HCMV-encoded G-protein-coupled receptors as constitutively active modulators of cellular signaling networks

Review

TRENDS in Pharmacological Sciences

Vol.27 No.1 January 2006

Constitutive Receptor Activity series

HCMV-encoded G-protein-coupled receptors as constitutively active modulators of cellular signaling networks Henry F. Vischer, Rob Leurs and Martine J. Smit Leiden/Amsterdam Center for Drug Research (LACDR), Division of Medicinal Chemistry, Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands

Several herpesviruses encode G-protein-coupled receptor (vGPCR) proteins that are homologous to human chemokine receptors. In contrast to chemokine receptors, many vGPCRs signal in a ligand-independent (constitutive) manner. Such constitutive signaling is of major significance because various pathologies are associated with activating GPCR mutations. Constitutive activity of the human herpesvirus 8-encoded GPCR (ORF74), for example, is essential for its oncogenic potential to cause angioproliferative Kaposi’s sarcomalike lesions. The human cytomegalovirus (HCMV) encodes four GPCRs, of which US28 and UL33 display constitutive activity in transfected, but also HCMVinfected, cells. In addition, US28 is activated by a broad spectrum of chemokines. Furthermore, both US28 and UL33 show promiscuous G-protein coupling, whereas chemokine receptors activate primarily Gi/o proteins. Thus, these vGPCRs are versatile signaling devices, reprogramming cellular signaling networks to modulate cellular function after infection. By these means, these HCMV-encoded receptors might contribute to HCMVrelated pathologies.

HCMV-encoded GPCRs Increasing evidence suggests that human cytomegalovirus (HCMV) contributes to the onset or progression of chronic inflammation, vascular diseases and infection with human immunodeficiency virus (HIV) [1]. HCMV is a widely spread b-herpesvirus, infecting 50–80% of the general population, and can persist lifelong in an asymptomatic latent form. However, primary infection or reactivation of the virus in immunocompromised hosts, such as the developing fetus, transplant recipients or acquired immune deficiency syndrome (AIDS) patients, can have severe implications and be fatal [1]. In fact, co-infection with HCMV and HIV accelerates the progression to AIDS [2]. Corresponding author: Smit, M.J. ([email protected]). Available online 13 December 2005

HCMV entry into host cells requires a series of events to take place between viral and cellular proteins. Glycoproteins gB and gH residing on the virion appear to interact first with cellular heparan sulfates and then with additional host membrane proteins [3,4]. HCMV infection increases host-cell responses, such as inositol phosphate hydrolysis, cAMP production, metabolism of arachidonic acid and activation of several transcription factors, including nuclear factor kB (NF-kB) and cAMP response element (CRE) [3]. Similar to other human b- and g-herpesviruses, HCMV appears to have ‘pirated’ genes encoding key regulatory cellular proteins, several of which exhibit high homology to cellular G-protein-coupled receptor (GPCRs) [5,6]. Because GPCRs are key regulators of cellular signaling, the HCMV-encoded GPCRs appear to be prime candidates for the observed increase of cellular signaling after infection. Thus, HCMV-encoded receptors can be regarded as an effective strategy of HCMV to elude the immune system, redirect cellular signaling networks, modulate cellular function and thus contribute to pathology. Studies using recombinant mouse and rat CMV strains (MCMV and RCMV, respectively) that lack GPCR-encoding genes support this hypothesis because reduced replication of the virus in salivary glands and a lower mortality in infected animals is observed [7–9]. Evolution of HCMV-encoded GPCRs GPCRs have key roles in cellular (patho)-physiology by responding to a diversity of external stimuli to generate an integrated cellular response via activation of heterotrimeric G proteins, which in turn can either stimulate or inhibit the activity of a wide range of cellular effectors [10]. In addition, many GPCRs can constitutively activate intracellular signaling pathways in the absence of external stimuli [11] (Box 1). Constitutive GPCR signaling is of major patho-physiological significance because various proliferative diseases are attributed to activating GPCR mutations [12]. The constitutive activity of the human herpesvirus 8-encoded receptor ORF74, for example, appears to have an essential role in the initiation and progression of Kaposi’s sarcoma-like lesions in ORF74

www.sciencedirect.com 0165-6147/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2005.11.006

Review

TRENDS in Pharmacological Sciences

Vol.27 No.1 January 2006

57

Box 1. HCMV-encoded G-protein-coupled receptor US28 GPCRs constitute the largest family of membrane-associated proteins involved in regulating intracellular signaling in response to chemically diverse extracellular stimuli [10]. All GPCRs are characterized by the presence of seven hydrophobic transmembrane helices, each consisting of w25–38 consecutive amino acid residues, linked by alternating intracellular or extracellular loops, and an extracellular Nterminus and intracellular C-terminus (Figure I). GPCRs signal through heterotrimeric G proteins consisting of an a- and a bg-subunit, which stimulate effector proteins and result in the activation or inhibition of the production of various second messengers. GPCRs can spontaneously adopt at least two different conformations: an inactive conformation in which receptors do not activate G proteins, and an active conformation in which receptors do activate G proteins to initiate an

intracellular response [11]. These receptor conformations exist in an equilibrium in which the inactive conformations are usually prevalent. Different classes of ligands can either stabilize the inactive receptor conformation (inverse agonists) or stabilize the active receptor conformation (agonists), resulting in decreased or increased receptormediated signaling, respectively. Interestingly, for some (mutant) receptors this equilibrium is shifted towards the active conformation, resulting in constitutive ligand-independent signaling, a condition that has been shown to cause pathological conditions. Currently, it is acknowledged that GPCRs bind, in addition to G proteins, GPCRinteracting proteins and multidomain scaffolding proteins that might regulate intracellular signaling and provide new ways to govern ligand recognition, signaling specificity and receptor trafficking.

