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In 1872, the famed Hungarian dermatologist Moritz Kaposi described the purple-coloured lesions found in the skin of 5 male patients as "idiopathic multiple pigmented sarcoma of the skin" (60), an entity that was later on known as classic Kaposi's sarcoma (KS), which predominantly affected older men of Mediterranean and eastern European descent. In the 1920s, it was observed that KS occurred more frequently in East and Central Africa. African endemic KS was more rapidly progressive over several months to years. The uneven geographical distribution led to the hypothesis that KS might be caused by an in-fectious agent (87). In 1972 Giraldo et al. identified herpesvirus-like particles in the tissue culture of KS (43). An abrupt increase in the number of cases of KS among previously healthy young homosexual men was first reported in 1981, ushering in a new era of aggressive, rapidly fatal KS (12, 38, 45, 53, 111). Since approximately 30% of acquired immunodefi-ciency syndrome (AIDS) patients presented with KS as their initial symptom of human immunodeficiency virus (HIV) infection, KS evolved into a defining characteristic of one of the most devastating infectious diseases in history (8, 44). In 1990, a landmark epide-miological study from Beral et al. reported that KS was 20 000 times more likely to occur in people with HIV than in the general population (9). KS was more common in those who had acquired HIV sexually than in those who had acquired it via other routes. The incidence of KS was not related to age or race, but showed a definite geographical distribution, with the highest prevalence in the areas that were the initial foci of the AIDS epidemic. The accumulation of the epidemiological evidence suggested the involvement of a sexually transmissible agent in the development of KS, which in western countries had spread mainly among homosexual men. Several groups attempted to identify the unknown agent, and in 1994, Chang et al. identified a new human herpsvirus called Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8) from an AIDS-KS lesion using representational difference analysis, which showed the presence of unique herpesviral sequences in the KS tissue (23).
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Based on biological characteristics and genomic organization, herpesviruses are classified into three subfamilies: alpha (α), beta (β), and gamma (γ). The gammaherpesvirinae are grouped into two classes: lymphocryptoviruses (γ-1) and rhadinoviruses (γ-2). Epstein-Barr virus (EBV) is a lymphocryptovirus while KSHV is a rhadinovirus (81). KSHV contains a large double-stranded DNA which is a closed circular episome in the nucleus during latency but is linear during lytic replication. Over 90 genes are encoded by a ~140 kilobase (kb) long unique region (LUR) with 53.5% G+C content, which is flanked by 20-35 kb terminal repeat regions composed of 801 base pair (bp) terminal repeat units (TR) with 84.5% G+C content (99, 101). The viral genes encoded by KSHV can be divided into three classes: 1) genes common to all herpesviruses, 2) genes unique to KSHV (these are generally given a "K" designation followed by number, and 3) KSHV-encoded genes that are homologous to cellular genes (these may be unique to KSHV or shared with other herpesviruses), and are likely to have usurped from the host genome during the course of evolution (85). Because of the KSHV genome complexity, the exact number of genes in the genome remain unknown.
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Similar to other herpesviruses, KSHV displays two patterns of infection: latent and lytic phase. KSHV genome contains genes coding for different proteins of the lytic and latent phases, which have functional roles in virion structure, viral replication, host cell gene regulation, and immune response (79).
During the latent phase of KSHV infection, 6 proteins are expressed from the viral episome: viral cyclin D/ vCyclinD (ORF72), latency-associated nuclear antigen 1/ LANA1 (ORF73), Kaposin A and B, the viral Fas-ligand interleukin-1β-converting enzyme (vFLICE) inhibitory protein/ vFLIP (K13), and the viral interferon regulatory factor 3/ vIRF3/ LANA2 (79, 123). The switch from latency to replicative phase is under tight control by a regulator of transcription [replication and transcription activator (RTA/ORF50)] (119). KSHV expresses 3 categories of lytic genes: immediate early (IE), early, and late genes (79). Early genes are expressed after IE genes and correspond to different ORF including K3, K5, K8, viral interleukin (vIL)-6, viral macrophage inhibitory protein (vMIP)-Ⅰ, vMIP-Ⅱ, vMIP-Ⅲ, vBCL-2, viral G protein-coupled receptor (vGPCR)/vIL-8R, and thymidilate cyclase (104, 119). Late lytic genes code for capsid, teguments, and membrane proteins (123). Lytic reactivation pro-motes the spread of the virions from the lymphoid reservoir to other target cell types such as endothelial cells (119).
