Signal transduction by human herpesvirus 8 viral interleukin-6 (vIL-6) is modulated by the nonsignaling gp80 subunit of the IL-6 receptor complex and is distinct from signaling induced by human IL-6

Human herpesvirus 8 (HHV-8) viral interleukin-6 (vIL-6) mediates signaling through the gp130 signal transducer but unlike human IL-6 (hIL-6) does not require the nonsignaling gp80 alpha subunit of the IL-6 receptor complex. By utilizing a gp80-refractory vIL-6 variant, vIL-6(R189L), we found that signal transduction, as measured by STAT1 and STAT3 activation and gp130 tyrosine phosphorylation in gp80+/gp130+ HEK293T cells, was modulated by gp80. Furthermore, the signaling and BAF-130 cell growth-promoting activities of vIL-6 and hIL-6 could be distinguished, and exogenous addition of soluble gp80 enhanced cell growth supported by vIL-6. Our findings demonstrate that gp80 can modulate vIL-6 activity and that vIL-6 and hIL-6 signaling are not directly equivalent.     

Intracellular signaling mechanisms and activities of human herpesvirus 8 interleukin-6

Human herpesvirus 8 (HHV-8)-encoded viral interleukin-6 (vIL-6) has been implicated as a key factor in virus-associated neoplasia because of its proproliferative and survival effects and also in view of its angiogenic properties. A major difference between vIL-6 and human IL-6 (hIL-6) is that vIL-6, uniquely, is largely retained and can signal intracellularly. While vIL-6 is generally considered to be a lytic gene, several reports have noted its low-level expression in latently infected primary effusion lymphoma (PEL) cultures, in the absence of other lytic gene expression. Thus, intracellular autocrine signal transduction by the viral cytokine may be of particular relevance to the growth and survival of latently infected cells and to pathogenesis. Here we report that most intracellular vIL-6 is located in the endoplasmic reticulum (ER), signals via the gp130 signal transducer in this compartment, and does so independently of the gp80 α-subunit of the IL-6 receptor, required for hIL-6 signal transduction. Signaling and biological assays incorporating ER-retained vIL-6 and hIL-6 confirmed vIL-6 activity, specifically, in this compartment. Knockdown of vIL-6 expression in PEL cells led to markedly reduced cell growth in normal culture, independently of extracellular cytokines. This could be reversed by reintroduction via virus vector of exclusively ER-retained vIL-6. These data indicate that in virus biology vIL-6 may act to support the growth and survival of cells latently infected with HHV-8 in an autocrine manner via intracrine signaling and that these activities may contribute to the maintenance of latently infected cells and to virus-induced neoplasia.     

Determinants of Secretion and Intracellular Localization of Human Herpesvirus 8 Interleukin-6

