The ubiquitin-specific protease USP7 modulates the replication of Kaposi's sarcoma-associated herpesvirus latent episomal DNA

Kaposi's sarcoma herpesvirus (KSHV) belongs to the gamma-2 Herpesviridae and is associated with three neoplastic disorders: Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). The viral latency-associated nuclear antigen 1 (LANA) is expressed in all latently KSHV-infected cells and is involved in viral latent replication and maintenance of the viral genome. We show that LANA interacts with the ubiquitin-specific protease USP7 through its N-terminal TRAF (tumor necrosis factor [TNF] receptor-associated factor) domain. This interaction involves a short sequence (amino acids [aa] 971 to 986) within the C-terminal domain of LANA with strong similarities to the USP7 binding site of the Epstein-Barr virus (EBV) EBNA-1 protein. A LANA mutant with a deletion of the identified USP7 binding site showed an enhanced ability to replicate a plasmid containing the KSHV latent origin of replication but was comparable to the wild-type LANA (LANA WT) with regard to the regulation of viral and cellular promoters. Furthermore, the LANA homologues of two other gamma-2 herpesviruses, MHV68 and RRV, also recruit USP7. Our findings suggest that recruitment of USP7 to LANA could play a role in the regulation of viral latent replication. The recruitment of USP7, and its role in herpesvirus latent replication, previously described for the latent EBNA-1 protein of the gamma-1 herpesvirus (lymphocryptovirus) EBV (M. N. Holowaty et al., J. Biol. Chem. 278:29987-29994, 2003), may thereby be a conserved feature among gammaherpesvirus latent origin binding proteins.     

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.     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

A Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 ORF50 deletion mutant is defective for reactivation of latent virus and DNA replication

Kaposi's sarcoma-associated herpesvirus (also called human herpesvirus type 8 [HHV8]) latently infects a number of cell types. Reactivation of latent virus can occur by treatment with the phorbol ester tetradecanoyl phorbol acetate (TPA) or with the transfection of plasmids expressing the lytic switch activator protein K-Rta, the gene product of ORF50. K-Rta expression is sufficient for the activation of the entire lytic cycle and the transactivation of viral genes necessary for DNA replication. In addition, recent evidence has suggested that K-Rta may participate directly in the initiation of lytic DNA synthesis. We have now generated a recombinant HHV8 bacterial artificial chromosome (BAC) with a large deletion within the ORF50 locus. This BAC, BAC36Delta50, failed to produce infectious virus upon treatment with TPA and was defective for DNA synthesis. Expression of K-Rta in trans in BAC36Delta50-containing cells was able to abolish both defects. Real-time PCR revealed that K-bZIP, ORF40/41, and K8.1 were not expressed when BAC36Delta50-containing cells were induced with TPA. However, the mRNA levels of ORF57 were over fivefold higher in TPA-treated BAC36Delta50-containing cells than those observed in similarly treated wild-type BAC-containing cells. In addition, immunohistochemical analysis showed that while the latency-associated nuclear antigen (LANA) was expressed in the mutant BAC-containing cells, ORF59 and K8.1 expression was not detected in TPA-induced BAC36Delta50-containing cells. These results showed that K-Rta is essential for lytic viral reactivation and transactivation of viral genes contributing to DNA replication.     

Kaposi's sarcoma-associated herpesvirus latent and lytic gene expression as revealed by DNA arrays

Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is associated with three human tumors, Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. KSHV encodes a number of homologs of cellular proteins involved in the cell cycle, signal transduction, and modulation of the host immune response. Of the virus complement of over 85 open reading frames (ORFs), the expression of only a minority has been characterized individually. We have constructed a nylon membrane-based DNA array which allows the expression of almost every ORF of KSHV to be measured simultaneously. A PEL-derived cell line, BC-3, was used to study the expression of KSHV during latency and after the induction of lytic replication. Cluster analysis, which arranges genes according to their expression profile, revealed a correlation between expression and assigned gene function that is consistent with the known stages of the herpesvirus life cycle. Furthermore, latent and lytic genes thought to be functionally related cluster into groups. The correlation between gene expression and function also infers possible roles for KSHV genes yet to be characterized.     

