Copyright © 1997, American Society for Microbiology
MINIREVIEW
Cell-Homologous Genes in the Kaposi’s Sarcoma-Associated
Rhadinovirus Human Herpesvirus 8: Determinants
of Its Pathogenicity?
FRANK NEIPEL, JENS-CHRISTIAN ALBRECHT,ANDBERNHARD FLECKENSTEIN*
Institut fu¨r Klinische und Molekulare Virologie, Universita¨t Erlangen-Nu¨rnberg, D-91054 Erlangen, Germany
The epidemiology of Kaposi’s sarcoma (KS) among patients with AIDS has suggested that a sexually transmitted infectious agent other than human immunodeficiency virus (HIV) must be involved in its pathogenesis. KS is about 20,000 times more common in patients with AIDS than in the general population of the United States, and other immunosuppressed groups develop KS approximately 600 times more frequently than the healthy population. During the first decade of the AIDS epi-demic, about 20% of homosexual and bisexual patients devel-oped KS, in contrast to about 1% of men with hemophilia. Women were more likely to have KS if their partners were bisexual rather than parenterally infected men (4). This led to a broad search by PCR in patients with KS for the presence of viruses known to affect humans, such as cytomegalovirus (53, 57), human herpesvirus 6 (HHV-6) (29), and BK virus (43). Some of these viruses were found frequently in KS biopsy specimens, but none of them was consistently present (25, 30). A new era of KS research began when Y. Chang, P. S. Moore, and their colleagues (14) detected by representational differ-ence analysis (35) two short DNA fragments from a herpesvi-rus that was distinct from all previously known herpesviherpesvi-ruses. Remarkably, more than 90% of KS tissues obtained from pa-tients with AIDS contained the virus. The viral sequences were not present in biopsy specimens from patients without AIDS but were found in 15% of non-KS tissue DNA from patients with AIDS. The new virus, tentatively termed KS-associated herpesvirus or HHV-8, was soon found to be common in all epidemiological forms of KS (24). Viral DNA is consistently present in AIDS-associated KS lesions (1, 36) and in the vast majority of classical European-Mediterranean KS lesions (1, 18), while it is far less frequent in uninvolved skin of patients with KS and in the various biopsy specimens from Caucasian patients without KS and HIV. AIDS-associated African KS specimens (92%) and non-AIDS-associated KS lesions in Uganda (85%) had the virus (16). Based on PCR, the preva-lence of HHV-8 appeared to be high in the general population in Uganda, while searches in non-KS tumors and in normal tissues showed that the virus is rarely detectable in Caucasians. While some lymphomas carry other herpesviruses, such as Ep-stein-Barr virus (EBV) and HHV-6 (22), those specimens did not contain HHV-8 DNA. However, one type of lymphoid tumor, the AIDS-associated body cavity-based lymphoma (BCBL), was positive for HHV-8 DNA by PCR and Southern
blotting (11, 12). Multifocal Castleman’s disease (MCD) is a rare lymphoproliferative disorder; it occurs more frequently in association with KS. HHV-8 DNA was always found in patients with AIDS-associated MCD, including the cases without de-tectable KS, and it was also seen in the MCD cases of HIV-negative patients (60). Thus, there are now three distinct dis-ease conditions for which PCR epidemiology has indicated that HHV-8 nearly always persists, leaving the question whether the few HHV-8-negative cases of KS are simply due to occa-sional technical problems in sample collection, DNA extrac-tion, or PCR unanswered. Although most PCR-based studies indicated that HHV-8 is rare in the healthy general population, a few studies were contradictory. The frequent detection of HHV-8 DNA in skin lesions of transplant patients (49) was questioned by others (8). Some studies frequently found viral transcripts in prostate tissues (61) or DNA in semen and pros-tate tissues of patients without AIDS and KS at a frequency between 20 and 90% (34, 42), while other studies found the virus only in semen and prostate tissues of patients at risk for KS (17, 36, 64).
The availability of B-lymphoid cell lines from BCBL harbor-ing the virus allowed the performance of the first seroepide-miology studies. Antibodies against nucleic antigens of HHV-8 were seen in 70 to 80% of patients with KS, but no more than 1% of normal healthy subjects had antibodies detectable by this assay (20, 28, 41). Similarly, enzyme-linked immunosor-bent assays using a procaryotically expressed small capsid pro-tein indicated that there was a high level of seroprevalence in KS cases but not in the general population (58). This was in contrast to an immunofluorescence-based serological study which determined that 25% of healthy adults had antibodies against a phorbol ester-induced BCBL lymphoid cell line (21, 32, 51).