Y

T P C V

A E D D Y D F E T T L E A T T T T P T M -N2H 16 14 12

C E S F D Y D Y L E M T S E K C S I L A S V K S K V W S Q D P S N T L N K L L K V Y C L K 277 M V D T I P D H T V F I L F H R N L L L T L T L A L A Q Y L I P E V M F C I A Y H M L L I Y P I L T L W V L E V A A G L P M A S F W I F I F V F A L C T W I V S A F I F V F W L P H C L F C L L L I C S F S D I V V I V L C T A L F S A A E N I A Y C I L A L P N L Y C L A R V K Q Y L F I R D I V R Y V V Y Y Y V G R I A D K S G 129 I F E C P S F H A E C V R R K Q H L R V F Y R Y M R R R I Q R G T I S L L R V A V S Q Q R L I Q S V R C A E D S L T D S S T E R R S P S S R R S F S M S H Y W S V D R S F 350 323

Y

L T D V L N Q K S V P L T F Y L V G F V L G F I S N G F V L F I I T W T

HOOC- P I

TRENDS in Pharmacological Sciences

Figure I. Two-dimensional serpentine representation of the HCMV-encoded US28 receptor, illustrating the N-terminal extracellular domain, the putative seven transmembrane helices and the C-terminal intracellular domain, as determined by amino acid alignment with the bovine rhodopsin structure. Conserved disulfide bridges present in chemokine receptors between the N-terminus and the third extracellular loop, and between the first and second extracellular loops, are indicated by filled circles and a connecting line. A conserved hexad motif present in the N-terminus of US28, and also found in CCR1, CCR2 and CX3CR1, is indicated by a grey box. The three amino acid residues (T12, F14 and Y16) within this motif that cooperatively contribute to the binding of CC-chemokines are highlighted in blue; only Y16 interacts with CX3CL1 [33]. Putative glycosylation sites are marked by a ‘Y’, whereas sulfation of Y16 is indicated by an exposed black circle. Acidic Glu277 (green) in transmembrane helix 7 is conserved in the majority of chemokine receptors and might represent a common interaction point for the basic region (e.g. piperidine) of nonpeptide compounds acting at chemokine receptors. The constitutive activity of US28 is abrogated upon mutation of Arg129 (purple) to alanine, within the ‘DRY’ motif. Mutational and phosphoamino acid analyses reveal that serine residues 323 and 350 are major phosphorylation sites [54,55].

transgenic mice because transgenic mice that express the inactive mutant of ORF74 fail to form Kaposi’s sarcomalike lesions (see references in [5]). HCMV encodes four GPCRs referred to as US27, US28, UL33 and UL78 [1], which show highest homology to the family of chemokine receptors (Figure 1). Chemokine receptors are involved in the regulation of the immune system [13,14] but are also implicated in various pathological processes (e.g. inflammation, atherosclerosis and oncogenesis); furthermore, some of these receptors act as crucial cellular entry factors for HIV [15]. In view of the fundamental role of GPCRs in general, and chemokine receptors in particular, the CMV-encoded receptors might be crucial determinants in CMV pathogenesis and chronic diseases. The GPCR-encoding gene clusters UL33 and UL78 are conserved in all sequenced CMVs, and have presumably been pirated from an ancient host by an ancestral CMV. www.sciencedirect.com

UL33 orthologs (M33 and R33 in MCMV and RCMV, respectively) are fairly well conserved, whereas amino acid sequences have diverged considerably among UL78 orthologs (M78 and R78 in MCMV and RCMV, respectively) [9,16–19]. Interestingly, only primate CMVs contain an additional GPCR-encoding gene cluster, which consists of two adjacent genes in human (H)CMV and chimpanzee (C)CMV (US27 and US28), and five juxtaposed genes in the rhesus macaque (Rh)CMV and African green monkey (S)CMV [16,18,19] (Figure 1). The strict human tropism of HCMV in combination with the uniqueness of two of the four HCMV-encoded GPCRs hampers the experimental analysis of their role in virulence in vivo. HCMV-encoded US27 and US28 proteins display highest sequence identity to the chemokine receptor CX3CR1, whereas UL33 is most similar to the chemokine receptor CCR10 (Figure 1). By contrast, UL78 has limited sequence identity to chemokine

58

Review

TRENDS in Pharmacological Sciences

Vol.27 No.1 January 2006

duffy h c rh t

UL78 r

m sORF3 rhUS28.1 sORF4 rhUS28.2 sORF7 rhUS28.4 sORF6 rhUS28.3 sORF5 h c

t

m r

US27

UL33 h c rh

D6 XCR1 h c

US28 rhUS28.5

CX3CR1 CCR8 CCR5 CCR2 CCR3 CCR1 CCR4 CXCR5 CXCR3 CCR10 CXCR2 CXCR1

CCR9 CCR7 CCR11 CCR6 CXCR6 CXCR4

TRENDS in Pharmacological Sciences

Figure 1. Phylogenetic relationship between host chemokine receptors and CMV-encoded GPCRs. Deduced amino acid (reference) sequences of mouse, rat and human chemokine receptors and CMV-encoded GPCRs were retrieved from the GenBank database at NCBI and analyzed using the ClustalW method (Gonnet series). Chemokine receptor orthologs of mouse, rat and human all cluster in a single branch per subtype, and are each presented as a single branch for clarity. CMV species are indicated as follows: African green monkey (S)CMV (s); chimpanzee, (C)CMV (c); human, (H)CMV (h); mouse (M)CMV (m); rat (R)CMV (r); rhesus macaque, (Rh)CMV (rh); and tupaia, (T)CMV (t).