Human herpesvirus 8 (HHV-8) interleukin-6 (vIL-6) is distinct from human and other cellular IL-6 proteins in that it does not require the nonsignaling α-receptor subunit for the formation of gp130-based signal transducing complexes and also is largely retained intracellularly rather than being secreted. We and others have reported that vIL-6 is retained and is active in the endoplasmic reticulum (ER) compartment, and data from our laboratory have demonstrated that intracellular vIL-6 is functional in the autocrine promotion of proliferation and survival of HHV-8 latently infected primary effusion lymphoma cells. It has also been reported that vIL-6 secretion in gp130-deficient cells can be enhanced by introduced gp130, thereby implicating the signal transducer in vIL-6 trafficking to the cell surface. We examine here the requirements for intracellular retention and localization of vIL-6. Using vIL-6-hIL-6 chimeric and point-mutated vIL-6 proteins, we identified regions and residues of vIL-6 influencing vIL-6 secretion. However, there was no correlation between vIL-6 secretion and gp130 interaction. We found that vIL-6, but not hIL-6, could associate stably with ER-resident chaperone protein calnexin. Glycosylation-dependent interaction of vIL-6 with calnexin correlated with proper protein folding, but there was no direct relationship between vIL-6-calnexin interaction and intracellular retention. While calnexin depletion had little influence on absolute amounts of secreted vIL-6, it led to markedly reduced levels of intracellular cytokine. This was reversed by gp130 transduction, which had no detectable effect on vIL-6 secretion, but redistributed vIL-6 into ER-distinct locations in calnexin-depleted cells, specifically. Our data reveal that calnexin plays a role in ER localization of vIL-6 and that gp130 promotes ER exit, but not secretion, of the viral cytokine.The viral homologue of interleukin-6, vIL-6, specified by human herpesvirus 8 (HHV-8) shows only 25% amino acid identity to human IL-6 (hIL-6) but is highly related structurally (2, 5). Despite the high degree of conservation of three-dimensional structure and equivalence of receptor interaction interfaces (1, 6), the viral cytokine can associate functionally with the gp130 signal transducer in the absence of the gp80 α-subunit, absolutely required for cellular IL-6 signaling through gp130. The nonsignaling gp80 subunit can be incorporated into vIL-6-induced signaling complexes and indeed seems to have a stabilizing effect that enhances signal transduction (1, 3, 11). Another major difference between vIL-6 and cellular IL-6 proteins, including hIL-6, is that the viral cytokine is very inefficiently secreted, retained largely within the endoplasmic reticulum (ER) compartment, where it is able to transduce signal via gp80-deficient vIL-6₂/gp130₂ tetrameric complexes, exclusively (4, 15). Thus, the unique ability of vIL-6 to signal intracellularly may be explained by its gp80 independence; hIL-6 cannot signal in the ER even when targeted to this compartment (4). The biological significance of intracellular, strictly autocrine signaling by vIL-6 was demonstrated recently in primary effusion lymphoma (PEL) cells, which are latently infected with HHV-8; these cells grew with markedly reduced kinetics and displayed higher rates of apoptosis upon shRNA-mediated vIL-6 depletion relative to cocultured untransduced cells (4). Thus, vIL-6 appears not only to be expressed in latently infected PEL cultures but also to be biologically active in this setting via intracrine signaling.Despite these findings and other mechanistic studies of vIL-6, the means by which the viral cytokine is retained in the ER and secreted so inefficiently is unknown. The elegant work of Meads and Medveczky (15) demonstrated the slow secretion kinetics of vIL-6 relative to hIL-6 and implicated gp130 as a necessary cofactor for vIL-6 secretion. Thus, vIL-6 expressed in gp130-negative Ba/F3 cells was able to be secreted only if gp130 was supplied via expression vector transduction. However, most cell types express gp130; thus, while the signal transducer may be involved in vIL-6 trafficking, the underlying explanation for the very slow rate of vIL-6 secretion must involve other factors.We report here investigations of the structural requirements for vIL-6 intracellular retention, the influence of gp130 on this process, and the possible involvement of ER-resident chaperon proteins for retention of vIL-6 in the ER. Our data identify effects of structural alterations and point mutations of vIL-6 on secretion efficiency, the lack of gp130 involvement in these observed effects, mechanistically relevant interactions of calnexin with the viral cytokine, and the influence of gp130 on vIL-6 subcellular localization and stability in the context of calnexin depletion. The results presented thus further advance our understanding of vIL-6-cellular protein interactions that impact upon its intracellular function.     

Detection of direct binding of human herpesvirus 8-encoded interleukin-6 (vIL-6) to both gp130 and IL-6 receptor (IL-6R) and identification of amino acid residues of vIL-6 important for IL-6R-dependent and -independent signaling

Human herpesvirus 8 (HHV-8) is associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease; in all of these diseases, interleukin-6 (IL-6) has been implicated as a likely mitogenic and/or angiogenic factor. HHV-8 encodes a homologue of IL-6 (viral IL-6 [vIL-6]) that has been shown to be biologically active in several assays and whose activities mirror those of its mammalian counterparts. Like these proteins, vIL-6 mediates its effects through the gp130 signal transducer, but signaling is not dependent on the structurally related IL-6 receptor (IL-6R; gp80) subunit of the receptor-signal transducer complex. However, as we have shown previously, IL-6R can enhance vIL-6 signal transduction and can enable signaling through a gp130 variant (gp130.PM5) that is itself unable to support vIL-6 activity, indicating that IL-6R can form part of the signaling complex. Also, our analysis of a panel of vIL-6 mutants in transfection experiments in Hep3B cells (that express IL-6R and gp130) showed that most were able to function normally in this system. Here, we have used in vitro vIL-6-receptor binding assays to demonstrate direct binding of vIL-6 to both gp130 and IL-6R and vIL-6-induced gp130-IL-6R complex formation, and we have extended our functional analyses of the vIL-6 variants to identify residues important for IL-6R-independent and IL-6R-dependent signaling through native gp130 and gp130.PM5, respectively. These studies have identified residues in vIL-6 that are important for IL-6R-independent and IL-6R-mediated functional complex formation between vIL-6 and gp130 and that may be involved directly in binding to gp130 and IL-6R.     

