Structural proteins of Kaposi's sarcoma-associated herpesvirus antagonize p53-mediated apoptosis.

The tumor suppressor p53 is a central regulatory molecule of apoptosis and is commonly mutated in tumors. Kaposi's sarcoma-associated herpesvirus (KSHV)-related malignancies express wild-type p53. Accordingly, KSHV encodes proteins that counteract the cell death-inducing effects of p53. Here, the effects of all KSHV genes on the p53 signaling pathway were systematically analyzed using the reversely transfected cell microarray technology. With this approach we detected eight KSHV-encoded genes with potent p53 inhibiting activity in addition to the previously described inhibitory effects of KSHV genes ORF50, K10 and K10.5. Interestingly, the three most potent newly identified inhibitors were KSHV structural proteins, namely ORF22 (glycoprotein H), ORF25 (major capsid protein) and ORF64 (tegument protein). Validation of these results with a classical transfection approach showed that these proteins inhibited p53 signaling in a dose-dependent manner and that this effect could be reversed by small interfering RNA-mediated knockdown of the respective viral gene. All three genes inhibited p53-mediated apoptosis in response to Nutlin-3 treatment in non-infected and KSHV-infected cells. Addressing putative mechanisms, we could show that these proteins could also inhibit the transactivation of the promoters of apoptotic mediators of p53 such as BAX and PIG3. Altogether, we demonstrate for the first time that structural proteins of KSHV can counteract p53-induced apoptosis. These proteins are expressed in the late lytic phase of the viral life cycle and are incorporated into the KSHV virion. Accordingly, these genes may inhibit cell death in the productive and in the early entrance phase of KSHV infection.

Multicenter comparison of serologic assays and estimation of human herpesvirus 8 seroprevalence among US blood donors.

BACKGROUND: As part of assessing the possibility of transfusion transmission of human herpesvirus 8 (HHV-8 or Kaposi's sarcoma-associated herpesvirus), HHV-8 seroprevalence was estimated among US blood donors, the performance of HHV-8 serologic tests was compared, and the presence of HHV-8 DNA was tested for in donated blood. STUDY DESIGN AND METHODS: Replicate panels of 1040 plasma specimens prepared from 1000 US blood donors (collected in 1994 and 1995) and 21 Kaposi's sarcoma patients were tested for antibodies to HHV-8 in six laboratories. HHV-8 PCR was performed on blood samples from 138 donors, including all 33 who tested seropositive in at least two laboratories and 22 who tested positive in at least one. RESULTS: The estimated HHV-8 seroprevalence among US blood donors was 3.5 percent (95% CI, 1.2%-9.8%) by a conditional dependence latent-class model, 3.0 percent (95% CI, 2.0%-4.6%) by a conditional independence latent-class model, and 3.3 percent (95% CI, 2.3%-4.6%) by use of a consensus-derived gold standard (specimens positive in two or more laboratories); the conditional dependence model best fit the data. In this model, laboratory specificities ranged from 96.6 to 100 percent. Sensitivities ranged widely, but with overlapping 95 percent CIs. HHV-8 DNA was detected in blood from none of 138 donors evaluated. CONCLUSIONS: Medical and behavioral screening does not eliminate HHV-8-seropositive persons from the US blood donor pool, but no viral DNA was found in donor blood. Further studies of much larger numbers of seropositive individuals will be required to more completely assess the rate of viremia and possibility of HHV-8 transfusion transmission. Current data do not indicate a need to screen US blood donors for HHV-8.

Cell surface heparan sulfate is a receptor for human herpesvirus 8 and interacts with envelope glycoprotein K8.1.

An immunodominant envelope glycoprotein is encoded by the human herpesvirus 8 (HHV-8) (also termed Kaposi's sarcoma-associated herpesvirus) K8.1 gene. The functional role of glycoprotein K8.1 is unknown, and recognizable sequence homology to K8.1 is not detectable in the genomes of most other closely related gammaherpesviruses, such as herpesvirus saimiri or Epstein-Barr virus. In search for a possible function for K8.1, we expressed the ectodomain of K8.1 fused to the Fc part of human immunoglobulin G1 (K8.1DeltaTMFc). K8.1DeltaTMFc specifically bound to the surface of cells expressing glycosaminoglycans but not to mutant cell lines negative for the expression of heparan sulfate proteoglycans. Binding of K8.1DeltaTMFc to mammalian cells could be blocked by heparin. Interestingly, the infection of primary human endothelial cells by HHV-8 could also be blocked by similar concentrations of heparin. The specificity and affinity of these interactions were then determined by surface plasmon resonance measurements using immobilized heparin and soluble K8.1. This revealed that K8.1 binds to heparin with an affinity comparable to that of glycoproteins B and C of herpes simplex virus, which are known to be involved in target cell recognition by binding to cell surface proteoglycans, especially heparan sulfate. We conclude that cell surface glycosaminoglycans play a crucial role in HHV-8 target cell recognition and that HHV-8 envelope protein K8.1 is at least one of the proteins involved.