receptors or any other GPCR, but shares some general conserved GPCR features. Spatiotemporal expression of HCMV-encoded GPCRs HCMV enters the body naturally via epithelial cells of the upper gastrointestinal, respiratory or urogenital tracts [1], and disseminates throughout the body by latently infected monocytes in the blood (see references in [20]). Allogenic stimulation of monocytes induces their differentiation into macrophages, which in latently infected www.sciencedirect.com

cells is accompanied by reactivation of HCMV, leading to the release of infectious virions [20]. Viral replication in permissive cells follows a well-orchestrated sequential cascade of gene expression. The US28- and UL78encoding genes are expressed with early kinetics and require immediate-early-protein-mediated transcriptional activation, whereas US27 and UL33 genes are transcribed with late kinetics after the onset of viral replication [21]. In addition, US28 transcripts have been detected in latently infected monocytic cells in vivo and

Review

TRENDS in Pharmacological Sciences

in vitro [22], suggesting that US28 has a prominent role in immune evasion and HCMV dissemination. Moreover, UL33 [23], UL78 [9], US28, rhUS28.5 [24] and presumably US27 are constituents of the virion. Colocalization of US28 [25], US27 and UL33 [26] with HCMV-encoded envelope glycoproteins gB and gH on virus-wrapping membranes in transfected or HCMVinfected cells indicates that these GPCRs are incorporated in the viral envelope. Although expression of CMVencoded GPCRs on the virion does not appear to be essential for infection of permissive cells in vitro, deletion of either R33/M33 [7,17] or R78/M78 [9,27] has a significant impact on viral dissemination in vivo. Ligands that bind HCMV-encoded GPCRs Despite their sequence similarity to chemokine receptors, UL33 [28] and US27 do not seem to interact with chemokines, and both receptors in addition to UL78 still

Vol.27 No.1 January 2006

59

need to be ‘deorphanized’ (i.e. their natural ligand identified). Interestingly, M33-mediated signaling was recently reported in response to mouse CCL5 [29]. By contrast, US28 binds CX3CL1 in addition to several inflammatory CC-chemokines (i.e. CCLs: 2, 3, 4, 5, 7, 11, 13, 26 and 28) [24]. The capacity of US28 to interact with a broad range of chemokines might contribute to efficient dissemination of HCMV throughout the body via migration of US28-expressing cells along multiple chemokine gradients. Moreover, adhesion of US28-expressing cells [30] and HCMV-virions [24,31] to membrane-associated CX3CL1 molecules, which are expressed on vascular endothelial cells in response to pro-inflammatory cytokines [32], might facilitate the extravasation of latently infected, US28-expressing monocytes into inflamed tissue [22] and viral infection of endothelial cells, respectively (Figure 2). Chemokines bind with high affinity to the conserved hexad motif within the N-terminus of US28 [33].

US28 Chemokine

2

1

3 5

αs

CX3CL1

αq

β γ

αi MAPK

CRE NFAT NF-κB InsP

7

6

α12 α13 Ca2+

8 αq

αi

CD4

4

AP-2 β-Arrestin-2

FAK

HCMV virion adhesion to cell through CX3CL1 –US28 tethering

Constitutive activity and G-protein promiscuity

Chemokine binding and scavenging by constitutive internalization

Cell–cell adhesion through CX3CL1 –US28 tethering

Chemotaxis

Potentiation of chemokine receptor signaling

HIV co-receptor together with CD4 mediates HIV entry

Facilitation of HCMV infection?

Modulation of cellular signaling networks

Evasion of immune system

Facilitation of HCMV infection?

Viral dissemination and potential cell recruitment to artherosclerotic lesion

Modulator of cellular responsiveness and signaling networks

HIV infection

TRENDS in Pharmacological Sciences

Figure 2. Putative roles of the HCMV-encoded US28 receptor. Expression of US28 on the virion envelope might facilitate viral infection by US28-mediated high-affinity binding to host-cell-expressing membrane-associated CX3CL1 (1) [24,31]. US28 modulates cellular signaling networks by constitutive (2) and/or chemokine-induced (3) activation of multiple signaling pathways by coupling to a variety of G proteins [35,42]. In addition, constitutive US28 signaling can potentiate signaling of Gi-coupled receptors (7) [49]. US28 is constitutively phosphorylated by GRKs and internalized via AP-2 adaptor complexes (4) [25,61]. In addition, binding of b-arrestins to the phosphorylated C-terminus of US28 enables the formation of scaffolding complexes that influence activation of several signaling pathways (4) [54]. The rapid and constitutive internalization of US28 enables the sequestration of a wide variety of US28-bound inflammatory chemokines from the surroundings of HCMV-infected cells, thereby limiting the inflammatory immune response (3). The interaction between membrane-associated CX3CL1 and US28 increases the adhesion of US28-expressing monocytes to vascular endothelial cells and facilitates their extravasion (5) [22,32]. US28 induces chemokine-directed chemotaxis of vascular smooth muscle cells (6), and might be involved in the migration of these cells into the vascular intima [36,66]. Both HIV R5 and X4 strains can use US28, when coexpressed with CD4, as a co-receptor for cellular entry (8) [5], which might account for the positive synergism between HCMV and HIV pathologies. Abbreviations: CRE, cAMP response element; FAK, focal adhesion kinase; InsP, inositol phosphate; NFAT, nuclear factor of activated T cells; NF-kB, nuclear factor kB. www.sciencedirect.com