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Many studies have been done to measure the preva-lence of antibodies to KSHV (24). A variety of techni-ques have been used in these studies, but to date there is no gold standard against which to measure the efficacy of these techniques, so some may overesti-mate the number of positives, and some may underesti-mate the number. Nonetheless, certain trends are clear (106). Unlike most human herpesviruses, KSHV is not spread universally among all human populations. The seroprevalence rates of infection with KSHV in the general population vary depending on the geographic origin of the subjects: it is relatively low in Asia (India, 3.7%; Thailand, Malaysia, and Sri Lanka less than 4.4%) (1), in the United States (5%-20%, but 20%-50% in populations infected with HIV-1), and in Europe [Italy, 2%-6.5%; The exception is Sweden, where 20% of blood donors were reported to test positive for KSHV antibodies (a wide variety of results depending on the assay used)] (25, 32, 39, 57, 94), and is high in the south of Italy (24%) (121), and in sub-Saharan Africa ( > 36%) (1, 33, 88). In East African countries the prevalence of serum antibodies to KSHV is invariably high (Tanzania, 33%-84%; Uganda, 53%-77%; and Zambia, 37.5%) (1, 29, 46, 63, 88). In black South African blood donors and patients with cancers other than KS, the seroprevalence is 32%; in KS patients, it rises to 83% (112). African countries have the highest prevalence of KS in the world (124).
KSHV seroprevalence is higher among HIV-serone-gative homosexual subjects compared with the serone-gative heterosexual population, ranging from 20% to 38% (6, 67, 86), and is even higher in HIV-seroposi-tive homosexual populations, ranging from 30% to 48% (1, 41, 67, 97). HIV-seropositive subjects with KS have at least a 95% KSHV seropositivity com-pared with 30% in HIV-seropositive subjects without KS (55). In homosexual men who are seropositive for both KSHV and HIV, the presence of KSHV DNA in peripheral blood mononuclear cells (PBMC) precedes and predicts the development of KS (80, 120), and between 30% and 50% of subjects in this group will develop KS within 10 years of contracting the dual infection (14, 56, 86).
Transmission of KSHV most likely includes both sexual and nonsexual routes. Sexual route is the most common route in lowprevalence developed countries, where homosexual men have the highest risk of contracting the virus (11, 67). In the endemic areas of Africa infection rates are high and both sexual and nonsexual transmission occurs. Most individuals get infected during childhood and high prevalence rates have been reported in infants and children reaching ≥50% in some studies (42, 70, 72). Epidemiological studies support the role of horizontal transmission of KSHV from mother to child and between siblings during close non-sexual contacts (30, 70, 72, 92, 93). KSHV DNA has been detected frequently in saliva, where virus titers are higher than in samples from other anatomic sites such as genital secretions (10, 91). Interestingly, also unaffected carriers can harbor infectious KSHV in their oral cavities and the virus from these carriers is able to infect cultured primary epithelial cells (31).
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The spindle cells isolated from KS lesions of HIV-1-infected individuals are generally hyperplastic non-transformed cells that proliferate in culture with inflammatory cytokines. These cells initially contain KSHV that is rapidly lost as the cells are passaged in culture. Cell lines composed of transformed cells have also been isolated from advanced KS lesions (3, 62). These cell lines, unlike AIDS-KS spindle cells, are transformed cells that grow in the absence of inflam-matory cytokines, contain cytogenetic abnormalities, and induce durable tumor lesions when inoculated into nude mice (58, 102). Similar to the hyperplastic KS spindle cells, the KS transformed cells have also lost the KSHV genomes. One possible explanation is that when cultured ex-vivo, KSHV-infected cells undergo epigenetic mutations or changes enabling them to grow without the virus. In addition, other host-specific factors not present in explants might be required for the maintenance of the virus. In cell culture, KSHV is capable of infecting a diverse range of human and animal cell lines including primary CD19+ B cells, human papilloma virus (HPV)-transformed human brain endothelial cells BB18 and 181GB1-4, primary neonatal capillary endothelial cells, human embryonic kidney 293 cells, Ln-Cap cells, human lung carcinoma A549 cells, CHELI (Chediak-Higashi syndrome) cells, squamous cell carcinoma SCC15 cells, human fibro-blast cells, human bladder carcinoma T24 cells, human prostate carcinoma DU145 cells, human cervical carcinoma HeLa cells, baby hamster kidney BHK-21 cells, owl monkey kidney OMK637 cells, green monkey fibroblasts (Vero) cells, green monkey kidney CV-1 cells, SLK cells (KS-spindle cells), and murine fibroblast 3T3 cells (7, 98).