Human Herpesvirus 8 Interleukin-6 Contributes to Primary Effusion Lymphoma Cell Viability via Suppression of Proapoptotic Cathepsin D,a Cointeraction Partner of Vitamin K Epoxide Reductase Complex Subunit 1 Variant 2

Human herpesvirus 8 (HHV-8) interleukin-6 (vIL-6) promotes cell proliferation and survival and is proangiogenic, implicating it as a contributor to virus-associated Kaposi''s sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman''s disease. Although predominantly lytically expressed, vIL-6 is also produced at low, functional levels during latency in PEL cells. Unlike other IL-6 cytokines, vIL-6 is secreted very inefficiently and localizes in the endoplasmic reticulum (ER). ER-localized vIL-6 supports PEL cell proliferation and survival, mediated in part through its interaction with the largely uncharacterized ER-resident protein vitamin K epoxide reductase complex subunit 1 variant 2 (VKORC1v2). Here, we report that the ER-transiting and functionally mitogenic secreted proenzyme (pCatD) form of cathepsin D (mature CatD), a proapoptotic lysosomal aspartate protease, is an interaction partner of VKORC1v2 and that vIL-6 promotes this interaction. Depletion of vIL-6 in PEL cells increased levels of the catalytically active, proteolytically cleaved form of CatD, corresponding with decreased PEL cell viability. Ectopic expression of CatD in PEL cells induced apoptosis, suggesting that CatD suppression by vIL-6 is biologically significant. In the context of high-density culture or reactivation of HHV-8 lytic replication in PEL cells, CatD depletion substantially reduced stress-induced apoptosis and increased virus production. In contrast, CatD overexpression, vIL-6 depletion, and peptide-mediated disruption of vIL-6–VKORC1v2 interaction inhibited replication and cell survival. Combined, our data identify pCatD as an interaction partner of VKORC1v2, demonstrate a role of vIL-6 in CatD suppression via VKORC1v2 in PEL cells, and identify a biologically significant mechanism of vIL-6 prosurvival and proreplication activities via VKORC1v2.     

Activation of STAT6 by STING is critical for antiviral innate immunity

STAT6 plays a prominent role in adaptive immunity by transducing signals from extracellular cytokines. We now show that STAT6 is required for innate immune signaling in response to virus infection. Viruses or cytoplasmic nucleic acids trigger STING (also named MITA/ERIS) to recruit STAT6 to the endoplasmic reticulum, leading to STAT6 phosphorylation on Ser(407) by TBK1 and Tyr(641), independent of JAKs. Phosphorylated STAT6 then dimerizes and translocates to the nucleus to induce specific target genes responsible for immune cell homing. Virus-induced STAT6 activation is detected in all cell-types tested, in contrast to the cell-type specific role of STAT6 in cytokine signaling, and Stat6(-/-) mice are susceptible to virus infection. Thus, STAT6 mediates immune signaling in response to both cytokines at the plasma membrane, and virus infection at the endoplasmic reticulum.     

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.     