Review

60

TRENDS in Pharmacological Sciences

(a) 125

% InsP production

100 75 50 25 0 AD169

US28

Mock

Infected HFFs (b)

% CRE activation

125 100 75 50 25 0 AD169

UL33

Mock

Infected U373 cells Figure 3. US28- and UL33-mediated signaling in HCMV-infected cells. (a) Human foreskin fibroblasts (HFFs) were either mock-infected or infected with wild-type HCMV (strain AD169) or US28 gene knockout HCMV (i.e. DUS28) and assayed (48 hours post-infection) for inositol phosphate (InsP) accumulation in the absence of ligands (green), or in the presence of 100 nM CX3CL1 (blue) or 10 mM VUF2274 (purple). Reproduced, with permission, from [64]. (b) Astrocytoma cells (U373) were either mock-infected or infected with wild-type HCMV (strain AD169) or UL33 gene knockout HCMV (i.e. DUL33) and assayed (48 hours post-infection) for CREdriven luciferase expression. Reproduced, with permission, from [28]. Cellular responses in AD169- and DUS28- or DUL33-infected cells indicate that US28 is responsible for virus-induced InsP accumulation whereas UL33 is at least in part responsible for virus-induced CRE activation.

Importantly, post-translational sulfation and O-glycosylation of tyrosine and threonine residues, respectively, within this motif is essential for chemokine recognition and the binding kinetics of US28 [33] and host chemokine receptors [34]. Versatile signaling devices Ligand-induced and constitutive signaling activity via promiscuous G-protein coupling To date, no constitutive and/or ligand-induced signaling has been reported for US27 and UL78. However, their presence on the virion either suggests a role during early stages of infection or infers modulation of cellular signaling pathways upon infection. Signaling has been reported for US28 and UL33 (Figure 2). CC chemokines, shown to bind to US28, induce increases in intracellular Ca2C levels and activation of mitogen-activated protein kinase (MAPK) in cells that www.sciencedirect.com

Vol.27 No.1 January 2006

express US28 [35]. CCL2 and CCL5 are also able to induce migration of US28-expressing or HCMV-infected vascular smooth muscle cells (vSMCs), providing a molecular basis for the involvement of HCMV in the progression of atherosclerosis [36]. Moreover, R33-induced vSMC migration contributes crucially to RCMV-accelerated cardiac allograft atherosclerosis and chronic rejection [37]. US28-mediated vSMC migration involves activation of Src and focal adhesion kinase [38], an important mediator of cytoskeletal rearrangement, in addition to activation of RhoA via G12/13a proteins [39]. MCMV-induced vSMC migration is mediated by M33 in response to mouse CCL5 through activation of Rac1 and extracellular signalregulated kinase 1,2 (ERK-1,2) [29]. Importantly, both US28 and UL33 also alter cellular signaling, including inositol phosphate production and activation of NF-kB, CRE and nuclear factor of activated T cells (NFAT), in a constitutive manner (Figure 2), whereas host chemokine receptors do not display, or display only limited, ligand-independent signaling [28,40–42]. This constitutive signaling is not only apparent in transfected cells but also 24–48 h after HCMV infection, as shown by impaired inositol phosphate production and activation of CRE mediated by US28 or UL33 gene knockout viruses, respectively (Figure 3). Thus, HCMV can effectively use these receptors, without the need of a ligand, to modulate multiple signaling networks via activation of effectors and transcription factors within infected cells. Importantly, the repertoire of intracellular signaling pathways activated by HCMV-encoded GPCRs (constitutively or ligand induced) is cell-type specific and dependent on the cellular signalosome. Moreover, because both receptors reside on the virion [24], they are likely to be incorporated into the membrane immediately after viral infection, and thus constitutive activation of these pathways, early after infection, might be of pathophysiological significance. Importantly, the immediate early promoter of HCMV, constituting the genetic switch for progression of viral infection and reactivation, contains four consensus CRE-binding sites and four NF-kB-binding sites. Binding of cognate transcription factors to these sites is required for efficient transactivation of the immediate early promoter [21,43,44]. Moreover, NF-kB is a ubiquitously expressed transcription factor that has a crucial role in the regulation of inducible genes involved in the immune response and inflammatory events associated with, for example, atherosclerosis [45]. NFAT is an important regulator of immune responses, developmental processes and angiogenesis [46]. US28, through constitutive activation of these transcription factors, might induce expression of viral immediate-early proteins and cellular proteins, leading to the alteration of the immune response in favor of viral survival and spreading, and thus might contribute to the onset, progression or enhancement of inflammatory disorders (Figure 2). Unexpectedly, however, US28-mediated constitutive signaling via multiple pathways does not modulate cellular gene expression significantly in HCMV-infected fibroblasts during the late phase of infection (50–98 hours post-infection) [47]. Moreover, US28 appears to activate