KSHV genome
KSHV life cycle
Epidemiology of KSHV
Cell culture of KSHV
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Four distinct clinical forms of KS have been described. Classic KS is a disease of elderly Mediterranean and Eastern European men (115). Endemic KS is found in parts of equatorial Africa such as Uganda and Zambia (67), where it is one of the most frequently occurring tumors. Endemic KS tends to be more aggressive than classic KS. Iatrogenic KS occurs after solid-organ transplantation in patients receiving immunosuppres-sive therapy and KS comprises an estimated 3% of all tumors associated with transplantation (73). Epidemic AIDS-KS is the most common neoplastic mani-festation of AIDS in the United States and Europe and is one of the diagnostic criteria for AIDS (68). Highly active antiretroviral therapy (HAART) has led to a substantial decline of AIDS-KS in the United States. However, even in the current post-HAART era, stan-dardized incidence rates for KS are higher than that of any other AIDS-defining or AIDS-associated cancers (71). This suggests that KS will remain a permanent health problem for years to come. In contrast to the indolent course of classic KS, AIDS-KS takes a much more aggressive course. AIDS-KS tends to dissemi-nate widely to mucous membranes and the visceral organs. Despite the different clinical manifestations of KS, the histology of lesions from skin, lymph nodes, respiratory tract, and intestines is very similar. The KS lesion is highly angiogenic and comprises spindle-shaped cells, slitlike endothelium-lined vasculature, and infiltrating blood cells. The spindle cells form the majority of the cell population, and are thought to arise from lymphatic endothelial cells. KS lesions are divided into patch, plaque, and nodular stages. KS lesions range from patches or plaques to nodules and there is evidence for both polyclonality and mono-clonality of the lesions (59, 95). It is thought that KS probably initiates as a polyclonal hyperplasia and develops into a clonal neoplasia.
In addition to KS, KSHV is also found in B lym-phoproliferative diseases: PEL and multicentric Castleman's disease (MCD) (21, 23, 114). PEL, also called body cavity-based lymphoma, is a rare non-Hodgkin's B-cell lymphoma commonly found in HIV-infected patients (22). This type of lymphoma is characterized as a malignant effusion in the pleural, pericardial, or peritoneal space, usually without a contiguous tumor mass (15, 22). Most PELs are KSHV-positive, and are often coinfected with EBV as well. These tumors are typically large-cell immuno-blastic or anaplastic large-cell lymphomas that express CD45, clonal immunoglobulin gene rearrangements, and lack c-myc, bcl-2, ras, and p53 gene alterations (2, 83). MCD is a B-cell lymphoproliferative disorder that is sometimes referred to as multicentric angiofolli-cular hyperplasia. There are two forms of MCD: 1) a plasmablastic variant form that is associated with lymphadenopathy and immune dysregulation and 2) a hyaline vascular form, which presents as a solid mass. KSHV-associated MCD belongs to the plasma cell variant subgroup of MCD. MCD is a polyclonal tumor and is highly dependent on cytokines such as IL-6. Contrary to KS and PELs, KSHV DNA sequences have been detected only in a subset of MCD-patients, most often those co-infected with HIV (114).
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KSHV is considered to be a human oncogenic virus. Its genome encodes not only proteins necessary for the reproduction and persistence of the virus but also pathogenic factors to promote tumor cell proliferation and survival, regulate angiogenesis, and modulate host immune response in favor of tumor growth.