Preliminary nucleotide sequence data indicated that HHV-8 belongs to the rhadinovirus subgroup of herpesviruses (14). Rhadinoviruses (gamma 2 herpesviruses) share a common ge-nome structure. The linear double-stranded DNA (about 165 kbp) has a central segment of low-GC DNA (L DNA) that is flanked by multirepetitive high-GC DNA (H DNA) (6). The extreme intragenomic GC heterogeneity, resulting in fragmen-tation by density centrifugation, led to the coining of the term rhadinovirus (p´adivo´z[Greek]5fragile) (52). The L region of every known rhadinovirus spans some 110 to 130 kb and con-tains about 75 reading frames. The first size estimation for HHV-8 DNA from two BCBL-derived cell lines suggested a genome size of approximately 270 kb (38, 45). In contrast, size measurements by pulsed-field gel electrophoresis, using lyti-cally infected cells and purified virus particles, suggested that * Corresponding author. Mailing address: Institut fu¨r Klinische und
Molekulare Virologie, Universita¨t Erlangen-Nu¨rnberg, Schlossgarten 4, D-91054 Erlangen, Germany. Phone: 853563. Fax: 49-9131-852101. E-mail: fleckenstein@viro.med.uni-erlangen.de.
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FIG. 1. Colinearity of the genetic maps of herpesvirus saimiri (HVS) and HHV-8 L DNAs. Protein coding regions are numbered from 1 to 75 (herpesvirus sai miri and HHV-8) or from K1 to K15 (HHV-8) and are indicated by arrows. Open arrows symbolize herpesvirus genes conserved in herpesvirus saimiri and HHV-8. Cell-homologous genes present in both HHV -8 and herpesvirus saimiri are indicated by grey arrows, whereas virus cell homologs that are present in either HHV-8 or herpesvirus saimiri, but not in both viral genomes, are filled in black. Open reading frames with out any homology identifiable by the BLAST suite of programs are displayed as hatched arrows (03, 12, 14, 51, K1, K4.2, K8, K8.1, K10, K10.1, K12, and K15). Herpesvirus saimiri open reading frames were numbered ac cording to the system of Albrecht et al. (3). HHV-8 reading frames that share homology with herpesvirus saimiri were assigned the same numbers, and HHV-8 genes without recognizable homologs in herpesvirus sa imiri were numbered separately and given the prefix K. HHV-8-specific reading frames not described by Russo and colleagues (54) were assigned decimal K numbers (K4.1, K4.2, K8.1, and K10.1). Abbreviation s: STP-A, saimiri transforming protein, strain A; CCPH, complement control protein homolog; MDBP, major DNA binding protein; gB, glycoprotein B; pol, DNA polymerase; vIL17, viral IL-17 homolog; vIL-6, vi ral IL-6 homolog; vbcl-2, viral bcl-2; TK, thymidine kinase; gH, glycoprotein H; MCP, major capsid protein; mCP, minor capsid protein; PK, protein kinase; EXO, alkaline exonuclease; gM, glycoprotein M; gL, gly coprotein L; IE 52k, 52-kDa immediate-early protein; RRs, ribonucleotide reductase, small subunit; RRI, ribonucleotide reductase, large subunit; TS, thymidine synthase; v-cyc, viral cyclin homolog; vIL -8R, viral IL-8 receptor; TR, terminal repeat; vMIP-1 a , viral MIP-1 a homologs; vMIP-1 b ,open reading frame with homology to MIP-1 b and macrophage chemoattractant protein; nut-1, nuclear tRNA-like transcript (66); v-IRF, interferon-responsive factor homolog.
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[image:2.612.60.560.68.721.2]HHV-8 DNA is in the genomic size range of other rhadinovi-ruses (165 kb) (50). The discordant size determinations might be explained by sequence rearrangements in episomally per-sisting rhadinovirus genomes of cell lines not producing infec-tious virus (27). The nearly complete nucleotide sequence of HHV-8 DNA was determined from a BCBL cell line (54) and from a KS biopsy specimen (47). This showed that it has the characteristic genome structure of rhadinoviruses, with numer-ous 801-nucleotide H DNA tandem repeats (84.5% GC) and a 140.5-kb L DNA (53.5% GC). The L DNA contains at least 80 putative reading frames that are in a colinear orientation with respect to the L DNA genes of herpesvirus saimiri (2), a New World primate tumor virus that is the biologically and molec-ularly well-characterized rhadinovirus prototype (19) (Fig. 1). Like other rhadinoviruses, HHV-8 has numerous open reading frames with striking homology to known cellular genes. All known rhadinoviruses have unspliced genes that seem to have been captured from the host cell during viral evolution. Typi-cally, they code for proteins that interfere with the immune system, for enzymes involved in nucleotide metabolism, and for putative regulators of cell growth (Table 1). Although the cell-homologous rhadinovirus genes are frequently located in equivalent genomic regions between conserved gene blocks, some have distinct patterns (Fig. 2). Several of the HHV-8 cell homologies are not shared by other rhadinoviruses; these in-clude the genes for viral analogs of interleukin-6 (IL-6), three CC chemokines, at least on interferon response factor, and the N-CAM family transmembrane protein ox-2. Two reading frames of HHV-8, for which equivalents are found in
herpes-virus saimiri, are related to the cellular genes cyclin D2 and
bcl-2.