Review

TRENDS in Pharmacological Sciences

pro-apoptotic signaling pathways in several cell types, providing US28 with an additional functional property [48]. Interestingly, US28-mediated constitutive signaling potentiates chemokine-induced signaling of the Gi-coupled CCR1 [49] (Figure 2). Because chemokine receptors are key components in leukocytes, smooth muscle and endothelial cells, all primary sites of HCMV infection, HCMV-encoded receptor expression might alter ligand-induced signaling via these receptors and contribute to the onset of CMV pathology. Characteristically, the CMV-encoded receptors show promiscuous G-protein coupling, whereas host chemokine receptors activate primarily Gi/o proteins [10]. The constitutive activation of inositol phosphate production and NF-kB activity by US28 and UL33 involve primarily activation of Gq proteins, whereas CRE-mediated gene transcription is Gs dependent [28,41,42]. R33 and M33 are also able to signal in a constitutively active manner [41,50]. The constitutive signaling of R33 differs from that of UL33 because R33 is only able to couple to Gi/o and Gq, whereas UL33 can activate the Gq, Gi/o and Gs classes. Interestingly, CC chemokines do not modulate the constitutive signaling of US28 in the inositol phosphate, NF-kB and CRE assays [42,51]. By contrast, CX3CL1 acts as inverse agonist in these assays [42]. Surprisingly, however, the same chemokines activate these signaling pathways when US28 loses its capacity to constitutively internalize (see later), while maintaining its constitutive signaling capacity, upon deletion of its C-terminal tail [52]. Hence, differential modulation of constitutive US28 internalization kinetics determines the efficacy of chemokines that act at this receptor. This implies that the cellular context in which US28 is expressed and the presence of differentially expressed adaptor, sorting and/ or scaffolding proteins that interact with the C-terminal tail can, in principle, modulate chemokine responsiveness through the regulation of US28 internalization. Dynamic regulation of US28 Ligand-induced GPCR activation is usually followed by G-protein-coupled receptor kinase (GRK)-mediated phosphorylation of the receptor and the subsequent recruitment of b-arrestins to attenuate G-protein coupling [53]. Interestingly, the C-terminal tail of US28 is constitutively phosphorylated by GRK2 and GRK5, which attenuates Gqa-mediated phospholipase C (PLC) signaling [54]. By contrast, US28-mediated constitutive activation of NF-kB appears to be unaffected by receptor phosphorylation [55]. Importantly, constitutive phosphorylation of the US28 is associated with rapid, agonist-independent receptor endocytosis (Figure 2) [25,55], and is independent of constitutive signaling [52,56]. Consequently, US28 is located primarily in perinuclear endosomes. Truncation or substitution of phosphorylation sites in the US28 C-terminal tail impairs internalization and leads to increased expression in the membrane. Moreover, the constitutive endocytotic property of the US28 C-tail is transposable to other GPCRs [52]. The broad chemokine binding profile in combination with rapid and constitutive internalization kinetics [25] enables US28 to sequester www.sciencedirect.com

Vol.27 No.1 January 2006

61

inflammatory chemokines efficiently from the environment of HCMV-infected cells [57–59] and might thereby reduce the recruitment of leukocytes and limit the inflammatory response (Figure 2) as recently described for the D6 chemokine decoy receptor [60]. US28 internalization is mediated via clathrin-coated vesicles and to a lesser extent via caveolae or lipid rafts, and requires the AP-2 adaptor complex and dynamin [56,61]. Interestingly, US28 internalization is independent of b-arrestins [56,61]. Nevertheless, b-arrestins are recruited to the phosphorylated US28 C-terminus and are required for maximal activation of the p38 signaling pathway [54]. Scaffolding proteins, such as G-protein-coupled-associated sorting protein (GASP) and sorting nexin 1 (SNX1), also bind the C-terminus, presumably regulating intracellular signaling and in particular receptor trafficking [62].

HCMV-encoded GPCRs: potential anti-viral drug targets? GPCRs constitute a highly ‘drugable’ class of membraneassociated proteins and account for w50% of protein targets for therapeutic interventions. The awareness that chemokines and their cognate receptors have a prominent role in numerous pathophysiological processes urges the quest for bioavailable small-molecule antagonists that specifically block chemokine receptor functioning [15]. The identification of non-peptide chemokine receptor antagonists is proceeding at a rapid pace, with some compounds entering Phase II or III clinical trials. In addition to CCR5 and CXCR4, other chemokine receptors, including US28, can serve as a co-receptor for HIV-1 infection in vitro (Figure 2) (see references in [5]). Co-infection of HIV and CMV has been detected in vivo in brain cells devoid of CCR5 expression, which suggests that a direct role exists for herpesviruses and, in particular, US28 in the pathogenesis of AIDS [63]. In view of the potential roles of CMV-encoded chemokine receptor homologs in viral dissemination, redirecting intracellular signaling pathways and immune evasion (Figure 2), small non-peptide compounds that inhibit constitutive signaling of UL33 or US28 and thus act as inverse agonists and/or inhibit chemokine or HIV binding to US28 can be considered as promising therapeutics for clinical anti-viral intervention. Recently, small non-peptide inverse agonists [e.g. VUF2274 (see Chemical names)] derived from a CCR1 antagonist {5-(4-(4-chlorophenyl)-4-hydroxypiperidin-yl)2,2-diphenylpentane-nitrile} have been identified [64,65] that dose-dependently inhibit: (i) US28-mediated constitutive activation of PLC in both transiently transfected cells and HCMV-infected fibroblasts; (ii) CCL5 binding to US28; and (iii) US28-mediated HIV entry in cells cotransfected with CD4. Importantly, VUF2274 inhibits CCL5 binding in a noncompetitive manner, thus acting as an allosteric modulator. Chemical names VUF2274: 5-(4-(4-chlorophenyl)-4-hydroxypiperidin-yl)-2,2diphenylpentane-nitrile