KSHV uses multiple mechanisms to promote cell survival and facilitate tumor development. Like other oncogenic DNA viruses, KSHV genes LANA-1, LANA-2, RTA, and vIRF-1 target both p53 and retinoblastoma (Rb) tumor suppressor pathways (37, 49, 84, 96, 100, 109, 110). Furthermore, KSHV can promote cell cycle progression from G1 to S-phase and accelerate cellular proliferation through vCyclin (27, 64, 103). The KSHV genome encodes several virus homologues of human antiapoptosis proteins. For instance, vBcl-2 is a KSHV homolog of human antiapoptosis protein Bcl-2 (26, 105). KSHV also encodes several cellular IRF homologues vIRFs that inhibit apoptosis (13, 40, 61, 66, 108, 109). Another unique mechanism that KSHV utilizes to promote tumor growth and cell survival is through activation of the nuclear factor κB (NF-κB) pathway. By activation of NF-κB, apoptosis is suppressed and regulation of the cell cycle is disturbed. Two KSHV genes, vFLIP and vGPCR have been shown to enhance cell growth and survival by activating the NF-κB pathway (36, 65, 69, 107). vFLIP is associated with anti-apoptotic activity, and activation of NF-kB by vFLIP is associated with prolonged survival of KSHV infected PEL cell lines (47, 116). vGPCR is a member of the CXC chemokine GPCR family that has significant homology to IL-8 receptor, but unlike the cellular GPCR, vGPCR exhibits ligand independent activity. vGPCR can activate PKC (protein kinase C), Akt [protein kinase B (PKB)], NF-κB, and MAPK (mitogen-activated protein kinase) to regulate the expression of growth factors [VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor)], cytokines (IL-1β, TNF-α, IL-6), and chemokines [IL-8, monocyte chemoattractant protein 1 (MCP-1)] (28, 78, 82, 90, 107, 113). The secretion of various factors could promote cell proliferation and stimulate angioproliferation through autocrine/paracrine signaling. KSHV-encoded vIL-6 also promotes cell survival. By coupling to the glycoprotein-130-receptor, the signal transducer and activator of transcription (STAT) cascade is activated and thus autocrine me-chanisms of tumorigenes is involved (51, 77, 89, 118).
KSHV utilizes two major strategies to evade the host immune response and achieve successful in-fections. The first is the so-called passive strategy. After a successful entry into host cell, KSHV establi-shes latency, during which only a minimum number of virus genes are expressed, thus reducing the number of antigens that are exposed to the immune systems. Secondly, during lytic replication or de novo infection, when most viral proteins are expressed and are susceptible to immune surveillance, the virus utilizes an active strategy that involves a number of its own unique genes to modulate the host immune response.
Diseases associated with KSHV
Mechanism of pathogenesis
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KSHV is a large (~170 kb) double-stranded DNA herpesvirus (99) and HIV-1 is a much smaller (~10 kb) retrovirus. Despite rather dramatic molecular dissimi-larities between the two viral genomes, many findings have provided compelling evidence that these two viruses interact to promote KS pathogenesis in dually infected individuals. Indeed, it is increasingly clear that, although the KSHV genome is invariably present in all described cases of KS (20), only a small proportion of infected people ever develop KS or KSHV-induced lymphomas. On the other hand, among KSHV-infected individuals, the risk of KS is much higher in those with HIV-1 infection than among those with other types of immunosuppression. Thus, a reasonable deduction is that KSHV is neces-sary but insufficient for producing KS and that HIV-1 is an important cofactor, which promotes KSHV induced KS.
HIV-1 probably exacerbates KSHV pathogenesis at multiple levels, including through immunosuppression, by priming of target cells and the tissue microenviron-ment for KSHV infection and replication, and by exerting direct effects on KSHV gene expression and viral replication. Direct and reciprocal interactions between HIV-1 and KSHV at the molecular level have received a lot of recent attention in the literature, they suggest mutual positive-feedback loops during replication of both viruses.
