Deregulation of cell growth by the K1 gene of Kaposi's sarcoma-associated herpesvirus.

At a position equivalent to the gene encoding the saimiri transforming protein (STP) of herpesvirus saimiri (HVS), Kaposi's sarcoma-associated herpesvirus (KSHV) contains a distinct open reading frame called K1. Although KSHV and HVS are related members of the rhadinovirus subgroup of gamma herpesviruses, K1 and STP exhibit no similarity in amino acid sequence or in structural organization. Since STP is required for the oncogenic potential of HVS, we investigated the functional consequence of K1 expression. Expression of the K1 gene in rodent fibroblasts produced morphologic changes and focus formation indicative of transformation. A recombinant herpesvirus in which the STP oncogene of HVS was replaced with K1, immortalized primary T lymphocytes to IL-2 independent growth and induced lymphoma in common marmosets. These results demonstrate the transforming potential of the K1 gene of KSHV.

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.

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.

Oncogenesis in HIV-infection.

The presence of human Kaposi's sarcoma associated herpesvirus-like sequences (KSHV) was examined in different epidemiological variants of Kaposi's sarcoma (KS) and in KS-derived cell cultures by polymerase chain reaction (PCR). KSHV DNA was present in all tumor biopsies of AIDS-associated KS (59 biopsies), endemic KS (26 biopsies; 21 African endemic KS, 5 Greek endemic KS), sporadic/classical KS (28 biopsies) and post-transplant/iatrogenic KS (6 of 7 biopsies). On the contrary, these sequences were only detected rarely in non-involved skin of KS patients (3 positive specimens of 12), in the peripheral blood mononuclear cells of HIV-infected patients (3 positive specimens of 54) and in lymphoma-biopsies (3 positive specimens of 47). Cell cultures derived from KS skin lesions were positive for KSHV DNA only in the first two passages. However, two longer-term positive cultures from a biopsy of a patient affected with sporadic KS and a biopsy of a patient affected with epidemic KS was identified. A strong association of KSHV with KS tissue was observed in all the different epidemiological variants of KS. Long-term positive KS-derived cell cultures will be an important tool to study the herpesvirus-like agent and to investigate its functional role in the initiation and progression of KS.

Presence of human herpesvirus type 6 in sporadic lymphoproliferative disorders. A comparative study.

A supportive or causal role for human herpesvirus 6 (HHV-6) in lymphoproliferative disorders is still controversial. Different results were obtained in both tissue-based and serological investigations. We investigated 243 lymph node and salivary gland tissue biopsies for the presence of viral DNA by using a newly developed, highly sensitive nested polymerase chain reaction method. HHV-6 was detected in 39% of the non-Hodgkin's lymphomas, in 52% of Hodgkin's diseases, 64% of non-neoplastic lymph nodes, 23% of tumor metastases, and 50% of salivary gland biopsies. When correlating the patients' ages with the occurrence of HHV-6, we found a significantly higher percentage of positive samples in patients younger than 60 years of age (54%) than in older patients (35%). This age-related difference was found in all the lymphoproliferative disorders studied as well as in salivary gland biopsies. Taking patient's ages into account, we found no significant difference between the various groups of disorders concerning the percentage of HHV-6-positive samples.

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.

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.

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.

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.

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.

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.

The unique region of the human herpesvirus 6 genome is essentially collinear with the UL segment of human cytomegalovirus.

The entire genome of human herpesvirus 6 (HHV-6) strain U1102 was cloned as overlapping fragments in cosmid and plasmid vectors. Cleavage maps were constructed for the restriction endonucleases BamHI, EcoRI, NotI and SmaI. The genome of HHV-6 U1102 is a linear dsDNA of 163 kbp, consisting of a long unique 142 kbp region flanked by direct terminal repeats of 10.5 kbp. Short stretches (290 to 470 nucleotides) of DNA, four from the terminal repeats and 55 from the UL region, were sequenced and compared by computer with the known herpesvirus amino acid sequences. Homologies were found for 10 open reading frames that are scattered over the UL region of HHV-6. Their relative positions and orientations indicate that the unique region of HHV-6 is essentially collinear with the UL region of human cytomegalovirus (HCMV), but is not collinear with the other human herpesviruses. It confirms and extends earlier observations that HHV-6 is more closely related to the beta-herpesvirus HCMV, suggesting that HHV-6 may be considered as the prototype of a new beta 2-herpesvirus subgroup.

Gene for the major antigenic structural protein (p100) of human herpesvirus 6.

A human herpesvirus 6 (HHV-6) structural protein of 100 kDa (p100) is the polypeptide most frequently and intensively reactive in immunoblotting analyses with human sera on HHV-6-infected cells or partially purified virions. The gene for p100 was identified by screening a bacteriophage lambda library with monospecific rabbit antisera. The gene codes for a polypeptide of 870 amino acids with a calculated molecular size of 97 kDa. Its amino-terminal third is weakly homologous to the immunogenic basic matrix phosphoprotein pp150 of human cytomegalovirus. Five fragments representing more than 93% of HHV-6 p100 were prokaryotically expressed. The antigenic epitopes of p100 were preliminary mapped by immunoblotting with human sera. They are located within the carboxy-terminal part which is neither homologous nor cross-reactive to pp150 of human cytomegalovirus. Availability of the gene for the immunodominant structural protein should provide tools for studies of pathogenesis by HHV-6.

Structure and transcription of an immediate-early region in the human herpesvirus 6 genome.