62

Review

TRENDS in Pharmacological Sciences

Future perspectives Exploitation of the chemokine receptor system through molecular mimicry appears to be an effective means to assist viruses in evading immune surveillance, contribute to viral dissemination and receptor-mediated entry. US28 is by far the best-characterized receptor. In accordance with the ability of US28 to respond to a broad spectrum of chemokines, and the ability of US28 and UL33 to signal in a constitutively active manner, these receptors appear to be versatile signaling devices that modulate cellular signaling networks, thereby reprogramming the cellular machinery to modulate cellular function after infection. The fact that US27 and UL78 both reside within the virion implies a potential role for these receptors, either early after infection or indirectly as modulators of cellular signaling. Further research needs to be performed to reveal their role. Although many attractive roles have been attributed to this class of receptors, little is known about their true (patho)physiological potential. The biological significance of members of the UL33 and UL78 family in the pathogenesis of CMV infections has been demonstrated in studies using recombinant rodent CMVs that carry a disrupted gene or lack the respective gene [7–9,17,27,37]. However, in vivo analysis of US27 and US28 is hampered by the strict higher primate tropism of the CMVs that acquired these genes. Development of adequate model systems that enable in vivo analyses of the (H)CMVencoded receptors, together with the availability of vGPCR-knockout strains of (H/C)CMV and specific inhibitors [inverse agonists or small interfering RNA (siRNA)], are essential to elucidate the contribution of HCMVencoded receptors and their constitutive activity to HCMV pathogenesis. Acknowledgements H.F.V. was supported by the Technology Foundation STW, and M.J.S. was supported by the Royal Netherlands Academy of Arts and Sciences.

References 1 Landolfo, S. et al. (2003) The human cytomegalovirus. Pharmacol. Ther. 98, 269–297 2 Deayton, J.R. et al. (2004) Importance of cytomegalovirus viraemia in risk of disease progression and death in HIV-infected patients receiving highly active antiretroviral therapy. Lancet 363, 2116–2121 3 Evers, D.L. et al. (2004) Cellular stress and signal transduction responses to human cytomegalovirus infection. Microbes Infect. 6, 1084–1093 4 Wang, X. et al. (2005) Integrin alphavbeta3 is a coreceptor for human cytomegalovirus. Nat. Med. 11, 515–521 5 Sodhi, A. et al. (2004) Viral hijacking of G-protein-coupled-receptor signalling networks. Nat. Rev. Mol. Cell Biol. 5, 998–1012 6 Couty, J.P. and Gershengorn, M.C. (2005) G-protein-coupled receptors encoded by human herpesviruses. Trends Pharmacol. Sci. 26, 405–411 7 Beisser, P.S. et al. (1998) The R33 G protein-coupled receptor gene of rat cytomegalovirus plays an essential role in the pathogenesis of viral infection. J. Virol. 72, 2352–2363 8 Beisser, P.S. et al. (1999) Deletion of the R78 G protein-coupled receptor gene from rat cytomegalovirus results in an attenuated, syncytium-inducing mutant strain. J. Virol. 73, 7218–7230 9 Oliveira, S.A. and Shenk, T.E. (2001) Murine cytomegalovirus M78 protein, a G protein-coupled receptor homologue, is a constituent of the virion and facilitates accumulation of immediate-early viral mRNA. Proc. Natl. Acad. Sci. U. S. A. 98, 3237–3242 www.sciencedirect.com