Kaposi's sarcoma-associated herpesvirus serology in Europe and Uganda: multicentre study with multiple and novel assays.

A multicentre study was undertaken to define novel assays with increased inter-assay concordance, sensitivity, specificity and predictive value for serological diagnosis of human herpesvirus type 8 (HHV-8) infection. A total of 562 sera from European and Ugandan human immunodeficiency virus (HIV)-infected or uninfected individuals with or without Kaposi's sarcoma (KS) and blood donors were examined under code by 18 different assays in seven European laboratories. Sera from KS patients and all non-KS sera found positive by at least 70%, 80%, or 90% of the assays were considered "true positive." The validity of the assays was then evaluated by univariate logistic regression analysis. Two immunofluorescence assays (IFA) for detection of antibodies against HHV-8 lytic (Rlyt) or latent (LLANA) antigens and two enzyme-linked-immunosorbent assays (ELISA) (M2, EK8.1) for detection of antibodies against HHV-8 structural proteins were found to be highly concordant, specific, and sensitive, with odds ratios that indicated a high predictive value. When used together, the two IFA (Rlyt-LLANA) showed the best combination of sensitivity (89.1%) and specificity (94.9%). The performance of these assays indicate that they may be used for the clinical management of individuals at risk of developing HHV-8 associated tumours such as allograft recipients.

Antibodies to human herpesvirus 8 latent and lytic antigens in blood donors and potential high-risk groups in Sweden: variable frequencies found in a multicenter serological study.

Human herpesvirus 8 (HHV-8) is a herpesvirus associated with Kaposi's sarcoma (KS). An immunofluorescence assay was used for detection of IgG, IgM, and IgA antibodies against lytic and latent HHV-8 antigens to analyse samples from KS patients (n = 8), healthy blood donors (n = 162), individuals with a high risk sexual behaviour (n = 114), and bone marrow transplant patients (with high risk for bloodborne infections) (n = 34) in Sweden. Of the KS patients, 88% had IgG antibodies to both lytic and latent antigens by immunofluorescence. In all other groups, antilatent antibodies were rare (0-2.6%). IgG antibodies to the lytic antigens were found, by immunofluorescence, in 20% of the blood donors, 31% of the high risk patients, and in 24 and 29% of the bone marrow transplant patients (pre- and post-transplant samples, respectively). For verification of the specificity of the anti-lytic antibodies, 170 of the samples were also tested blindly at different laboratories world-wide with five other assays shown previously to detect HHV-8 antibodies in most KS patients. By using two recombinant HHV-8 proteins (ORF65/vp17 and K8.1/gp 35-37) in ELISA, a whole-virion ELISA and two immunofluorescence assays confirmation of the reactivity against lytic viral antigens was sought. The comparison of the different methods suggested the K8.1 ELISA to be highly specific and also showed a good agreement between two of the immunofluorescence assays. However, generally there was a poor correlation for positive results, indicating the need of further methodological development.

Generation of monoclonal antibodies directed against the immunogenic glycoprotein K8.1 of human herpesvirus 8.

