HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Rezaee, S. A. R. Articles by Blackbourn, D. J. Search for Related Content PubMed PubMed Citation Articles by Rezaee, S. A. R. Articles by Blackbourn, D. J. Agricola Articles by Rezaee, S. A. R. Articles by Blackbourn, D. J.

Kaposi's sarcoma-associated herpesvirus immune modulation: an overview

¹ Cancer Research UK Institute for Cancer Studies, University of Birmingham, Vincent Drive, Birmingham B15 2TT, UK
² MRC Virology Unit, Church Street, Glasgow G11 5JR, UK

Correspondence
David J. Blackbourn
d.j.blackbourn{at}bham.ac.uk

	ABSTRACT

TOP ABSTRACT REFERENCES

 
Kaposi's sarcoma-associated herpesvirus (KSHV) is the most recently discovered human herpesvirus. It is the aetiological agent of Kaposi's sarcoma (KS), a tumour frequently affecting AIDS patients not receiving treatment. KSHV is also a likely cause of two lymphoproliferative diseases: multicentric Castleman's disease and primary effusion lymphoma. The study of KSHV offers exciting challenges for understanding the mechanisms of virus pathogenesis, including those involved in establishing infection and dissemination in the host. To facilitate these processes, approximately one-quarter of KSHV genes encode cellular homologues or unique proteins that have immunomodulatory roles in cytokine production, apoptosis, cell signalling and the immunological synapse. The activities of these molecules are considered in the present review and the positions of their genes are mapped from a complete KSHV genome sequence derived from a KS biopsy. The understanding gained enables the significance of different components of the immune response in protection against KSHV infection to be evaluated. It also helps to unravel the complexities of cellular and immunological pathways and offers the potential for exploiting viral immunomodulators and derivatives in disease therapy.

Published online ahead of print on 30 March 2006 as DOI 10.1099/vir.0.81919-0.

Introduction
Establishment of virus infection in higher vertebrates necessitates penetration of the host's complex defence barriers against microbial invasion. Innate immunity provides the first line of defence. Antiviral innate immunity involves several effector mechanisms, including type I interferons (IFNs), phagocytes, natural killer (NK) cells and complement activities (reviewed by Goodbourn et al., 2000; Blue et al., 2004; Lodoen & Lanier, 2005). These mechanisms operate while adaptive immune responses, including humoral and cell-mediated immunity, develop. Innate responses also promote development of adaptive immune responses. To establish infection and disseminate in vivo, viruses have to evade these innate and adaptive immune responses.

Viral strategies of immune evasion
Viral strategies for evading the immune response can be considered as passive or active. In this regard, members of the family Herpesviridae, to which Kaposi's sarcoma-associated herpesvirus (KSHV) belongs, can enter into either of two transcriptional programmes: latency or lytic reactivation. Latency is an example of a passive strategy of immune evasion, when a minimal number of gene products are expressed, thus reducing the number of antigens that can be presented to the immune system and invoke a response. Reactivation is the re-entry to productive, lytic replication from latency. During lytic replication, when the bulk of viral proteins are expressed and are susceptible to immune surveillance, as well as upon virus entry to a cell during de novo infection, active evasion strategies are necessary.

Herpesviruses have the genomic capacity to encode numerous genes that actively modulate immune responses. Many of these genes have cellular homologues and are therefore considered as having been captured by the virus, thereafter evolving separately. Table 1 shows a list of KSHV immunomodulatory genes.

View larger version (33K): [in this window] [in a new window]   Fig. 1. KSHV gene map. The genome in virions consists of a unique region (U) flanked at each end by variable numbers of a direct repeat (TR) totalling 35–45, giving a genome size of approximately 170 kbp. Each genome end terminates with a partial copy of TR, such that circularization would create a complete copy. The sequence is depicted here as U followed by a single copy of TR, which is shown in a broader format. Protein-coding regions are indicated by coloured arrows grouped according to the key, with gene nomenclature below and names of the encoded immunomodulatory proteins above (see Table 1). Introns are shown as narrow white bars. The sequence represented was derived by using custom primers from a set of overlapping cosmids obtained from a classic KS biopsy provided by Professor T. F. Schulz, Institut für Virologie, Medizinische Hochschule Hannover, Germany (strain GK18; GenBank accession no. AF148805). Genetic features were derived from the previous annotations (Neipel et al., 1997b; Russo et al., 1996) and analyses published subsequently. The sequence of one cosmid, containing from nt 115410 to the right genome end, was reported by Glenn et al. (1999).

KSHV immune modulation.
Establishment of KSHV latency in vitro occurs with low efficiency and is dependent upon epigenetic factors such as modification of the chromatin structure (Grundhoff & Ganem, 2004). These observations provided a model in which there is a need for continuous rounds of lytic infection in vivo in order to recruit additional latently infected cells, which would otherwise decline as episomes are lost upon cell division. Lytic replication is also necessary for the initial establishment of infection and dissemination in the host. KSHV proangiogenic and proinflammatory gene products (see below) could provide a microenvironment conducive to host-cell proliferation and necessary for viral persistence, secondarily inducing inflammatory responses and pathogenesis. In this context, the evolutionary pressure driving incorporation of immunomodulatory genes into the KSHV genome can be appreciated: lytic replication, which is necessary to maintain infection and replication of the virus, invokes immune responses that, unless negatively modulated, could yield KSHV clearance and lack of persistence. KSHV latency, at least in the context of KS, is therefore only a partially effective strategy of immune evasion and, although arguably sufficient for persistence in B cells, may be defective for KSHV persistence in endothelial cells (Grundhoff & Ganem, 2004). In this regard, the separation of latent and lytic gene-expression profiles may be less demarcated than originally appreciated, as the transcriptional profiles of some genes overlap with both replicative phases. Examples include K9 (Chen et al., 2000; Pozharskaya et al., 2004) and K3 (Taylor et al., 2005). Added complexity was revealed by Krishnan et al. (2004), who reported the concurrent expression of further latent and lytic genes in de novo-infected cells. These genes included some [open reading frames (ORFs) K4, K5, K7 and K11.1/K11] that specify immunomodulatory proteins (Table 1). The import of virion-associated RNAs explains the presence of some (Bechtel et al., 2005). The presence of the remainder is apparently due to their de novo transcription through selective or abortive lytic replication, in which an incomplete lytic cascade of gene expression occurs, but is regulated in ways that we do not yet understand.

Taken together, these observations imply the exertion of selective pressure upon KSHV for the evolution of gene products participating in active immune modulation. These proteins are the subjects of the present review.

KSHV immunomodulatory proteins
Modulators of cytokines and cytokine receptors
The cytokine network constitutes a communication circuit that links and orchestrates the early innate inflammatory responses and the subsequent developing adaptive immune responses to infections. Cytokines also stimulate the development of haematopoietic cells. The anti-cytokine strategies of viruses inhibit either cytokine production or cytokine activity, for example through repressing signal transduction with cytokine and cytokine-receptor mimics (Alcami, 2003). The KSHV-related diseases, particularly KS, are associated with deregulation of the inflammatory-cytokine network (Ensoli & Stürzl, 1998; Nicholas, 2003; Milligan et al., 2004), suggesting the capacity of this virus to intervene in normal cytokine responses. The current section focuses on KSHV modulation of cytokine activities.

