Kaposi's sarcoma-associated herpesvirus and Kaposi's sarcoma

Kaposi's sarcoma-associated herpesvirus (KSHV) is present in all epidemiologic forms of Kaposi's sarcoma (KS). The KSHV genome contains several open reading frames which are potentially implicated in the development of KS. Some are unique to KSHV; others are homologous to cellular genes. The putative role of these genes in the genesis of KS is discussed.     

Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 RTA reactivates murine gammaherpesvirus 68 from latency

Murine gammaherpesvirus 68 (MHV-68), Kaposi's sarcoma-associated herpesvirus (HHV-8), and Epstein-Barr virus (EBV) are all members of the gammaherpesvirus family, characterized by their ability to establish latency in lymphocytes. The RTA protein, conserved in all gammaherpesviruses, is known to play a critical role in reactivation from latency. Here we report that HHV-8 RTA, not EBV RTA, was able to induce MHV-68 lytic viral proteins and DNA replication and processing and produce viable MHV-68 virions from latently infected cells at levels similar to those for MHV-68 RTA. HHV-8 RTA was also able to activate two MHV-68 lytic promoters, whereas EBV RTA was not. In order to define the domains of RTA responsible for their functional differences in viral promoter activation and initiation of the MHV-68 lytic cycle, chimeric RTA proteins were constructed by exchanging the N-terminal and C-terminal domains of the RTA proteins. Our data suggest that the species specificity of MHV-68 RTA resides in the N-terminal DNA binding domain.     

Host range of Kaposi's sarcoma-associated herpesvirus in cultured cells

Difficulties in efficiently propagating Kaposi's sarcoma-associated herpesvirus (KSHV) in culture have generated the impression that the virus displays a narrow host range. Here we show that, contrary to expectation, KSHV can establish latent infection in many adherent cell lines, including human and nonhuman cells of epithelial, endothelial, and mesenchymal origin. (Paradoxically, the only lines in which we have not observed successful latent infection are cultured lymphoma cell lines.) In most latently infected lines, spontaneous lytic replication is rare and (with only two exceptions) is not efficiently induced by phorbol ester treatment-a result that explains the failure of most earlier studies to observe efficient serial transfer of infection. However, ectopic expression of the KSHV lytic switch protein RTA from an adenoviral vector leads to the prompt induction of lytic replication in all latently infected lines, with the production of infectious KSHV virions. These results indicate (i) that the host cell receptor(s) and entry machinery for KSHV are widely distributed on cultured adherent cells, (ii) that latency is the default pathway of infection, and (iii) that blocks to lytic induction are frequent and largely reside at or upstream of the expression of KSHV RTA.     

Long-term-infected telomerase-immortalized endothelial cells: a model for Kaposi's sarcoma-associated herpesvirus latency in vitro and in vivo

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease. Most KS tumor cells are latently infected with KSHV and are of endothelial origin. While PEL-derived cell lines maintain KSHV indefinitely, all KS tumor-derived cells to date have lost viral genomes upon ex vivo cultivation. To study KSHV latency and tumorigenesis in endothelial cells, we generated telomerase-immortalized human umbilical vein endothelial (TIVE) cells. TIVE cells express all KSHV latent genes 48 h postinfection, and productive lytic replication could be induced by RTA/Orf50. Similar to prior models, infected cultures gradually lost viral episomes. However, we also obtained, for the first time, two endothelial cell lines in which KSHV episomes were maintained indefinitely in the absence of selection. Long-term KSHV maintenance correlated with loss of reactivation in response to RTA/Orf50 and complete oncogenic transformation. Long-term-infected TIVE cells (LTC) grew in soft agar and proliferated under reduced-serum conditions. LTC, but not parental TIVE cells, formed tumors in nude mice. These tumors expressed high levels of the latency-associated nuclear antigen (LANA) and expressed lymphatic endothelial specific antigens as found in KS (LYVE-1). Furthermore, host genes, like those encoding interleukin 6, vascular endothelial growth factor, and basic fibroblast growth factor, known to be highly expressed in KS lesions were also induced in LTC-derived tumors. KSHV-infected LTCs represent the first xenograft model for KS and should be of use to study KS pathogenesis and for the validation of anti-KS drug candidates.     

