent with its classification as a lymphotropic gammaherpesvirus, KSHV is also tightly linked to certain B-cell lymphomas, termed primary effusion lymphomas (9, 45). Although KSHV infection of KS spindle cells is predominantly latent, lytic replication is also evident in the tumor (28,35,36,46), and growing evidence suggests that the lytic cycle contributes importantly to tumorigenesis. For example, the incidence of KS is greatly decreased when AIDS patients at risk for KS are treated with ganciclovir, a drug that specifically blocks lytic viral replication (25). Moreover, several lytic-cycle gene products can stimulate inflammatory and angiogenic responses in surrounding cells and tissues (2, 3, 7). Lytic replication has also been posited to be required for KSHV spread from its presumed lymphoreticular reservoir to its endothelial targets (24). Thus, the study of the KSHV lytic cycle (and the switch from latency to lytic growth) is important not only to fully characterize the molecular basis of viral replication but also to f...

No abstract

Viruses are obligate intracellular pathogens whose biological success depends upon replication and packaging of viral genomes, and transmission of progeny viruses to new hosts. The biological success of herpesviruses is enhanced by their ability to reproduce their genomes without producing progeny viruses or killing the host cells, a process called latency. Latency permits a herpesvirus to remain undetected in its animal host for decades while maintaining the potential to reactivate, or switch, to a productive life cycle when host conditions are conducive to generating viral progeny. Direct interactions between many host and viral molecules are implicated in controlling herpesviral reactivation, suggesting complex biological networks that control the decision. One viral protein that is necessary and sufficient to switch latent Kaposi’s sarcoma-associated herpesvirus (KSHV) into the lytic infection cycle is called K-Rta. K-Rta is a transcriptional activator that specifies promoters by binding DNA directly and interacting with cellular proteins. Among these cellular proteins, binding of K-Rta to RBP-Jk is essential for viral reactivation. In contrast to the canonical model for Notch signaling, RBP-Jk is not uniformly and constitutively bound to the latent KSHV genome, but rather is recruited to DNA by interactions with K-Rta. Stimulation of RBP-Jk DNA binding requires high affinity binding of Rta to repetitive and palindromic “CANT DNA repeats” in promoters, and formation of ternary complexes with RBP-Jk. However, while K-Rta expression is necessary for initiating KSHV reactivation, K-Rta’s role as the switch is inefficient. Many factors modulate K-Rta’s function, suggesting that KSHV reactivation can be significantly regulated post-Rta expression and challenging the notion that herpesviral reactivation is bistable. This review analyzes rapidly evolving research on KSHV K-Rta to consider the role of K-Rta promoter specification in regulating the progression of KSHV reactivation.

Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) is a lymphotropic virus strongly linked to the development of KS, an endothelial cell neoplasm frequent in persons with AIDS. Reactivation from latency in B cells is thought to be an important antecedent to viral spread to endothelial cells during KS pathogenesis. Earlier experiments have posited a role for the transcriptional activator encoded by KSHV open reading frame 50 (ORF50) in such reactivation, since ectopic overexpression of this protein induces reactivation in latently infected B cells. Here we have explored several aspects of the expression, structure, and function of this protein bearing on this role. The ORF50 gene is expressed very early in lytic reactivation, before several other genes implicated as candidate regulatory genes in related viruses, and its expression can upregulate their promoters in transient assays. The protein is extensively phosphorylated in vivo and bears numerous sites for phosphorylation by protein kinase C, activators of which are potent stimulators of lytic induction. The C terminus of the ORF50 protein contains a domain that can strongly activate transcription when targeted to DNA; deletion of this domain generates an allele that expresses a truncated protein which retains the ability to form multimers with full-length ORF50 and functions as a dominant-negative protein. Expression of this allele in latently infected cells ablates spontaneous reactivation from latency and strikingly suppresses viral replication induced by multiple stimuli, including phorbol ester, ionomycin, and sodium butyrate. These results indicate that the ORF50 gene product plays an essential role in KSHV lytic replication and are consistent with its action as a putative molecular switch controlling the induction of virus from latency.