Review

Kaposi’s sarcoma-associated herpesvirus andKaposi’s sarcoma

Henri Gruffat*, Alain Sergeant, Evelyne Manet

Laboratoire de Virologie Humaine, U412 Inserm, ENS-Lyon, 46 allée d’Italie, 69364 Lyon cedex 07, France

ABSTRACT – Kaposi’s sarcoma-associated herpesvirus (KSHV) is present in all epidemiologic formsof Kaposi’s sarcoma (KS). The KSHV genome contains several open reading frames which arepotentially implicated in the development of KS. Some are unique to KSHV; others are homologous tocellular genes. The putative role of these genes in the genesis of KS is discussed. © 2000 Éditionsscientifiques et médicales Elsevier SAS

human herpesvirus 8 / KSHV / rhadinovirus / Kaposi’s sarcoma / cell transformation

1. IntroductionKaposi’s sarcoma (KS) is a multifocally developing

tumour which predominantly affects the skin but also, insome cases, visceral organs and lymph nodes. Four differ-ent forms of KS are commonly distinguished, mainly onepidemiological grounds: the sporadic (classic) form, theendemic (African) form, the epidemic (AIDS-related) formand a form associated with immunosuppression (usuallyin transplant recipients). However, the histological fea-tures of these different forms of KS are very similar and arecharacterized by prominent angiogenesis together withnumerous infiltrating leucocytes and the presence of theso-called ’KS spindle cells’. This spindle cell populationappears to be composed of a heterogeneous mixture ofcell types from different origins. Moreover, the growth ofthe KS lesion may be regulated by paracrine signals gen-erated by various KS cell populations. IL-1b, IL-6, VEGF(vascular endothelial growth factor), MCP-1 and bothisoforms of platelet-derived growth factor have been foundto be synthesized in the KS lesions (for a review see [1]).

In 1994, Chang et al. detected the presence of a thenunknown human γ-herpesvirus in KS tissues [2]. This viruswas initially called Kaposi’s sarcoma-associated herpesvirus (KSHV) and classified in the rhadinovirus genus, butis also referred to as human herpes virus eight (HHV8). Thevirus has been shown to be closely related to herpesvirussaimiri (HVS). KSHV has been found systematically asso-ciated with all forms of KS. It is also associated with twoother neoplastic disorders: primary effusion lymphoma(PEL) also termed body cavity-based lymphoma (BCBL),and multicentric Castleman’s disease (MCD). KSHV-infected B lymphoma cell lines (often dually infected with

EBV) have been established from these PELs. In these celllines the viral transcription pattern is mainly latent, i.e.,there is no production of viral particles, but the productivecycle can be activated by treatment of the cells withchemical agents such as sodium butyrate or phorbol esters(see [3] and the references therein).

The completion of the entire KSHV genomic sequenceand its analysis revealed that in addition to the pattern ofgenes usually expressed by γ-herpesviruses, KSHV con-tains a set of genes which resemble those that controlcellular growth. This review will survey the more recentdata concerning the molecular biology of the virus and thepossible role of the expression of the different KSHVspecific genes in the genesis and evolution of KS.

2. KSHV genomic organization andstructure

In the virion, the KSHV genome is a linear double-stranded DNA molecule, but it is stably maintained inlatently infected B cells (PEL origin) as episomal mono-meric circles.

The full genome of KSHV (around 165 kb) has beenmapped and sequenced from contiguous, overlappingviral DNA fragments of a genomic library made from a PELcell line [4]. KSHV has also been sequenced from a KSbiopsy, and the genome was found to be almost identical[5].

The KSHV genomic structure is similar to that of otherγ-herpesviruses (figure 1), with a central unique segmentof 140.5 kb long (53.5% GC) flanked by numerous tan-demly repeated units of noncoding GC-rich DNA (84.5%GC) approximately 800 bp long. Each KSHV moleculeharbours 35 to 45 such repeats asymmetrically distributed* Correspondence and reprints.

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at each end of the molecule [6]. The long unique regioncontains about 80 putative open reading frames (ORFs).

A comparison of KSHV genome sequences obtainedfrom different KSHV isolates, coming from geographicallydistinct locations, shows that most of the KSHV genome ishighly conserved except in two small regions at theextremities of the DNA molecule. For example, at the 5’end of the genome, a difference at the protein sequencelevel of up to 20% is found in ORFK1 between differentisolates. This variability allows the definition of four majorvirus subtypes: A, B, C and D. In some isolates, the 3’ endof the genome sequence also shows a high degree ofvariability, which allows the definition of two furthersubtypes [7, 8]. It is interesting to note that in the case ofHVS, sequence variabilities are observed in a region of thegenome encoding proteins required for T-cell transforma-tion, and a good correlation exists between the subtypeand the pathogenicity of the virus.