Kaposi's sarcoma-associated herpesvirus-encoded viral interleukin-6 is secreted and modified differently than human interleukin-6: evidence for a unique autocrine signaling mechanism

Viral interleukin-6 (vIL-6) is a homolog of cellular IL-6 that is encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) genome. vIL-6 binds to the IL-6 signal transducer gp130 without the cooperation of the IL-6 high affinity receptor to induce STAT3 DNA binding and cell proliferation. Although vIL-6 is believed to be important in the pathogenesis of KSHV-induced diseases, its secretion and post-translational modifications have not previously been characterized. Pulse-chase analysis revealed that the half-time of vIL-6 secretion is approximately 8-fold longer than that of human IL-6. Yet, the vIL-6 signal sequence targets human IL-6 secretion to nearly wild-type levels. Surprisingly, vIL-6 was not secreted from a cell line that does not express gp130 but expression of human gp130 in these cells enabled the secretion of vIL-6. Consistent with this observation, complete maturation of gp130 N-glycans is inhibited by vIL-6 coexpression, suggesting that the binding of the receptor to vIL-6 occurs intracellularly in early or pre-Golgi compartments. Furthermore, a vIL-6 mutant containing an endoplasmic reticulum retention signal is not secreted but does still induce receptor activation and signaling. Secreted vIL-6 is completely glycosylated at both possible N-glycosylaton sites and contains a large proportion of immature high-mannose glycans that is not typical of cytokines. These findings suggest that vIL-6 may induce gp130 signaling by an exclusively autocrine mechanism that relies on intracellular binding to its receptor. During KSHV infection, vIL-6 may only induce signaling in KSHV-infected cells to benefit the viral life cycle and promote oncogenic transformation.     

Human herpesvirus 8 (HHV-8)-encoded cytokines induce expression of and autocrine signaling by vascular endothelial growth factor (VEGF) in HHV-8-infected primary-effusion lymphoma cell lines and mediate VEGF-independent antiapoptotic effects

The potential roles of human herpesvirus 8 (HHV-8) cytokines in HHV-8 pathogenesis were investigated by determining the expression of the HHV-8 chemokines viral macrophage inflammatory protein 1A (vMIP-1A) and vMIP-1B in primary effusion lymphoma (PEL)-derived cell lines and examining the signaling activities of these chemokines and HHV-8-encoded vIL-6 in these cells. Secreted vMIP-1A and vMIP-1B were detected in biologically significant concentrations following tetradecanoyl phorbol acetate treatment, which induces productive replication. vIL-6 and vMIP-1A, added exogenously to cultures of four different PEL cell lines, induced the expression of vascular endothelial growth factor type B (VEGF-B) and VEGF-A, respectively. These cells were found to express VEGF receptor 1 (Flt-1) protein, and signaling by recombinant VEGF-A(165) was demonstrated for two of the PEL cell lines, indicating the potential for autocrine, as well as paracrine, effects of viral cytokine-induced VEGF. In addition, vMIP-1A and vMIP-1B, but not VEGF-A(165), were found to inhibit chemically induced apoptosis in PEL cells. Our data suggest that vIL-6 and vMIP-1A may influence PEL through VEGF autocrine and paracrine signaling that promotes PEL cell growth and extravascular effusion and that vMIP-1A and vMIP-1B can act independently of VEGF as antiapoptotic factors.     

Human herpesvirus 8 viral interleukin-6 interacts with splice variant 2 of vitamin K epoxide reductase complex subunit 1

Viral interleukin-6 (vIL-6) specified by human herpesvirus 8 is, unlike its cellular counterpart, secreted very inefficiently and can signal via vIL-6(2):gp130(2) signaling complexes from the endoplasmic reticulum (ER) compartment. Intracellular, autocrine activities of vIL-6 are important for proproliferative and prosurvival activities of the viral cytokine in latently infected primary effusion lymphoma (PEL) cells. However, the molecular determinants of vIL-6 ER localization and function are unclear. Using yeast two-hybrid analysis, we identified the database-documented but uncharacterized splice variant of vitamin K epoxide reductase complex subunit 1 (VKORC1), termed VKORC1 variant 2 (VKORC1v2), as a potential interaction partner of vIL-6. In transfected cells, epitope-tagged VKORC1v2 was found to localize to the ER, to adopt a single-transmembrane (TM) topology placing the C tail in the ER lumen, and to bind vIL-6 via these sequences. Deletion mutagenesis and coprecipitation assays mapped the vIL-6-binding domain (vBD) of VKORC1v2 to TM-proximal residues 31 to 39. However, while sufficient to confer vIL-6 binding to a heterologous protein, vBD was unable to induce vIL-6 secretion when fused to (secreted) hIL-6, suggesting a VKORC1v2-independent mechanism of vIL-6 ER retention. In functional assays, overexpression of ER-directed vBD led to suppression of PEL cell proliferation and viability, effects also mediated by VKORC1v2 depletion and, as reported previously, by vIL-6 suppression. The growth-inhibitory and proapoptotic effects of VKORC1v2 depletion could be rescued by transduced wild-type VKORC1v2 but not by a vIL-6-refractory vBD-altered variant, indicating the functional relevance of the vIL-6-VKORC1v2 interaction. Notably, gp130 signaling was unaffected by VKORC1v2 or vBD overexpression or by VKORC1v2 depletion, suggesting an alternative pathway of vIL-6 activity via VKORC1v2. Combined, our data identify a novel and functionally significant interaction partner of vIL-6 that could potentially be targeted for therapeutic benefit.     