Vol.27 No.1 January 2006

10 Offermanns, S. (2003) G-proteins as transducers in transmembrane signalling. Prog. Biophys. Mol. Biol. 83, 101–130 11 Milligan, G. (2003) Constitutive activity and inverse agonists of G protein-coupled receptors: a current perspective. Mol. Pharmacol. 64, 1271–1276 12 Schoneberg, T. et al. (2004) Mutant G-protein-coupled receptors as a cause of human diseases. Pharmacol. Ther. 104, 173–206 13 Moser, B. et al. (2004) Chemokines: multiple levels of leukocyte migration control. Trends Immunol. 25, 75–84 14 Romagnani, P. et al. (2004) CXC chemokines: the regulatory link between inflammation and angiogenesis. Trends Immunol. 25, 201–209 15 Johnson, Z. et al. (2005) Multi-faceted strategies to combat disease by interference with the chemokine system. Trends Immunol. 26, 268–274 16 Davison, A.J. et al. (2003) The human cytomegalovirus genome revisited: comparison with the chimpanzee cytomegalovirus genome. J. Gen. Virol. 84, 17–28 17 Davis-Poynter, N.J. et al. (1997) Identification and characterization of a G protein-coupled receptor homolog encoded by murine cytomegalovirus. J. Virol. 71, 1521–1529 18 Hansen, S.G. et al. (2003) Complete sequence and genomic analysis of rhesus cytomegalovirus. J. Virol. 77, 6620–6636 19 Sahagun-Ruiz, A. et al. (2004) Simian Cytomegalovirus Encodes Five Rapidly Evolving Chemokine Receptor Homologues. Virus Genes 28, 71–83 20 Streblow, D.N. and Nelson, J.A. (2003) Models of HCMV latency and reactivation. Trends Microbiol. 11, 293–295 21 Mocarski, E.S. and Courcelle, C.T. (2001) Cytomegalovirus and their replication. In Fields Virology (Knipe, D. and Howley, P., eds), pp. 2629–2673, Lippincott, Williams and Wilkins 22 Beisser, P.S. et al. (2001) Human cytomegalovirus chemokine receptor gene US28 is transcribed in latently infected THP-1 monocytes. J. Virol. 75, 5949–5957 23 Margulies, B.J. et al. (1996) Identification of the human cytomegalovirus G protein-coupled receptor homologue encoded by UL33 in infected cells and enveloped virus particles. Virology 225, 111–125 24 Penfold, M.E. et al. (2003) Characterization of the rhesus cytomegalovirus US28 locus. J. Virol. 77, 10404–10413 25 Fraile-Ramos, A. et al. (2001) The human cytomegalovirus US28 protein is located in endocytic vesicles and undergoes constitutive endocytosis and recycling. Mol. Biol. Cell 12, 1737–1749 26 Fraile-Ramos, A. et al. (2002) Localization of HCMV UL33 and US27 in endocytic compartments and viral membranes. Traffic 3, 218–232 27 Kaptein, S.J. et al. (2003) The rat cytomegalovirus R78 G proteincoupled receptor gene is required for production of infectious virus in the spleen. J. Gen. Virol. 84, 2517–2530 28 Casarosa, P. et al. (2003) Constitutive signaling of the human cytomegalovirus-encoded receptor UL33 differs from that of its rat cytomegalovirus homolog R33 by promiscuous activation of G proteins of the Gq, Gi, and Gs classes. J. Biol. Chem. 278, 50010–50023 29 Melnychuk, R.M. et al. (2005) Mouse cytomegalovirus M33 is necessary and sufficient in virus-induced vascular smooth muscle cell migration. J. Virol. 79, 10788–10795 30 Haskell, C.A. et al. (2000) Unique role of the chemokine domain of fractalkine in cell capture. Kinetics of receptor dissociation correlate with cell adhesion. J. Biol. Chem. 275, 34183–34189 31 Kledal, T.N. et al. (1998) Selective recognition of the membrane-bound CX3C chemokine, fractalkine, by the human cytomegalovirusencoded broad-spectrum receptor US28. FEBS Lett. 441, 209–214 32 Bazan, J.F. et al. (1997) A new class of membrane-bound chemokine with a CX3C motif. Nature 385, 640–644 33 Casarosa, P. et al. (2005) CC and CX3C chemokines differentially interact with the N terminus of the human cytomegalovirus-encoded US28 receptor. J. Biol. Chem. 280, 3275–3285 34 Fernandez, E.J. and Lolis, E. (2002) Structure, function, and inhibition of chemokines. Annu. Rev. Pharmacol. Toxicol. 42, 469–499 35 Billstrom, M.A. et al. (1998) Intracellular signaling by the chemokine receptor US28 during human cytomegalovirus infection. J. Virol. 72, 5535–5544 36 Streblow, D.N. et al. (1999) The human cytomegalovirus chemokine receptor US28 mediates vascular smooth muscle cell migration. Cell 99, 511–520

Review

TRENDS in Pharmacological Sciences

37 Streblow, D.N. et al. (2005) Rat cytomegalovirus-accelerated transplant vascular sclerosis is reduced with mutation of the chemokinereceptor R33. Am. J. Transplant. 5, 436–442 38 Streblow, D.N. et al. (2003) Human cytomegalovirus chemokine receptor US28-induced smooth muscle cell migration is mediated by focal adhesion kinase and Src. J. Biol. Chem. 278, 50456–50465 39 Melnychuk, R.M. et al. (2004) Human cytomegalovirus-encoded G protein-coupled receptor US28 mediates smooth muscle cell migration through Galpha12. J. Virol. 78, 8382–8391 40 Minisini, R. et al. (2003) Constitutive inositol phosphate formation in cytomegalovirus-infected human fibroblasts is due to expression of the chemokine receptor homologue pUS28. J. Virol. 77, 4489–4501 41 Waldhoer, M. et al. (2002) Murine cytomegalovirus (CMV) M33 and human CMV US28 receptors exhibit similar constitutive signaling activities. J. Virol. 76, 8161–8168 42 Casarosa, P. et al. (2001) Constitutive signaling of the human cytomegalovirus-encoded chemokine receptor US28. J. Biol. Chem. 276, 1133–1137 43 Keller, M.J. et al. (2003) Role of the human cytomegalovirus major immediate-early promoter’s 19-base-pair-repeat cyclic AMP-response element in acutely infected cells. J. Virol. 77, 6666–6675 44 DeMeritt, I.B. et al. (2004) Activation of the NF-kappaB pathway in human cytomegalovirus-infected cells is necessary for efficient transactivation of the major immediate-early promoter. J. Virol. 78, 4498–4507 45 Kumar, A. et al. (2004) Nuclear factor-kappaB: its role in health and disease. J. Mol. Med. 82, 434–448 46 Horsley, V. and Pavlath, G.K. (2002) NFAT: ubiquitous regulator of cell differentiation and adaptation. J. Cell Biol. 156, 771–774 47 Hertel, L. and Mocarski, E.S. (2004) Global analysis of host cell gene expression late during cytomegalovirus infection reveals extensive dysregulation of cell cycle gene expression and induction of Pseudomitosis independent of US28 function. J. Virol. 78, 11988–12011 48 Pleskoff, O. et al. (2005) The human cytomegalovirus-encoded chemokine receptor US28 induces caspase-dependent apoptosis. FEBS J 272, 4163–4177 49 Bakker, R.A. et al. (2004) Constitutively active Gq/11-coupled Receptors Enable Signaling by Co-expressed Gi/o-coupled Receptors. J. Biol. Chem. 279, 5152–5161 50 Gruijthuijsen, Y.K. et al. (2002) The rat cytomegalovirus R33-encoded G protein-coupled receptor signals in a constitutive fashion. J. Virol. 76, 1328–1338 51 McLean, K.A. et al. (2004) Similar activation of signal transduction pathways by the herpesvirus-encoded chemokine receptors US28 and ORF74. Virology 325, 241–251 52 Waldhoer, M. et al. (2003) The carboxyl terminus of human cytomegalovirus-encoded 7 transmembrane receptor US28 camouflages agonism by mediating constitutive endocytosis. J. Biol. Chem. 278, 19473–19482