Similarly to other herpesviruses, the KSHV genomecontains genes encoding proteins which are required forthe replication and assembly of new virions. These genesare arranged in four clusters and are distributed in colinearposition and orientation with respect to those of otherherpesviruses. Among the 80 KSHV ORFs, 66 sharehomologies with ORFs from HVS, which is the rhadinovi-rus prototype. Interestingly, the KSHV genome also con-tains numerous ORFs with striking homology to knowncellular genes.

3. KSHV replication

Latently infected cells harbour multiple copies of circu-larized KSHV genome maintained as episomes. Similarlyto other γ-herpesviruses, such as EBV, the episomes persistin the cells through a cis-acting DNA sequence calledOri-P (origin of plasmid replication). KSHV Ori-P wasmapped near the 5’ end of the genome. It contains threecopies of the terminal repeat sequence and up to 600 bp ofunique sequence (figure 1) [9]. No sequence homologywas detected between the KSHV Ori-P and those of HVS

or EBV. However, KSHV contains a gene, orf73, whosefunction is reminiscent of that of EBNA1 which is requiredfor EBV episomal maintenance. orf73 encodes a high-molecular weight protein of 1162 amino acids (222–234kDa), called LNA (latent nuclear antigen) or latency-associated nuclear antigen (LAMA).

LNA is able to mediate the episomic persistance ofKSHV Ori-P-harbouring plasmids, independently of otherKSHV proteins [9]. In addition, immunofluorescenceexperiments using LNA-specific antisera, applied to PELcell lines or orf73-transfected mammalian cells, gave char-acteristic speckled nuclear staining [10]. Such character-istic staining is also found in KS spindle cells. Moreover,investigation by confocal microscopy shows that LNAcolocalises with KSHV DNA in multiple, small and dis-crete subnuclear dots ([9] and references therein). Thefinding that LNA is restricted to KSHV DNA localization ininterphase is indicative of specific DNA recognition byLNA, but the DNA recognition site is still unknown. How-ever, whether LNA, like EBNA1, has transactivation andtransformation properties is as yet unknown. orf73 isexpressed from a tri-cistronic mRNA harbouring the cod-ing sequences for ORF73, ORF72 (v-cyclin) and ORFK13(v-Flip). In line with its suspected role in the maintenanceof the KSHV episomal genome, orf73 mRNA is expressedboth in uninduced PEL cell lines and in the majority of KSspindle cells and represents an abundant latent transcriptwhose expression is downregulated upon induction of thelytic cycle.

During the lytic cycle of γ-herpesviruses, replication isdirected from origins different from Ori-P and is depen-dent upon viral proteins (table I). KSHV also encodes anumber of enzymes involved in nucleotide metabolismwhich play an ancillary role in DNA synthesis. Some ofthese proteins, like the viral thymidilate synthase or theviral thymidine kinase, could be targets for antiviral agents.

The replication origins that function during the lyticcycle of EBV, referred to as OriLyt, have been character-ized in the EBV genome as two inverted repeatedsequences called DR-L and DR-R. Two regions of theKSHV genome share some structural homologies with the

Figure 1. Genomic organisation of KSHV. This scale diagram shows the relative size. Genes homologous between herpes viruses are drawnas hatched boxes. Open arrows indicate the unique KSHV genes (noted K1 to K15) or genes whose functions are discussed in the text.DR–L and DR–R: duplicated region left and right, respectively. Ori–P: latent origin of replication.

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DR sequences of EBV (figure 1). Although they have beensuggested to act as the OriLyt for KSHV [7], there is as yetno experimental data to support this hypothesis.

4. KSHV genes with homology tocellular genes

As already mentioned, in addition to the genes involvedin the replication of the KSHV genome and the productionof virions, several KSHV genes have been found to have anextended homology with cellular genes. These genes haveprobably been captured from the host cell during viralevolution: they code for proteins that may interfere withthe immune system, or enzymes involved in nucleotidemetabolism, or putative regulators of cell growth. Althoughseveral of these genes are unique to KSHV, some have alsobeen found in either HVS or EBV genomes, suggesting thatthey have coevolved with viruses of the γ-herpesvirusfamily. Moreover, KSHV codes for a set of genes with noknown cellular homologues and to which no function hasbeen allocated (table II). These genes are good candidatesfor playing a role in KSHV-related pathogenesis.