The amino acid sequence of the HHV-8 IL-6 homolog (viral IL-6 [vIL-6]) is 24.7% identical to human IL-6 (hIL-6) (48). The highest degree of conservation is in the IL-6 domain known to be involved in receptor binding, and preliminary studies indicate that the gene product supports growth of IL-6-dependent cell lines (44, 48). hIL-6 had been suspected to be involved in KS pathogenesis. Cultured KS cells have been reported to respond to human recombinant IL-6 with in-creased growth (39, 56); however, this has not been confirmed by others (62). A possible role for vIL-6 in KS pathogenesis is supported by the finding that KS spindle cells express the high-affinity IL-6 receptor in vivo (40). vIL-6 is found in KS lesions to a limited extent, while it is clearly expressed in HHV-8-associated lymphoproliferative disorders such as BCBL (44). hIL-6 is a tumor-promoting factor in multiple myeloma, acting by suppression of apoptosis. The vIL-6 of HHV-8 has been shown to prevent apoptosis of the mouse hybridoma cell line B9 (44). This supports the notion that vIL-6 contributes to pathogenesis of HHV-8-associated B-cell lymphomas and, possibly, of the plasma cell variant of Castle-man’s disease.
Two genes of HHV-8 have sequence homologies to the CC chemokine macrophage inflammatory protein 1a (MIP-1a). They exhibit some 30% amino acid sequence identity to the equivalent cellular chemokines. A third HHV-8 reading frame is related to the CC chemokine family, but sequence data did not allow an assignment discriminating between MIP-1band macrophage chemoattractant protein; it may well be derived from another member of the CC chemokine family. There are no functional data on the viral CC chemokines; thus, their role in pathogenesis is open for speculation. Perhaps they are in-volved in the chemoattraction of hematopoietic cells into KS lesions.
Like herpesvirus saimiri, HHV-8 codes for a type D cyclin (13, 15); it displays 32% amino acid identity (53% similarity) to v-cyclin of herpesvirus saimiri and 31% identity (53% similar-ity) to cellular cyclin D2 (33). Cellular D-type cyclins are as-sociated with at least four cyclin-dependent kinases, Cdk2, Cdk4, Cdk5, and Cdk6. Like v-cyclin of herpesvirus saimiri, the cyclin of HHV-8 associates predominantly with Cdk6 (33). The catalytic subunit for type D cyclin-dependent kinase activity in macrophages and fibroblasts is mainly Cdk4, while Cdk6 activ-ity is predominantly found in lymphoid cells. HHV-8 is present in the lymphocytes and spindle cells of KS lesions and in the transformed B cells of patients with BCBL. The specificity of HHV-8 v-cyclin for the kinase Cdk6 would thus be consistent with the lymphotropic or lymphocyte-transforming properties of the virus.
Induction of apoptosis is a typical response of the host cell to virus infection. A number of viruses have been shown in the past to carry apoptosis-inhibiting genes which may help these viruses to escape the host response. Like EBV and herpesvirus saimiri, HHV-8 carries a gene (open reading frame 16 [ORF16]) with homology to cellular bcl-2. Cellular Bcl-2 binds to Bax, Bik, and other proteins of this family (9, 55). The heterodimerization of Bcl-2 with Bax is important in prevent-ing Bax-mediated apoptosis. The homology between HHV-8 ORF16 and members of the bcl-2 family may suggest that it prolongs the life span of persistently infected cells. Disregu-lated bcl-2 expression has been shown to occur in a variety of human cancers, and overexpression of bcl-2 may contribute to carcinogenesis through its antiapoptotic effect.