Viral IFN-regulatory factors (vIRFs).
The term interferon (IFN) derives from the ability of these cytokines to interfere with virus infection (Isaacs & Lindenmann, 1957). IFNs are a family of multifunctional cytokines that activate transcription of subsets of genes, the induced products of which are responsible for IFN antiviral, anti-proliferative and immunomodulatory properties. There are two types of IFN, I and II (reviewed by Pestka et al., 2004): type I is composed of several classes, but the principal ones that are induced directly by virus infection of many cell types are IFN- and IFN-. Although structurally different, they bind to the same cell-surface receptor and represent the first line of innate immune defence against viruses. Antigen-activated T cells also stimulate mononuclear phagocytes to synthesize type I IFNs. The single type II IFN, IFN-, is produced by NK cells, but more abundantly by activated T cells; it is important in cell-mediated immunity against intracellular microbes. Thus, IFN- is a major component of the adaptive immune response and is involved in upregulation of class I and II major histocompatibility complex (MHC) and other costimulatory molecules, promotion of naïve CD4⁺ T-helper cell (Th) differentiation into type 1 cells (Th1), macrophage activation and B-cell antibody class switching into IgG. The Th1 subset of T cells is fundamental for driving optimal cellular responses against intracellular pathogens and, hence, antiviral immune responses, whilst the CD4⁺ Th2 subset promotes responses such as humoral immunity against extracellular pathogens; each subset may suppress the development or activity of the other (O'Garra & Arai, 2000; McGuirk & Mills, 2002).

A new family of IFNs includes interleukin (IL)-28A, IL-28B (IL-28) and IL-29, also called IFN-1–3, respectively. Their expression is induced by viral infection and they have antiviral functions (Kotenko et al., 2003; Sheppard et al., 2003). IFN-s share homology with IFN- and -, but signal through their own receptor to IFN-stimulated response element (ISRE)-containing promoters, through formation of the IFN-stimulated gene factor-3 (ISGF-3) transcription complex (reviewed by Vilcek, 2003).

The type I IFNs induce the innate immune response to viral infection, known as the ‘antiviral state’ in both infected cells (through autocrine activities) and uninfected cells (through paracrine activities). The main actions of type I IFNs are inhibition of virus replication, increased expression of class I MHC molecules, development of Th1 cells in humans, inhibition of cell proliferation and promotion of apoptosis (reviewed by Goodbourn et al., 2000). Activation of type I IFN can be considered to result in a defence mechanism that bridges innate and T cell-mediated adaptive immunity.

The regulation of the type I IFN-responsive genes, and the type I IFN genes themselves, involves the IFN-regulatory factor (IRF) family of transcription factors that, in humans, contains at least nine members (reviewed by Nguyen et al., 1997; Harada et al., 1998; Pitha et al., 1998; Tanaka & Taniguchi, 2000), the most important of which are arguably IRF-3 and IRF-7 (Taniguchi & Takaoka, 2002). IRFs bind to cognate DNA sequences that include the ISRE present in the promoters of type I IFN-responsive genes and also to positive-regulatory domains (PRD) I and II of the IFN- promoter (King & Goodbourn, 1994; Decker et al., 1997; Stark et al., 1998). Fig. 2 presents an outline of the type I IFN signalling pathways.

View larger version (36K): [in this window] [in a new window]   Fig. 2. An overview of the type I IFN signalling pathways. The mechanisms behind the regulation of IFN-pathway genes are understood with increasing clarity, at least in response to RNA virus infection. A cellular mechanism senses virus infection and triggers the IFN type I pathway to respond (reviewed by Sen & Sarkar, 2005b), as shown at the left side of this illustration. Theinduction and assembly of the IFN- enhanceosome are shown, as it is the prototype complex for understanding IFN-responsive gene activation. It consists of IRF-3, NF-B and ATF-2/c-Jun. These proteins are expressed constitutively. They are activated in response to infection by post-translational modification and assemble on the promoter at their cis-acting cognate recognition sequences, originally identified as positive-regulatory domains (PRDs) I–IV (Goodbourn et al., 2000). Here they act coordinately, but they can also function separately to induce antiviral gene expression. HMG-I(y), CBP/p300 and RNA polymerase II are then recruited to the IFN- enhanceosome as the pre-initiation complex evolves. C-terminal phosphorylation of IRF-3 is a pivotal step between cellular sensing of virus infection and the response to that infection. It occurs by a ‘virus-activated kinase’ (VAK) (Servant et al., 2001; Smith et al., 2001) that promotes translocation of IRF-3 from the cytoplasm to the nucleus. The components of VAK that phosphorylate IRF-3 include the IB kinase homologues IB kinase-epsilon (IKK) and TANK-binding kinase-1 (TBK1) (Fitzgerald et al., 2003; Sharma et al., 2003). The pathway leading to activation of IRF-3 depends on the route of virus entry, which determines whether the viral RNA is ‘sensed’ either by Toll-like receptor (TLR) 3 or one of the RNA helicases, RIG-I (Yoneyama et al., 2004) or mda5 (Andrejeva et al., 2004). If the recognition event occurs through TLR3, this protein interacts with TRIF via the Toll/IL-1 receptor (TIR) domains in each protein. If recognition occurs through RIG-I or mda5, these proteins interact with IPS1 via their respective caspase-recruitment domains (CARD) (reviewed by Crozat & Beutler, 2004; Levy & Marié, 2004; Sen & Sarkar, 2005b). The choice of pathways by IPS1 is cell type-specific. IRF-3 can also form the double-stranded RNA (dsRNA)-activated factor 1 (DRAF-1) inassociation with p300 (Weaver et al., 1998) in cells activated through treatment with dsRNA, which positively regulates IFN-stimulated genes (not shown). Whether KSHV induces similar pathways, for example through the production of dsRNA molecules during gene expression, is unknown and the role of TLR3 in sensing virus infection is controversial (Sen & Sarkar, 2005a). However, KSHV targeting of IRF-3 activity, for example by vIRF-2, implicates the involvement in KSHV entry or replication of at least some of the illustrated components of the ‘cellular-sensing’ mechanism of virus infection. The mechanism could involve TLRs other than TLR3. TLR4 is expressed on the cell surface and uses TRIF and the TRIF-related adaptor molecule (TRAM) to activate IRF3. Other possibilities include TLR2 and TLR9, known to recognize other herpesviruses (e.g. envelope glycoproteins and CpG DNA motifs, respectively) and signal through MyD88, although IRF-3 may still be activated (reviewed by Boehme & Compton, 2004). Once the cell has responded to the infection through the production of type I IFNs, they are secreted and can act in autocrine and paracrine ways to initiate the remainder of the IFN response (right side of illustration). This initiation occurs through the production of IFN-stimulated gene factor (ISGF)-3 and concomitant expression of genes that have IFN-stimulated response element (ISRE)-containing promoters. The products of these genes establish the antiviral state in the infected cell and uninfected bystander cells (reviewed by Stark et al., 1998). This process involves the recruitment and phosphorylation of signal transducer and activator of transcription (STAT)-1 and STAT-2 by IFN receptor-associated tyrosine kinases upon IFN binding. ISGF-3 is formed by the heterodimerization of phosphorylated STAT-1 and -2, followed by their recruitment of p48 (IRF-9). As the ISRE is recognized by IRF-3 and ISGF-3, the genes activated with early kinetics in response to virus infection and those induced by the type I IFNs overlap. IRF-7, inactivated by KSHV ORF45, RTA and possibly LANA-2 (vIRF-3) (see text), is not shown in the figure. This figure was compiled from the reviews by Goodbourn et al. (2000) and Sen & Sarkar (2005a, b).