The unique segment of the human herpesvirus 6 (HHV-6) genome is essentially collinear to the unique long DNA segment of another betaherpesvirus, the human cytomegalovirus (HCMV). However, the HHV-6 genomic section that is analogous in position to the major immediate-early (IE) locus of HCMV does not exhibit recognizable sequence homologies. The respective HHV-6 region of 5.5 kbp is flanked on one side by 25 to 28 incomplete tandem repeats of 105 to 110 bp that contain, with one exception, a single KpnI restriction site (KpnI repeats). About 250 reiterations of the sequence motif CACATA are located on the other end. We identified two open reading frames of 375 and 2,595 nucleotides, respectively, on one strand. Strand-specific Northern blot analyses with RNA harvested from HHV-6 (strain U1102)-infected HSB-2 cells or cord blood lymphocytes revealed two transcripts of about 3.5 and 4.7 kb in the corresponding orientation. Sequence analyses of the respective cDNA clones and primer extension experiments were used to map the mRNAs. The two transcripts are coterminal and multiply spliced and code for the same putative 104.6-kDa protein, but they are initiated from different promoters. Characterization of smaller cDNA clones and Northern blotting with other strand-specific probes showed that singly spliced mRNAs of 1.0 and 1.5 kb are transcribed from the opposite strand; they could code for a 17.2-kDa polypeptide. Blocking experiments with cycloheximide led to the conclusion that only the 3.5-kb mRNA is synthesized in the absence of protein biosynthesis upon infection with cell-free virus. This identifies a single IE gene of HHV-6 at the genomic position corresponding to the major IE region of HCMV, although the coding content and transcriptional regulation are quite different for these two herpesvirus IE regions.

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.

The glycoprotein B homologue of human herpesvirus 6.

The gene for the homologue of herpesvirus glycoprotein B (gB) has been identified in the genome of human herpesvirus 6 (HHV-6), strain U1102, and the nucleotide sequence was determined. The open reading frame encodes a protein of 830 amino acids (93.2K) with the characteristics of a transmembrane glycoprotein and close similarity to the gp58/116 complex of human cytomegalovirus (HCMV). Monoclonal antibodies 2D10 and 2B9 have been shown previously to react with an HHV-6 glycoprotein of apparent M(r) 112K, and its proteolytic cleavage products of M(r) 64K and 58K. We show that both monoclonal antibodies detect prokaryotically expressed carboxy-terminal fragments of the HHV-6 gB homologue. This indicates that the HHV-6 gB homologue is probably processed by proteolytic cleavage similar to its equivalents in HCMV and various other herpesviruses.

Detection of herpesvirus type 6 by polymerase chain reaction in blood donors: random tests and prospective longitudinal studies.

In order to evaluate the prevalence of HHV-6 in blood donors, we examined 112 persons by polymerase chain reaction (PCR) and ELISA. HHV-6 antibodies could be detected in 107/111 (96.4%) of the donors. The median ELISA antibody level was 0.451 (range 0.056-0.914). 14 individuals (12.5%) were PCR positive in either oral lavage fluid, urine or buffy coat. Six persons (5.4%) were PCR positive in buffy coat samples. The prospective longitudinal analysis of 11 donors for periods between 7 and 13 weeks revealed that 4/6 persons who were initially PCR negative had positive tests in 9/63 weeks studied. Two persons were consistently PCR positive over the whole observation period of 12 and 13 weeks. HHV-6 variants could be determined in 14 persons as variant A in nine and variant B in five cases. These observations emphasize the high prevalence of HHV-6 and suggest that some blood donors carry detectable concentrations of the virus and therefore may be a source for transmission of HHV-6. The finding of positive PCR in antibody negative individuals suggests that antibody determination may not be sufficient to identify potentially infectious persons.

Identification and mapping of the gene encoding the glycoprotein complex gp82-gp105 of human herpesvirus 6 and mapping of the neutralizing epitope recognized by monoclonal antibodies.

Monoclonal antibodies (MAbs) 2D4, 2D6, and 13D6 against human herpesvirus 6 (HHV-6) variant A strain GS recognized virion envelope glycoprotein complex gp82-gp105 and neutralized the infectivity of HHV-6 variant A group isolates. A 624-bp genomic fragment (82G) was identified from an HHV-6 strain GS genomic library constructed in the lambda gt11 expression system by immunoscreening with MAb 2D6. Rabbit antibodies against the fusion protein expressed from the genomic insert recognized glycoprotein complex gp82-gp105 from HHV-6-infected cells, thus confirming that the genomic fragment is a portion of the gene(s) that encodes gp82-gp105. This genomic insert hybridized specifically with viral DNAs from HHV-6 variant A strains GS and U1101 under high-stringency conditions but hybridized with HHV-6 variant B strain Z-29 DNA only under low-stringency conditions. DNA sequence analysis of the insert revealed a 167-amino-acid single open reading frame with an open 5' end and a stop codon at the 3' end. Hybridization studies with HHV-6A strain U1102 DNA localized the gp82-gp105-encoding gene to the unique long region near the direct repeat at the right end of the genome. To locate the neutralizing epitope(s) recognized by the MAbs, a series of deletions from the 3' end of the gene were constructed with exonuclease III, and fusion proteins from deletion constructs were tested for reactivity with MAbs in a Western immunoblot assay. Sequencing of deletion constructs at the reactive-nonreactive transition point localized the epitope recognized by the three neutralizing MAbs within or near a repeat amino acid sequence (NIYFNIY) of the putative protein. This repeat sequence region is surrounded on either side by two potential N-glycosylation sites and three cysteine residues.