Reactive oxygen species hydrogen peroxide mediates Kaposi's sarcoma-associated herpesvirus reactivation from latency

Kaposi''s sarcoma-associated herpesvirus (KSHV) establishes a latent infection in the host following an acute infection. Reactivation from latency contributes to the development of KSHV-induced malignancies, which include Kaposi''s sarcoma (KS), the most common cancer in untreated AIDS patients, primary effusion lymphoma and multicentric Castleman''s disease. However, the physiological cues that trigger KSHV reactivation remain unclear. Here, we show that the reactive oxygen species (ROS) hydrogen peroxide (H₂O₂) induces KSHV reactivation from latency through both autocrine and paracrine signaling. Furthermore, KSHV spontaneous lytic replication, and KSHV reactivation from latency induced by oxidative stress, hypoxia, and proinflammatory and proangiogenic cytokines are mediated by H₂O₂. Mechanistically, H₂O₂ induction of KSHV reactivation depends on the activation of mitogen-activated protein kinase ERK1/2, JNK, and p38 pathways. Significantly, H₂O₂ scavengers N-acetyl-L-cysteine (NAC), catalase and glutathione inhibit KSHV lytic replication in culture. In a mouse model of KSHV-induced lymphoma, NAC effectively inhibits KSHV lytic replication and significantly prolongs the lifespan of the mice. These results directly relate KSHV reactivation to oxidative stress and inflammation, which are physiological hallmarks of KS patients. The discovery of this novel mechanism of KSHV reactivation indicates that antioxidants and anti-inflammation drugs could be promising preventive and therapeutic agents for effectively targeting KSHV replication and KSHV-related malignancies.     

A novel viral mechanism for dysregulation of beta-catenin in Kaposi's sarcoma-associated herpesvirus latency

The Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is expressed in all KSHV-associated tumors, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). We found that beta-catenin is overexpressed in both PEL cells and KS tissue. Introduction of anti-LANA small interfering RNA (siRNA) into PEL cells eliminated beta-catenin accumulation; LANA itself upregulated expression of beta-catenin in transfected cells. LANA stabilizes beta-catenin by binding to the negative regulator GSK-3beta, causing a cell cycle-dependent nuclear accumulation of GSK-3beta. The LANA C terminus contains sequences similar to the GSK-3beta-binding domain of Axin. Disruption of this region resulted in a mutant LANA that failed to re-localize GSK-3beta or stabilize beta-catenin. The importance of this pathway to KSHV-driven cell proliferation was highlighted by the observation that LANA, but not mutant LANA, stimulates entry into S phase. Redistribution of GSK-3beta can therefore be a source of beta-catenin dysregulation in human cancers.     

Activation of plasmacytoid dendritic cells by Kaposi's sarcoma-associated herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with multiple human malignancies, including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Following primary infection, KSHV typically goes through a brief period of lytic replication prior to the establishment of latency. Plasmacytoid dendritic cells (pDCs) are the major producers of type 1 interferon (IFN), primarily in response to virus infection. Toll-like receptors (TLRs) are key components of the innate immune system, and they serve as pathogen recognition receptors that stimulate the host antiviral response. pDCs express exclusively TLR7 and TLR9, and it is through these TLRs that the type 1 interferon response is activated in pDCs. Currently, it is not known whether KSHV is recognized by pDCs and whether activation of pDCs occurs in response to KSHV infection. We now report evidence that KSHV can infect human pDCs and that pDCs are activated upon KSHV infection, as measured by upregulation of CD83 and CD86 and by IFN-α secretion. We further show that induction of IFN-α occurs through activation of TLR9 signaling and that a TLR9 inhibitor diminishes the production and secretion of IFN-α by KSHV-infected pDCs.     

Molecular virology of Kaposi's sarcoma-associated herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV), the most recently discovered human tumour virus, is the causative agent of Kaposi's sarcoma, primary effusion lymphoma and some forms of Castleman's disease. KSHV is a rhadinovirus, and like other rhadinoviruses, it has an extensive array of regulatory genes obtained from the host cell genome. These pirated KSHV proteins include homologues to cellular CD21, three different beta-chemokines, IL-6, BCL-2, several different interferon regulatory factor homologues, Fas-ligand ICE inhibitory protein (FLIP), cyclin D and a G-protein-coupled receptor, as well as DNA synthetic enzymes including thymidylate synthase, dihydrofolate reductase, DNA polymerase, thymidine kinase and ribonucleotide reductases. Despite marked differences between KSHV and Epstein-Barr virus, both viruses target many of the same cellular pathways, but use different strategies to achieve the same effects. KSHV proteins have been identified which inhibit cell-cycle regulation checkpoints, apoptosis control mechanisms and the immune response regulatory machinery. Inhibition of these cellular regulatory networks app ears to be a defensive means of allowing the virus to escape from innate antiviral immune responses. However, due to the overlapping nature of innate immune and tumour-suppressor pathways, inhibition of these regulatory networks can lead to unregulated cell proliferation and may contribute to virus-induced tumorigenesis.     