KSHV encodes four vIRF genes with homology to cellular IRFs (Russo et al., 1996; Neipel et al., 1997a). These genes have probably evolved to subvert cellular IRF signalling, but other activities cannot be excluded and may therefore explain why the virus carries so many of these genes. It is also possible that certain of the vIRF genes are expressed preferentially in different cell types or during different stages of the virus life cycle (Dittmer, 2003). The vIRF genes are not unique to KSHV, as rhesus rhadinovirus encodes nine, none of which appears to be spliced (Searles et al., 1999; Alexander et al., 2000). KSHV vIRF1, vIRF2 and vIRF3 have been cloned and characterized functionally, whilst vIRF4 (K10/K10.1) has been detected by gene array (Jenner et al., 2001), Northern blot and RT-PCR analyses (Cunningham et al., 2003), but the protein remains to be characterized. A description of our current understanding of the three characterized vIRF proteins follows.

   vIRF-1.
K9, specifying the vIRF-1 protein, was the first viral member of the IRF family to be described (Moore et al., 1996). K9 is an unspliced, lytic-cycle gene expressed at a low level in PEL cell lines in those cells in which KSHV reactivation is occurring spontaneously; expression can be enhanced by treatment with TPA (Moore et al., 1996; Sarid et al., 1998; Jenner et al., 2001; Paulose-Murphy et al., 2001; Cunningham et al., 2003). K9 transcripts have been detected in KS biopsies by RT-PCR (Dittmer, 2003), but not by Northern blot analysis (Gao et al., 1997), and analyses of KSHV-associated tumour material revealed that the vIRF-1 protein was only detectable in MCD (Parravicini et al., 2000).

vIRF-1 negatively regulates IFN signalling in the cell. Thus, in reporter-gene studies, vIRF-1 inhibited IFN signalling from type I and type II IFN-responsive reporter genes, although not by a mechanism that involves DNA binding (Gao et al., 1997; Flowers et al., 1998; Zimring et al., 1998). Cellular IRFs contain a conserved N-terminal DNA-binding domain (Escalante et al., 1998) (as well as a C-terminal regulatory region) that is partially conserved in vIRF-1, but apparently does not serve this function. vIRF-1 inhibits IFN induction of responsive genes by suppressing the transcriptional activity of IRF-1 and IRF-3, interacting with them directly or competing for their binding to the transcriptional coactivator p300 (Burek et al., 1999b; Lin et al., 2001). The vIRF-1 protein may also inhibit the histone acetyltransferase activity of p300, restricting chromatin remodelling and therefore transcriptional activity of cellular genes, including those encoding cytokines (Li et al., 2000). Nevertheless, it is debatable whether the kinetics of K9 expression in KSHV-infected cells are consistent with an effective anti-IFN response (Pozharskaya et al., 2004). The multifunctional nature of vIRF-1 can be appreciated from other studies indicating that, in addition to its role in inhibiting transcription, this protein can act as a transcriptional activator (Roan et al., 1999).

Furthermore, vIRF-1 has transforming activity; it reduced the inducible cyclin-dependent kinase inhibitor (CDKI) p21^WAF1/CIP1 and transformed NIH3T3 cells to become tumorigenic in nude mice (Gao et al., 1997). Further evidence in support of an oncogenic role is the fact that vIRF-1 suppresses the transcription and pro-apoptotic activities of p53 (Nakamura et al., 2001; Seo et al., 2001); it also facilitates degradation of p53 by the proteasome (Shin et al., 2006). The involvement of vIRF-1 in immune escape, as well as modulating the cell cycle, is supported by its inhibition of Fas ligand (CD95L) expression and concomitant T-cell antigen receptor (TCR)/CD3-mediated activation-induced cell death (Kirchhoff et al., 2002). Moreover, by binding to and suppressing the activity of the smad3 and smad4 transcriptional components of the TGF- signalling pathway, vIRF-1 suppresses this ‘anti-tumour’ cellular-defence strategy (Seo et al., 2005).

   vIRF-2.
The first functional studies of this protein were performed with a 163-residue protein encoded by K11.1, the first exon of vIRF2 (Burek et al., 1999a). This protein bound to a consensus NF-B-binding site, but not to the ISRE, and suppressed IRF-1- and IRF-3-driven activation of an IFN- reporter promoter in cells infected with Newcastle disease virus. In pull-down assays, this fragment of the vIRF-2 protein also interacted with cellular IRF-1 and weakly with p300/CREB-binding protein (CBP), p65, IRF-2 and IFN consensus sequence-binding protein (ICSBP)/IRF-8; it did not bind IRF-3. This group went on to show that K11.1 is a 20 kDa protein that exerts its anti-IFN effect in part by binding to, and suppressing, double-stranded RNA-activated protein kinase R (PKR) (Burek & Pitha, 2001). Other workers showed that, like vIRF-1, K11.1 inhibited apoptosis by transcriptional repression of CD95L (Kirchhoff et al., 2002).

vIRF2 is now known to encode an inducible, 2.2 kbp, spliced transcript representing the two exons K11.1 and K11 (Jenner et al., 2001; Cunningham et al., 2003) from which full-length vIRF-2 protein is translated. Others have also found vIRF-2 to be inducible (Sarid et al., 1998; Paulose-Murphy et al., 2001; Fakhari & Dittmer, 2002). In contrast, some suggested that expression of this gene is constitutive (Burek et al., 1999a; Burek & Pitha, 2001). Our functional studies indicate that the full-length vIRF-2 protein inhibits both IRF-3- and type I IFN-driven signalling pathways, as well as signalling induced by IFN- family members (Fuld et al., 2006). These functions are consistent with the expression of vIRF2 being detectable as early as 2 h (the earliest time point studied) following experimental infection of cells (Krishnan et al., 2004) and with vIRF-2 mitigating the innate IFN response during KSHV de novo cell infection. IRF-3 deregulation by viruses is not unprecedented; other examples include, but are not limited to, Hepatitis C virus (Foy et al., 2003) and Bunyamwera virus (Weber et al., 2002).

   vIRF-3.
This protein is encoded by a transcript spliced from K10.5 and K10.6 (Lubyova & Pitha, 2000; Jenner et al., 2001; Rivas et al., 2001; Cunningham et al., 2003). As with vIRF2, there is controversy in the literature as to whether vIRF3 is inducible in PEL cell lines: two groups claim that it is (Lubyova & Pitha, 2000; Jenner et al., 2001) and three that it is not (Rivas et al., 2001; Fakhari & Dittmer, 2002; Cunningham et al., 2003). However, only two published studies have documented the function of the vIRF-3 protein. It was observed initially that vIRF-3 decreased the transcription of the type I IFN genes by targeting IRF-3 and IRF-7 (Lubyova & Pitha, 2000). Subsequently, this group found that vIRF-3 transactivated genes under the transcriptional control of IRF-3 and IRF-7 (Lubyova et al., 2004), thus contradicting the concept that the vIRF proteins inhibit the IFN response.