5. KSHV membrane proteins5.1. orfK12

In KS tumour spindle cells and in PEL latently infectedcell lines, only a few KSHV genes are expressed. Amongthese, a small, highly hydrophobic protein of 60 amino

Table I. Trans-acting replication genes and EBV coun-terparts.

KSHVORF

EBV ORF Function in EBV DNA replication.

ORF9 BALF5 DNA polymerase5′-3′ polymerase activity3′-5′ exonuclease/proof reading activity5′-3′ exonuclease activity (RNase Hlike)

ORF6 BALF2 Single-stranded-DNA binding proteinUnspecific binding to a single-strandDNADestabilizing DNA helixColocalizes with Pol at the nuclearmatrix

ORF56 BSLF1 PrimaseORF44 BBLF4 DNA helicase

ORF40/41 BBLF2/3 Primase-associated factorCopurifies with helicase-primasecomplexPrimer stabilization?

ORF59 BMRF1 Polymerase processivity factorEquimolar complex with Pol asheterodimerIncreases processivityPhosphorylated protein

ORFK8? BZLF1 Transcription/replication factorSpecific binding to ZRE sites in EBVOriLyt

Table II. KSHV genes potentially implicated in KS development and their functions.

KSHV geneHVS

homologueEBV

homologueHomologous

cellular genesPutative function

orfK1 – +/– (LMP) – Membrane protein. ITAM motiforfK2 – – IL6 CytokineorfK3 – – – Unknown functionorfK4 – – MIP ChemokineorfK4.1 – – MIP ChemokineorfK5 – – – Unknown functionorfK6 – – MIP ChemokineorfK7 – – – Unknown functionorfK8 – +/– (EB1) – bZIP protein. Unknown functionorfK9 – – IRF Signal transductionorfK10 – – – Unknown functionorfK11 – – – Unknown functionorfK12 – – – Membrane protein, Golgi apparatus and reticulum

endoplasmic localisationorfK13 + – Flip Apoptosis inhibitororfK14 + – NCAM Adhesion moleculeorfK15 – +/– – Membrane protein. TRAF binding motiforf4 + – CCP Complement control proteinorf2 + – DHFR Dihydrofolate reductaseorf70 + – TS Thymidylate synthaseorf21 + + TK Thymidine kinaseorf16 + + Bcl–2 Apoptosis inhibitororf72 – – D cyclin Cell cycle controlorf74 + – IL8–R (GPCR) Chemokine receptor

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acids (12 kDa) termed K12 or kaposin is expressed. TheK12 protein can be detected by immunofluorescence inKS cells and in PEL cell lines. In these cells the protein islocalized in the Golgi apparatus and the endoplasmicreticulum as well as at the cell surface. It has been shownthat K12 expressed in Rat-3 cells induces cell transforma-tion, and the transformed cells produce highly vascularand undifferentiated sarcomas upon subcutaneous injec-tion in athymic nu/nu mice [11]. The exact role of K12 inthis process is not as yet determined, but it has beensuggested that the kaposin protein is homologous to the E5oncoprotein of bovine papilloma virus, which is also asmall and highly hydrophobic protein localized in theGolgi apparatus and the endoplasmic reticulum. E5 hasbeen shown to bind to a 16-kDa component of the vascu-lar H + -ATPase. This complex is important in the process-ing of growth factor receptors, resulting in their constitu-tive activation. Like E5, K12 may play an important role inthe development of oncogenesis.

In most KS tumours, the transcriptional pattern of theK12 region is complex and several unrelated proteins canpotentially be produced. One of these, kaposin B, is thepredominant product and does not contain the K12 ORF[12]. The biological function of these different proteinsremains to be discovered.

5.2. orfK15

At the 3’ end of the KSHV genome, in a region of highvariability, there is a familly of alternatively spliced tran-scripts of around 7.5 kb, expressed in latently infected celllines but also induced after activation of the lytic cycle.These mRNAs encode a family of proteins, K15 (35 to 60kDa), which differ by the number of transmembranedomains (up to 12). The cytoplasmic hydrophilicC-terminal domain contains several tyrosine-rich motifswith homologies to known TRAF binding sites [13, 14].The presence of these motifs, some aspects of the splicingpattern of the transcripts, and the location of the K15 ORFon the KSHV genome, are reminiscent of two EBV latentproteins: LMP-2A and LMP-1. LMP-2A interacts with theSrc familly of protein tyrosine kinases, but is not essentialfor B-cell immortalisation. In contrast, LMP-1 is essentialfor B-cell immortalisation, and interacts with TRAF-1,TRAF-2, TRAF-3 and TRADD. In this way, it can inducethe activation of both NF-jB and c-Jun N-terminal kinase-mediated gene expression. However, for ORFK15 func-tion, no experimental data is as yet available.