Structural requirements for gp80 independence of human herpesvirus 8 interleukin-6 (vIL-6) and evidence for gp80 stabilization of gp130 signaling complexes induced by vIL-6

Human herpesvirus 8 interleukin-6 (vIL-6) displays 25% amino acid identity with human IL-6 (hIL-6) and shares an overall four-helix-bundle structure and gp130-mediated STAT/mitogen-activated protein kinase signaling with its cellular counterpart. However, vIL-6 is distinct in that it can signal through gp130 alone, in the absence of the nonsignaling gp80 alpha-subunit of the IL-6 receptor. To investigate the structural requirements for gp80 independence of vIL-6, a series of expression vectors encoding vIL-6/hIL-6 chimeric and site-mutated IL-6 proteins was generated. The replacement of hIL-6 residues with three vIL-6-specific tryptophans implicated in gp80 independence from crystallographic studies or the A and C helices containing these residues did not confer gp80 independence to hIL-6. The N- and C-terminal regions of vIL-6 could be substituted with hIL-6 sequences with the retention of gp80-independent signaling, but substitutions of other regions of vIL-6 (helix A, A/B loop, helix B, helix C, and proximal half of helix D) with equivalent sequences of hIL-6 abolished gp80 independence. Interestingly, the B helix of vIL-6 was absolutely required for gp80 independence, despite the fact that this region contains no receptor-binding residues. Point mutational analysis of helix C, which contains residues involved in physical and functional interactions with gp130 domains 2 and 3 (cytokine-binding homology region), identified a variant, VI120EE, that was able to signal and dimerize gp130 only in the presence of gp80. gp80 was also found to stabilize gp130:g130 dimers induced by a distal D helix variant of vIL-6 that was nonetheless able to signal independently of gp80. Together, our data reveal the crucial importance of overall vIL-6 structure and conformation for gp80-independent signaling and provide functional and physical evidence of the stabilization of vIL-6-induced gp130 signaling complexes by gp80.     

Human herpesvirus 8 encodes a homolog of interleukin-6.

Kaposi's sarcoma is a multifocal lesion that is reported to be greatly influenced by cytokines such as interleukin-6 (IL-6) and oncostatin M. DNA sequences of a novel human gammaherpesvirus, termed human herpesvirus 8 (HHV-8) or Kaposi sarcoma-associated herpesvirus, have been identified in all epidemiological forms of Kaposi's sarcoma with high frequency. The presence of HHV-8 DNA is also clearly associated with certain B-cell lymphomas (body cavity-based lymphomas) and multicentric Castleman's disease. Sequence analysis of a 17-kb fragment revealed that adjacent to a block of conserved herpesvirus genes (major DNA-binding protein, glycoprotein B, and DNA polymerase), the genome of HHV-8 encodes structural homolog of IL-6. This cytokine is involved not only in the pathogenesis of Kaposi's sarcoma but also in certain B-cell lymphomas and multicentric Castleman's disease. The viral counterpart of IL-6 (vIL-6) has conserved important features such as cysteine residues involved in disulfide bridging or an amino-terminal signal peptide. Most notably, the region known to be involved in receptor binding is highly conserved in vIL-6. This conservation of essential features and the remarkable overlap between diseases associated with HHV-8 and diseases associated with IL-6 disregulation clearly suggest that vIL-6 is involved in HHV-8 pathogenesis.     