The vIRF-3 protein has also been named latency-associated nuclear antigen 2 (LANA-2), consistent with its expression kinetics and cellular location and in order to distinguish it from the ORF73-encoded LANA (Rivas et al., 2001). These authors found LANA-2 in the nuclei of B cells of PEL and MCD; it was not expressed in KS and they showed that it inhibited p53-induced transcription and apoptosis.

ORF45 and RTA.
The ORF45 virion-associated (tegument) protein blocks phosphorylation, nuclear translocation and therefore the function of IRF-7 (Zhu et al., 2002; Zhu & Yuan, 2003). ORF45 inhibits the activation of type I IFNs and their genes during viral infection, as IRF-7 is a fundamental component orchestrating the IFN response (Honda et al., 2005). Its introduction into the cell as part of the virion ensures that ORF45 is available at the very earliest stages of infection, when deregulating IRF-7 activity is key. It may complement the activities of the vIRF proteins, which are expressed during virus replication. In addition, as cellular IRFs are multifunctional, also being involved in cell-cycle regulation, apoptosis and tumour suppression or promotion (Taniguchi & Takaoka, 2002), the role of ORF45 in regulating IRF-7 activity may not be limited to negatively regulating type I IFNs. The apparent importance to KSHV of the ability to modulate IRF-7 activity is evident from the observation that the RTA protein also negatively regulates IRF-7, by targeting it for proteasome-mediated degradation (Yu et al., 2005).

vIL-6 (K2).
Human IL-6 is a multifunctional cytokine with effects on a wide variety of cell types, particularly those involved in both innate and adaptive immunity. Overexpression of IL-6 has been implicated in the pathology of a number of proliferative diseases, including multiple myeloma, Castleman's disease and some autoimmune diseases, such as rheumatoid arthritis, psoriasis, post-menopausal osteoporosis and colitis. In response to microbes and to other cytokines, particularly IL-1 and tumour necrosis factor (TNF), IL-6 is synthesized by mononuclear phagocytes, vascular endothelial cells, fibroblasts and other cells. The receptor for IL-6 consists of a cytokine-binding protein, IL-6R (gp80), and a signal-transducing subunit, gp130. Receptor engagement by any member of the IL-6 family results in homodimerization of gp130 or heterodimerization of gp130 and gp80. The ensuing JAK/STAT signalling pathway (reviewed by Hodge et al., 2005) in turn induces the synthesis of acute-phase proteins, contributing to the systemic effects of inflammatory reactions during innate immunity. In adaptive immunity, IL-6 may be involved in the development of some autoimmune diseases; also, it stimulates the growth of B lymphocytes that have differentiated into antibody producers. In addition, human IL-6 has anti-apoptotic activity in some cells, mediated by upregulation of apoptotic antagonists such as BCL-X_L (Schwarze & Hawley, 1995; reviewed by Hodge et al., 2005).

The role of IL-6 in KS and MCD had been suspected prior to the discovery of KSHV. Thus, IL-6 enhanced the proliferation of KS cells in culture and elevated levels of IL-6 correlated with disease development in MCD (Miles et al., 1990; Burger et al., 1994). Hence, the discovery of a KSHV homologue (vIL-6) of this cytokine, encoded by K2 and sharing 25 % identity with human IL-6 (Moore et al., 1996; Neipel et al., 1997b; Nicholas et al., 1997b; Burger et al., 1998), implicated the viral cytokine in KSHV pathogenesis. However, the kinetics of expression are complex: although KSHV vIL-6 is expressed constitutively in PEL cell lines in vitro and is minimally inducible (Parravicini et al., 2000), in biopsy material, the expression profile is restricted to haematopoietic cells (Moore et al., 1996; Staskus et al., 1999). Thus, vIL-6 is rarely detectable in KS, expressed in <5 % of cells in PEL tumours and present in up to 25 % of LANA-1-positive cells from MCD (Cannon et al., 1999; Staskus et al., 1999; Parravicini et al., 2000). In other studies, vIL-6 acted as an angiogenic factor through the induction of vascular endothelial growth factor (VEGF) (Aoki et al., 1999; Hideshima et al., 2000; Müllberg et al., 2000) and was abundant in the effusions of PEL (Aoki et al., 2000). Many myeloma cells secrete IL-6 as an autocrine growth factor, which led to the erroneous aetiological link between KSHV and multiple myeloma. The first in vivo evidence for immune modulation per se by vIL-6 was provided by a model of peritoneal inflammation that mimics bacterial peritonitis, in which administration of vIL-6 inhibited chemokine-driven recruitment of neutrophils (Fielding et al., 2005).

Unlike human IL-6, vIL-6 binds directly to the shared human cytokine-signalling receptor gp130, independently of gp80 (Molden et al., 1997; Chatterjee et al., 2002). However, vIL-6 can also form a hexameric vIL-6–gp80–gp130 complex with enhanced signalling potency (Boulanger et al., 2004), activating the STAT and MAPK signalling pathways (Osborne et al., 1999).

Thus, in terms of pathogenesis, the lack of gp80 requirement for signalling suggests that vIL-6 has broader cell specificity than its human equivalent, potentially substituting for or synergizing with IL-6 and contributing to KSHV-associated MCD and perhaps PEL by promoting cell survival, driving proliferation and preventing apoptosis (Nicholas et al., 1997b). At least for MCD, this hypothesis is consistent with the established relationship between elevated IL-6 and MCD (reviewed by Waterston & Bower, 2004). However, in the absence of abundant vIL-6 expression in KS lesions, it seems unlikely that it contributes significantly to the pathogenesis of this tumour.

Viral macrophage inflammatory proteins (vMIPs, vCCLs).
The chemokines form a large family of cytokines that co-ordinate leukocyte movement and regulate the recruitment of leukocytes to the site of inflammation (reviewed by Rot & von Andrian, 2004). At least 43 human chemokines have been identified, excluding isoforms. They mediate their biological effects by binding to and signalling through a family of G protein-coupled receptors, which numbers 19 so far. The response common to most cells carrying receptors following their ligation by the majority of chemokines is that of chemotaxis.