[image:3.612.57.297.89.323.2]EBV is the closest known relative of HHV-8 in humans. The EBV nuclear antigens (EBNAs) and latent membrane proteins TABLE 1. Cellular homologs of rhadinoviruses and
the gamma 1 herpesvirus EBV
Host cell geneb
Presence or absence of homologous open reading frame in virusa:
HHV-8 HVS HVA AHV-1 BHV-4 EHV-2c EBV
CCPH 1 1 1 2 2 2 2
vIL-6 1 2 2 2 2 2 2
DHFR 1 1 2 2 2 2 2
TS 1 1 1 2 2 1 2
CC chemokines 3 2 2 2 2 2 2
Bcl-2 family protein 1 1 1 (1) 1 2 1
Interferon-responsive
factor 1 2 2 2 2 2 2 D-type cycline 1 1 1 2 2 2 2
N-CAM family
protein 1 2 2 2 2 2 2 IL-8R (gcr) 1 1 1 (1) 2 1 2
U-RNAs 2 #7 2 2 2 2 2
Tyrosine
kinase-inter-acting protein 2 6 1 2 2 2 2 Protein with
Colla-gen motifs 2 1 1 2 2 2 2 IL-17 2 1 2 2 2 2 2
CD59 2 1 2 2 2 2 2
IL-10 2 2 2 2 NKd 1 1
Semaphorin 2 2 2 1 2 2 2 a1, protein or RNA present;2, protein or RNA absent. Numbers represent the number of homologous reading frames and are given if more than one is present in the virus. Abbreviations: HVS, herpesvirus saimiri; HVA, herpesvirus ateles; AHV-1, alcelaphine herpesvirus 1 (18a); BHV-4, bovine herpesvirus 4; EHV-2, equine herpesvirus 2 (65).
bAbbreviations: CCPH, complement control protein homolog; DHFR, dihy-drofolate reductase; IL-8R (gcr), IL-8 receptor or (in parentheses) other G protein-coupled receptor homolog; TIP, tyrosine kinase-interacting protein; IL-17, IL-17 homolog; CD59, CD59 homolog; IL-10, IL-10 homolog.
cPositionally conserved regions were sequenced.
dNK, not known.
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(LMPs) of EBV are known to be crucial for the maintenance of viral latency and for growth transformation of the host cell by this virus. EBNA- and LMP-Homologous genes are entirely missing in all rhadinoviruses, including HHV-8. However, there is a striking correlation between the cell-homologous genes in the HHV-8 genome and cellular genes induced by EBV through EBNA and LMP proteins. EBV induces hIL-6 (63), cyclin D (59), an IL-8 receptor (5, 10), cellular Bcl-2 (23), and the complement-controlling protein CR-2 (31). Viral ho-mologs of all of these genes have been identified in the HHV-8 genome. Apparently, HHV-8 and EBV have developed differ-ent strategies to achieve the same goal: to overcome cell cycle arrest, apoptosis, and activation of cellular immunity, which are typical host responses to virus infection. Thus, it is not unlikely that two different strategies converged to contribute to malignant B-cell growth transformation by both herpesviruses of humans.
Seroepidemiology and PCR studies have strongly suggested that HHV-8 is associated with KS, MCD, and BCBL. IL-6,
[image:4.612.143.474.71.308.2]originally termed B-cell growth factor, is an essential cytokine required for growth differentiation of B lymphocytes and B-cell-derived lymphomas. Thus, it is not unlikely that virus-encoded IL-6 contributes to malignant growth of HHV-8-pos-itive B-cell lymphomas by an autocrine-paracrine loop. Increased levels of IL-6 have been detected in Castleman’s disease, and the cytokine seems to play a functional role. Clin-ical improvement correlates with decreasing serum levels of IL-6, and monoclonal antibodies against IL-6 have been used successfully for therapy. The pathogenesis of KS is largely enigmatic. On the basis of studies of continuous cell lines derived from KS spindle cells, cytokines have been incrimi-nated as being major factors in cell proliferation. They include IL-6 and the structurally related oncostatin M (37, 39, 40). However, the role of these cytokines in tumor growth has remained controversial (46). The discrepancies may be related to different cell culture conditions, resulting in distinct types of cultured cells (26, 62). More confusingly, cell lines derived from KS lesions are negative for HHV-8 DNA (1), while in the FIG. 2. Equivalent positions of cell-homologous genes in rhadinovirus genomes. The genomes of HHV-8, herpesvirus saimiri (HVS), herpesvirus ateles (HVA), alcelaphine herpesvirus 1 (AHV-1) (18a), and equine herpesvirus 2 (EHV-2) (65) are depicted in an orientation that aligns conserved herpesvirus genes (core gene blocks). Cell-homologous genes present in the nonconserved regions are indicated by arrowheads. Open arrowheads indicate modulators of the immune system (complement control protein homolog [CCPH]; ORF14, of herpesvirus saimiri, with homology to mouse mammary tumor virus superantigen; viral CD59 homolog [vCD59]). Arrowheads shaded light grey depict genes of the nucleotide metabolism (dihydrofolate reductase [DHFR], thymidylate synthase [TS]). Arrowheads shaded dark grey indicate open reading frames with homology to regulators of cell growth or apoptosis (viral cyclin D homolog [cyclin], viral bcl-2 homolog [bcl-2]). Genes related to cytokines or cytokine signal transduction are shown as filled black arrowheads (vIL-6, MIP1a, MIP1b[open reading frame with homology to MIP1band macrophage chemoattractant protein], IRF [interferon-responsive factor], vIL-8R [viral IL-8 receptor], vIL-17, gcra/b[G protein-coupled receptora/b]). sema., semaphorin.