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Studies have demonstrated that KSHV can modu-late HIV-1 replication and KSHV might be a cofactor for HIV progression. HIV-1 replication significantly increases in the presence of KSHV both in vitro and in vivo model systems (76). KSHV coinfection markedly increases HIV replication in primary or transformed monocytic and endothelial cells (19). KSHV infection also induces HIV reactivation in chronically infected cell lines and in PBMCs from patients with asympto-matic HIV (19). ORF50, an IE gene of KSHV, encodes RTA necessary for virus reactivation and lytic replication. KSHV ORF50 can enhance HIV repli-cation in T and B cells (Jurkat and BC-3 cells) (17, 18). Transfection of ORF50 into nonsusceptible B and glial cells [BCBL-1 (body cavity-based lymphoma 1, latently infected with KSHV) and A172, respectively] increases cell susceptibility to infection and results in transient permissiveness to HIV replication (17, 18). KSHV ORF50 also interacts synergistically with HIV-1 transactivative transcription protein (Tat) in the transactivation of HIV-1 long terminal repeat (LTR) both in BCBL-1 cells and in HL3T1 cells (an epithelial cell line non-permissive to KSHV infection) (16). In addition, KSHV-encoded vFLIP (K13) is able to activate HIV-1 LTR via the activation of the classical NF-κB pathway and coexpression of HIV-1 Tat with K13 leads to synergistic activation of HIV-1 LTR (117). KSHV LANA can also transactivate the HIV-1 LTR in the human B-cell line BJAB, human monocytic cell line U937, and the human embryonic kidney fibroblast cell line 293T. Moreover, LANA cooperates with HIV Tat in activation of the LTR in a dose-response fashion with increasing amounts of LANA (54). KSHV ORF57, encoding a post-trans-criptional regulator, also enhances Tat-induced trans-activation of HIV-1 LTR in BCBL-1 cells (16).
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HIV-1 has an active role in the development of KSHV-associated diseases. HIV-1 not only activates lytic replication of KSHV but also induces secretion of a number of cytokines and growth factors, further promoting tumor cell proliferation and survival, as well as tumor angiogenesis. In BC-3 cells, HIV-1 infection leads to reactivation of latent KSHV genomes (74). Although recombinant HIV-1 gp120 fails to enhance herpesvirus expression, transient transfection of the HIV-1 Tat suffices to reactivate latent KSHV (74). HIV-1 Tat activates lytic cycle replication of KSHV, JAK/STAT signaling partially contributes to this process (122). HIV-1-encoded viral protein r (Vpr) also increases the expression of KSHV genes (52). Full-length HIV-1 Tat and a 13-amino-acid peptide corresponding to the basic region of Tat can specifically enhance the entry of KSHV into endothelial and other cells, probably by concentrating virions on cell surface (5). The specific cytokines produced by or in response to HIV-1-infected T cells in the coculture system (HIV-1-infected T cells and the KSHV-infected BCBL-1 cell line), particularly Oncostatin M, hepatocyte growth factor/scatter factor, and interferon-gamma, can play an important role in the initiation and progression of KS through reactivation of KSHV (75). HIV and its Tat protein induced inflammatory cytokines including TNF-α, IL-1β, IL-8, and IL-6 can trigger lesion formation by inducing the activation of endothelial cells that leads to adhesion and tissue extravasation of lymphomono-cytes, spindle cell formation, angiogenesis and KSHV reactivation that, in turn, leads to virus spread to all circulating cell types and virus dissemination into tissues (34, 35, 75). The other viral proteins of HIV, e. g. gp120 and nef also lead to paracrine secretion of cytokines and thus to stimulation of tumor growth and angiogenesis (4). HIV-1 Tat stimulates angiogenesis via specifically binding and activating the Flk-1/ kinase insert domain receptor (Flk-1/KDR), a VEGF-A tyrosine kinase receptor (50). The combination of inflammatory cytokines (IL-1β, TNF-α, and IFN-γ) increased in KS lesions synergizes with Tat to promote the development of angioproliferative KS-like lesions in nude mice. Inflammatory cytokines induce the tissue expression of both VEGF and bFGF, two angiogenic molecules highly produced in primary KS lesions. Tumorigenesis by KSHV vGPCR is accelerated by HIV-1 Tat via activating NF-κB and NF-AT (48).
KSHV acts to HIV
HIV acts to KSHV
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As discussed above, KSHV is associated with KS, PEL, and MCD, malignancies occurring more fre-quently in AIDS patients. The aggressive nature of KSHV in the context of HIV infection suggests that interactions between the two viruses enhance patho-genesis. KSHV encodes several genes that have the capacity to stimulate angiogenesis by inducing an angiogenic phenotype of KSHV-infected cells, or by the production of inflammatory cytokines, growth factors and chemokines, that can activate and promote angiogenesis. However, our current recognition and understanding of KSHV-related malignancies and its molecular mechanisms are still poorly developed. Further studies are needed to reveal the unique mechanisms used by this viral pathogen, which is crucial for the development of targeted-therapies for KSHV-associated malignancies.
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