KSHV ORFs K6, K4 and K4.1 encode three chemokine homologues (Russo et al., 1996; reviewed by Nicholas, 2005), formerly referred to as viral macrophage inflammatory proteins (vMIP-I, vMIP-II and vMIP-III) and now known as vCCL-1, vCCL-2 and vCCL-3, respectively. They are lytic-cycle genes (Nicholas et al., 1997a; Jenner et al., 2001; Paulose-Murphy et al., 2001). KSHV is not the only virus to have evolved mechanisms for interacting with the chemokine network; other examples include the use of chemokine receptors as coreceptors for cell infection by human immunodeficiency virus (HIV) (Fauci, 1996), the production of soluble decoy proteins that sequester chemokines by certain poxviruses (Graham et al., 1997) and murine gammaherpesvirus-68 (MHV-68) (Parry et al., 2000), and the expression of a chemokine homologue by human cytomegalovirus (Penfold et al., 1999; reviewed by Lalani et al., 2000; Murphy, 2001; Nicholas, 2005). The KSHV chemokines share up to 41 % identity at the amino acid sequence level with the CC chemokine macrophage inflammatory protein (MIP)-1, whilst vCCL-1 and vCCL-2 are related more closely to each other, sharing 48 % amino acid identity, than they are to vCCL-3 (Moore et al., 1996; Boshoff et al., 1997). Thus, the cellular homologues from which these viral genes arose probably have yet to be identified (McGeoch, 2001).

Cells of the Th1 and Th2 subsets express different chemokine receptors, hence the predominant chemokine profile can polarize the Th response and consequently the effector T-lymphocyte response. The CC chemokine receptors CCR4 and CCR8, and perhaps CCR3, are thought to be involved in the chemotaxis and polarization of Th2 lymphocytes (reviewed by Cosmi et al., 2001) and the KSHV vCCLs are preferential agonists for these receptors (reviewed by Nicholas, 2005). vCCL-2, but not vCCL-1, activated and triggered the chemotaxis of eosinophils and Th2-like T cells by engaging CCR3 (Boshoff et al., 1997; Weber et al., 2001). Moreover, vCCL-2 functioned as an antagonist of CCR1 and CCR5, inhibiting the recruitment of Th1-like lymphocytes. Consistent with these data, the authors observed a predominance of Th2-type, CCR3⁺ cells in KS lesions (Weber et al., 2001). Likewise, vCCL-1 is an agonist for CCR8, although vCCL-2 is a dominant antagonist (Sozzani et al., 1998; Dairaghi et al., 1999), and vCCL-3 is an agonist of CCR4 (Stine et al., 2000). Overall, these results indicate that, as Th2-cell chemokine-receptor agonists, the KSHV vCCLs can polarize the adaptive immune response toward a predominantly Th2-type (i.e. humoral) response at sites of KSHV infection, potentially reducing the efficacy of the antiviral response.

Aside from their apparent immunomodulatory roles, the vCCLs have at least two other activities. First, vCCL-2 inhibited infection of CD4⁺ cells by dual-tropic, syncytium-inducing HIV strains via CCR5 and CXCR4 and more strongly via CCR3 (Boshoff et al., 1997; Kledal et al., 1997). Second, all three vCCLs are angiogenic in the chick chorioallantoic-membrane assay (Boshoff et al., 1997; Stine et al., 2000). Thus, they may contribute to the development of KSHV-associated diseases (Boshoff et al., 1997; Stine et al., 2000; Liu et al., 2001), cooperating with other growth factors such as the potently angiogenic VEGF that is abundant in KS, where it may function as an autocrine and paracrine growth factor (Cornali et al., 1996; Masood et al., 1997; Nakamura et al., 1997), not least because expression of the gene encoding its receptor is abundant in KS lesions (Brown et al., 1996).

Viral G protein-coupled receptor (vGPCR).
Another homologue of the chemokine system encoded by KSHV is the vGPCR encoded by ORF74. This protein shares similarity with members of the CXC chemokine-receptor family, especially the IL-8 (or CXCL8) receptor, and, like them, is a seven transmembrane-spanning protein (Cesarman et al., 1996; Russo et al., 1996). This viral-receptor homologue is constitutively active, signalling independently of agonist (Arvanitakis et al., 1997), but signalling can be increased in a chemokine-dependent manner (Gershengorn et al., 1998). Interestingly, vGPCR binds both CC and CXC chemokines (Arvanitakis et al., 1997). In HEK 293 epithelial cells, vGPCR activated two of the three major mitogen-activated protein kinase pathways: p38 MAPK and JNK/SAP (not ERK), which are activated by angiogenic and inflammatory cytokines (Bais et al., 1998). vGPCR immortalized endothelial cells through constitutive VEGF receptor-2 expression and activation of the downstream phosphatidylinositol 3-kinase (PI3K)/AKT anti-apoptotic pathway by a VEGF-mediated autocrine loop (Bais et al., 2003). It also induced VEGF-associated KS-like lesions in transgenic animals (Yang et al., 2000).

Taken together, these data suggest that vGPCR is a major determinant of KSHV pathogenesis, having broad signalling activity that transactivates pro-inflammatory and pro-angiogenic cytokine and growth-factor gene expression (Pati et al., 2001; Schwarz & Murphy, 2001). In part, this activity is mediated through NF-B, activator protein-1 (AP-1) and nuclear factor of activated T cells (NFAT) via the small G protein Rac1 (Montaner et al., 2004). Clearly, vGPCR orchestrates complex and cell type-dependent signalling pathways, as demonstrated by work in PEL cells, where it activated AP-1, NFAT and CREB in part through the ERK-1/-2 MAPK pathway (Cannon & Cesarman, 2004).

However, vGPCR is an early lytic-cycle protein, whereas KSHV is predominantly latent in KS and lytic replication leads to host shutoff (see below). Therefore, the role of vGPCR in viral pathogenesis, particularly in KS, is controversial (Cesarman et al., 2000). A possible explanation for this paradox is that the putative low level of continuous lytic replication provides sufficient vGPCR to effect pathogenesis (Grundhoff & Ganem, 2004). Furthermore, vGPCR may negatively regulate the latent to lytic switch in KSHV replication, whilst still exerting paracrine effects on infected and uninfected cells (Cannon et al., 2006).

Nevertheless, KSHV has evolved to use the IL-8R signalling cascade in an agonist-independent manner, as agonist (IL-8) is one of the major mediators of the inflammatory response. Whilst the intracellular-signalling consequences of IL-8 may benefit survival of KSHV-infected cells, as determined by studies of vGPCR, abundant levels of this pro-inflammatory chemokine may be disadvantageous to KSHV, perhaps explaining why IL-8 production is suppressed by another KSHV immune modulator, vOX2 (Rezaee et al., 2005) (see below).

