Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8)

Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus 8, HHV-8) are members of gamma-herpes virus family. Both viruses infect to B cells and cause malignancies such as lymphoma. Since EBV and HHV-8 are so-called 'oncovirus', their oncogenecities have been focused in the researches on EBV and KSHV for a long time. EBV was discovered in 1964, whereas KSHV was identified in 1994. However, KSHV was analyzed rapidly in these fifteen years. One of the recent progresses in the research on EBV and KSHV is that virus-encoded small RNAs were identified in their genomes and characterized. EBV is the first human virus in whose genome microRNA was identified. The oncogenecity of EBV and KSHV remains unclear. Here, I discuss the pathogenesis by EBV and KSHV with special reference to recent progress in this field.     

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.     

Inflammatory cytokines inhibit Kaposi's sarcoma-associated herpesvirus lytic gene transcription in in vitro-infected endothelial cells

The response of Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) to inflammatory cytokine treatment of experimentally infected endothelial cells was investigated. The cytokines inhibited spontaneous KSHV lytic gene expression but not the level of infection. The data suggest that if inflammatory cytokines present in KS lesions contribute to KSHV pathogenesis, they do so in part by promoting latent KSHV infection of the endothelial cells.     

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.     

Activation of Kaposi's sarcoma-associated herpesvirus lytic gene expression during epithelial differentiation

The oral cavity has been identified as the major site for the shedding of infectious Kaposi's sarcoma-associated herpesvirus (KSHV). While KSHV DNA is frequently detected in the saliva of KSHV seropositive persons, it does not appear to replicate in salivary glands. Some viruses employ the process of epithelial differentiation for productive viral replication. To test if KSHV utilizes the differentiation of oral epithelium as a mechanism for the activation of lytic replication and virus production, we developed an organotypic raft culture model of epithelium using keratinocytes from human tonsils. This system produced a nonkeratinized stratified squamous oral epithelium in vitro, as demonstrated by the presence of nucleated cells at the apical surface; the expression of involucrin and keratins 6, 13, 14, and 19; and the absence of keratin 1. The activation of KSHV lytic-gene expression was examined in this system using rKSHV.219, a recombinant virus that expresses the green fluorescent protein during latency from the cellular EF-1alpha promoter and the red fluorescent protein (RFP) during lytic replication from the viral early PAN promoter. Infection of keratinocytes with rKSHV.219 resulted in latent infection; however, when these keratinocytes differentiated into a multilayered epithelium, lytic cycle activation of rKSHV.219 occurred, as evidenced by RFP expression, the expression of the late virion protein open reading frame K8.1, and the production of infectious rKSHV.219 at the epithelial surface. These findings demonstrate that KSHV lytic activation occurs as keratinocytes differentiate into a mature epithelium, and it may be responsible for the presence of infectious KSHV in saliva.     

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.     

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.     

Spontaneous activation of the lytic cycle in cells infected with a recombinant Kaposi's sarcoma-associated virus

The genetic analysis of human herpesvirus 8 (HHV8), also termed Kaposi's sarcoma-associated virus, has been hampered by severe difficulties in producing infectious viral particles and modifying the viral genome. In this article, we report the successful cloning of the HHV8 complete genome onto a prokaryotic F-plasmid replicon which allows the propagation of the recombinant viral DNA in Escherichia coli. The insertion of the F-plasmid into the HHV8 genome interrupts the ORF56 gene, whose expression product-by homology with the Epstein-Barr virus BSLF1 gene--is supposed to be necessary for lytic DNA replication. After introduction of the recombinant HHV8 DNA into 293 cells, early viral antigens are expressed, suggesting that spontaneous lytic replication is initiated. However, completion of the lytic program is prevented by the absence of the ORF56 protein, and a quasi-latent state is established. Upon reintroduction of the ORF56 viral gene, the block is overcome and infectious HHV8 virions are produced. As the recombinant HHV8 genome can be easily modified in E. coli, this experimental system opens the way to an extensive genetic analysis of other HHV8 functions.     

Productive lytic replication of a recombinant Kaposi's sarcoma-associated herpesvirus in efficient primary infection of primary human endothelial cells

Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to the development of Kaposi's sarcoma (KS), a vascular spindle cell tumor primarily consisting of proliferating endothelial cells. Although KSHV has been shown to infect primary human endothelial cells and convert them into spindle shapes, KSHV infection is largely latent, and efforts to establish a highly efficient and sustainable infection system have been unsuccessful. A recombinant KSHV, BAC36, that has high primary-infection efficiency in 293 cells has been obtained (F. C. Zhou, Y. J. Zhang, J. H. Deng, X. P. Wang, H. Y. Pan, E. Hettler, and S. J. Gao, J. Virol. 76:6185-6196, 2002). BAC36 contains a green fluorescent protein cassette which can be used to conveniently monitor viral infection. Here, we describe the establishment of a KSHV lytic-replication-permissive infection cell model using BAC36 virions to infect primary human umbilical vein endothelial cell (HUVEC) cultures. BAC36 infection of HUVEC cultures has as high as 90% primary-infection efficiency and consists of two phases: a permissive phase, in which the cultures undergo active viral lytic replication, producing a large number of virions and concomitantly resulting in large-scale cell death, and a latent phase, in which the surviving cells from the permissive phase switch into latent infection, with a small number of cells undergoing spontaneous viral lytic replication, and proliferate into bundles of spindle cells with KS slit-like spaces. An assay for determining the KSHV titer in a virus preparation has also been developed. The cell model should be useful for examining KSHV infection and replication, as well as for understanding the development of KS.     

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.