TABLE 2. Five hypothetical scenarios for involvement of HHV-8 in the pathogenesis of KS
Scenario no. Description Role of virus
1 Passenger hypothesis HHV-8 is a frequent persisting virus, solely reactivated in tumor tissue but not contributing to pathogenesis.
2 Risk factor model HHV-8 is a frequent virus that may contribute to, but is not necessary for, KS pathogenesis. 3 Virus as necessary cofactor HHV-8 is a frequently observed virus required for KS development; other factors are important
to explain tumor epidemiology.
4 Dual-factor scenario HHV-8 is a sexually transmitted, relatively infrequently observed virus; it is a determinant of KS in conjunction with immunosuppression (AIDS) and/or genetic predisposition.
5 Intrinsically transforming virus HHV-8 is a rare virus, and infection with it is sufficient for KS induction.
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[image:4.612.58.559.634.727.2]original tumors both endothelial and spindle cells contain HHV-8 DNA (7), and a latency-associated HHV-8 transcript of 0.7 kb is expressed abundantly in the vast majority of spindle cells in situ (61, 62a). Despite all of the controversies, local cytokine disregulation is assumed to be the major factor in multifocal growth of KS lesions. A unifying concept for the role of HHV-8 in KS has to consider epidemiology, the consistent presence of viral DNA in tumor tissues at relatively high con-centrations, and the evidence for HHV-8 seroconversion prior to tumor development.
In general, the presence of HHV-8 allows at least five the-oretical scenarios for its role in KS (Table 2). Scenario 1, in which HHV-8 is merely a passenger, seems unlikely in view of the high virus loads in the tumors and the regular presence of HHV-8 in the majority of KS spindle cells. The patterns of viral cytokines and cytokine receptors suggest that it contributes to KS pathogenesis through cytokine disregulation. Scenario 2, a risk factor model, would not apply if careful studies confirm that HHV-8 is always present in the tumors. Scenario 5, in which HHV-8 is a transforming virus and the sole determining factor of KS, can be excluded because of the natural history of KS and seroepidemiological data available today. HHV-8 does not possess genes with homology to the transforming genes Stp and Tip of other rhadinoviruses. However, the HHV-8 tran-scripts that are expressed most abundantly in KS lesions are not homologous to any known gene. Thus, it remains to be elucidated whether the nonhomologous reading frames of HHV-8 possess transforming properties. As long as epidemi-ological data are conflicting, we cannot decide between the two other scenarios. Scenario 3, in which HHV-8, although rela-tively widespread in the general population ($20%), would be a necessary factor for KS, could not yet explain the specific epidemiology of AIDS-associated KS, which appears to be sexually transmitted through a relatively rare agent (!20%); thus, scenario 3 would require a third infectious agent that determines the chance of contracting KS. Scenario 4, the dual-factor hypothesis, appears most attractive as it can explain KS epidemiology by HHV-8; seroepidemiological data available today hint at sexual modes of transmission of HHV-8. How-ever, if the dual-factor model should apply, all means must be used to rule out the possibility that HHV-8 is common in the general population. According to the dual-factor model, infec-tion with HHV-8 is the essential predisposing factor for KS; concomitant immunosuppressive events, such as AIDS, could favor increasing virus loads and focal virus accumulation and trigger the multifocal disease KS.
Nucleotide sequence accession number.The nucleotide se-quence of KS-associated HHV-8 has been submitted to Gen-Bank and has been assigned accession no. U93872.
ACKNOWLEDGMENTS
We thank Ronald C. Desrosiers for critical reading of the manu-script.
This work was supported by the Mildred Scheel Foundation for Cancer Research (grant W134/94/FL2).
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