Kaposin B.
This protein is encoded by the K12 locus, which was first described as encoding a very abundant transcript of 0.7 kb, called T0.7, and specifying a 60-residue hydrophobic protein (Russo et al., 1996; Zhong et al., 1996). K12-containing mRNAs are detectable by in situ hybridization in most spindle cells of all stages of KS and in PEL cells during latency and lytic replication (Staskus et al., 1997; Stürzl et al., 1997). Further work clarified that the K12 (or kaposin) locus is complex and that the K12 transcript is always much longer than 0.7 kb, encoding K12 and two different, upstream, GC-rich, direct-repeat sequences termed DR1 and DR2. The number of DR1 and DR2 repeats varies between different KSHV isolates, resulting in variable sizes of the K12 transcript (Sadler et al., 1999). Complex translational regulation of the K12 transcript, including initiation from non-AUG (i.e. CUG) codons, yields a minimum of three protein species, of which kaposin B is the most abundant, at least in the PEL cell line BCBL-1, and encoded by the sequences upstream of K12, but not by K12 itself. Kaposin C is a chimaera of DR1, DR2 and K12, whilst kaposin A is the predicted product of K12, which would need to be initiated from an AUG codon in the absence of an apparent internal ribosome entry site (Sadler et al., 1999).

Kaposin A might be involved in cell transformation (Muralidhar et al., 1998; Tomkowicz et al., 2005), perhaps by activating the ERK-1/2 MAPK signalling pathway (Kliche et al., 2001). However, kaposin B has a distinct immunomodulatory function: increasing the expression of certain cytokines by stabilizing their mRNAs (McCormick & Ganem, 2005). Kaposin B binds to and activates the kinase MK2 in the nucleus through its reiterated DR2 repeats. MK2 kinase is a target of the p38 MAPK pathway and, when activated, inhibits the decay of AU-rich elements (AREs) in 3'-untranslated regions of mRNAs. Inflammatory signals (that activate the p38 MAPK pathway) induced nuclear export of both proteins (MK2 and kaposin B) together, resulting in increased levels of two proteins encoded by ARE-containing mRNAs: IL-6 and granulocyte–macrophage colony-stimulating factor. The molecular mechanisms behind this increase have yet to be elucidated in further detail, but could involve either the selective inhibition of the degradation of ARE-containing transcripts or the selective stimulation of their translation; p38 MAPK may also be recruited to the kaposin B–MK2 complex. The effect was extended to the stabilization of a model ARE-containing transcript in KSHV-infected cells (McCormick & Ganem, 2005). Also of interest is the recent identification of microRNAs encoded within this region of the KSHV genome, including the kaposin locus (Cai et al., 2005; Pfeffer et al., 2005; Samols et al., 2005), which may be involved in the regulation of viral and cellular gene expression, perhaps including genes of the immune system.

Modulation of apoptosis as an effector component of the immune system
Apoptosis, an overview of which is provided in Fig. 3, is an important component of immune-system homeostasis, maintaining immunological unresponsiveness and providing a regulatory mechanism for innate and adaptive immune responses as apoptotic cells are internalized by phagocytes (Albert, 2004). It can be triggered by a variety of inducers. Ligands of the cell-surface death receptors cause them to form the death-inducing signalling complex (DISC) and activate the extrinsic apoptotic pathway. These receptors include the tumour necrosis factor receptor (TNFR) superfamily, one member of which is Fas (CD95). Alternatively, the intrinsic mitochondrial programme of cell death is activated by signals such as peroxides produced by macrophages or neutrophils, cell-cycle inhibitors, DNA damage, viruses and oncogenes.

View larger version (40K): [in this window] [in a new window]   Fig. 3. Apoptosis pathways. The extrinsic pathway of apoptosis (left side of illustration) is triggered by the binding of ligands to death-inducing membrane proteins. The two most well-described death domain-containing receptors are CD95 (Fas) and the type I tumour necrosis factor receptor (TNFR). The extrinsic pathway is initiated by the binding and trimerization of death receptors containing intracellular death domains, to form the death-inducing signalling complex (DISC). This event leads to the recruitment of Fas-associated death receptor (FADD). FADD in turn binds procaspase 8 and activates it, generating the ‘initiator’ caspase 8. Consequently, caspase 8 activates ‘effector’ caspases (caspases 3, 6 and 7). They then cleave proteins responsible for maintaining cellular and genome integrity, as well as repressors of pro-death enzymes, such as inhibitor of caspase-activated DNase (iCAD), thereby releasing CAD that fragments DNA. The result is cell death and phagocytic engulfment. The type I TNFR activates a similar mechanism of cell death. However, this receptor does not bind FADD directly, but rather via the TNFR-associated death domain (TRADD), which in turn recruits FADD to the receptor complex. The extrinsic pathway can be regulated by the intracellular FLICE inhibitory protein (FLIP), which binds to FADD and competes with DISC for caspase 8. FLICE is the acronym for FADD-like interleukin-1-converting enzyme, the name by which caspase 8 is also known. KSHV vFLIP operates similarly. Cellular FLIP was identified through the recognition and study of viral FLIP proteins (Irmler et al., 1997). During conditions of cellular stress, such as DNA damage and growth-factor deprivation, the intrinsic apoptosis pathway (the pathway that is activated most frequently by the tumour-suppressor protein p53) is activated (right side of illustration). The intrinsic apoptotic pathway converges on the disruption of mitochondrial membranes. Sequestered within the intermembrane space of these organelles are pro-apoptotic proteins that, once released, signal the cell-death programme. The prototypical example is cytochrome c. When cytochrome c binds to apoptotic protease-activating factor 1 (APAF-1), pro-caspase 9 is recruited via caspase-recruitment domains (CARD), forming the apoptosome. In this complex, the initiator caspase 9 is auto-activated and, in turn, activates effector caspases, such as caspase 3, releasing CAD. The balance of the pro- and anti-apoptotic bcl-2 family proteins determines whether or not apoptosis proceeds. They are classified based on structural determinants: bcl-2 homology (BH) domains. These proteins include the pro-apoptotic BAX and BAK proteins, which, when activated, oligomerize and increase the permeability of the mitochondrial outer membrane. Anti-apoptotic members of the bcl-2 family, including bcl-2 itself and BCL-XL, regulate these proteins and therefore the intrinsic pathway. Granzyme B, one of the components of cytotoxic granules produced by CD8+ cytotoxic T lymphocytes and NK cells, can trigger apoptosis by activating effector caspase 3. However, it can also operate in a caspase-independent manner, for example by activating CAD directly. There is overlap between the intrinsic and extrinsic apoptotic pathways, ascaspase 8 can cleave and activate the bcl-2 family protein BID, forming the pro-apoptotic truncated BID (tBID). tBID translocates to the mitochondria where it causes oligomerization of BAX and BAK. BID belongs to the ‘BH3-only’ subclass of bcl-2 proteins. Other BH3-only pro-apoptotic proteins include NOXA and PUMA. They are under the transcriptional control of p53 and invoke apoptosis through the intrinsic pathway in response to DNA damage. Regulation of both the intrinsic and extrinsic pathways can also occur through the activities of members of the caspase-inhibitor family, ‘inhibitors of apoptosis' (IAPs), that can regulate both initiator and effector caspases, binding and inhibiting active caspases 3, 7 and 9. One of the most studied of the mammalian members of the IAP family is survivin (Altieri, 2003), not least because of its potential as a therapeutic target for certain malignancies (Schimmer & Dalili, 2005). The mechanism of KSHV vIAP inhibition of apoptosis is unclear, but it localizes to the mitochondria and ER. The mechanism behind the HAX-1 anti-apoptotic function is also still tobe determined fully, but it is cleaved within the mitochondria in response to pro-apoptotic stimuli (Cilenti et al., 2004). K15 may augment thisanti-apoptotic activity. This outline was compiled from recent reviews (Lieberman, 2003; Danial & Korsmeyer, 2004; Lowe et al., 2004).