Epidemiology and pathogenesis of Kaposi's sarcoma-associated herpesvirus

Kaposi's sarcoma (KS) occurs in Europe and the Mediterranean countries (classic KS) and Africa (endemic KS), immunosuppressed patients (iatrogenic or post-transplant KS) and those with acquired immune deficiency syndrome (AIDS), especially among those who acquired human immunodeficiency virus sexually (AIDS-KS). KS-associated herpesvirus (KSHV or HHV-8) is unusual among herpesviruses in having a restricted geographical distribution. Like KS, which it induces in immunosuppressed or elderly people, the virus is prevalent in Africa, in Mediterranean countries, among Jews and Arabs and certain Amerindians. Distinct KSHV genotypes occur in different parts of the world, but have not been identified as having a differential pathogenesis. KSHV is aetiologically linked to three distinct neoplasms: (i) KS, (ii) primary effusion lymphoma, and (iii) plasmablastic multicentric Castleman's disease. The histogenesis, clonality and pathology of the tumours are described, together with the epidemiology and possible modes of transmission of the virus.     

Virion proteins of Kaposi's sarcoma-associated herpesvirus

The proteins that compose a herpesvirus virion are thought to contain the functional information required for de novo infection, as well as virion assembly and egress. To investigate functional roles of Kaposi's sarcoma-associated herpesvirus (KSHV) virion proteins in viral productive replication and de novo infection, we attempted to identify virion proteins from purified KSHV by a proteomic approach. Extracellular KSHV virions were purified from phorbol-12-tetradecanoate-13-acetate-induced BCBL-1 cells through double-gradient ultracentrifugation, and their component proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thirty prominent protein bands were excised and subjected to high-performance liquid chromatography ion trap mass spectrometric analysis. This study led to the identification of 24 virion-associated proteins. These include five capsid proteins, eight envelope glycoproteins, six tegument proteins, and five proteins whose locations in the virions have not yet been defined. Putative tegument proteins encoded by open reading frame 21 (ORF21), ORF33, and ORF45 were characterized and found to be resistant to protease digestion when purified virions were treated with trypsin, confirming that they are located within the virion particles. The ORF64-encoded large tegument protein was found to be associated with capsid but sensitive to protease treatment, suggesting its unique structure and array in KSHV virions. In addition, cellular beta-actin and class II myosin heavy chain type A were found inside KSHV virions and associated with tegument-capsid structure. Identification of KSHV virion proteins makes it possible to study the functional roles of these virion proteins in KSHV replication and pathogenicity.     

Analysis of viral cis elements conferring Kaposi's sarcoma-associated herpesvirus episome partitioning and maintenance

Maintenance of Kaposi's sarcoma-associated herpesvirus (KSHV) episomes in latently infected cells is dependent on the latency-associated nuclear antigen (LANA). LANA binds to the viral terminal repeats (TR), leading to recruitment of cellular origin recognition complex proteins. Additionally, LANA tethers episomes to chromosomes via interactions with histones H2A and H2B (A. J. Barbera et al., Science 311:856-861, 2006). Despite these molecular details, less is known about how episomes are established after de novo infection. To address this, we measured short-term retention rates of green fluorescent protein-expressing replicons in proliferating lymphoid cells. In the absence of antibiotic selection, LANA significantly reduced the loss rate of TR-containing replicons. Additionally, we found that LANA can support long-term stability of KSHV replicons for more than 2 months under nonselective conditions. Analysis of cis elements within TR that confer episome replication and partitioning revealed that these activities can occur independently, and furthermore, both events contribute to episome stability. We found that replication-deficient plasmids containing LANA binding sites (LBS1/2) exhibited measurable retention rates in the presence of LANA. To confirm these observations, we uncoupled KSHV replication and partitioning by constructing hybrid origins containing the Epstein-Barr virus (EBV) dyad symmetry for plasmid replication and KSHV LBS1/2. We demonstrate that multiple LBS1/2 function in a manner analogous to that of the EBV family of repeats by forming an array of LANA binding sites for partitioning of KSHV genomes. Our data suggest that the efficiency with which KSHV establishes latency is dependent on multiple LANA activities, which stabilize viral genomes early after de novo infection.     