Human Herpesvirus 8 (HHV-8) is clearly associated with Kaposi's sarcoma (KS), body cavity-based lymphomas (BCBL), and certain forms of multifocal Castleman's disease (MCD). It appears to be the sexually transmissible agent involved in the development of AIDS-associated KS. HHV-8 genomes are invariably present in BCBL-derived cell lines where lytic replication of the virus can be induced by phorbol esters (PE). First-generation HHV-8 serological assays were based on these cell lines. More recently, several genes encoding HHV-8 antigens have been identified. One of the most reactive antigens is encoded by HHV-8 open reading frame K8.1. Although K8.1 does not exhibit overt sequence homology to any other known gene, it is likely to be analogous to gp220/350 of Epstein-Barr or gp150 of murine herpesvirus-68, virion-envelope glycoproteins involved in target cell recognition. Mice were immunized with purified GST-K8.1 fusion protein expressed in E. coli. After fusion of murine plasma cells with the myeloma cell line P3-X63-Ag8. monoclonal antibodies (MAbs) were generated, which are specifically directed against K8.1 protein. The binding site for each MAb was identified by deletion mutant analysis using recombinant GST-K8.1 mutants and K8.1-specific peptides. Without exception, the epitopes recognized by these MAbs were located within the N-terminal part of the protein [amino acids (aa) 29 to 80], thus identifying a highly immunogenic region. These antibodies will not only be useful tools for HHV-8 diagnostics, but will also facilitate the analysis of K8.1 function.

Clinical presentations of infection by the human herpesvirus-7 (HHV-7).

Reports on the clinical picture of primary infection with the human herpesvirus-7 (HHV-7) are scarce. A heterogenous population of 478 patients (1 month-14 years) was examined for the presence of an acute HHV-7 infection. A variety of clinical pictures can be observed during primary infection with HHV-7, such as exanthema subitum and mononucleosis-like syndrome. The authors describe in two children, for the first time, the presentation of HHV-7-infection as an acute exacerbation or relapse of a patient's chronic disease. The hematological changes for the respective clinical presentations during HHV-7 infection are reported.

Human herpesvirus 8: is it a tumor virus?

Human herpesvirus 8 (HHV-8), also termed Kaposi's sarcoma-associated herpesvirus, was identified in Kaposi's sarcoma (KS) biopsy specimens in 1994. The epidemiological data available to date indicate a strong association of HHV-8 with KS. It appears that HHV-8 is necessary for KS development. HHV-8 DNA is invariably found in all epidemiological forms of KS and primary effusion lymphomas. In contrast, HHV-8 DNA is rarely found in various tumor and nontumor tissues from patient groups not at risk of KS. Although current serology does not allow us to assess the HHV-8 prevalence in the general population, high titers of HHV-8 antibodies are almost exclusively found in KS risk groups. In addition, HHV-8 seroconversion has been shown to precede KS development. The mechanisms and genes involved in HHV-8 pathogenesis are less clear. HHV-8 belongs to a family of transforming viruses, and several candidate oncogenes have been identified by using rodent fibroblast transformation assays. However, expression of most of these genes could not be shown in latently infected tumor cells. As the HHV-8 genome encodes several cytokines and cytokine receptor homologues, HHV-8 may also promote KS pathogenesis through paraendocrine mechanisms.

Human interleukin-6 is in vivo an autocrine growth factor for human herpesvirus-8-infected malignant B lymphocytes.

Human interleukin-6 (hIL-6) acts as a growth factor in several human B lymphoid cancers. As human herpesvirus-8 (HHV-8) encodes for a viral IL-6 (vIL-6), the viral cytokine may be responsible for several manifestations of HHV-8-related disorders. Using an anti-hIL-6 mAb (B-E8) which does not recognize vIL-6, we investigated the involvement of the human cytokine in the proliferation of HHV-8-positive primary effusion lymphoma (PEL) cells. In vitro, 5/5 PEL cell lines produced hIL-6 (4 to 1,200 pg/ml). The EBV- HHV-8+ cell line (BCBL-1) was adapted to grow in SCID mice. hIL-6 was detected in the serum of mice with grafts, as well as human soluble CD138 (sCD138) and human IL-10 (hIL-10). The serum level of these mediators increased with tumor progression. The effect of treatment with the B-E8 mAb on the tumor progression and survival was evaluated. This treatment significantly slowed down the tumor development: on day 54, there were more mice with low levels of sCD138 and hIL-10 in the treated group than in controls (p = 0.03 and 0.02, respectively); treatment also delayed death (median date of death was day 65 for control mice and day 84 for anti-hIL-6 mAb-treated mice; p < 0.02). Thus, hIL-6 is expressed in addition to vIL-6 in HHV-8-positive malignant B lymphocytes, and the viral cytokine does not totally substitute for human IL-6 in promoting tumor progression.

Development of recombinant diagnostic reagents based on pp85(U14) and p86(U11) proteins to detect the human immune response to human herpesvirus 7 infection.