Expression of K15 in vivo in KS is not clear, but 2 KSpatients out of 20 tested had some antibodies directedagainst the C-terminal domain of K15, suggesting that theprotein is expressed [13].

5.3. orfK1

orfK1 encodes for a type I membrane protein with anN-terminal signal sequence, a single transmembraneregion, and a small, 37-amino acid, C-terminal cytoplas-mique tail. K1 is thought to be present in oligomeric formsat the cell surface [15]. A highly conserved region of thecytoplasmic domain of K1 is structurally and functionallyhomologuous to the immunoreceptor tyrosine-based acti-vation motif (ITAM). The ITAM sequence is necessary for

signal transduction: its tyrosine residues are phosphory-lated by Src kinases upon stimulation, allowing subse-quent binding of SH2-containing proteins, such as Syk,Vav, Lyn, leading to calcium-dependent signal transduc-tion in B cells [16]. However, unlike other ITAM-basedreceptors, K1 signalling is independent of external stimu-lation, suggesting that K1 homo-oligomerisation drivesthis process. Homodimerisation can occur via the cysteine-rich extracellular domain of the protein or by an endog-enous ligand constitutively present on the cell surface[17].

K1 has transforming properties when expressed in Rat-1cells. In addition, when the saimiri transforming proteingene of HVS, which is required for the transforming poten-tial of HVS, is replaced by K1, the recombinant virus canimmortalise primary T lymphocytes in vitro [16].

Whether K1 is expressed in KS spindle cells in vivo hasnot yet been demonstrated. However, K1 mRNAs are notexpressed in PEL cell lines in vitro but are upregulatedduring lytic viral replication induced by tetradecanoylphorbol acetate (TPA) treatment. Thus the role of K1 in thepathogenesis of KSHV-associated neoplasmas remains tobe established. KSHV reactivation in cultured cells hasbeen shown to be upregulated by drugs such as ionomycinthat upregulate intracellular calcium. This could be theresult of K1 action. Enhancement of ITAM signalling in Bcells may therefore result in viral and/or cellular geneactivation. In addition, K1 signalling pathways could pro-vide paracrine signals that stimulate proliferation or angio-genesis in neighbouring cells.

6. v-IRFs

Transcriptional regulation by interferon signalling ismediated through interferon regulatory factors (IRFs), afamily of cellular DNA-binding proteins that act as activa-tors or repressors of promoters of genes containing specificinterferon-stimulated response elements. KSHV potentiallyencodes four ORFs (v-IRFs) that show partial homologywith cellular transcription proteins from the IRF family [4](figure 1 and table II). So far, the function of two of thesev-IRFs has been studied: v-IRF-1 and v-IRF-2. v-IRF-1 is a449-amino acid protein with 13% overall homology withmembers of the IRF family. The best homology (70%identity) is found between a region of v-IRF-1 (amino acid88 to 121) and the conserved N-terminal DNA-bindingregion of the IRF family of proteins, although only two offour conserved tryptophan thought to be involved in DNA-binding are positionally conserved.

v-IRF-1 was the first KSHV protein shown to havetransforming effects [18]. At least part of this effect seemsto be due to v-IRF-1 interfering with IFN signalling byinhibiting IRF-1- and IRF-3-mediated transcription [19,20]. Despite the homology of part of v-IRF-1 with theDNA-binding region of cellular IRFs, v-IRF-1 does notseem to bind DNA. However, v-IRF-1 can associate withcertain members of the IRF protein family, such as IRF-1 orthe interferon consensus sequence binding protein [20]. Italso targets the transcriptional co-activators p300 and CBP(CREB-binding protein) [20, 21]. CBP/p300 are essential

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co-activators of a variety of transcriptional activators,including several members of the IRF family, such as IRF-1and IRF-3. p300 and CBP have generally indistinguishableactivities. Interestingly, although vIRF-1 synergizes withCBP to transactivate the mouse myc promoter, p300 sup-presses v-IRF-1 transactivation [21]. Thus, v-IRF-1 couldexert its effects by direct heterodimerization with membersof the IRF family or by competitive interaction with cellu-lar transcription factors binding to CBP/p300 co-activators.