Vol.27 No.1 January 2006

53 Lefkowitz, R.J. and Shenoy, S.K. (2005) Transduction of receptor signals by beta-arrestins. Science 308, 512–517 54 Miller, W.E. et al. (2003) G-protein-coupled receptor (GPCR) kinase phosphorylation and beta-arrestin recruitment regulate the constitutive signaling activity of the human cytomegalovirus US28 GPCR. J. Biol. Chem. 278, 21663–21671 55 Mokros, T. et al. (2002) Surface expression and endocytosis of the human cytomegalovirus-encoded chemokine receptor US28 is regulated by agonist-independent phosphorylation. J. Biol. Chem. 277, 45122–45128 56 Droese, J. et al. (2004) HCMV-encoded chemokine receptor US28 employs multiple routes for internalization. Biochem. Biophys. Res. Commun. 322, 42–49 57 Bodaghi, B. et al. (1998) Chemokine sequestration by viral chemoreceptors as a novel viral escape strategy: withdrawal of chemokines from the environment of cytomegalovirus-infected cells. J. Exp. Med. 188, 855–866 58 Billstrom, M.A. et al. (1999) Depletion of extracellular RANTES during human cytomegalovirus infection of endothelial cells. Am. J. Respir. Cell Mol. Biol. 21, 163–167 59 Randolph-Habecker, J.R. et al. (2002) The expression of the cytomegalovirus chemokine receptor homolog US28 sequesters biologically active CC chemokines and alters IL-8 production. Cytokine 19, 37–46 60 Jamieson, T. et al. (2005) The chemokine receptor D6 limits the inflammatory response in vivo. Nat. Immunol. 6, 403–411 61 Fraile-Ramos, A. et al. (2003) Endocytosis of the viral chemokine receptor US28 does not require beta-arrestins but is dependent on the clathrin-mediated pathway. Traffic 4, 243–253 62 Heydorn, A. et al. (2004) A library of 7TM receptor C-terminal tails. Interactions with the proposed post-endocytic sorting proteins ERMbinding phosphoprotein 50 (EBP50), N-ethylmaleimide-sensitive factor (NSF), sorting nexin 1 (SNX1), and G protein-coupled receptor-associated sorting protein (GASP). J. Biol. Chem. 279, 54291–54303 63 Nelson, J.A. et al. (1988) HIV and HCMV coinfect brain cells in patients with AIDS. Virology 165, 286–290 64 Casarosa, P. et al. (2003) Identification of the first nonpeptidergic inverse agonist for a constitutively active viral-encoded G proteincoupled receptor. J. Biol. Chem. 278, 5172–5178 65 Hulshof, J.W. et al. (2005) Synthesis and structure–activity relationship of the first nonpeptidergic inverse agonists for the human cytomegalovirus encoded chemokine receptor US28. J. Med. Chem. 48, 6461–6471 66 Streblow, D.N. et al. (2001) The HCMV chemokine receptor US28 is a potential target in vascular disease. Curr. Drug Targets Infect. Disord. 1, 151–158

Previously published articles in the Constitutive Receptor Activity series The physiological significance of constitutive receptor activity (Editorial) Terry Kenakin, 26, 603–605 (Dec 2005) Historical review: Negative efficacy and the constitutive activity of G-protein-coupled receptors Tommaso Costa and Susanna Cotecchia, 26, 618–624 (Dec 2005) Physiological relevance of constitutive activity of 5-HT2A and 5-HT2C receptors Kelly A. Berg, John A. Harvey, Umberto Spampinato and William P. Clarke, 26, 625–630 (Dec 2005) Coming next month: Recent developments in constitutive receptor activity and inverse agonism, and their potential for GPCR drug discovery Richard A. Bond and Ad P. IJzerman www.sciencedirect.com

63