Human antibodies raised in response to human herpesvirus 7 (HHV-7) infection are directed predominantly to one or more HHV-7-infected cell proteins with apparent molecular masses of about 85 to 89 kDa. The genes that encode these proteins are unknown. However, several HHH-7 genes that possibly encode proteins in this molecular mass range have been identified. Thus, the proteins encoded by open reading frame U14 (85 kDa) and U11 (86 kDa) were expressed as recombinant proteins in bacteria. Of 13 human serum specimens that recognized the 85- to 89-kDa protein(s) of HHV-7-infected cells by immunoblotting, 12 were also reactive with recombinant pp85(U14) and 8 were reactive with p86(U11). It is concluded that (i) the HHV-7 immunodominant protein is pp85(U14) and (ii) the lack of posttranslational modifications in procaryotically expressed pp85 does not adversely affect the reactivity of human sera. Monoclonal antibody (MAb) 5E1 is an HHV-7-specific MAb directed to pp85(U14). Here, the HHV-7-specific epitope in pp85(U14) was finely mapped to the C' terminal region between amino acid residues 484 and 502. However, as indicated by the low level of reactivity of human sera with the HHV-7-specific epitope recognized by MAb 5E1, human sera recognize additional epitopes of pp85(U14) that are required for their full reactivity.

Comparison of the immunoglobulin-G-specific seroreactivity of different recombinant antigens of the human herpesvirus 8.

The open reading frames ORF 52, ORF 65, K12, and K8.1 of the human herpesvirus 8 (HHV8) were expressed as glutathione-S-transferase (GST) fusion proteins and analysed by Western blotting (WB) and enzyme-linked immunosorbent assay (ELISA). The open reading frame (ORF) 65 and K8.1 antigens gave the highest reactivity (71%) in sera from HIV-dependent Kaposi's sarcoma (KS) patients. Therefore both antigens appear to be essential for HHV8 diagnostics, whereas ORF K12 and ORF 52 were of minor importance. Using polymerase chain reaction (PCR) out of the peripheral blood of these KS patients, 48% were detected as positive. By testing an N-terminal-deleted construct (amino acid 80-171) of ORF 65, we could show that the N-terminal region of this protein is essential to mediate full immunogenic reactivity. By analysing different deletion mutants of ORF K8.1, the major epitope was found to be located between aa 29 and 101. The prevalence of antibodies directed against the different antigens was determined for healthy blood donors to be 3-6%. The different antibody patterns obtained in HIV-patients with and without KS support the hypothesis that different antibody profiles develop during the course of KS.

Identification of human herpesvirus 8-specific cytotoxic T-cell responses.

Human herpesvirus 8 (HHV-8) (or Kaposi's sarcoma-associated herpesvirus) is implicated in the etiopathogenesis of Kaposi's sarcoma (KS) and certain lymphoproliferations. The introduction of more effective therapies to treat human immunodeficiency virus infection has led to a decline in the incidence of KS and also in the resolution of KS in those already affected. This suggests that cellular immune responses including cytotoxic T lymphocytes (CTLs) could play a vital role in the control of HHV-8 infection and in KS pathogenesis. Here we elucidate HLA class I-restricted, HHV-8-specific cellular immune responses that could be important in the control of HHV-8 infection and subsequent tumor development. We show the presence of CTLs against HHV-8 latent (K12), lytic (K8.1), and highly variable (K1) proteins in infected individuals.

The role of HHV-8 in Kaposi's sarcoma.

The epidemiology of Kaposi's sarcoma (KS) amongst North American and Northern European patients with AIDS suggests that an infectious agent other than HIV is involved in its pathogenesis. Several lines of evidence indicate that human herpesvirus 8 (HHV-8), also termed Kaposi's sarcoma associated herpesvirus, is the sought after agent. DNA of HHV-8 is invariably found in all forms of KS where the virus is present in the KS spindle cell. In contrast, HHV-8 DNA is not regularly detected in most other malignancies. Antibodies against HHV-8 are more frequently found in groups at risk of KS, and HHV-8 seroconversion precedes KS development. Several HHV-8 genes have been identified that exhibit transforming potential in cell culture systems. In addition, the virus encodes and induces several cytokines and angiogenic factors. This is of particular interest as models of KS pathogenesis developed before the discovery of HHV-8 emphasized the importance of inflammatory cytokines. Although the expression pattern of viral genes in KS is not certain yet, it appears likely that the pathogenetic role of HHV-8 in KS may be rather complex and differs from other virus-induced malignancies. 1999 Academic Press.