A second KSHV-encoded v-IRF protein, v-IRF-2,recently characterized by Burysek et al. [22], encodes a163-amino acid protein. V-IRF-2 is a DNA-binding proteinwith a binding specificity distinct from that of cellular IRFs,as it binds to NF-κB binding sites, but not to interferon-stimulated response elements. Recombinant v-IRF-2 formshomodimers but also interacts in vitro with cellular IRFssuch as IRF-1, IRF-2 and interferon consensus sequencebinding protein and with p300 and NF-κB. In transienttransfection assays v-IRF-2 exerts an inhibitory effect bothon IRF-1/IRF-3-mediated transcriptional activation of theinterferon-g gene promoter and on the NF-κB-activation ofthe HIV long terminal repeat [22]. It has not yet beenreported whether v-IRF-2 is a transforming protein likev-IRF-1.

Thus, v-IRF-1 and v-IRF-2 have both common andnon-overlapping functional properties. In addition to thesefunctional differences, the expression profile of theirrespective mRNAs is also different: v-IRF-1 is expressedfrom a unique 1.5-kb mRNA that is constituvely expressedin PEL cells and upregulated after treatment with TPA, thispattern being consistent with expression of this mRNAduring the lytic phase. On the other hand, v-IRF-2 mRNAs,are expressed at low levels in PEL cells and their expres-sion is not increased by TPA treatment. v-IRF-1 and v-IRF-2could thus exert their function at different moments duringthe KSHV viral cycle [22].

7. v-cyclin

The KSHV-encoded cyclin (v-cyclin) is a 257-aminoacid protein with strong homology to cellular type Dcyclins. It shows 31–32% identity (53–54% similarity)with cyclin D2 [23]. Cellular cyclins are proteins thatregulate cell proliferation and cell cycle progression byassociating with and activating specific cyclin-dependentserine/threonine kinases (CDKs). Different CDK-cyclincomplexes regulate the passage through sequential cellcycle transitions. The KSHV v-cyclin associates predomi-nantly with Cdk6 and forms an active complex that phos-phorylates GST-Rb and histone H1 in vitro, whereas Cdk6activated by cellular D cyclins phosphorylates only Rb.The association of v-cyclin with Cdk6 has been shown toalter the substrate specificity of the Cdk6 kinase [23, 24].Furthermore, and again in contrast to the D-cyclin/Cdk6complexes, v-cyclin/Cdk6 complexes are resistant to inhi-bition by CDK inhibitors such as p21Cip, p27Kip andP16INK4a. Ectopic expression of the KSHV cyclin preventsG1 arrest imposed by each inhibitor and stimulates cellcycle progression of quiescent fibroblasts [25]. The resis-tance of the v-cyclin-Cdk6 complexes to inhibition by

p27Kip appears to be due to the inability of the inhibitor tointeract efficiently with the cyclin subunit [25]. Further-more, the v-cyclin-Cdk6 complex phosphorylates p27Kip

on a specific C-terminal threonine. This phosphorylationleads to the destabilization of p27 Kip and is necessary toachieve full cell cycle progression [26]. Thus, KSHV-cyclin associated with Cdk6 can first initiate the cells toenter the S phase even in the presence of cdkI and second,by its inactivation of p27 Kip, can also stimulate full cell-cycle transit, probably through other cyclin-dependentkinases.

v-cyclin transcripts can be detected by reversetranscription-PCR in biopsy specimens from both AIDS-related or classical KS and by in situ hybridization in 1% ofspindle cells from early patch lesions and approximately60% of the spindle cells in late nodular lesions of KS [27].Furthermore, a v-cyclin specific activity, i.e., phosphory-lation of both Rb and p27 Kip, has been detected in extractsof two PEL cell lines and a primary KS biopsy, whichsuggests the implication of this gene in the oncogenicproperties of the virus [26].

8. KSHV cytokines8.1. v-IL-6

The KSHV interleukin-6 homologue (v-IL-6) is a 204-amino acid protein encoded by orfK2. It has conservedimportant features of cellular IL-6, such as cysteine resi-dues involved in disulphide bridging of an amino-terminalsignal peptide and the region involved in receptor binding[28].

v-IL-6 binds and promotes proliferation of an IL-6-dependent human myeloma cell line, INA-6 [29] andinduces acute-phase gene expression in hepatic cell lines[30]. Similarly to cellular IL-6, v-IL-6 activates specificJAK/STAT signalling via interactions with the gp130 signaltransducing subunit, but independently of the IL-6Rα chain[31]. However, the IL-6R component does augment v-IL-6activity and enables signal transduction by v-IL-6 througha gp130 mutant that is otherwise nonfunctional [32].