Kaposi's sarcoma-associated herpesvirus and mechanisms of oncogenesis

Kaposi's sarcoma-associated herpesvirus (KSHV, also known as human herpesvirus 8), is well known to be responsible for Kaposi's sarcoma, the most common AIDS-related cancer. KSHV is also associated with the B cell malignancies primary effusion lymphoma and multicentric Castleman's disease. Cellular signaling pathways regulate the proliferation and differentiation during normal development and a small number of signaling pathways are involved in tumors. KSHV utilize those pathways, such as pRb-E2F, Wnt and Notch pathways, to promote driving of cell cycle and to regulate their own life-cycles (i.e., latency and lytic cycle). This review focuses on signaling pathways which KSHV gene products manipulate and discusses their contributions to tomorigenesis and regulation of viral life-cycles.     

Immune evasion by Kaposi's sarcoma-associated herpesvirus

To efficiently establish a persistent infection, Kaposi's sarcoma-associated herpesvirus (KSHV; also known as HHV8) dedicates a large amount of its coding potential to produce proteins that antagonize the immune system of its host. These viral immunomodulators interfere with both the innate and adaptive immune responses and most of them are homologous to cellular proteins, suggesting that they have been pirated from the host during viral evolution. In this Review, I present recent advances in the understanding of immune evasion by KSHV, with a particular focus on the virally encoded modulators of immune responses that are unique to this virus.     

Novel Kaposi's sarcoma-associated herpesvirus homolog in baboons

Kaposi's sarcoma (KS) and lymphoproliferative diseases induced by KS-associated herpesvirus (KSHV/human herpesvirus 8) cause substantial morbidity and mortality in human immunodeficiency virus-infected individuals. To understand KSHV biology it is useful to investigate closely related rhadinoviruses naturally occurring in nonhuman primates. Here we report evidence for a novel KSHV homolog in captive baboon species (Papio anubis and other). Using degenerate PCR we identified a novel rhadinovirus, PapRV2, that has substantial sequence identity to two essential KSHV genes, the viral polymerase and thymidylate synthase. A subset of animals exhibited detectable PapRV2 viral load in peripheral blood mononuclear cells. Extensive serological analysis of nearly 200 animals in the colony demonstrated that the majority carried cross-reacting antibodies that recognize KSHV or macaque rhadinovirus antigens. Seroreactivity increased with age, similar to the age-specific prevalence of KSHV in the human population. This establishes baboons as a novel resource to investigate rhadinovirus biology, which can be developed into an animal model system for KSHV-associated human diseases, vaccine development, and therapy evaluation.     

The molecular pathology of Kaposi's sarcoma-associated herpesvirus

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is the eighth and most recently identified human herpesvirus (HHV-8). KSHV was discovered in 1994 by Chang et al. who used representational difference analysis to search for DNA sequences present in AIDS-associated KS but not in adjacent normal skin [1]. The virus has since been shown to be specifically associated with all forms of this disease and has fulfilled all of Hill's criteria for causation (reviewed in ). KSHV is also found in all cases of primary effusion lymphoma and in a plasmablastic variant of multicentric Castleman's disease. Over the last few years a wealth of data has been gained on the role of KSHV genes during infection. This review is an attempt to assemble this information into a more complete picture of how KSHV may cause disease.     

Virion-wide protein interactions of Kaposi's sarcoma-associated herpesvirus

Herpesvirus virions are highly organized structures built through specific protein-protein interactions. Thus, revelation of the protein interactions among virion proteins will shed light on the processes and the mechanisms of virion formation. Recently, we identified 24 virion proteins of Kaposi's sarcoma-associated herpesvirus (KSHV), using a proteomic approach (F. X. Zhu et al., J. Virol. 79:800-811, 2005). In the current study, a comprehensive analysis of protein-protein interaction between KSHV virion proteins was carried out using yeast two-hybrid (Y2H) and coimmunoprecipitation (co-IP) approaches. Every pairwise combination between KSHV tegument and capsid proteins, between tegument and envelope proteins, and among tegument proteins was tested for possible binary interaction. Thirty-seven protein-protein interactions were identified by both Y2H and co-IP analyses. The results revealed interactions between tegument and capsid proteins such as that of open reading frame 64 (ORF64) with ORF25 (major capsid protein [MCP]), ORF62 (triplex-1 [TRI-1]), and ORF26 (TRI-2). Many interactions were detected among the tegument proteins. ORF64 was found to interact with several tegument proteins including ORF11, ORF21, ORF33, ORF45, ORF63, ORF75, and ORF64 itself, suggesting that ORF64 may serve as a hub protein and play a role in recruiting tegument proteins during tegumentation and virion assembly. Our investigation also revealed redundant interactions between tegument proteins and envelope glycoproteins. These interactions are believed to contribute to final envelopment in virion assembly. Overall, this study allows us to establish a virion-wide protein interaction map, which provides insight into the architecture of the KSHV virion and sets up a foundation for exploring the functions of these proteins in viral particle assembly.