Suppression of HHV-8 viremia by foscarnet in an HIV-infected patient with Kaposi's sarcoma and HHV-8 associated hemophagocytic syndrome.

Human herpes virus 8 (HHV-8) seems to be involved in the pathogenesis of Kaposi s sarcoma. In vitro, antiviral drugs with activity against herpes viruses also can suppress HHV-8, however, little is known about the antiviral activity against HHV-8 in vivo. In this report we describe the effects of foscarnet on HHV-8 viremia in an HIV-infected patient with disseminated Kaposi s sarcoma and a presumably HHV-8 associated hemophagocytic syndrome. HHV-8 DNA could be detected in this patient by PCR in peripheral blood mononuclear cells (PBMC), in bronchoalveolar fluid and tumor biopsies. After initiation of foscarnet because of a severe hemophagocytic syndrome HHV-8 PCR turned negative in PBMC, but stayed positive in pleural effusions and in a tumor biopsy. After termination of foscarnet therapy HHV-8 DNA in PBMC persistently reappeared. Under treatment with foscarnet the hemophagocytic syndrome dramatically improved, suggesting that HHV-8 had a pathogenetic role in this syndrome.

Detection of HHV-8 DNA in a German patient with classical Kaposi's sarcoma may allow an estimation of the incubation period.

DNA of HHV-8 (Kaposi's sarcoma-associated Herpes virus [KSHV]) was detected in a biopsy of a Kaposi's sarcoma in an elderly male patient from Saxony (East Germany). The diagnosis of classical Kaposi's sarcoma was first made in 1986. During World War II, the patient had been on active service on the Greek Islands of Crete and Rhodes only, he did not travel outside East Germany after the war. It is assumed that the patient was infected during his stay on the islands of Crete or Rhodes, where classical Kaposi's sarcoma is endemic. If so, the incubation period of classical Kaposi's sarcoma could be as long as 40 years.

Human herpesvirus 8--the first human Rhadinovirus.

Kaposi's sarcoma (KS)-associated herpesvirus, also known as human herpesvirus 8 (HHV-8), is the first known human member of the genus Rhadinovirus. It is regularly found by polymerase chain reaction in all forms of KS, in certain types of Castleman's disease, and in body cavity-based B-cell lymphoma. Other members of this virus group occur in nonhuman primates, ungulates, rabbits, and mice and cause in part fulminant lymphomas and other neoplastic disorders of the hematopoietic system. Rhadinoviruses share a typical genome structure; most characteristically, they contain numerous sequences that appear to be sequestered from cellular DNA. We cloned and sequenced almost the complete genome of HHV-8 from a single KS biopsy specimen. Although this procedure revealed collinear organization and extensive homologies with the open reading frames of herpesvirus saimiri, genes with homology to the known oncoproteins (Stp, Tip) were not identified in the HHV-8 genome. However, HHV-8 reading frame K1, the positional analogue of Stp/Tip, was found to be significantly variable between different strains. We found, in addition, the reading frames for homologues of cellular interleukin 6, macrophage inflammatory proteins alpha and beta (MIP1 alpha and MIP1 beta, respectively), an interferon-responsive factor, and two inhibitors of apoptosis. Several of these cell-homologous genes of HHV-8 have already been shown to code for functional proteins.

Human herpesvirus type 6 variants identified by single-strand conformation polymorphism analysis.

Six human herpesvirus 6 (HHV-6) variants were analyzed for heterogeneity using the polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP). Two independent DNA regions were selected: a fragment of the gene U11 (position 18966-21578) coding for a basic phosphoprotein, the major antigenic structural protein pp100; and a fragment from an open reading frame (ORF) area of the gene U67, previously referred to as 13R (position 102458-103519), coding for a product of unknown function. The two PCR systems based on the above DNA sequences yielded products of 187 bp and 223 bp, respectively. DNA obtained from three laboratory reference strains (U1102, R104 and St.W.) and from HHV-6 infected peripheral white blood cells of bone marrow transplant patients and blood donors was used to test the applicability of two different SSCP analysis systems for the identification of HHV-6 variants using amplicons derived by PCR from the two genomic regions described above (U11 [pp100], U67 [13R-ORF]). The generation of characteristic SSCP patterns enables the rapid differentiation of HHV-6 A and B strains for the classification of variants derived from clinical samples, reducing the need for expensive and time-consuming direct sequencing analyses.