Molecular mechanisms for viral mimicry of a human cytokine: activation of gp130 by HHV-8 interleukin-6

Kaposi's sarcoma-associated herpesvirus (KSHV, or HHV-8) encodes a pathogenic viral homologue of human interleukin-6 (IL-6). In contrast to human IL-6 (hIL-6), viral IL-6 (vIL-6) binds directly to, and activates, the shared human cytokine signaling receptor gp130 without the requirement for pre-complexation to a specific alpha-receptor. Here, we dissect the biochemical and functional basis of vIL-6 mimicry of hIL-6. We find that, in addition to the "alpha-receptor-independent" tetrameric vIL-6/gp130 complex, the viral cytokine can engage the human alpha-receptor (IL-6Ralpha) to form a hexameric vIL-6/IL-6Ralpha/gp130 complex with enhanced signaling potency. In contrast to the assembly sequence of the hIL-6 hexamer, the preformed vIL-6/gp130 tetramer can be decorated with IL-6Ralpha, post facto, in a "vIL-6-dependent" fashion. A detailed comparison of the viral and human cytokine/gp130 interfaces indicates that vIL-6 has evolved a unique molecular strategy to interact with gp130, as revealed by an almost entirely divergent structural makeup of its receptor binding sites. Viral IL-6 appears to utilize an elegant combination of both convergent, and unexpectedly divergent, molecular strategies to oligomerize gp130 and activate similar downstream signaling cascades as its human counterpart.     

IL-6 receptor independent stimulation of human gp130 by viral IL-6

The genome of human herpes virus 8, which is associated with Kaposi's sarcoma, encodes proteins with similarities to cytokines and chemokines including a homologue of IL-6. Although the function of these viral proteins is unclear, they might have the potential to modulate the immune system. For viral IL-6 (vIL-6), it has been demonstrated that it stimulates IL-6-dependent cells, indicating that the IL-6R system is used. IL-6 binds to IL-6R, and the IL-6/IL-6R complex associates with gp130 which dimerizes and initiates intracellular signaling. Cells that only express gp130 but no IL-6R cannot be stimulated by IL-6 unless a soluble form of the IL-6R is present. This type of signaling has been shown for hematopoietic progenitor cells, endothelial cells, and smooth muscle cells. In this paper we show that purified recombinant vIL-6 binds to gp130 and stimulates primary human smooth muscle cells. IL-6R fails to bind vIL-6 and is not involved in its signaling. A Fc fusion protein of gp130 turned out to be a potent inhibitor of vIL-6. Our data demonstrate that vIL-6 is the first cytokine which directly binds and activates gp130. This property points to a possible role of this viral cytokine in the pathophysiology of human herpes virus 8.     

Cellular tropism and viral interleukin-6 expression distinguish human herpesvirus 8 involvement in Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease

Human herpesvirus 8 (HHV-8) infection has been implicated in the etiology of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD), three diseases that frequently develop in immunocompromised, human immunodeficiency virus-positive individuals. One hypothesis that would account for different pathological manifestations of infection by the same virus is that viral genes are differentially expressed in heterogeneous cell types. To test this hypothesis, we analyzed the localization and levels of expression of two viral genes expressed in latent and lytic infections and the viral homologue of interleukin-6 (vIL-6). We show that PEL parallels KS in the pattern of latent and lytic cycle viral gene expression but that the predominant infected cell type is a B cell. We also show that MCD differs from KS not only in the infected cell type (B-cell and T-cell lineage) but also in the pattern of viral gene expression. Only a few cells in the lesion are infected and all of these cells express lytic-cycle genes. Of possibly greater significance is the fact that in a comparison of KS, PEL, and MCD, we found dramatic differences in the levels of expression of vIL-6. Interleukin-6 is a B-cell growth and differentiation factor whose altered expression has been linked to plasma cell abnormalities, as well as myeloid and lymphoid malignancies. Our findings support the hypothesis that HHV-8 plays an important role in the pathogenesis of PEL and MCD, in which vIL-6 acts as an autocrine or paracrine factor in the lymphoproliferative processes common to both.