Kaposi's sarcoma-associated herpesvirus ori-Lyt-dependent DNA replication: involvement of host cellular factors

Herpesvirus lytic DNA replication requires both the cis-acting element, the origin, and trans-acting factors, including virally encoded origin-binding protein, DNA replication enzymes, and auxiliary factors. Two lytic DNA replication origins (ori-Lyt) of Kaposi's sarcoma-associated herpesvirus (KSHV) have been identified, and two virally encoded proteins, namely, RTA and K8, have been shown to bind to the origins. In this study, we sought to identify cellular factors that associate with ori-Lyt by using DNA affinity purification and mass spectrometry. This approach led to identification of several cellular proteins that bind to KSHV ori-Lyt. They include topoisomerases (Topo) I and II, MSH2/6, RecQL, poly(ADP-ribose) polymerase I (PARP-1), DNA-PK, Ku86/70 autoantigens, and scaffold attachment factor A (SAF-A). RecQL appears to associate with prereplication complexes and be recruited to ori-Lyt through RTA and K8. Topoisomerases, MSH2, PARP-1, DNA-PK, and Ku86 were not detected in prereplication complexes but were present in replication initiation complexes on ori-Lyt. All these cellular proteins accumulate in viral replication compartments in the nucleus, indicating that these proteins may have a role in viral replication. Topo I and II appear to be essential for viral DNA replication as inhibition of their activities with specific inhibitors (camptothecin and ellipticine) blocked ori-Lyt-dependent DNA replication. Furthermore, inhibition of PARP-1 with chemical inhibitors (3-aminobenzamide and niacinamide) resulted in decreased ori-Lyt-dependent DNA replication, whereas hydroxyurea, which raises PARP-1 activity, caused an increase in the DNA replication, suggesting a positive role for PARP-1 in KSHV lytic DNA replication.     

Activation of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) lytic replication by human cytomegalovirus

The majority of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells identified in vivo contain latent KSHV, with lytic replication in only a few percent of cells, as is the case for the cells of Kaposi's sarcoma (KS) lesions. Factors that influence KSHV latent or lytic replication are not well defined. Because persons with KS are often immunosuppressed and susceptible to many infectious agents, including human cytomegalovirus (HCMV), we have investigated the potential for HCMV to influence the replication of KSHV. Important to this work was the construction of a recombinant KSHV, rKSHV.152, expressing the green fluorescent protein (GFP) and neo (conferring resistance to G418). The expression of GFP was a marker of KSHV infection in cells of both epithelial and endothelial origin. The rKSHV.152 virus was used to establish cells, including human fibroblasts (HF), containing only latent KSHV, as demonstrated by latency-associated nuclear antigen expression and Gardella gel analysis. HCMV infection of KSHV latently infected HF activated KSHV lytic replication with the production of infectious KSHV. Dual-color immunofluorescence detected both the KSHV lytic open reading frame 59 protein and the HCMV glycoprotein B in coinfected cells, and UV-inactivated HCMV did not activate the production of infectious KSHV-GFP. In addition, HCMV coinfection increased the production of KSHV from endothelial cells and activated lytic cycle gene expression in keratinocytes. These data demonstrate that HCMV can activate KSHV lytic replication and suggest that HCMV could influence KSHV pathogenesis.     

Disruption of Kaposi's sarcoma-associated herpesvirus latent nuclear antigen leads to abortive episome persistence

Latent nuclear antigen (LNA) is implicated in Kaposi's sarcoma-associated herpesvirus (KSHV) episome persistence. LNA colocalizes with KSHV episomes on chromosomes in metaphase, and it maintains the stability and replication of KSHV terminal repeat-containing plasmids. In this study, we examined the function of LNA in episome persistence in the context of full-length KSHV genome by mutagenesis analysis. We generated a KSHV mutant, BAC36-DeltaLNA, with LNA disrupted by transposon-based mutagenesis with a KSHV BAC clone, BAC36, as a template. Immunofluorescence antibody staining revealed that the insertion of a transposon cassette into LNA disrupted its expression but had no effect on the expression of two adjacent genes, the vCyclin and vFLIP genes. Using a green fluorescent protein (GFP) cassette as a tracking marker for the KSHV episome, we found 8.7-fold-fewer GFP-positive cells in BAC36-DeltaLNA cultures than in wild-type BAC36 cultures at the early stage following episome delivery into 293 cells by transfection, which could be partially rescued by cotransfection with a LNA expression plasmid but not a control plasmid. Cells harboring BAC36-DeltaLNA with or without transient complementation rapidly lost episomes and became virus-free after 2 weeks of culture based on GFP expression and Gardella gel analysis and quantitative PCR assays for detecting KSHV genomes. In contrast, BAC36 episomes were stably maintained during the same period. Stable cultures with close to 100% of cells harboring KSHV episomes were readily established by hygromycin selection for BAC36 but not for BAC36-DeltaLNA. These results conclusively indicate that LNA is essential for the establishment and persistence of KSHV episomes in mammalian cells.     

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.     

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.