In athymic mice, NIH3T3 cells expressing v-IL-6 giverise to tumours more rapidly than do control cells; thesetumours are more vascularised than the controls and asso-ciated with a higher level of VEGF. Such inoculated micedisplay increased haematopoiesis in the myeloid, eryth-roid and megakaryocytic lineages [33].

v-IL-6 transcripts are induced by TPA in PEL cell lines,which associates them with the lytic cycle of the virus.Whereas high levels of v-IL-6 RNAs are found in PEL andMCD, v-IL-6 expression cannot be consistently detected inspindle tumour cells in KS lesions [34]. The high expres-sion of v-IL-6 in PEL and MCD, together with the fact thatcellular IL-6 is a B-cell growth and differentiation factorwhose altered expression has been linked to myeloid andlymphoid malignancies, suggests that v-IL-6 could play anessential role in the pathogenesis of PEL and MCD as anautocrine and paracrine factor.

8.2. v-MIP

Chemokines are a superfamily of chemotactic proteinswith a role in regulating leukocyte trafficking. Three KSHV

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genes, orfK6, orfK4 and orfK4.1, referred to respectively asv-MIP-I, v-MIP-II and v-MIP-III, have homologies withhuman �-chemokines. The highest homology is foundwith the human macrophage inflammatory protein-1α(huMIP-1α). v-MIP-I and v-MIP-II share only 49% aminoacid identity. However, they are more closely related toeach other than to any cellular protein of the family, whichsuggests that they may have evolved by gene duplication.

v-MIP-II is similar to human MIP chemokines in that itblocks infection of human immunodeficiency virus-type I(HIV-1) [35]. v-MIP-II was shown to inhibit cell entry ofHIV-1 mediated by CCR3, CCR5 or CXCR4 coreceptors[36]. v-MIP-II appears thus to bind to a wider spectrum ofhuman CC and CXC chemokine receptors than any of themammalian chemokines. v-MIP-II has been suggested toact as an agonist for CCR3: it can activate and chemoat-tract human eosinophils by the way of CCR3 [35]. v-MIP-IIis also a selective chemoattractant for T helper 2 (Th2)cells and for monocytes. It shows agonist activity forCCR8, a receptor selectively expressed on polarized typeII cells [37]. Furthermore, v-MIP-II has been reported to actas a potent antagonist for other receptors, including CCR5[36].

If v-MIP-II is promiscuous in its binding profile, v-MIP-Ion the contrary, selectively engages CCR8, a receptor thatis preferentially expressed on Th2 T cells [38, 39]. v-MIP-Iinduces calcium mobilization in cells expressing CCR8,and these cells respond strongly to v-MIP-I in an in vitrochemotaxis assay [38]. In agreement with its restrictedbinding profile, v-MIP-I, unlike v-MIP-II, has no effect onCCR5-mediated HIV infection [38].

Interestingly, both v-MIP-I and v-MIP-II induce angio-genesis to the same extent as VEGF, in chick embryos,whereas cellular CC chemokines MIP-1α and RANTEShave no effect [35].

Given the different properties of the v-MIPs describedabove, it is tempting to hypothesize an important para-crine role for these chemokines in the development of KSwhich is associated with an important angiogenesis. More-over, accumulation of Th2 T cells has been reported inKaposi’s sarcoma lesions [40]. In PEL cells, v-MIP tran-scripts are associated with the virus lytic cycle.

8.3. v-GPCR

orf74 encodes a 342-amino acid protein that has homol-ogy to many mammalian G-protein coupled receptors(GPCRs). The highest homology is with CXCR1 andCXCR2, receptors for interleukin-8 (IL-8), an endothelialcell chemokine and angiogenic factor. KSHV-GPCR exhib-its constitutive signalling via activation ofphosphoinositide-specific phospholipase C and stimulatescell proliferation [41]. KSHV-GPCR expression in NIH3T3cells leads to cell transformation and the transformed cellsare tumorigenic in nude mice. v-GPCR constituvely acti-vates two protein kinases, JNK/SAPK and p38 MAPK [42].Activation of JNK/SAPK appears to be mediated by KSHV-GPCR inducing tyrosine phosphorylation of RAFTK, therelated adhesion focal tyrosine kinase [43]. KSHV-GPCRalso induces the secretion of the angiogenic factor, VEGF.KSHV-GPCR could therefore participate in autocrine or

paracrine stimulation resulting in the induction of the KSangiogenic and proliferative phenotype [42].