vFLIP (K13/ORF71).
ORF71, which is also called K13, encodes the KSHV FLIP protein (Thome et al., 1997). The vFLIP gene is expressed in KS and PEL cells from a polycistronic mRNA encompassing the latency locus (Talbot et al., 1999). As such, vFLIP may be an important component of KSHV pathogenesis. Indeed, this protein can inhibit the extrinsic apoptosis pathway by preventing the activation of caspases, including caspase 8 (Djerbi et al., 1999).

The potential role of vFLIP in KSHV pathogenesis is also reflected in its ability to drive cell transformation in vitro (Sun et al., 2003) and in vivo, as mice transgenic for the gene had a 6.5-fold increased incidence of lymphoma compared with their non-transgenic littermates (Chugh et al., 2005). In ectopic-expression studies, the mechanism appeared to involve constitutive NF-B activation (Chaudhary et al., 1999). PEL cells (both primary and from established lines), naturally infected with KSHV, also have constitutive NF-B activity that appears to be essential for cell proliferation (perhaps via IL-6 production) and apoptosis inhibition (Keller et al., 2000). Although other KSHV proteins, including K1, vGPCR and K15, can activate NF-B, it is vFLIP that drives the constitutive NF-B activation in PEL cells and is responsible for their survival in vitro and in vivo (Guasparri et al., 2004; Godfrey et al., 2005). vFLIP activates NF-B through the IB kinase (IKK) complex. It does so via members of the TNF receptor-associated factor (TRAF) family, which are adaptor molecules involved in transducing the signal from TNF receptors to the IKK complex that activates NF-B. Specifically, TRAF2 and TRAF3 are required for vFLIP signalling, but only TRAF2 recruits vFLIP to the IKK complex (Guasparri et al., 2006). Thus, vFLIP is implicated in the pathogenesis of PEL and perhaps KS, and can be considered a tumour growth factor with dual roles of apoptosis prevention and tumour progression.

vBcl-2 (ORF16).
The KSHV vBcl-2 protein is encoded by ORF16 (Russo et al., 1996). It is expressed in late-stage KS lesions, but in PEL cells, only the transcript is detectable and not the protein (Widmer et al., 2002). vBcl-2 shares 15–20 % amino acid identity with human cellular homologues and inhibits apoptosis induced by virus infection and the pro-death protein BAX (Cheng et al., 1997; Sarid et al., 1997). Cellular members of the bcl-2 family often either homodimerize or heterodimerize with other family members, but there are mixed reports as to the capability of vBcl-2 in this regard (Cheng et al., 1997; Sarid et al., 1997; Huang et al., 2002). One possibility is that vBcl-2 does not heterodimerize with other bcl-2 family members, obviating negative regulation by such proteins (Cheng et al., 1997).

Therefore, as a lytic protein, vBcl-2 presumably inhibits apoptosis induced as a consequence of KSHV infection, ensuring that the cell survives long enough for the virus to assemble progeny. It may also counter any pro-apoptotic consequences of other KSHV proteins, such as the ORF72-encoded cyclin D homologue vCyclin (Ojala et al., 2000).

vIAP (K7).
The product of KSHV K7 was identified as a survivin, or inhibitor of apoptosis (IAP), homologue by in silico analyses, followed by ectopic-expression studies that confirmed the apoptosis-inhibiting activity. For these reasons, it was named vIAP. Northern blot and RT-PCR studies of PEL cells revealed K7 to be a lytic gene. The protein localizes to the mitochondria and the ER, although the anti-apoptotic mechanism is unclear; it may operate as a molecular adaptor, recruiting activated caspases to anti-apoptotic regulatory proteins, like Bcl-2 (Wang et al., 2002).

p53 suppression.
The p53 protein is one activator of the intrinsic apoptotic pathway and, not surprisingly, KSHV encodes several proteins that deregulate it. One of the most highly documented is LANA-1, which, in the context of KSHV replication, binds to the viral genome and an acidic region of the histone H2A–H2B dimer of the nucleosome, thereby tethering the episome to chromatin during mitosis and segregating it into daughter nuclei (Ballestas et al., 1999; Barbera et al., 2006). LANA-1 interacts with p53, repressing its transcriptional activity and its ability to induce apoptosis, thereby promoting the cell cycle (Friborg et al., 1999). Indeed, LANA-1 can promote S-phase entry (An et al., 2005), in part by driving the cell cycle through positive regulation of the retinoblastoma (RB) protein/E2F transcriptional pathway (Radkov et al., 2000).

Several other KSHV proteins have been reported to repress the transcriptional activity of p53. They include vIRF-1 and LANA-2 (vIRF-3) (see above), K-bZIP (the product of K8) (Park et al., 2000) and the lytic-switch protein RTA (Gwack et al., 2001).

Taken together in the context of tumour formation, there may be functional cooperation between these proteins that regulate p53 activity and other KSHV proteins. For example, whilst vCyclin is oncogenic, it is so only in the absence of functional p53 (Verschuren et al., 2002).

Regulation of complement
KSHV complement-control protein (KCP).
Complement bridges innate and adaptive immune responses as well as humoral and cell-mediated immunity, and is antiviral. Complement effects on virus infection include (i) lysis of infected cells and enveloped virus via formation of a pore or ‘membrane-attack complex’ in the cell or viral surface, (ii) coating of infected cells and virions with component C3b to enhance phagocytosis or block viral infection and (iii) production of potent anaphylatoxins, which exert a variety of effects on the immune system, including recruitment of inflammatory cells to the site of infection.

Complement activation occurs through the cleavage of pro-enzymes to enable the formation of the C3 and C5 convertase enzymic complexes, with the release of smaller chemoattractant and anaphylatoxin fragments. The covalent attachment of C4b and C3b to pathogen and infected-cell surfaces also enhances recognition by phagocytes and increases the humoral response to those pathogens (Watanabe et al., 2003; Bower et al., 2004). For detailed diagrams of the complement-activation pathways and their regulation, the reader is referred to the review by Blue et al. (2004).

To protect host cells from autologous complement attack, soluble and membrane-bound complement regulators have evolved to limit inflammation to the infected site. An important group of these regulators is encoded in the regulators of complement activation (RCA) gene cluster at chromosome 1 (locus 1q32). All of these proteins, including membrane cofactor protein (MCP; CD46), complement receptor 1 (CR1; CD35), decay-accelerating factor (DAF; CD55), factor H (FH) and C4b-binding protein (C4BP), contain between four and 35 short-consensus-repeat (SCR) domains and share significant homology, as well as complement-inhibition mechanisms (Kirkitadze & Barlow, 2001).

Complement does not require previous antigen exposure to be fully effective and accelerates and enhances the generation of an adaptive immune response to pathogens. Therefore, it represents a potentially important antiviral immune response. This possibility is borne out by the diversity of complement-evasion strategies adopted by viruses that we are now beginning to understand (reviewed by Blue et al., 2004).

The ORF4 gene of KSHV encodes a lytic-cycle protein called KSHV complement-control protein (KCP) that inhibits activation of the complement cascade. KCP consists of four N-terminal SCR domains, a dicysteine motif, a long serine (S)- and threonine (T)-rich domain and a C-terminal hydrophobic element sufficient to act as a transmembrane anchor (Spiller et al., 2003a, b). KCP has also been named kaposica (Mullick et al., 2003). The full protein is 550 residues in length, including a 19-residue signal peptide, but PEL cells naturally infected with KSHV also express two isoforms of KCP in which either the S/T region or the S/T region plus the dicysteine motif is removed through alternative splicing of the ORF4 transcript. All three isoforms retain the four SCR domains and the transmembrane region and all can regulate complement through two separate mechanisms (Spiller et al., 2003a): firstly, by directly accelerating the decay of the classical pathway C3 convertase enzyme complex, and secondly, by acting as a cofactor for factor I-mediated inactivation of C3b and C4b, two components of the C3 and C5 convertases (Blue et al., 2004). Decay of the classical pathway C3 convertase and cofactor activity for C4b inactivation depend on three N-terminal SCR domains (SCR1–3; Spiller et al., 2006) and a positively charged linker region between SCR1 and 2. There is no evidence for an abundant soluble form of KCP, unlike, for example, that encoded by the related rhadinovirus herpesvirus saimiri (Albrecht & Fleckenstein, 1992).

Structurally and functionally, KCP belongs to the RCA protein family. However, KCP may be multifunctional. In addition to being expressed on the surface of naturally and experimentally infected cells, KCP is associated with the envelope of purified KSHV virions, where it potentially protects them from complement-mediated immunity. Furthermore, recombinant KCP binds heparin, an analogue of the known KSHV cell-attachment receptor heparan sulphate, via the SCR1–2 region where it can facilitate KSHV binding to target cells (Spiller et al., 2006).

Deregulating cell–cell contact: the central event of T cell-dependent immune responses
Modulators of the immunological synapse.
Unlike B cells, T cells are not able to recognize soluble antigens. Instead, they recognize antigenic determinants associated with self MHC molecules on the surface of antigen-presenting cells (APCs). When T cells recognize antigenic peptides on APCs, the TCR complex and accessory molecules often colocalize at the site of cell–cell contact. The accessory molecules include those involved in adhesion and coactivation, an intracellular signal-transduction complex and elements of the cytoskeleton. This cluster of molecules at the site of T cell–APC contact has been called either a supramolecular activation cluster (SMAC) (Monks et al., 1998) or an immunological synapse (IS) (Grakoui et al., 1999) and is considered a dynamic structure at which information exchange occurs between the participating cells (Trautmann & Valitutti, 2003; Friedl et al., 2005). Whilst the concept was originally applied to the CD4⁺ T helper-cell synapse, it can also be considered in the context of cytotoxic T lymphocytes (CTLs) (Stinchcombe et al., 2001), B-cell acquisition of antigen (Batista et al., 2001), NK-cell surveillance (Davis et al., 1999) and myeloid lineage-cell interactions (e.g. monocytes/macrophages) (Barclay et al., 2002).

Given the nature of the components, the IS is probably responsible for initiating and sustaining TCR signalling to orchestrate the appropriate immune response, as suggested by Trautmann & Valitutti (2003). The IS may also ensure polarized T-cell cytokine secretion, directing either a Th1 or Th2 response. As the IS is the site of cell–cell contact and the main signalling event that sets thresholds for T-cell activation, these thresholds must be met to ensure either suitable immune responses against pathogens and toxins, including differentiation into effector and memory cells or maintenance of immunological tolerance. If thresholds are set incorrectly, the host is either susceptible to pathogen escape or, conversely, autoimmune diseases. Full T-cell activation, necessary for antigen elimination, occurs once the TCR-induced signals reach a critical threshold, which is dependent on the antigenic-peptide dose, its agonistic activity and the T-cell microenvironment (reviewed by Friedl et al., 2005). Therefore, the T-cell response is also influenced by the nature of the APCs that display peptide–MHC complexes and costimulators to the T cells. For example, mature dendritic cells express high levels of the MHC molecules and the costimulators that provide second signals to T cells. Some APCs may even engage in active processes to manipulate IS formation and stabilization. Nevertheless, following cognate receptor–ligand interaction at the IS, physiological T-cell activation can occur and the process can be divided into a series of temporal stages: T-cell polarization, initial adhesion, IS formation (initial signalling) and IS maturation (sustained signalling).

Thus, IS formation in general, and the signal-transduction process more specifically, are potential targets for intervention to modulate immune responses by modifying either the microenvironment or gene-expression profiles in inducer or responder cells. Concomitantly, the IS could represent a target for viruses to modulate the first stages of immune activation and provide immunological unresponsiveness as an adaptive evasion strategy (Table 2). One aspect of differences in biology between different types of APC concerns the site of the KSHV reservoir. Although not incontrovertible, as other sites are possible, current evidence supports the concept that the major reservoir is the B cell, not least because this is the predominant cell type in the peripheral blood infected with KSHV (Ambroziak et al., 1995). It is conceivable that evolutionary pressure has selected B cells as the site of KSHV latency due to their relative inability to recruit patrolling leukocytes, including T cells (Friedl et al., 2005), another manifestation of KSHV immune evasion.

KSHV modulators of MHC and accessory molecules.
CTLs are the main protagonists of virus elimination, killing virus-infected cells before virions are produced (see Fig. 4). Thus, in the context of virology, their principal function is surveillance against virus infection. Most virus-specific CTLs are CD8⁺ T cells that recognize cytosolic, usually endogenously synthesized, viral antigens in association with class I MHC on any nucleated cell. Full activation and differentiation of CTLs as effector cells, like other lymphocytes, requires at least two signals. The first is the antigenic peptide–MHC complex and the second is either produced by Th cytokines or costimulators expressed on infected cells. Therefore, if a virus inhibits the MHC class I-restricted antigen-presentation pathway, that virus-infected cell would become invisible to CTL surveillance. Even reduced antigen presentation might restrict CTL-activation thresholds from being reached, although as few as three to ten peptide–MHC complexes within the IS can induce target-cell elimination (Purbhoo et al., 2004). However, NK cells are activated by, and can eliminate, those cells displaying reduced MHC class I density (see below). In addition, IFN upregulation of MHC class I protein expression may