Signalling by KSHV-GPCR in COS-1 cells and inNIH3T3 cells can be inhibited by coexpression of certainGPCR-specific kinases and activation of protein kinase C[44]. Moreover, v-GPCR binds a wide range of CXC andCC chemokines and its activity can be modulated by thesechemokines: interleukin-8 (IL-8) and the growth-relatedprotein-alpha have been shown to activate KSHV-GPCRbeyond constitutive levels [45]; HuIP-10 (humaninterferon-γ-inducible protein 10) and the stromal cell-derived factor 1 alpha (SDF-1α) specifically inhibit KSHV-GPCR-induced signalling [46, 47]. Interestingly, the KSHV-encoded vMIP-II protein also inhibits KSHV-GPCRsignalling [47]. The N-terminus of v-GPCR has been shownto be important for chemokine regulation of GPCR signal-ling but not for constitutive activity [48].

The v-GPCR gene is expressed early during the lyticcycle. It is encoded by a single bicistronic mRNA togetherwith K14 (figure 1). The positioning of GPCR at the 3’ endof the bicistronic mRNA suggests that its expression maynot be very efficient. As overexpression of v-GPCR in Coscells has been shown to induce cell death it may beimportant to keep a low level of v-GPCR in the cells [49].In KS, v-GPCR expression has been shown to be associ-ated with lytic gene expression in a subpopulation ofspindle cells. The majority of spindle cells, however, donot express v-GPCR mRNAs [49].

9. Antiapoptotic proteins9.1. v-Flip

KSHV Flip belongs to a new class of proteins withantiapoptotic properties. It was first characterised inviruses, and thus referred to as viral FLICE-inhibitory pro-tein (v-Flip). More recently, several cellular homologueshave been identified [50]. These proteins contain twodeath-effector domains which interact with the FADDprotein. This interaction inhibits the recruitment and acti-vation of the FLICE protease. Cells expressing v-Flip (fromEHV-2: equine herpes virus 2) are protected against apo-ptosis induced through CD95 or the death receptorsTRAMP and TRAIL-R. Although similar experiments withKSHV Flip have not as yet been reported, it is likely to havethe same properties.

The KSHV Flip protein is encoded by orfK13, frommulticistronic mRNAs: a tricistonic cDNA contains thelatency-associated nuclear antigen, v-cyclin and v-Flipgenes and a bicistronic mRNA 2-kb species presumablyencodes v-cyclin and v-Flip [10]. Sequences encodingv-Flip are expressed at low level in early stages of KS butincrease dramatically in late-stage KS. This increase isassociated with a diminution of apoptosis in KS lesions[51].

9.2. v-Bcl-2

orf16 of KSHV has been proposed as a potential homo-logue of Bcl-2 by sequence analysis [52]. The homologybetween ORF16 and Bcl-2 is not high (15–20%), but isconcentrated within two conserved motifs: Bcl-2 homol-

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ogy 1 and 2 (BH1 and BH2). These motifs have beenshown to be essential for the death-repressor activity ofBcl-2 and Bcl-XL [53].

Accordingly, overexpression of v-Bcl-2 inhibits apopto-sis induced by Sindbis virus as efficiently as Bcl-2 andBcl-XL. However, v-Bcl-2 does not homodimerize or het-erodimerize with other Bcl-2 homologues such as Bax orBak that are known to negatively regulate the antiapop-totic activity of Bcl-XL [53].

KSHV from primary cells established from KS biopsiescan be transmitted to the EBV-negative B-lymphoma cellline Louckes and replicates in these cells (referred to asBKS-1 cells), although only low virus titres are obtained.Interestingly, 293 cells infected with BKS-1-derived virusesundergo apoptosis: 82% cells in apoptosis three dayspostinfection. However, 293 cells constitutively express-ing the KSHV-Bcl-2 protein are protected from KSHV-induced apoptosis [54]. Thus, v-Bcl2 can actually protectcells from apoptosis induced by KSHV infection. As v-Bcl2appears to be expressed only during lytic replication [53],it may be essential to prolong cell survival until completevirions are released from the cells.

10. KSHV reactivation

KSHV-associated pathologies are the results of an equi-librium between latent and lytic infection. One KSHVgene involved in the disruption of viral latency and acti-vation of the lytic cycle is the orf50 immediate early gene[55]. orf50 is a homologue of the EBV BRLF1 gene encod-ing the R transcription factor [56]. orf50 activates KSHVearly genes implicated in lytic viral DNA replication. Italso activates expression of v-IL-6, K12 and other viralgenes thought to be implicated in cell transformation.ORF50 is a phosphorylated nuclear protein of 691 aminoacids (110 kDa on SDS-PAGE).

In addition to the BRLF1 gene, EBV expresses a gene,called BZLF1, whose product, the EB1/Z protein, is alsoresponsible for the switch from latency to the lytic cycle[57]. Furthermore, Z trans-activates OriLyt-dependentDNA replication. Characterization of the transcriptionpattern in the KSHV region colinear with the BRLF1/BZLF1coding region (figure 2), led to the identification of aputative Z homologue, called K8. Like EBV Z, K8 is amember of the bZip protein family and forms homodimersin solution [58]. However, no function has as yet beenascribed to this protein, either in the activation of KSHVgenes or in the activation of KSHV replication.

In addition to ORF50 and K8, KSHV also encodes twoother putative transcription factors, ORFK3 and ORFK5,which have homology to the bovine herpes virus immedi-ate early genes. The function of these proteins is stillunknown.

11. KSHV in vitro infection models

In vitro, most cell lines established from KS tissues loseKSHV during continuous propagation, which suggests thatif KSHV is involved in cell transformation, it is no longer

essential for cell propagation in vitro. However, in vitrocellular transformation by KSHV has recently beenreported by two groups: Flore et al. [59] have infectedhuman primary endothelial cells with purified KSHV andfound that the cells could proliferate in the long term.Interestingly, these authors found that KSHV was presentin only a subset of cells, and that conditioned mediumfrom these cell cultures could induce the expression ofVEGF from human primary endothelial cells. These resultsconfirmed the hypothesis that a paracrine mechanismcould contribute to the development of Kaposi’s sarcoma.Moreover, the infected cells formed colonies in soft agarand acquired telomerase activity, showing that the infectedcells are transformed. Moses et al. [60] also succeeded ininfecting dermal endothelial cells with KSHV. In theirstudy however, the endothelial cells were first immortal-ised with the E6 and E7 genes of HPV type 16. In thismodel, the majority of the cells were latently infected byKSHV, whereas a minority of the cells were spontaneouslyentering the lytic cycle. This pattern of KSHV expression isreminiscent of what is observed in vivo in the KS spindlecell population. In addition, the infected cells acquired thespindle morphology seen in Kaposi’s sarcoma. Thus, suchin vitro systems should be very useful to study the contri-bution of KSHV in the development of Kaposi’s sarcomaand to define the role of KSHV individual genes in cellproliferation.

12. Conclusion

Two hypotheses, not as yet resolved, have been pro-posed to define how KSHV can induce cell transforma-tion. Some observations are in favour of a direct role ofKSHV in the infected cells, whereas others are in favour ofan indirect role: the release of paracrine factors that affectneighbouring cells. By in situ hybridization with strand-specific RNA probes and by immunohistochemistry withspecific antibodies, it has been shown that KSHV isdetected at all stages of the KS lesion. However, expressionof KSHV genes in the early KS lesion is low or not detect-able, suggesting that either KSHV is not the only agentresponsible for cell transformation or that only a fewKSHV-infected cells promote the initiation of the lesion[61].

From this review, it is evident that a number of genesexpressed by KSHV have oncogenic properties. However,only a very limited subset of KSHV genes (LNA, v-cyclin,v-Flip and K12) are expressed in most KS spindle cellslatently infected in vivo. Thus, most of the genes describedas having a role in cell regulation and signalisation will beexpressed only during the lytic/productive cycle of thevirus and consequently in cells destined to die. Interest-ingly, several KSHV genes expressed during the lytic cycleof the virus have paracrine properties or induce cellularparacrine factors such as VEGF, and their release may thusbe important for the development of KS. Accordingly, thenumber of spindle cells supporting KSHV lytic replicationis more important at late stages of KS.

We have also discussed several models of infection/transformation of primary endothelial cells that have been

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described. These models display some of the characteris-tics of the KS tumour. Infection of primary endothelial cellsby mutated viruses is now essential to evaluate the contri-bution of individual KSHV genes to this cell transforma-tion.

Acknowledgments

We apologise in advance for having not cited all thedata which has been published on the topic discussed inthe review. We also wish to thank Dr R. Buckland forreading the manuscript. Our lab is supported by the Insermand by grants supplied by the ‘Association pour la Recher-che contre le Cancer’.

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